Open Collections

UBC Theses and Dissertations

UBC Theses Logo

UBC Theses and Dissertations

Investigations into a biological control strategy for Lodgepole Pine Dwarf Mistletoe Ramsfield, Tod Donald 2002

Your browser doesn't seem to have a PDF viewer, please download the PDF to view this item.

Item Metadata

Download

Media
831-ubc_2002-75071X.pdf [ 8.76MB ]
Metadata
JSON: 831-1.0090452.json
JSON-LD: 831-1.0090452-ld.json
RDF/XML (Pretty): 831-1.0090452-rdf.xml
RDF/JSON: 831-1.0090452-rdf.json
Turtle: 831-1.0090452-turtle.txt
N-Triples: 831-1.0090452-rdf-ntriples.txt
Original Record: 831-1.0090452-source.json
Full Text
831-1.0090452-fulltext.txt
Citation
831-1.0090452.ris

Full Text

Investigations into a Biological Control Strategy for Lodgepole Pine Dwarf Mistletoe By Tod Donald Ramsfield B.Sc. University of Victoria, 1994 M.Sc. Simon Fraser University, 1998 A THESIS SUBMITTED IN P A R T I A L F U L F U L L M E N T OF T H E REQUIREMENTS FOR THE D E G R E E OF DOCTOR OF PHILOSOPHY In THE F A C U L T Y OF G R A D U A T E STUDIES (Faculty of Forestry, Department of Forest Sciences) We accept this thesis as conforming to the required standard  i  THE UNIVERSITY OF BRITISH C O L U M B I A May 2002 © Tod Ramsfield,, 2002  U B C Rare Books and Special Collections - Thesis Authorisation Form  Page 1 of 1  In p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t of the requirements f o r an advanced degree at the U n i v e r s i t y of B r i t i s h Columbia, I agree t h a t the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e and study. I f u r t h e r agree t h a t p e r m i s s i o n f o r e x t e n s i v e c o p y i n g of t h i s t h e s i s f o r s c h o l a r l y purposes may be g r a n t e d by the head of my department or by h i s or her r e p r e s e n t a t i v e s . I t i s understood t h a t c o p y i n g or p u b l i c a t i o n of t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l not be a l l o w e d without my w r i t t e n p e r m i s s i o n .  Department of  /^^S7~  Sr£<r~ *Z c  The U n i v e r s i t y of B r i t i s h Columbia Vancouver, Canada  Date  Si Ay  Z-^^oo  <L.  http://www.library.ubc.ca/spcoll/mesauth.html  ' 5/29/02  Abstract A n inundative biological control strategy for lodgepole pine dwarf mistletoe (Arceuthobium americanum) parasitizing lodgepole pine (Pinus contorta var. contorta) in British Columbia was investigated in this study. Of 36 collection sites in British Columbia and Alberta, Caliciopsis arceuthobii was observed at 8 sites and Colletotrichum gloeosporioides was collected from 23 sites and 187 isolates were recovered in culture. It was decided to focus on C. gloeosporioides because it damaged all parts of male and female A. americanum infections, grew readily in culture, produced abundant inoculum in culture and its distribution coincided with the range of A. americanum that was sampled in this study. A n isolate of C. gloeosporioides was selected based on growth characteristics and formulated using the 'Stabileze' method for inoculation of A. americanum in a field trial. Two months after inoculation, the average disease rating of A. americanum infections treated with C. gloeosporioides was significantly higher than the controls. One year after inoculation, the average number of fruit present on A. americanum swellings that were treated with C. gloeosporioides was reduced, but the difference between the treatments and controls was not significant. The effect of C. gloeosporioides on the endophytic system of A. americanum was determined through culturing and histopathologic^ examination. Colletotrichum gloeosporioides was cultured from the basal cup region but not from woody tissues. No fungal hyphae were observed within the endophytic tissues of A. americanum; however, two different types of hyphae were observed in the outer dead bark and on the bark surface. Analysis of the distribution of C. gloeosporioides within the canopy of lodgepole pine suggested that the presence of C. gloeosporioides was not related to crown position; under natural conditions, all A. americanum was susceptible to C. gloeosporioides. A study designed 5  to follow C. arceuthobii infection of A. americanum over time found that the fungus caused an average fruit reduction of 57% each year over the first three years, and a predicted reduction of 39% in the fourth year of the study and that the fungus was able to naturally infect disease free A. americanum. The maximum biocontrol treatment periodicity required for prevention of fruit production was determined to be 3 years, based on the interval between shoot removal and fruit production.  ii  Table of Contents Abstract  ii  Table of Contents  iii  List of Tables  xi  List of Figures  xiii  Acknowledgements  xviii  Chapter 1: Literature Review  1  1.1 Background  1  1.2 Dwarf mistletoes in British Columbia  1  1.2.1 Arceuthobium americanum  1  1.2.2 Arceuthobium tsugense  2  1.2.3 Arceuthobium laricis  3  1.2.4 Arceuthobium douglasii  4  1.3 Life cycle of A. americanum  4  1.4 Effects of dwarf mistletoe infection  6  1.5 Dwarf mistletoes as agents of biodiversity  8  1.6 Silvicultural control of dwarf mistletoe  9  1.7 Biological control  12  1.8 Examples of inundative biological control agents  14  1.9 Past dwarf mistletoe biological control studies  17  iii  1.10 Fungi that infect Arceuthobium americanum  19  1.10.1 Caliciopsis arceuthobii  19  1.10.2 Cylindrocarpon gillii  20  1.10.3 Colletotrichum gloeosporioides  21  1.10.4 Resin Disease  22  1.10.5 Associations with rust fungi 1.11 Research objectives  22 23  Chapter 2 - Development of a biological control strategy for Arceuthobium americanum, Part I: Discovery and Development  25  2.1 Introduction  25  2.2 Materials and Methods  25  2.2.1 Collection of diseased A. americanum and hyperparasite isolation  25  2.2.2 Growth characteristics of Colletotrichum gloeosporioides  27  2.2.3 Sodium alginate - kaolin clay formulation  28  2.2.4 Inoculation of Arceuthobium tsugense with sodium alginate formulated Colletotrichum gloeosporioides under greenhouse conditions 2.3 Results  30 32  2.3.1 Collection of fungal hyperparasites of A. americanum  32  2.3.2 Growth characteristics of Colletotrichum gloeosporioides 2.3.3 Formulation  41 46  2.3.4 Inoculation of Arceuthobium tsugense with sodium alginate formulated Colletotrichum gloeosporioides under greenhouse conditions 2.4 Discussion  46 47  iv  2.4.1 Collection of diseased A. americanum and hyperparasite isolation  47  2.4.2 Growth characteristics of Colletotrichum gloeosporioides 2.4.3 Formulation  49 50  2.4.4 Inoculation of Arceuthobium tsugense with Colletotrichum gloeosporioides under greenhouse conditions  51  Chapter 3 - Development of a biological control strategy for Arceuthobium americanum, Part II: Field trial  53  3.1 Introduction  53  3.2 Materials and Methods  55  3.2.1 'Stabilize' formulation  55  3.2.2 Site description  56  3.2.3 Experimental design  56  3.2.4 Pre-treatment assessment  58  3.2.5 Treatment application dates  59  3.2.6 Treatment assessment  60  3.2.7 Sampling at one year  60  3.2.8 Statistical analysis  61  3.3 Results  62  3.3.1 'Stabileze' formulation  62  3.3.2 Establishment of C. gloeosporioides  62  3.3.3 Cut shoot inoculation - Treatment 2 3.3.4 Dead replicates one year following inoculation 3.3.5 Effect of C. gloeosporioides on A. americanum fruit production v  63 64 64  3.3.6 Number offemale flowers  66  3.3.7 Effect of C. gloeosporioides on A. americanum shoot height  67  3.3.8 Effect of C. gloeosporioides on the number of A. americanum shoots  67  3.3.9 Effect of C. gloeosporioides on A. americanum bud production  68  3.3.10 Analysis of disease rating  69  3.3.11 Vigour rating analysis  71  3.3.12 Effect of increased humidity  72  3.3.13 Sampling in 2001  72  3.4 Discussion  73  Chapter 4 - Cultural and histopathological examination of the infection of the endophytic system of Arceuthobium americanum by Colletotrichum gloeosporioides  77  4.1 Introduction  77  4.2 Materials and Methods  80  4.2.1 A. americanum naturally infected with C. gloeosporioides  80  4.2.2 Artificial infection of A. americanum with C. gloeosporioides  81  4.2.3 Paraffin embedding.  82  4.2.4 Staining  83  4.2.5 Observation and photomicrography 4.3 Results  83 83  4.3.1 Culturing andfield observation of C. gloeosporioides inoculated A. americanum  83  4.3.1.1 A. americanum naturally infected with C. gloeosporioides vi  83  4.3.1.2 Artificial infection of A. americanum with C. gloeosporioides 4.3.2 Histopathological examination  84 86  4.3.2.1 C. gloeosporioides infected A. americanum shoots  86  4.3.2.2 C. gloeosporioides infected A. americanum parasitizing P. contorta var. latifolia branch tissue  89  4.3.2.3 C. gloeosporioides inoculated A americanum 4.4 Discussion  94 94  4.4.1 Culturing  94  4.4.2 Histopathology  95  4.4.3 Potential C. gloeosporioides exclusion mechanism 4.4.4 Conclusion  96 96  4.4.5 Future research  97  Chapter 5: Incidence of Colletotrichum gloeosporioides on Arceuthobium americanum in the crown of lodgepole pine  98  5.1 Introduction  98  5.2 Materials and Methods  99  5.2.1 Stand and tree selection  99  5.2.2 Data collection  99  5.2.2.1 Trees  99  5.2.2.2 Arceuthobium americanum 5.2.3 Statistical Analysis  99 100  5.3 Results  100  5.3.1 Individual trees  100 vii  5.3.2 Crown position  102  5.3.3 Canopy position  103  5.3.4 Canopy aspect  105  5.3.5 A. americanum sex and the presence of C. gloeosporioides  106  5.3.6 A. americanum horizontal canopy position 5.4 Discussion  106 107  Chapter 6 - Infection of Arceuthobium americanum by Caliciopsis arceuthobii  Ill  6.1 Introduction  111  6.2 Materials and Methods  111  6.2.1 Stand characteristics  Ill  6.2.2 Arceuthobium americanum selection 6.2.3 Data collection and analysis  112 112  6.3 Results  114  6.3.1 Caliciopsis arceuthobii infection of Arceuthobium americanum  114  6.3.2 Fruit production on A. americanum  115  6.3.3 Movement in the stand  117  6.4 Discussion  118  Chapter 7 - Arceuthobium americanum response to shoot removal.... 121 7.1 Introduction  121  7.2 Materials and Methods  122  7.2.1 Partial shoot removal experiment. viii  123  7.2.2 Statistical analysis  124  7.2.3 Total shoot removal  124  7.3 Results  126  7.3.1 Partial shoot removal experiment  126  7.3.1.1 Effects of treatment on growth of the endophytic system  126  7.3.1.2 New shoot production  127  7.3.1.3 Fruit production  129  7.3.1.4 Cut shoots  130  7.3.1.5 Control  131  7.3.2 Total shoot removal  131  7.3.2.1 Fruit production  133  7.3.2.2 Endophytic system growth  135  7.3.2.3 Shoot production  135  7.4 Discussion  136  7.4.1 Endophytic system growth  136  7.4.2 New shoot production  137  7.4.3 Fruit production  137  7.4.4 Response of shoots to die backfromthe tip  138  7.4.5 Shoot characteristics over time  138  7.4.6 Biological control treatment periodicity  139  7.4.7 Conclusions  140  Chapter 8 - Conclusions  141  Literature Cited  145 ix  Appendix 1 - Temperature on inoculation day (July 23, 2000) and immediately following  157  Appendix 2 - Relative humidity on inoculation day and immediately following  158  Appendix 3 - Histological staining methods  159  Appendix 4 - ANOVA tables for canopy study, Chapter 6  162  Appendix 5 - Diagrammatic representation of the lifecycle of Caliciopsis arceuthobii and fruit production by Arceuthobium americanum  163  Appendix 6 - Presence of Caliciopsis arceuthobii on Arceuthobium americanum  165  x  List of Tables Table 2-1. Treatments formulated in sodium alginate - kaolin clay and number of replicates applied to A. tsugense infecting T. heterophylla  32  Table 2-2. Sites visited during the collection phase of the research program and the number of Colletotrichum gloeosporioides isolates collected from, and where Caliciopsis arceuthobii was observed on, Arceuthobium americanum parasitizing Pinus contorta var. latifolia  34  Table 3-1. Treatment description and number of replicates per treatment in the field trial designed to test the efficacy of C. gloeosporioides as a biological control agent for A. americanum  57  Table 3-2. Vigour rating classes developed to classify individual A. americanum infections  58  Table 3-3. Number of living dwarf A. americanum infections prior to treatment and one year following treatment application  64  Table 3-4. Distribution of the number of fruit present on A. americanum infections in 2000 and 2001  66  Table 3-5. Number of A. americanum infections of each disease rating (DR) for all treatments two months after inoculation  70  Table 3-6. Number of A. americanum infections of each disease rating (DR) for all treatments one year after inoculation  71  Table 4-1. Recovery of fungi in culture from A. americanum subjected to treatment 2 and 3 during the field trial. A total of 10 replicates of the treatment and 1 of the control were randomly selected  86  Table 5-1. Characteristics of individual trees surveyed in this study  101  Table 5-2. Infection of A. americanum by C. gloeosporioides at different tree crown thirds  102  Table 6-1. Average reduction in fruit production caused by Caliciopsis arceuthobii infection of Arceuthobium americanum  116  Table 7-1. Treatments applied at the partial shoot experiment and number of replicates. 124 Table 7-2. Mortality two years after treatment xi  126  Table 7-3. Partial shoot removal experiment. Average number of fruit present on female A. americanum infections of each treatment at every assessment and average number of flowers present at the final assessment  129  Table 7-4. Average response of each variable measured up to 3 years following treatment application at both experimental locations. A l l measurements are recorded in centimeters. Trt = treatment, Ck = control  132  Table 7-5. Number of control replicates of A. americanum in each category to record consecutive crops of fruit at Canal Flats and Lytton  135  Table appendix 4-1. Two-way analysis of variance to compare trees and canopy thirds. 162 Table appendix 4-2. Two-way analysis of variance to compare trees and canopy aspect. 162 Table appendix 6-1. Number of Caliciopsis arceuthobii infected Arceuthobium americanum flowers observed at the Knife Creek Block of the Alex Fraser Research Forest, near 150 Mile House, British Columbia  xii  165  List of Figures Figure 2-1. Laboratory apparatus for sodium alginate - kaolin clay formulation of C. gloeosporioides. 1. Magnetic stir plate; 2. sodium alginate - kaolin clay + C. gloeopsorioides;3. peristaltic pump, 4. Pasteur pipettes; 5. 0.25M CaCL; solution and pellets  29  Figure 2-2. Geographic location of: A . Arceuthobium americanum collection sites, B . Sites where Colletotrichum gloeosporioides isolates were collected and, C. Where Caliciopsis arceuthobii was observed during this study  37  Figure 2-3. Colletotrichum gloeosporioides infection of Arceuthobium americanum and morphological characteristics. 1. Lesion on shoot, scale bar represents 1 mm, E = epidermis. 2. Acervuli exuding conidia (C), scale bar represents 1 mm. 3. Conidia (C) production on diseased fruit, scale bar represents 1 mm. 4. Conidia (C), scale bar represents 100 um. 5. Appressoria (A), scale bar represents 100 um. 6. Culture on M E A  39  Figure 2-4. Perithecia (P) of Caliciopsis arceuthobii infecting female flowers of Arceuthobium americanum. Scale bar represents 1 mm  41  Figure 2-5. C. gloeosporioides colony diameter (mm) after 12 days incubation at temperatures ranging from 0°C to 35°C. Points plotted are the mean +/- standard error of the mean  42  Figure 2-6. Colony growth. Increase in colony diameter of C. gloeosporioides incubated at 20°C over 20 days. Mean +/- standard error of the mean  43  Figure 2-7. Percent conidial germination of C. gloeosporioides isolates at temperatures ranging from 0°C to 35°C  44  Figure 2-8. Germ tube elongation at temperatures ranging from 0°C to 35°C after 18 hours incubation. Mean +/- standard error of the mean  45  Figure 3 -1. Typical A. americanum infections of each vigour rating. 1. Vigour rating 1. 2. Vigour rating 2. 3. Vigour rating 3. 4. Vigour rating 4. 5. Vigour rating 5... 59 Figure 3-2. Infection of A. americanum by C. gloeosporioides as a result of application of treatment 1, 'Stabileze' formulated C. gloeosporioides sprayed to run-off (DR = disease rating, V R = vigour rating). This replicate was not enclosed with a polyethylene bag. A . Pre-inoculation, D R = 0, V R = 5. B . One month postxm  inoculation, D R = 1, V R = 5. C, Two months post-inoculation, D R = 2, V R = 5. D. One year post-inoculation, D R = 5, V R = 0  63  Figure 3-3. Average number of fruit per female A. americanum infection of each treatment prior to treatment application in 2000 and one year following application in 2001. Treatment 2 excluded from 2001 A N O V A analysis. Mean +/- standard error of the mean  65  Figure 3-4. Average number of female flowers present one year after inoculation. Treatment 2 excluded from A N O V A . Mean +/- standard error of the mean  66  Figure 3-5. Average maximum shoot height (mm) of A. americanum swellings prior to treatment application in 2000 and one year following application in 2001. Treatment 2 excluded from 2001 A N O V A . Mean +/- standard error of the mean. 67 Figure 3-6. Average number of shoots per A. americanum infection prior to treatment application in 2000 and following inoculation in 2001. Treatment 2 excluded from A N O V A in 2001. Mean +/- standard error of the mean  68  Figure 3-7. Average number of buds (shoots <5mm high) present on A. americanum infections prior to treatment in 2000 and one year following treatment in 2001. Treatment 2 included in A N O V A in 2001. Mean +/- standard error of the mean... 69 Figure 3-8. Average disease rating of A. americanum infections two months and one year following inoculation. Mean +/- standard error of the mean  70  Figure 3-9. Average vigour rating of A. americanum prior to treatment application and at one year following application. Treatment 2 excluded from A N O V A in 2001. Mean +/- standard error of the mean  72  Figure 4-1. Colletotrichum gloeosporioides infected Arceuthobium americanum shoots. Safranin - Picro=analine stain. Scale bars represent 100 um. 1. Acervulus (A) of C. gloeosporioides producing condia (C). Longitudinal section. 2. Acervulus of C. gloeosporioides. Cross section. Note: hyphae present in the center of the shoot at the xylem core. Epidermis (E), xylem (X). 3. Acervulus of C. gloeosporioides prior to breaking through the epidermal layer. Acervuli is integrated with host mesophyll cells. Hypha (H). 4. Longitudinal section of diseased shoot with intercellular hyphae (H) of C. gloeosporioides. Cells are disrupted and not organized. 5. Longitudinal section of healthy A. americanum shoot tissue with xiv  healthy, ordered mesophyll cells. Epidermis (E). 6. Diseased and healthy A. americanum tissue, showing the extent of necrosis. Acervuli (A) are present on the upper and lower shoot surfaces, close to the node (node is not in micrograph). Epidermis (E)  87  Figure 4-2. Transverse sections of healthy and C. gloeosporioides infected A. americanum tissues on and in lodgepole pine bark. Scale bars represents 100 urn. 1. Spores (S) of melanized hyphae, on the surface of the bark? 2. Melanized hyphae (MH) on bark surface. 3. Melanized hyphae outside of bark tissue. 4. Melanized hyphae (MH) penetrating into the outer bark tissue. 5. Putative intercellular C. gloeosporioides hyphae (Cg H) present near bark surface. 6. C. gloeosporioides (Cg H) and melanized hyphae (MH) present in the outer bark tissue of lodgepole pine. 7. A. americanum shoot base with C. gloeosporioides hyphae present between shoot base and bark (arrow). 8. C. gloeosporioides hyphae (Cg H) in region shown in plate G  90  Figure 4-3. Xylem cells in various endophytic tissues of Arceuthobium americanum. Pair of micrographs in transmitted light and birefringence to show xylem cells. Scale bar represents 100 jam  92  1,2. Long section of sinker in host xylem. Micrograph is oriented horizontally. X=host xylem, BP=borderd pit, RT=ray trachied  92  3, 4. Cross section of sinker crossing the vascular cambium. S=sinker, VC=vascular cambium, P=phloem, X=xylem  92  5, 6. Cross section of a radial cortical strand in a longitudinal section of bark. X=A. americanum xylem  92  7, 8. Longitudinal section of a cortical strand in a longitudinal section of bark. X=A. americanum xylem  92  Figure 5-1. Percent infection of A americanum by C. gloeosporioides in different tree crown thirds. Mean +/- standard error of the mean  103  Figure 5-2. Percent infection of A. americanum by C. gloeosporioides at different locations in the stand canopy. Mean +/- standard error of the mean  104  Figure 5-3. Distribution of A. americanum within the canopy. Numbers above bars represent the number of trees having crowns within the interval xv  105  Figure 5-4. Percent infection of A. americanum by C. gloeosporioides at different aspects. Mean +/- standard error of the mean  106  Figure 5-5. Distribution of C. gloeosporioides infected and uninfected A. americanum on the host branch. Values close to 0 represent infections close to the main stem; values close to 1 represent infections close to the branch tip  107  Figure 6-1. Arceuthobium americanum infected by Caliciopsis arceuthobii. Photo taken August 17, 2001 at the Knife Creek block of the Alex Fraser Research Forest, near 150 Mile House, British Columbia. 1. Caliciopsis arceuthobii perithecia present on A. americanum flower infected in 2001. 2. Arceuthobium americanum flower that escaped infection in 2001. 3. Maturing fruit of A. americanum that escaped infection in 2000. 4. Arceuthobium americanum flower that was infected by C. arceuthobii in 2000  115  Figure 6-2. Scatter to compare the number of C. arceuthobii infected A. americanum flowers at year x with the number of infected flowers at year x+1. In the 1999 vs 2000 plot, 6 data points are located at the origin, while in the plot of 2000 vs 2001,2 data points are at the origin  117  Figure 7-1. Change in A. americanum swelling diameter and length over the course of the partial shoot removal experiment. Treatments pooled. Mean +/- standard error of the mean  127  Figure 7-2. Bud production one and two years after treatment. Male and female infections pooled. Mean number of buds per A americanum infection +/- standard error of the mean  128  Figure 7-3. Average number of fruit per shoot over the course of the experiment for every treatment. Mean +/- standard error of the mean  130  Figure 7-4. Observation of control A. americanum infections. Number of shoots and maximum shoot length of male and female infections pooled. Mean +/- standard error of the mean for all plots  131  Figure appendix 1-1. Temperature on inoculation day and following. Near Lytton, British Columbia. Maximum temperature on inoculation day was 22°C at 3:19 P M . 157  xvi  Figure appendix 1-2. Temperature at inoculation site from July 30 to August 9, 2001. Near Lytton, British Columbia  157  Figure appendix 2-1. Relative humidity on inoculation day and immediately following. Minimum relative humidity at inoculation was 46.6% at 3:19 P M . Near Lytton, British Columbia  158  Figure appendix 2-2. Relative humidity at inoculation site from July 30 to August 9, 2001. Near Lytton, British Columbia  158  Figure appendix 5-1. Timing of ascospore release and new perithecia production by Caliciopsis arceuthobii infecting Arceuthobium americanum Figure appendix 5-2. Fruit production on Arceuthobium americanum  xvii  163 164  Acknowledgements Many people and organizations have provided support to me throughout the course of the research conducted for this degree and I want to thank them all individually because without their support, this research could not have been completed. My supervisory committee provided input throughout the research to insure that my research stayed focused and on track. My supervisor, Dr. Bart van der Kamp, was always readily available to me and provided me with many challenging questions that made me carefully consider all aspects of this research. He treated me honestly and fairly and always considered my best interests; I am honored to have been one of his students. My research supervisor, Dr. Simon Shamoun, provided much more than the materials and supplies necessary for me to conduct my research; he made me feel at home in his laboratory and I value our friendship. Dr. Will Hintz always kept his door open to me and provided me with encouragement and advice throughout my degree program, which I very much appreciate. Dr. Steve Mitchell provided the silvicultural expertise necessary to keep the development of a biological control agent relevant to the forest industry. M y committee was composed of individuals who shared their enthusiasm and expertise without hesitation, and I truly thank them for making my graduate experience enjoyable. The research involved with this degree had a very large field component and the forest health personnel of the British Columbia Ministry of Forests district offices in Invermere and Lillooet were particularly helpful. Mr. Emile Begin of the Invermere forest district allowed me to establish a research plot and insured that the plot was preserved throughout the course of the experiment. The help of Mr. Ed Senger of the Lillooet forest district was greatly appreciated. He came on collection trips with me, provided research plots and made us feel at home during our stay in Lillooet during the summers of 2000 and 2001. Ms. Claire Trethewey of the U B C Alex Fraser Research forest toured me through the forest and allowed me to establish a field trial within the forest. I appreciate her time and enthusiasm. Dr. and Mrs. Dick Smith welcomed me into their home and allowed me to stay with them while I was traveling through B C during 1998 and 1999. Dick spent time with me collecting diseased A. americanum and his expertise was very valuable.  xviii  Preparation of samples and staining for the histopathological study described in Chapter 3 was conducted in Ms. Lesley Manning's laboratory. Her advice, as well of the help of Mr. Terry Holmes and Mr. Garry Jensen is greatly appreciated. Funding for this research was provided by a G R E A T award from the Science Council of British Columbia, MycoLogic Inc., and Forest Renewal British Columbia. My lab mates Shannon, Peter, Carmen, Jennifer, Grace, Rob, Anna-Mary, Brad, Lea, Sue, Chris and Cheryl provided encouragement and friendship that made the hours spent together very enjoyable. This work could not have been completed without the constant support of my family. Mom, Dad and Leanne, and my in-laws Peter, Pat and Christina have always believed in me and supported me, no matter what. M y wife and best friend Theresa, and Abby and Ethan have endured some stressful times, but they have always loved me and stood beside me. We traveled this road together and my accomplishment is due, in a large part, to their unconditional love.  xix  Chapter 1: Literature Review 1.1 Background Dwarf mistletoes of the genus Arceuthobium (Viscaceae) are obligate parasitic flowering plants of conifers within the families Pinaceae and Cupressaceae; they rely upon the host for support, mineral nutrients, a portion of their required carbon compounds, water and possibly other growth factors. Thirty-four New World species and eight Old World species are presently recognized. In North America, the greatest species diversity is located within northwestern Mexico and the western United States where twenty-eight of the thirty-four New World species are present. Five species of dwarf mistletoe have ranges that extend into Canada, A. americanum Nuttall ex Engelmann in Gray, A. tsugense (Rosendahl) G.N. Jones, A. laricis (Piper) St. John, A. douglasii Engelmann and A. pusillum Peck. Of the five species in Canada, all but A. pusillum are present in British Columbia (BC) (Hawksworth and Wiens, 1996).  1.2 Dwarf mistletoes in British Columbia Dwarf mistletoe host specificity has been categorized as "principal", "secondary", "occasional" and "rare" and tree species that are not parasitized are classified as "immune". These categories are based on the percent infection of host species inside a 6meter radius plot centered on a heavily parasitized tree. In heavily infected older stands, a host is considered to be a principal host i f greater than 90% of the individuals of a species within the plot are parasitized. In areas where the principal host is at least 80% infected, secondary hosts are 50% to 90% parasitized and occasional hosts are 5% to 50% parasitized. A host is classified as a rare host i f less than 5% of individuals of a species are parasitized (Hawksworth and Wiens, 1972).  1.2.1 Arceuthobium americanum Arceuthobium americanum, lodgepole pine dwarf mistletoe, is the most widely distributed species within B C . It occurs east of the Coast Mountain Range to beyond the Alberta / British Columbia border and from the border with the United States to as far north as 57° latitude. The most heavily infected stands of lodgepole pine occur in the region south of Spillimacheen in the Columbia River Valley and in the area north of 1  Clinton to Prince George, extending westward to Anahim Lake in the Chilcotin (Baranyay and Smith, 1972). The principal host is lodgepole pine (Pinus contorta Dougl. ex Loud, var latifolia Engelm.), ponderosa pine (Pinus ponderosa P. Laws. Ex C. Laws.) is a secondary host and occasional hosts include white spruce (Picea glauca (Moench) Voss), Engelmann spruce (Picea engelmannii Parry ex Engelm) and Douglas-fir (Pseudotsuga menziesii (Mirb.) Franco). Immune species include grandfir(Abies grandis (Dougl. ex D. Don) Lindl.) and mountain hemlock (Tsuga mertensiana (Bong.) Carriere). Aerial shoots have verticillate branching (whorled) with height of 5 to 9 (max. 30) cm and colour ranging from yellow to green. Flowering occurs from March to June (peak in May) with seed release from July to October (peak in late August and early September), approximately 16 months after pollination (Hawksworth and Wiens, 1996).  1.2.2 Arceuthobium tsugense Arceuthobium tsugense, western hemlock dwarf mistletoe, is restricted to the west side of the Coast Mountain Range; including Vancouver Island and the Queen Charlotte Islands. The most heavily infected stands are located on the Queen Charlotte Islands, northern Vancouver Island, Texada Island, and near Prince Rupert and Vancouver (Baranyay and Smith, 1972). Two subspecies of A tsugense are recognized in British Columbia: A. tsugense (Rosendahl) G.N. Jones subsp. tsugense and A. tsugense (Rosendahl) G.N. Jones subsp. mertensianae Hawksworth and Nickrent. Arceuthobium tsugense subsp. tsugense has been broken down into two races, the western hemlock race and the shore pine race depending upon principal host association. Western hemlock (Tsuga heterophylla (Raf.) Sarg.) is the principal host of the western hemlock race and grand fir and lodgepole pine are occasional hosts. Rare hosts include Engelman spruce, western white pine (Pinus monticola Dougl. ex D. Don), Douglas fir and mountain hemlock (Hawksworth and Wiens, 1996). The shore pine race of A. tsugense subsp. tsugense occurs principally on shore pine (Pinus contorta Dougl. ex Loud. var. contorta) and is morphologically very similar to the western hemlock race. The race distinction is based upon host relationships as determined by inoculation trials (Smith and Wass, 1979).  2  Mountain hemlock dwarf mistletoe, A. tsugense subsp. mertensianae, has a limited range in B C ; herbarium specimens present in the forest pathology herbarium at the Pacific Forestry Centre were collected from North Vancouver, West Vancouver and Port Alberni. The primary hosts are mountain hemlock, amabilis fir {Abies amabilis (Dougl. ex Loud) Dougl. ex J. Forbes) and subalpine fir {Abies lasiocarpa (Hook.) Nutt.). The secondary host is whitebark pine {Pinus albicaulis Engelm.) and western white pine is an occasional host, while grand fir, lodgepole pine and western hemlock are rare hosts (Hawksworth and Wiens, 1996). Arceuthobium tsugense shoots are flabellately (fan-like) branched and green to reddish in colour. Shoot height of A tsugense subsp. tsugense ranges from 3 to 13 cm, while the shoots of A. tsugense subsp. mertensianae are typically shorter. Flowering and seed dispersal of the two subspecies are slightly different. Both subspecies flower from July to October, but subsp. tsugense peaks 1 to 2 weeks earlier, while seed dispersal occurs from August to December in subsp. tsugense and from August to November in subsp. mertensianae. Fruit maturation requires 13 to 14 months in subsp. tsugense and 12 to 13 months in subsp. mertensianae (Hawksworth and Wiens, 1996).  1.2.3 Arceuthobium laricis The range of A. laricis, larch dwarf mistletoe, is restricted to southeastern B C in a triangle bounded by Osoyoos, St. Leon (Upper Arrow Lake) and Moyie. Heavily infected stands are present near Trail and the Creston-Kimberley area (Baranyay and Smith, 1972). Western larch {Larix occidentalis Nutt.) and mountain hemlock are principal hosts, lodgepole pine is a secondary host and occasional hosts include grand fir, Engelmann spruce, western white pine and whitebark pine {Pinus albicaulis Engelm.). The shoots of A laricis average 4 (max. 6) cm, are flabellately branched and are dark purple. Flowering occurs from June to September (peak from mid-July to August) and seed dispersal occurs from July to October (peak in September), thus fruit require 13 to 14 months for maturation (Hawksworth and Wiens, 1996).  3  1.2.4 Arceuthobium douglasii Arceuthobium douglasii, Douglas-fir dwarf mistletoe, is widely distributed in the United States and Mexico, but is limited in B C to the Okanagan and Similkameen valleys and the Creston area. Isolated infected stands are located near Lytton, Sicamous and Rossland. Arceuthobium douglasii is not present in the coastal forests of B C (Baranyay and Smith, 1972). The principal host is Douglas-fir and no secondary hosts are present. Occasional hosts that occur in B C are grand fir and Engelmann spruce. The shoots of A. douglasii are olive green in colour, average 2 (max. 8) cm and have flabellate branching. Flowering peaks in April and May, but ranges from February to July, while seed dispersal occurs from late August to late September, thus fruit maturation requires 17 to 18 months (Hawksworth and Wiens, 1996).  1.3 Life cycle of A. americanum Dwarf mistletoes are dioecious, obligate parasites, requiring a living host to complete their life cycle. Initiation of new dwarf mistletoe infection begins when a mature seed is cast from the aerial shoot of a female dwarf mistletoe plant onto susceptible host tissue in the fall. Dwarf mistletoe seeds are dispersed using a hydrostatic discharge mechanism (Hinds et al., 1963) that propels the seed at speeds up to 27 meters per second (Hinds and Hawksworth, 1965). The maximum recorded seed dispersal distance of A. americanum was 13.7 meters (Muir, 1970 In Hawksworth and Johnson, 1989). The seed is coated with a sticky substance called viscin, which serves to adhere the seed to any object that it lands upon (Mathiasen, 1996). Viscin is a hygroscopic compound that swells after rain and lubricates the seed, allowing the seed to slide to the needle base where it adheres to the twig when the viscin dries (Hawksworth and Wiens, 1996). Not all seeds are successfully relocated to the needle base; if the needle is pointing down, the seed will slide off of the needle onto foliage below or through to the ground (Shaw and Loopstra, 1991). Germination of the seed, indicated by radicle emergence, occurs in the spring following seed dispersal and successful infection normally occurs on branches less than five years old (Hawksworth and Wiens, 1996), likely as a result of increased bark thickness of older branches (Sproule, 1996). After germination, when the radicle contacts the needle base or some other obstruction, a 4  holdfast structure is formed which contains a penetration wedge. The penetration wedge forces into the host tissue and initiates the formation of the root-like endophytic system consisting of sinkers in the xylem and cortical strands in the inner bark (Hunt et al., 1996). The development of the endophytic system results in the formation of a swelling on the host branch at the point of infection. A n incubation period of 2-8 years precedes shoot production (Hawksworth and Wiens, 1996). Shoot production is initiated by meristematic cells in the outer cortical strand that push the buds through the bark to the branch surface (Cohen, 1954). Flowering begins the year after shoot emergence and fruit mature over 16 months after pollination to be released from late August to early September. Individual shoots can produce multiple crops of flowers and fruit in successive years (Hawksworth and Wiens, 1996). Completion of the life cycle of A. americanum, from seed dispersal to fruit maturation, averages 6 years (Hawksworth and Johnson, 1989). The endophytic system of A. americanum consists of radially oriented sinkers that become embedded in the xylem as the vascular cambium divides to add xylem tissue and strands that are longitudinally and tangentially oriented in the cortex and phloem (Hunt et al., 1996). To remain connected to the strands, the sinkers must divide in conjunction with the host cambium (Srivastava and Esau, 1961). Elongation of the strands occurs by transverse division of an apical cell to create a new row of segments that divide longitudinally. The new cells then undergo anticlinal and periclinal divisions to create longer strands of increased diameter (Bhandari and Nanda, 1970). Intensification is the process by which dwarf mistletoe infection is increased in abundance and distribution within the stand. The rate of intensification is influenced by stand density, host growth rate and disease severity; the rate of intensification decreases over time as the stand reaches crown closure or dwarf mistletoe reaches maximum levels (Geils and Mathiasen, 1990). Long distance introduction of dwarf mistletoe into new stands occurs via birds and small mammals that vector the seeds. Satellite infections, infections that have arisen at distances greater than could be achieved by explosive seed dispersal, and infections in the tops of otherwise uninfected trees are presumed to be the result of seeds that have  5  adhered to the feathers of birds or the fur of small mammals and have been deposited on a susceptible host (Hawksworth and Wiens, 1996).  1.4 Effects of dwarf mistletoe infection The most obvious symptom of dwarf mistletoe infection is stimulation of the host to produce witches' brooms, which are composed of dense masses of host branches; however, every dwarf mistletoe infection does not result in the development of witches' brooms, many infections remain localized. The production of witches' brooms by diseased trees can cause drastic changes in the canopy structure of the stand (Mathiason, 1996). Witches' brooms increase the potential for stand clearing fires because the lush foliage of the witches' broom acts as ladder fuel to bridge fire from the understory to the overstory (Anonymous, 1995). Two types of witches' brooms are formed: systemic (isophasic) brooms and non-systemic (anisophasic) brooms. In systemic brooms, the growth of the endophytic system occurs at the same rate as the growth of the cambial and apical portions of the host branch, resulting in dwarf mistletoe shoots that are scattered along the length of the branch or at each branch node. Arceuthobium americanum and Arceuthobium douglasii produce systemic witches' brooms. In non-systemic witches' brooms, the endophytic system remains localized; Arceuthobium tsugense and Arceuthobium laricis form non-systemic brooms (Kuijt, 1960). The formation of witches' brooms is hypothesized to be induced by an imbalance of plant growth regulators in the infected host tissue. Cytokinin and indole acetic acid (IAA) concentration of infected branches was higher and abscisic acid (ABA) was lower than uninfected branches of the same age of black spruce (Picea mariana (Mill) B.S.P.) infected by A. pusillum (Livingston et al., 1984). The exact mode of action of these hormones on witches' broom development is not clear. Increased cytokinins and I A A , or one hormone independently, may disrupt the apical dominance of the meristem of infected branches, allowing a proliferation of lateral twigs if dormant meristems are present. Alternatively, decreased A B A may have the same affect. It is also not known i f the hormones are produced by the dwarf mistletoe or if the dwarf mistletoe stimulates the host to change the production of these compounds (Mathiasen, 1996).  6  The physiological effects of dwarf mistletoe infection on the host with respect to wood quality, volume production and carbohydrate metabolism have been studied. When lumber from Arceuthobium abietinum Engelmann ex Munz f. sp. concoloris Hawksworth & Wiens infected and uninfected white fir from California was graded, a greater proportion of high grade boards were cut from logs that derived from dwarf mistletoe infected trees. The authors concluded that either dwarf mistletoe does not affect lumber quality or that the quality control procedures utilized were unable to detect differences (Wilcox et al., 1973). A n alternative explanation for this observation is that the most affected wood is the outer sapwood, which is removed during slabbing (Hawksworth and Johnson, 1989). Dwarf mistletoe infection of the host tree has been found to affect wood quality throughout the entirety of infected tree, not only in regions that are directly parasitized (Piirto et al. 1974). When microscopic examination of dwarf mistletoe infected wood was conducted to compare infected and non-infected wood from diseased trees and wood from healthy trees, it was found that the percentage of latewood was lower in the diseased trees than the controls. Microfibril angle was increased in infected wood (leading to increased shrinkage of dried lumber), alcohol-benzene extractive content was three times higher while trachied length, modulus of elasticity, modulus of rupture and work to proportional limit were all reduced (Piirto et al. 1974). It was suggested by Piirto et al. (1974) that these microscopic differences may not be noticed at the sawmill, as was indicated by Wilcox et al. (1973). In a study designed to compare lumber yields and visual grades of dwarf mistletoe infected lodgepole pine and healthy lodgepole pine, Dobie and Britneff (1975) found no differences in lumber grade and recovery; however, dwarf mistletoe infected trees were older and shorter than noninfected trees, suggesting that dwarf mistletoe infection severely retards volume production. Dwarf mistletoe infections on the main stem may also lead to breakage due to failure of the wood in the infected region. It has been hypothesized that dwarf mistletoe causes a reduction in volume by acting as a carbon sink. Carbohydrate production by dwarf mistletoe through photosynthesis is not enough to supply the entire plant; the host supplies the balance of carbohydrates (Leonard and Hull, 1965). The increased carbohydrate production induced by the dwarf mistletoe results in the infected branch acting as a carbon sink (Clark and 7  Bonga, 1970) causing carbon to be diverted from other portions of the tree and resulting in decreased volume (Broshot and Tinnin, 1986). Volume losses to dwarf mistletoe have been estimated at 3.8 million cubic meters annually in western Canada and 11.3 million cubic meters in the western United States. The economic impact of this volume loss is difficult to calculate exactly, but it has been suggested that it totals several billion dollars annually (Hawksworth and Weins, 1996). The impact of dwarf mistletoes in British Columbia is ranked in the top three forest diseases in British Columbia (Nevill and Winston, 1994). Dwarf mistletoe infection of young trees results in high mortality while infection of older trees results in decreased needle length, decreased length of needle bearing branches, decreased needle surface area and a decrease in the total number of needles. As dwarf mistletoe severity increases, diameter and height growth decrease resulting in reduced volume production. In some host-parasite combinations mortality is increased by dwarf mistletoe infection (summarized from Hawksworth and Wiens, 1996).  1.5 Dwarf mistletoes as agents of biodiversity The presence of dwarf mistletoe in a stand being managed for timber production has a negative economic impact; however, where timber production is not the primary management objective, the presence of dwarf mistletoe has positive effects. Several insects have been observed to feed on dwarf mistletoe shoots. Most feeding occurs by insects that feed incidentally and opportunistically; however, insects that are obligate feeders on dwarf mistletoe shoots do exist. Obligate feeders include: larvae of Mitoura spinetorum (Lycaenidae), the thicket hairstreak butterfly, Dasypyga alternosqualmella (Pyralidae), Filatima natalis (Gelechidae), Neoborella tumida (Miridae), and Pityophthorus arceuthobii (Scolytidae). Several mites and spiders have also been associated with dwarf mistletoes; however, their role has not been thoroughly explored (Hawksworth and Wiens, 1996). Bird species have been observed to utilize dwarf mistletoe fruits as a food source and to utilize witches' brooms as nesting sites. The relationship between bird habitat and dwarf mistletoe was studied in Colorado within ponderosa pine that was parasitized by A. vaginatum subsp. cryptopodum (Engelmann) Hawksworth and Wiens. It was found that 8  bird species diversity and density was correlated with dwarf mistletoe abundance. Dwarf mistletoe infection often results in snag creation and the density of cavity nesting birds was found to increase with increasing dwarf mistletoe infection (Hawksworth and Wiens, 1996). Small mammals also feed on dwarf mistletoe shoots, as well as dwarf mistletoe infected branches; however, no mammals are obligate feeders on dwarf mistletoes. The red squirrel has been observed to eat the living bark tissues of dwarf mistletoe swellings and to nip off small dwarf mistletoe infected branches and carry the branches to seed caches (Baranyay, 1968). In a study of wildlife usage of A. douglasii induced witches' brooms, Parks et al. (1999) found that the nests that were observed in the witches' brooms were all mammal nests and they hypothesized that the presence of mammals prevented bird nesting. Use of dwarf mistletoe stands by large mammals has been studied and mule deer and elk in Colorado preferred dwarf mistletoe infected stands (Bennetts et al., 1991 in Hawksworth and Wiens, 1996). Parks et al. (1999) observed that the area under large brooms was used as a resting site for large mammals because the brooms provided shelter from rain and snow.  1.6 Silvicultural control of dwarf mistletoe Stand management objectives dictate which, if any, dwarf mistletoe control strategies will be utilized. Dwarf mistletoe causes reduced volume production, loss of wood quality and increased mortality, which are not desirable in stands managed for timber production. Selection of the proper management regime must be carefully considered because forest harvesting and regeneration can limit or enhance dwarf mistletoe spread and intensification (Anonymous, 1995). Since dwarf mistletoe is an obligate parasite, it can be destroyed by killing infected parts of the host tree or all infected trees. This characteristic allows dwarf mistletoe management to be integrated into normal forest management practices, resulting in control of dwarf mistletoe with little extra cost (Baranyay and Smith, 1972), but it does result in limiting the silvicultural options. Other characteristics of dwarf mistletoes that make them amenable to silvicultural control include: host specificity, long life cycles, slow spread within stands  9  and easy diagnosis due to the presence o f witches' brooms (Hawksworth and Johnson, 1989). Factors that effect dwarf mistletoe spread and intensification within a stand include stand density, host vigour, tree age, stand composition and stand history. D w a r f mistletoe spread occurs more rapidly i n open stands because as density increases, seed interception increases, slowing spread. Stand density is related to tree vigour, i n that open stands result in greater light penetration and less competition for nutrients. This results in trees with fuller crowns, therefore increasing seed interception. Additionally, increased host vigour results in increased dwarf mistletoe vigour. Tree age is important in dwarf mistletoe spread because young trees are small and are more likely to escape infection. M i x e d stands have lower rates o f spread because non-hosts serve to intercept seeds and reduce infection on susceptible species. Stand history, especially fire, affects the dwarf mistletoe presence in a stand. If a dwarf mistletoe infected stand is not burned during a fire, it serves as an inoculum source to invade the adjacent replacement stand. Witches' brooms act as ladder fuel to carry fire from a low intensity ground fire up into the crown, resulting in a stand clearing fire. This has the effect o f removing dwarf mistletoe from the stand, but also perpetuating serai species that are susceptible to dwarf mistletoe (Parmeter, 1978). Prevention o f dwarf mistletoe establishment in regenerating stands is a much more effective management tool than removal o f infected trees from infected stands after stand establishment (Hawksworth and Johnson, 1989). Cutblock design provides an opportunity to prevent establishment o f dwarf mistletoe i n the new stand. Knowledge o f the distribution o f dwarf mistletoe within the stand prior to cutting is essential so that cutblock boundaries do not intersect the dwarf mistletoe infected areas. Natural boundaries to dwarf mistletoe spread, such as roads, utility rights-of-way and resistant species can be utilized as cut-block boundaries to prevent spread o f dwarf mistletoe into the new stand from the edge. Where dwarf mistletoe free boundaries are not possible, large clear-cut blocks reduce the ratio o f perimeter to edge and reduce the proportion o f the area invaded by dwarf mistletoe. In such situations, narrow strips should be avoided when designing cut blocks because infection o f the new stand from the stand edge w i l l occur rapidly (Baranyay and Smith, 1972).  10  If alternative silvicultural systems, such as shelterwood, variable retention, or a seed tree system are to be utilized, steps can be taken to avoid dwarf mistletoe infection of the regenerating stand. If the seed-tree system is being used for establishment of the new stand, the seed trees left after harvesting should be dwarf mistletoe free or lightly infected. If infected trees must be left, they should be removed or girdled as soon as seedlings are established (Anonymous, 1995). If the shelterwood system is being used, the overstory trees should be non-host trees or trees with little or no infection, and as with the seed tree system, infected residuals should be removed as soon as the stand is established (Anonymous, 1995). If the stand is being managed under an uneven-aged selection system, non-host species and trees with little or no dwarf mistletoe infection should be left as residuals (Anonymous, 1995). The host specificity of dwarf mistletoe can be exploited when establishing a new stand in dwarf mistletoe infected areas. Planting immune tree species at the edge of a cut block boundary will reduce ingress of the dwarf mistletoe into the stand. When the silvicultural system utilized requires retention of the overstory, planting immune tree species in the understory will reduce dwarf mistletoe in the future stand (Baranyay and Smith, 1972). Planting immune conifer species as a barrier to dwarf mistletoe infection is not always effective since susceptible species may naturally regenerate from a seed bed. The resulting mixed stand will likely allow dwarf mistletoe spread into the pure stand, especially in the case of A. americanum and lodgepole pine because lodgepole pine has great early height growth and rapidly overtops the non-hosts (van der Kamp, personal communication). In British Columbia, the Forest Practices Code of 1995 legislated smaller cutblock size, increased riparian reserves and more partial cutting, reducing the options for silvicultural control of dwarf mistletoe. Riparian areas serve as refugia for dwarf mistletoe because the damp environment resists fire; increased riparian reserves may result in increased dwarf mistletoe entry into regenerating stands (van der Kamp, personal communication). Constrained silvicultural options for dwarf mistletoe control in partial cuts has rekindled the search for alternative dwarf mistletoe management strategies. Biological control has been studied in the past; however, a suitable biological control strategy has 11  not been developed. Chemical control has also been investigated and almost 60 different formulations, mostly mixtures of 2,4-D or 2,4,5-T, have been tested. None were able to control the dwarf mistletoe without coincidental damage to the host and none affected the endophytic system (Hawksworth and Wiens, 1996). The only chemical that reduces the rate of spread of dwarf mistletoe is the plant growth regulator ethephon (2-chloroethyl phosphoric acid), which causes shoot abscission. The chemical does not affect the endophytic system and rapid shoot resprouting occurs following shoot removal; 52% of treated female A americanum infections bore fruit after 5-years (Nicholls, 1988). Ethephon is registered by the Environmental Protection Agency in the United States (Hawksworth and Wiens, 1996) under the name Florel® (Rhone-Poulenc A g Co), but it is not registered for forestry use in Canada. Genetic resistance to dwarf mistletoes has been noted in the western hemlock - A. tsugense pathosystem (Smith et al., 1993) and in the ponderosa pine -A. campylopodum pathosystem (Roth, 1974). Clonal propagation of western hemlock with a range of susceptibility to A. tsugense allowed increased numbers of genetically identical replicates for testing. Seed germination rate did not differ significantly between clones, but two of the selected clones had low levels of infection. The resistance mechanism was not determined, however, it appeared that the penetrating structure of the dwarf mistletoe was unable to enter the host cortex (Smith et al., 1993). The mode of resistance of ponderosa pine to A. campylopodum is hypothesized to be a result of foliar and physiological characteristics (Roth, 1974). Although genetic resistance has been suggested, no genetically resistant seed stock is available for plantation establishment (Hawksworth and Wiens, 1996).  1.7 Biological control The biological control strategy is the deliberate use of living organisms to suppress or reduce the effects of a pest to acceptable levels (Mortensen, 1998). Advantages of biological control include specificity to the target, no persistent residue or toxicity hazards because biological control agents and their metabolites are biodegradable, a potentially sustainable effect because the biological control agent might become a persistent component of the ecosystem and, because multiple genes are 12  involved in pathogenesis, a low probability of resistance development (Holdenrieder and Greig, 1998; Wilson, 1969). Disadvantages of biological control include sensitivity to climatic conditions, limited shelf life and possible effects on non-target organisms, which may result in a shift in the biodiversity of the system (Holdenrieder and Greig, 1998). Furthermore, because the specificity of biological control agents limits their market potential, a limited market must pay the development and registration costs (Mathre et al., 1999). Two main approaches to biological control have been utilized: classical (or inoculative) biological control and inundative biological control (inundative biological control agents are often referred to as bioherbicides or mycoherbicides) (Mortensen, 1998). Classical biological control assumes that the target has escaped its natural enemies, thus allowing it to proliferate. Natural enemies are sought out in other regions and introduced into areas where they are absent and the target is a problem, resulting in a reduction in the competitive ability of the target and a reduction in the target population (Mortensen, 1986). The ultimate goal of classical biological control is to establish a natural, self-regulating balance between the host and the pathogen (Hasan and Ayres, 1990). Classical biological control agents must cause severe damage to the target organism without damaging other organisms in the area of introduction. Thorough host range testing under greenhouse conditions must be conducted to ensure the safety of native, non-target, species (Mortensen, 1998). The inundative biological control approach utilizes indigenous strains of fungi or other organisms that are well adapted to the local environment. This approach overcomes many of the barriers of classical biological control, such as spatial, temporal and environmental constraints to establishment. The advantage of the inundative biological control strategy over the classical biological control strategy is that efficacy is not reliant upon the agent being self-sustaining; long term survival of the agent is not as important because inundative biological control agents are typically re-applied in years following initial treatment (Hintz et al., 2001). The agent is released by a single, timely application of inoculum at a climatically suitable time that results in infection, disease development and eventual death of the specific host (Templeton, 1992). The release of a large  13  concentration of inoculum can overcome the natural constraints that limit epidemic development under natural conditions (Mortensen, 1998). The strategy for development of inundative biological control agents consists of three major stages: (1) discovery; (2) development and; (3) deployment. During the discovery stage, the organism of interest is identified and extensive field collections of diseased samples are made. Under laboratory conditions, the pathogens present are isolated from the organism and identified. Pure cultures of the pathogens are then applied to healthy and uninfected hosts under greenhouse conditions and the disease process is monitored. Once the symptoms are described, each pathogen is re-isolated from the diseased tissue and identified. The identity of the pathogen isolated from the diseased plant must be same as that which the plant was initially inoculated with and result in the same symptoms to prove Koch's postulates (Manion, 1981), and thereby prove that the potential biological control agent is the causal agent of the disease. The development stage involves determination of conditions for optimum inoculum production, disease development, host range and evaluation of the efficacy of the pathogen as a potential biocontrol agent. During the deployment stage, mass production strategies for scale-up, formulation, regulatory, and marketing strategies for the biocontrol agent are developed (Watson and Wall, 1995). During these stages, the essential criteria of: (1) ability to produce abundant and durable inoculum in artificial culture; (2) genetic stability of the inoculum and specificity to the target organism; and (3) ability to infect and kill the host under variable environmental conditions, are all assessed (Templeton, 1982; Sands et al., 1990). Failure of the potential biological control agent to meet any of these criteria will result in the search for alternative pathogens.  1.8 Examples of inundative biological control agents  Several inundative biological control agents have been commercialized for weed control in agriculture and forestry. In conifer regeneration sites, competition for light, water and nutrients from hardwood species such as red alder (Alnus rubra Bong.) slows the growth of regenerating trees; therefore, these hardwoods are considered weeds (Biring et al., 1996). Chondrostereum purpureum (Pers.:Fr) Pouzar, is a basidiomycete fungus that is able to rapidly colonize cambium and sapwood of inoculated trees (Wall, 14  1991), and it has been observed to control red alder and other weedy hardwood species with the same efficacy as chemical herbicide when applied to the cut stump (Shamoun et al., 1996; Harper et al., 1999; Pitt et al., 1999). This fungus has been registered under the trade name BioChon™ in the Netherlands by Koppert Biological Systems for control of American black cherry (Prunus serotina Erhr.) and in North America under the trade name Myco-Tech™ in Quebec and is currently being registered by MycoLogic Innovative Biologicals Inc. under the trade name Chontrol™ (Shamoun, Personal Communication). In South Africa, Cylindrobasidium laveae (Pers.:Fr.) Chamuris has been registered as Stumpout™ for control of Acacia spp. (Lennox et al., 2000). Other inundative biological control strategies for competitors of forest regeneration include Fusarium avenaceum (Fr.) Sacc, Colletotrichum dematium (Pers.) Grove and Phomopsis sp. for control of Rubus spp. (Oleskevich et al., 1998) and C. dematium for fireweed control (Chamerion angustifolium L . ssp. mgustifolium) (Winder and Watson, 1994). Inundative biological control of forest diseases has also been investigated. Heterobasidion annosum (Fr.:Fr.) Bref, the causal agent of annosus root and butt rot, infects stumps of susceptible trees via spores, colonizes the stump and is able to infect adjacent trees through root grafts. Inoculation of stumps with Peniophora gigantea (Fr.) Massee (syn. Phlebiopsis gigantea (Fr.) Jiilich or Phlebia gigantea (Fr.) Donk or Phanerochataete gigantea (Fr.:Fr.) Rattan, Abdullah & Ismail) following cutting results in colonization of the stump by P. gigantea, preventing colonization by H. annosum through competitive exclusion. Several other fungi have also been utilized for H. annosum control and these include: Bjerkandera adusta (Willd.:Fr.) P. Karst., Fomitopsis pinicola (Sw.:Fr.) P. Karst., Resinicium bicolour (Albertini & Schwein.:Fr.) Parmasto, Hypholoma spp., Melanotus proteus (Kalchbr.) Singer, Armillaria spp., Trichoderma spp., and Scytalidium spp. (Holdenrieder and Greig, 1998; Mercer, 1988). Peniophora gigantea has been registered in Europe and was commercialized as RotStop™ in Finland (Shamoun, Personal Communciation). Innundative biological control of Armillaria ostoyae (Romagn.) Herink through competitive exclusion by Hypholoma fasciculare (Huds. Ex. Fr.) has been investigated in British Columbia (Chapman and Xiao, 2000). Placing H. fasciculare in close proximity to Douglas-fir or  15  pine stumps resulted in colonization of the stumps although the direct impact on A. ostoyae has not yet been quantified. Although examples of inundative biological control strategies for utilization in forestry scenarios exist, the majority of research on inundative biological control is targeted at weeds in agricultural systems. In China, since 1966, C. gloeosporioides f. sp. cuscutae, referred to as Luboa™, has been applied to control dodder (Cuscuta chinensis Lam. and C. australis R. br.) parasitizing soybeans (Glycine max (L.) Merr.) (Watson et al., 2000). Round-leaved mallow (Malvapusilla Sm.) is a common farmyard and garden weed that has become a problem in field crops. The fungus Colletotrichum gloeosporioides (Penz.) Sacc. f. sp. malvae was isolated from round-leaved mallow and tested as an inundative biological control agent (Mortensen, 1988). The isolate was registered as BioMal™ in 1992, and the potential of the agent was high (Templeton, 1992), but it was never marketed due to the high cost of inoculum production (Watson et al., 2000). The taxonomic classification of the BioMal™ isolate was reassessed, based on ribosomal D N A and the infection process, and changed to C. orbiculare (Berk. & mont.) von Arx (syn. C. lagenarium (Pass.) Ell. & Halst.) (Bailey et al., 1996). This change in classification was recently supported by studies of the infection process of the Biomal™ isolate, which was found to utilize the same infection process as C. orbiculare (Shen et al., 2001). Another forma specialis of Colletotrichum gloeosporioides that has been registered as an inundative biological control agent is C. gloeosporioides f. sp. aeschynomene, under the trade name Collego™ for the control of northern jointvetch (Aeschynomene virginica (L.) B.S.P.) in rice and soybean fields (Templeton, 1992). The product was marketed from 1982 to 1992 through three successive producers and not reregistered due to low perceived market potential. Collego™ was re-registered in 1997 and approximately 5,000 hectares were treated in 1998 (Watson et al., 2000). In South Africa, C. gloeosporioides has been registered to control silky needlebush (Hakea sericea Schrader) (Watson et al., 2000). Several other inundative biological control studies have investigated the potential of Colletotrichum, including: control of hemp sesbania (Sesbania exaltata (Rydb.) ex. A.W. Hill) with Colletotrichum truncatum (Schw.) Andrus & Moore. (Boyette et al., 1993), control of Koster's curse (Clidemia hirta (L.)) in Hawaiian forests with Colletotrichum gloeosporioides f. sp. clidemiae (Norman and  16  Trujillo, 1995). Other species of fungi have also been tested for their efficacy as inundative biological control agents, including Phytophthora palmivora (Butler) Butler (registered as DeVine™) for stranglervine (Morrenia odorata Lindl.), Alternaria spp., Cercospora rodmanii Conway, Fusarium spp., Nectria ditissima (Tul) for red alder and Puccinia canaliculata (Schw.) Lagerh. (registered as Dr. Biosedge™) for yellow nutsedge (Cyperus esculentus L.) (Mortensen, 1998), and Valdensinia heteroxa Peyronel for control of salal (Shamoun et al., 2000). Commercial success for inundative biological control agents that utilize Colletotrichum spp. has been limited. There are many reasons for this, including: expectation that inundative biological control would replace chemical herbicides, limited shelf-life, extended dew requirements, low fecundity, low virulence and restricted niche markets. Current research in inundative biological control is shifting away from isolating new strains of fungi to solving production, storage and efficacy problems. Future usage of inundative biological control agents will likely occur as an integrated pest management approach in association with chemical or biological compounds and silvicultural tools such as manual brushing to increase efficacy to a satisfactory level (Watson and Wall, 1995; Watson etal., 2000).  1.9 Past dwarf mistletoe biological control studies The attributes of a successful biological control agent for dwarf mistletoes were outlined by Wicker and Shaw (1968) and included: 1. distribution which coincides with that of the target pathogen; 2. ecologic amplitude sufficient to ensure persistence within its host range; 3. production of abundant inoculum for establishment of epiphytotics; 4. high infectivity; 5. high virulence and; 6. efficient mode of action for curtailing development of the target host. These criteria incorporate attributes of both classical and inundative biological control agents. Several fungi, including Caliciopsis arceuthobii (Peck) Barr, Cylindrocarpon gillii (D.E. Ellis) Muir and Colletotrichum gloeosporioides (Penz.) Penz. & Sacc. have been observed to infect dwarf mistletoe and many authors have suggested that these fungi be utilized as biological control agents (Muir, 1967; Parmeter et al. 1959; Wicker and Shaw, 1968). In spite of this, there is a paucity of literature describing inoculation trials. 17  Parmeter et al. (1959) inoculated A. campylopodum f. abietinum with conidia of C. gloeosporioides, Knutson and Hutchins (1979) inoculated A. douglasii with Caliciopsis arceuthobii and Ellis (1946) inoculated A. douglasii with Cylindrocarpon gillii. These inoculation studies were conducted to prove Koch's postulates; only one field trial was conducted to test the utility of a fungus, Cylindrocarpon gillii, as a biological control agent (Mielke, 1959). When biological control of dwarf mistletoes was initially proposed (Ellis, 1946), and the only real attempt to establish a fungus as a biological control agent was initiated (Mielke, 1959), it was envisioned that biological control would occur following the classical biological control model. In the case of dwarf mistletoes, the dwarf mistletoe host and fungal pathogen are indigenous organisms that have co-evolved (Hawksworth and Wiens, 1996); therefore, it is not surprising that Mielke (1959) found no evidence of C. gillii 3 years after inoculation. Likely, there is a natural equilibrium that exists between the dwarf mistletoe and the fungal hyperparasite, analogous to the system proposed by van der Kamp and Blenis (1996) to regulate the western gall rust (Endocronartium harknessii (J.P. Moore) Hirat.) / hard pine pathosystem. Muir (1977), quantified the natural occurrence of C. gloeosporioides on A. americanum in Alberta, and observed a 35% infection of A. americanum at one site and 75% infection of A. americanum at another site. Muir (1977) concluded that although C. gloeosporioides caused a significant reduction in dwarf mistletoe shoot and fruit production, the fungus would not significantly reduce the impact of A. americanum because the number of new dwarf mistletoe plants was not significantly reduced by natural infection of A. americanum by C. gloeosporioides. There are many scenarios in which inundative biological control may prove to be a useful option to the forest manager. If timber production with retained overstory is the primary stand management objective, prevention of dwarf mistletoe entry into the regenerating stand from overstory and adjacent trees using inundative biological control may allow retention of the overstory trees without the risk of dwarf mistletoe infection. Under the forest practices code of British Columbia, reduced clearcut size and increased riparian reserves will lead to increased dwarf mistletoe spread and intensification; however, inclusion of biological control in the silvicultural treatment of the stand may 18  reduce this spread. In areas such as parks, where large witches' brooms increase the hazard rating of trees (Hadfield et al., 2000), application of a biological control agent to prevent new dwarf mistletoe infections in trees surrounding camping or picnicking sites would prevent dwarf mistletoe spread and intensification while retaining the infected host tree.  1.10 F u n g i that infect  Arceuthobium americanum  The fungi that parasitize dwarf mistletoes have been termed hyperparasites because they cause disease on dwarf mistletoe, an obligate parasite. The hyperparasites described below cause disease on A, americanum and have been observed to regulate the dwarf mistletoe through interfering with the dwarf mistletoe life cycle by reducing seed production or shoot mortality.  1.10.1  Caliciopsis arceuthobii Caliciopsis arceuthobii (ex. Wallrothiella arceuthobii (Peck) Saccardo) is a  highly specialized ascomycete that infects the female flowers of the spring flowering dwarf mistletoes A. pusillum (Peck, 1875 in Weir, 1915), A. douglasii (Weir, 1915), A. americanum (Dowding, 1931) and A vaginatum sub sp. cryptopodium (Hawksworth and Wiens, 1996). In Canada, this fungus has been collected from A. americanum in British Columbia (Kuijt, 1969; Wood, 1986), Alberta and Manitoba (Dowding, 1931) and A. pusillum in Quebec (Pomerleau, 1942 in Kuijt, 1969), however, the Quebec population was recorded as extinct by Kuijt (1969). The presence of C. arceuthobii is readily identified by the presence of 40 - 50 black perithecia on the female flower. Ascospores mature in the asci within the perithecia in March and April and are then released during the dwarf mistletoe flowering period. It is hypothesized that insects transport the ascospores to susceptible female flowers, where the ascospore germinates and infects the stigmatic tissues. Infection of the female flower prevents development of the seed and perithecia are present on A. americanum in the fall of the year the infection was initiated (Kuijt, 1969). The life cycle of C. arceuthobii therefore requires two seasons for completion (Knutson and Hutchins, 1979). 19  Caliciopsis arceuthobii has been cultured on medium that contained glucose, asparagine and 0.2% yeast extract (Parker, 1970) as well as potato dextrose agar (PDA) + 1% yeast extract (Knutson and Hutchins, 1979). On both media, the fungus grew very slowly, radial growth was 20 mm after 6 months on the glucose - asparagine media (Parker, 1970) and on PDA, a diameter of 5 mm was reached after 4 months (Knutson and Hutchins, 1979). The difference in growth rates may be attributable to the origin of C. arcuethobii; Parker (1970) isolated the fungus from A americanum while Knutson and Hutchins (1979) isolated the fungusfromA. douglasii. The colonies became black with age in both studies. No perithecia or asci were produced in culture (Knutson and Hutchins, 1979), although Dowding (1931) recovered "sprout cells" from surface sterilized stromata tissue. Parker (1970) suggested that these "sprout cells" were a contaminant. The presence of another fungus on the perithecia of C. arceuthobii has been recorded (Dowding, 1931; Kuijt, 1969; Knutson and Hutchins, 1979) and was identified as Cladosporium sp. in Kuijt's (1969) study. Knutson and Hutchins (1979) identified the other fungus as Aureobasidium pullulans (de Bary) Arn. Kuijt (1969) observed, and Knutson and Hutchins (1979) isolated, C. arceuthobiifromA americanum and A. douglasii, respectively, possibly accounting for the difference in the fungal associate. Dowding (1931) believed that the fungus arisingfromthe stomata tissue was the imperfect state of C. arceuthobii; however, Knutson and Hutchins (1979) did not recover conidiafromstromata that arosefromascospore cultures. The imperfect state of C. arceuthobii, if it exists, is unknown.  1.10.2 Cylindrocarpon gillii Cylindrocarpon gillii (ex. Septogloeum gillii D.E. Ellis) was first described by Gill (1935) on A. tsugense and A. abietinum Engelmann ex Munz in Washington and A. cyanocarpum (A. Nelson ex Rydberg) Coulter & Nelson, A. microcarpum (Engelmann) Hawksworth and Wiens and A. abietinum in Arizona although it was not named. Ellis (1939) observed the fungus on A. douglasii in Arizona and tentatively placed it in the genus Fusarium. Later, Ellis (1946) named the fungus as Septogloeum gillii and extended the host range to include A. blumeri A. Nelson, A. divaricatum Engelmann and A. campylopodum Engelmann in Gray. Gill (1952) observed C. gillii on A. americanum 20  in Montana. In Canada, C. gillii has been recorded in British Columbia on A. americanum, A. douglasii and A. tsugense (Wood, 1986). Muir (1973) reclassified the fungus as Cylindrocarpon based on conidiophore morphology and spore formation. Cylindrocarpon gillii primarily infects the dwarf mistletoe shoots and occasionally the fruit. Small yellow-white spots are initially formed in the spring, which converge and erupt through the epidermal layer during the summer to expose white spore masses (Ellis, 1946). On the host, phialospores are non- to three-septate, 12-41 x 3-5 urn and in culture, phialospores are non- or one-septate, 6-14 x 3-4 um. Phialides were 30-80 x 1-3 um and chlamydospores of 10-14 um diameter occurred singly or in groups of up to four and are present in older cultures but not hosts. The fungus grows slowly, reaching 5 mm diameter after 14 days (Muir, 1973).  1.10.3 Colletotrichum gloeosporioides Unlike the hyperparasites described above, Colletotrichum gloeosporioides is a pathogen that occurs on a wide variety of plant hosts and crops worldwide (Prusky et al., 2000). It was first described on Arceuthobium abietinum infecting red fir (Abies magnifica Murr.) in California (Parmeter et al., 1959). Colletotrichum gloeosporioides also infects A. americanum and A. campylopodum in California (Scharpf, 1964), Idaho and Washington (Wicker, 1967; Wicker and Shaw, 1968) and A. douglasii and A. laricis in Idaho and Washington (Wicker and Shaw, 1968). In Canada, it has been observed on A. americanum in British Columbia (Wood, 1986), Alberta and Saskatchewan (Muir, 1967). Recently, C. gloeosporioides was isolated from A tsugense subsp. tsugense on Vancouver Island (Kope et al., 1997). Colletotrichum gloeosporioides has also been isolated from European mistletoe (Viscum album subsp. typicum Beck.) (Stojanovic, 1989). Symptoms of Colletotrichum gloeosporioides infection first appear as dark brown to black lesions, usually at the shoot nodes. The lesions enlarge and acervuli rupture through the epidermis and under moist conditions, salmon to cream coloured masses of conidia are exuded from the acervuli. No setae were observed in acervuli on infected shoots (Parmeter et al., 1959). Conidia are hylaine, 15-18 x 4.5-5.5 um, single celled becoming two celled at germination. Appresoria are produced on germ tubes (Parmeter 21  et al., 1959). Infection of the endophytic system of Arceuthoium sp. has been reported (Parmeter et al., 1959; Wicker and Shaw, 1968) although the pathological affects of infection of the endophytic system have not been described. In culture, isolates of C. gloeosporioides from different Arceuthobium hosts had different growth rates, colony colour and sporulation rates but cross inoculation indicated that the isolates were not host specific (Scharpf, 1964). The sexual stage of Colletotrichum gloeosporioides is Glomerella cingulata (Stonem.) Spaulding & Schrenk (Mordue, 1971).  1.10.4 Resin Disease  Resin disease is a disease syndrome of Arceuthobium americanum that is hypothesized to be the result of the combination of 11 different fungi, the most common being Alternaria alternata (Fries) Keissler and Aureobasidiumpullulans. The disease has been found in Colorado, Idaho, Montana, Utah and Wyoming and is characterized by excessive resin exudation from the swellings, necrotic and discoloured bark, formation of a necrophylactic periderm and retention of dead, resin filled, needles. The formation of the necrophylactic periderm is hypothesized to be the mechanism of shoot death because it isolates the shoots and cortical strands from the sinkers. The authors suggest that the resin disease complex would be a good biological control method for A. americanum (Mark et al., 1976). It is doubtful, however, that the fungi isolated from diseased swellings are the sole cause of resin disease because the fungi implicated were endophytic fungi that were commonly isolated from healthy A. americanum during a survey of the mycoflora associated with A americanum (Gilbert, 1984).  1.10.5 Associations with rust fungi  Arceuthobium americanum has often been located in association with Cronartium comandrae Pk., a rust fungus, on lodgepole pine. A n experiment was designed to investigate the possibility that A. americanum may be acting as a telial host of C. comandrae. Inoculation of A. americanum did not result in infection by C. comandrae, indicating that it is likely not a host for C. comandrae (Peterson, 1966). Peridermium bethelii Hedgecock & Long, is very similar to C. comandrae and is almost always found in association with dwarf mistletoes, including A. americanum. The  22  distinguishing feature that separates these rust fungi is the morphology of the aeciospores; aeciospores of P. bethelii are smaller and subglobose in shape. No alternate hosts for P. bethelii have been observed and inoculation of comandra (the alternate host of C. comandrae) was unsuccessful. Field observation suggests that P. bethelii is a pineto-pine rust. It is possible that P. bethelii represents an autoecious, microcyclic ecotype of C. comandrae. The exact details of the relationship between P. bethelii and A. americanum have not been established (Hawksworth et al., 1983).  1.11 Research objectives The research conducted in this study was focused toward the development of an inundative biological control strategy for Arceuthobium americanum infecting Pinus contorta var. latifolia in British Columbia. The research milestones are outlined below. 1. To survey A. americanum in British Columbia and to collect and describe hyperparasitic fungi that have been reported to cause disease on A. americanum. 2. To select a lead isolate of the candidate hyperparasite based on mycelial growth over a range of temperatures, conidial germination over a range of temperatures and conidia production in culture in the absence of experimental units of A. americanum for greenhouse testing. 3. To formulate the selected lead isolate in different formulations and determine which formulation will provide the best inoculation system for a field trial. Formulation is necessary because it protects the hyperparasite against desiccation, provide nutrients, and aids in application, ultimately leading to increased efficiency. 4. To conduct a field trial to test the selected hyperparasite and assess the potential of the fungus as a biological control agent for A. americanum. 5. To describe the pathological effects of the selected hyperparasite on the endophytic system of A. americanum to elucidate the pathogenesis process of the selected hyperparasite using histopathological methods. 6. To describe the natural distribution of the hyperparasite within the canopy of A. americanum infected lodgepole pine trees to determine if there are any constraints to hyperparasite development on A. americanum at different canopy positions. 23  7. To conduct trials that mimic the action of a biological control agent by shoot removal to determine how the dwarf mistletoe responds, and particularly, the time required for new shoot and fruit production. These data are necessary to develop guidelines for inundative biological treatment interval required to prevent A. americanum seed production.  These studies were conducted to increase the understanding of the role of hyperparasitic fungi in regulating the life cycle of A. americanum and to investigate the potential of an inundative biological control strategy to mitigate the impact of A. americanum on lodgepole pine stands.  Note: In the following chapters the word "infection" has been utilized to describe both part of the pathogenesis process of A. americanum on lodgepole pine and hyperparasites on A. americanum as well as the dwarf mistletoe plant on the host, i.e. "Spread to A. americanum infections that escaped initial biological control application..."  24  Chapter 2 - Development of a biological control strategy for  Arceuthobium americanum, Part I: Discovery and Development 2.1 Introduction Prevention of dwarf mistletoe entry into newly established stands can be achieved by effective silvicultural planning (Baranyay and Smith, 1972; Van Sickle and Wegwitz, 1978; Hawksworth and  Johnson, 1989).  In British Columbia however, current forest  practices are moving towards smaller cut block size, increased riparian reserves and increased use of partial harvesting systems that will promote dwarf mistletoe spread. The changes in forest management have led to a renewed search for alternative management strategies, such as inundative biological control, to minimize dwarf mistletoe impact on regenerating stands. The ability to interfere with the life cycle of dwarf mistletoe, and thus reduce spread, is the most important characteristic of an inundative biological control agent. This chapter details studies that were conducted during the initial discovery and development phases of an inundative biological control approach for Arceuthobium americanum infecting Pinus contorta var. latifolia in British Columbia. The objectives of this research phase were to: 1. collect diseased A. americanum to isolate hyperparasitic fungi (pathogens of dwarf mistletoe) that may be utilized as biological control agents; 2. to study the growth characteristics of the selected hyperparasite; 3. to formulate the hyperparasite in a medium to allow long term storage and to increase efficacy and; 4. to test the inoculation system under greenhouse conditions in advance of a field trial.  2.2 Materials and Methods 2.2.1 Collection of diseased A americanum and hyperparasite isolation Diseased A. americanum shoots, fruit and swellings were collected in the province of British Columbia during the summer of 1998. Forest health officers of the British Columbia Ministry of Forests directed me to A. americanum infected lodgepole pine stands within their districts. Lodgepole pine stands in the area bounded by Quesnel in the north, the Canada - United States border in the south, the British Columbia 25  Alberta border in the East and Coast Mountains in the west were visited and inspected for the presence of A. americanum. Additionally, A. americanum in a stand of lodgepole pine near Nordegg, Alberta and a stand of Jack pine (Pinus banksiana) near Bruderheim, Alberta were inspected for diseased A. americanum. If dwarf mistletoe was located in the stand, the presence of fungal disease on the shoots was assessed and diseased samples were collected for later fungal isolation in the laboratory. Dead A. americanum swellings on dead branches were not collected in order to avoid isolation of saprophytic fungi; swellings that were collected had either obvious signs of infection or they had shoots with necrotic and healthy tissue. At the laboratory, diseased A. americanum shoots were surface sterilized by placing the diseased material in 95% ethanol for 3 minutes, then 10% sodium hypochlorite for 3 minutes, followed by 3 washes in sterile distilled water for 3 minutes per wash. The surface sterilized pieces were then transferred to potato dextrose agar (PDA, Difco Laboratories, Detroit, MI) and incubated at 20°C. If hyphae emerged from the dwarf mistletoe material, a piece of media containing hyphal tips was subcultured onto a new P D A plate and incubated at 20°C. If characteristic Colletotrichum gloeosporioides lesions were present on the diseased shoot, the lesions were wetted with sterile distilled water for five minutes and then a sterile loop was used to streak the conidia onto a P D A plate. Plates were then incubated at 20°C and observed for the production of single conidia colonies. Single conidia colonies were transferred to new P D A plates and the culture identity was confirmed based on morphological characteristics. For long term storage, cultures were placed on 1.5% malt extract agar slants and stored at 5°C. Attempts to isolate Caliciopsis arceuthobii from parasitized female flowers were conducted as follows: 1. perithecia were aseptically removed from the female flower and attached to the lid of a petri plate so that ascospores could be cast onto media below; 2. perithecia were crushed in sterile distilled water and streaked onto media; 3. perithecia were surface sterilized in ethanol and sodium hypochlorite and placed directly on media. In all cases, the fungus was placed on glucose-asparagine media (glucose, 30.0 g, asparagine, 1.0 g, MgSCvTFbO, 0.5 g, KH2PO4, 1.5 g, water, 1 L) modified by the addition of 0.2% yeast extract and 1.5% agar (Parker, 1970). 26  2.2.2 Growth characteristics of Colletotrichum gloeosporioides. The growth response of C. gloeosporioides  at temperatures ranging from 0°C to  35°C, increasing in 5°C increments, was determined. The rate of linear mycelial growth, percent conidial germination and germ tube length at each temperature were measured. To measure mycelial growth rate at various temperatures, isolate PFC-4277 collected at Logan Lake, B C , isolate PFC-4278 collected from Nordegg, A B , and isolates PFC-4493 and PFC-4501 from Canal Flats, B C were selected. To measure mycelial growth, a mycelial plug was placed in the center of a PDA plate and incubated at each temperature in the dark. There were five replicates of each isolate at each temperature. Every other day, the plates were placed under a dissecting microscope and the leading edge of the hyphal front was drawn on the plate using a permanent fine point marker. The experiment was concluded after 21 days incubation, when the hyphal margin of the fastest growing isolate contacted the edge of the petri plate, at which time the growth marks were measured. Growth on each plate was measured by averaging two diameters at right angles to each other. Conidia for the germination studies were produced in 250 ml Erlenmeyer flasks by inoculating 20 g of millet grain that was soaked in 20 ml H2O and autoclaved for 20 minutes with 2 mycelial plugs of each isolate. The inoculated grain was allowed to incubate for 2 weeks at room temperature with frequent agitation. There were 2 replicate flasks per isolate. The conidia were washed from the grain by adding 100 ml of sterile dFbO to each flask and placing the flask on a rotary shaker at 225 rpm for 30 minutes. The contents of each flask were then poured through sterile cheesecloth into sterile beakers. The filtered conidia were placed in multiple 50 ml centrifuge tubes, centrifuged at 2500 xg for 10 minutes, the supernatant was decanted and the conidia were resuspended in 2 ml of sterile distilled water by vortexing. The contents of all centrifuge tubes from each flask were then combined so that all of the conidia of each replicate flask were in one centrifuge tube. The tubes were centrifuged as before, the supernatant was decanted and the conidia resuspended in 2 ml of sterile dtl^O by vortexing. The two tubes for each isolate, each tube representing the conidia of one flask, were combined, centrifuged as before, the supernatant was decanted and the conidia were finally resuspended in 10 ml of sterile dri^O. Conidial production of each isolate was quantified 27  by making a 10" dilution of the stock suspension and then using a hemacytometer to determine the concentration of conidia in the stock suspension. Conidia were diluted to 1 x 10 conidia per ml and then 500 ul was spread onto a 5  1.5% water agar plate for the germination and germ tube length study. One plate per isolate was incubated at temperatures ranging from 0°C to 35°C, increasing in 5°C intervals, for 18 hours in the dark. A l l isolates were incubated in the same incubators over the same period of time to insure uniform incubation conditions. To insure that each plate had 18 hours growth prior to measurement, all plates were placed at 4°C after 18 hours incubation to slow growth to a negligible amount. Conidial germination was recorded as percent germination of approximately 300 conidia surveyed on each plate. Germ tube length was calculated by averaging the germ tube length of 10 randomly selected germinated conidia on each plate. Conidia were selected for measurement if the direction of germ tube elongation matched the plane of the ocular micrometer in the microscope eyepiece. The germ tube was considered germinated i f germ tube length was greater than the diameter of the conidia.  2.2.3 Sodium alginate - kaolin clay formulation The formulation method utilized was encapsulation of conidia and mycelial fragments within sodium alginate pellets based on the protocol of Walker and Connick (1983). Isolate PFC-4277 was grown in 250 ml of modified Richard's media, containing 50 g commercial sucrose, 10 g K N 0 , 5g K H P 0 , 2.5 g M g S 0 . 7 H 0 , 0.02g FeCl ,150 3  2  4  4  2  3  ml V-8 juice and distilled water to 1 litre. The pH of the medium was adjusted to 6.0 using 50% NaOH (Templeton, 1992). The media was inoculated with mycelial fragments and placed on a rotary shaker at 125 rpm for 1 week at room temperature. After incubation, the culture was poured into an autoclaved blender and pulsed for approximately 30 seconds. After blending, the culture was examined under the microscope for damage to conidia and conidia were quantified using the hemacytometer. The total number of conidia harvested was calculated to be 3.1 x 10 resulting in a final 9  concentration of conidia in the formulated product of 2.2 x 10 conidia per ml. The 6  contents of the blender flask, as well as 125 mg of streptomycin sulfate (Fisher Scientific, Fair Lawn, NJ), were then transferred into a sterile solution of 16.625 g sodium alginate 28  (BDH, Toronto, ON) and 125 g kaolin clay (Sigma, St. Louis, MO) suspended in 1 litre of distilled water. The suspension had been autoclaved the previous day and placed on a stirrer over night to insure that the kaolin was well mixed and that the suspension was cool prior to inoculation. After addition of the fungal culture and antibiotic, the suspension was stirred for 45 minutes for complete homogenization. To form individual pellets, the suspension was pumped out of the flask using a peristaltic pump and dropped through Pasteur pipettes into a 0.25 M solution of CaCL; (Figure 2-1). The chemical reaction between sodium alginate and CaCL; resulted in the formation of pellets 3 mm in diameter as soon as the droplet entered the CaCL; solution. The pellets were removed from the CaCb solution using a strainer and washed with sterile distilled water to remove excess CaCL. and then placed on foil covered trays and allowed to air dry.  Figure 2-1. Laboratory apparatus for sodium alginate - kaolin clay formulation of C. gloeosporioides. 1. Magnetic stir plate; 2. sodium alginate - kaolin clay + C. gloeopsorioides;3. peristaltic pump, 4. Pasteur pipettes; 5. 0.25M CaCb solution and pellets.  Colletotrichum gloeosporioides isolate PFC-2329 collected from A. tsugense by Dr. Simon Shamoun (Canadian Forest Service, Pacific Forestry Centre) was also formulated at a final concentration of 1.15 x 10 conidia per ml. A sterile control was 5  formulated by adding sterile modified Richard's media and streptomycin sulfate to the 29  kaolin / sodium alginate mix, followed by dropping the suspension into CaCh and air drying. To test the viability of the fungus after formulation, fresh and dried pellets were placed on PDA and incubated at 20°C. Viability was determined by the emergence of fungal hyphae from the pellet. Dried pellets were also ground to a fine powder using a mortar and pestle and placed on PDA to assess emergence from the ground formulation. Dried pellets were stored at 4°C until required. Viability of pellets stored at room temperature and 4°C was verified by plating out on PDA after 108 days storage. The number of conidia within each pellet of the PFC-4277 formulation was calculated by dropping 1 ml of the solution into CaCb and the resultant number of pellets counted. A total of 26 pellets were formed from 1 ml of solution, therefore each pellet contained 85,500 conidia. To achieve a concentration of 1.0 x 10 conidia per ml, 6  approximately 12 pellets were used for each ml of inoculum, or 0.055 g of dried pellets per ml. To suspend the pellets for application, and to provide protection from desiccation (Boyette et al., 1993), a series of corn oil and water emulsions were made, with oil concentration of 100%, 75%, 50%, 25%, 20%, 15%, 10% and 5% by volume (Egley and Boyette, 1995). Pellets were removed from storage at 4°C and ground to a fine powder using a mortar and pestle. To each 1 ml emulsion, 0.055 g of powdered pellets were added. The powder was suspended by vortexing and four 100 ul drops were placed on each PDA plate, with 3 replicate plates per emulsion. The plates were then incubated at 20°C and the emergence of the fungus was observed.  2.2.4 Inoculation of Arceuthobium tsugense with sodium alginate formulated Colletotrichum gloeosporioides under greenhouse conditions. A small-scale greenhouse trial was conducted to test the efficacy of the sodium alginate formulated C. gloeosporioides. Experimental material was limited to western hemlock (Tsuga heterophylld) seedlings that were inoculated with Arceuthobium tsugense 37 months prior to the trial. A total of 16 A tsugense infections were present on 12 trees and prior to inoculation, the number of shoots and maximum shoot length of each A. tsugense swelling were measured. Six treatments were applied, with 5 treatments having 3 replicates per treatment and 1 treatment with 1 replicate as outlined in table 2-1. 30  Two isolates of C. gloeosporioides were formulated and applied; PFC-2329 was isolated from A. tsugense and PFC-4277 was isolated from A. americanum. As an alternative to corn oil emulsion, a carrier solution containing 2% sucrose and 0.5% gelatin (Trujillo et al., 1994) was utilized. A sterile sucrose - gelatin solution was placed in a spray atomizer and treatment 1 was applied by spraying the shoots to run off. Treatments 2,4 and 5 were performed by first spraying the A. tsugense infection with the sucrose gelatin solution to run off followed by sprinkling the aerial shoots and swelling with the sodium alginate - kaolin clay formulation that had been ground to a fine powder with a mortar and pestle. After sprinkling the formulated C. gloeosporioides onto the dwarf mistletoe, the inoculated A. tsugense was misted with the sucrose - gelatin solution to hydrate the formulation. Treatment 3 was applied by spraying a suspension of PFC-4277 conidia at 1 x 10 conidia per ml suspended in the sucrose - gelatin solution to run off 6  with a spray atomizer. Treatment 6 was applied by cutting the dwarf mistletoe shoots 5 mm from the shoot base followed by immediate application of 10 to 20 ul of the suspension used for treatment 3. After treatment application, a sample of each treatment was placed on PDA and incubated in the dark at 20°C to check the viability of the formulated conidia. For 60 hours following inoculation, the humidity in the greenhouse chamber was raised to near 100% with a humidifier and continual soaking of the cement floor.  31  Table 2-1. Treatments formulated in sodium alginate - kaolin clay and number of replicates applied to A. tsugense infecting T. heterophylla. Treatment #  Treatment description  # replicates  Tree#  1  Control 1: sucrose - gelatin only  3  5  2  Control 2: sucrose - gelatin and sterile  3  12,15,20  sodium alginate pellets 3  Conidia of PFC-4277 at 1.0 x 10 / ml  3  6, 16  4  Formulation of PFC-4277  3  9,11  5  Formulation of PFC-2329  3  2,7  6  Shoots cut 5mm from base and conidia of  1  3  6  PFC-4277 at 1.0 x 10 / ml applied to cut 6  shoots  A disease rating system was developed that was based on the percent area of the dwarf mistletoe aerial shoots covered by lesions and necrotic tissue to quantify the amount of disease. A value from 0 to 5 was assigned based on the percent necrosis as follows: 0 = no necrosis, 1 = 1% to 25%, 2 = 26% to 50%, 3 = 51% to 75%, 4 = 76% to 100%, 5 = dead. The term of the experiment was 55 days and disease rating was assessed every other day for the first 23 days and then weekly during the remaining 22 days. A t the end of the experimental period, the number of shoots and maximum shoot height were recorded.  2.3 Results 2.3.1 Collection of fungal hyperparasites of A. americanum. Diseased A. americanum was collected from lodgepole pine stands within southern interior of the province of British Columbia and in Nordegg, Alberta during the summer of 1998. A stand of jack pine near Bruderheim, Alberta was also sampled. A total of 36 sites were visited (Table 2-2, Figure 2-2 panel A ) and 504 fungal isolates were collected and lodged in Dr. Simon Shamoun's culture collection at the Pacific Forestry Centre. Of the 504 isolates collected, 187 were isolates of Colletotrichum gloeosporioides. The remainder of the fungi isolated included Cladosporium sp. and 32  Sclerophoma pithyophila, as well as other fungi that were not identified. Colletotrichum gloeosporioides was collected throughout the region sampled, except from Bruderheim, A B (Figure 2-2 panel B). The distribution of Caliciopsis arceuthobii in British Columbia was limited (Figure 2-2 panel C), and no isolates of the fungus were recovered. Cylindrocarpon gillii symptoms were not observed, nor was the fungus isolated in culture from any of the sites that were surveyed in 1998.  33  Table 2-2. Sites visited during the collection phase of the research program and the number of Colletotrichum gloeosporioides isolates collected from, and where Caliciopsis arceuthobii was observed on, Arceuthobium americanum parasitizing Pinus contorta var. latifolia.  Site  1  Location"  150 Mile House  GPS (site)  52° 02'52"N  Date  Number of C.  C. arceuthobii  (m/d/y)  gloeosporioides observed (yes/no)  5/13/98  5  Yes  5/13/98  0  Yes  5/14/98  17  No  5/14/98  0  No  5/14/98  6  No  5/16/98  0  No  6/11/98  2  No  6/11/98  6  Yes  6/11/98  0  No  6/12/98  2  No  6/12/98  0  No  6/12/98  5  No  121° 48' 39"W 2  150 Mile House  52°02'19"N 121° 46' 54"W  3  Riske Creek  51°56' 00"N 122° 47' 07"W  4  Riske Creek  51° 58' 10"N 122° 47' 33"W  5  Riske Creek  51°59'41"N 122° 50' 30"W  6  100 Mile House  51° 30' 40"N 121° 22' 23"W  7  Beaverdell  49° 22' 51"N 119° 06' 06"W  8  Beaverdell  49° 29' 54"N 119° 07' 35"W  9  Beaverdell  49° 30' 59"N 119° 06' 02"W  10  Greenwood  49° 04' 49"N 118° 35' 25"W  11  Greenwood  49° 04' 05"N 118° 36' 21"W  12  Greenwood  49° 09' 29"N 118° 37' 30"W 34  Site  13  Location*  Cranbrook  GPS (site)  49° 26' 01"N  Date  Number of C.  C. arceuthobii  (m/d/y)  gloeosporioides observed (yes/no)  6/13/98  0  No  6/14/98  0  No  6/14/98  16  No  6/15/98  0  No  6/16/98  13  No  6/16/98  8  Yes  6/16/98  0  No  6/16/98  4  No  6/17/98  0  No  7/5/98  12  No  7/5/98  22  No  7/7/98  1  Yes  7/7/98  5  No  7/7/98  3  No  7/8/98  0  No  115° 54' 00"W 14  Cranbrook  49° 22' 15"N 116° 04' 19"W  15  Cranbrook  49°23'19"N 115° 35' 33"W  16  Bull River  49°29' 17"N 115° 26' 58"W  17  Invermere  50° 27' 48"N 116° 14' 06"W  18  19  20  Radium Hot  50° 39' 34"N  Springs  116°13'32"W  Radium Hot  50° 30' 28"N  Springs  115° 40' 49"W  Canal Flats  50° 12' 50"N 116° 02' 20"W  21  Moyie  49° 16' 06"N 115° 36' 45"W  22  Logan Lake  50° 29' 34"N 120° 29' 43"W  23  Logan Lake  50° 29' 06"N 120° 52' 34"W  24  Riske Creek  51°46'43"N 122° 39' 10"W  25  Riske Creek  51°43' 53"N 122° 58' 11"W  26  Riske Creek  51°43'49"N 123° 02' 05"W  27  Big Lake  52° 27' 34"N 35  Site  Location  GPS (site)  t  Date  Number of C.  C. arceuthobii  (m/d/y)  gloeosporioides  observed (yes/no)  7/8/98  11  Yes  7/8/98  2  Yes  7/9/98  3  No  8/13/98  14  No  8/14/98  0  0  8/17/98  6  No  8/18/98  6  Yes  8/18/98  5  No  8/12/98  12  No  6/9/98  1  No  122° 08' 49" W 28  Quesnel  52°32'19"N 122° 38' 25"W  29  Quesnel  52° 31' 15"N 122° 42' 13"W  Lytton  30  50° 28' 20"N 121° 37' 39"W  31  Nordegg, A B  52° 00'14"N 116° 39' 45"W  32  §  Bruderheim, A B  53° 52'32"N 112° 58' 36"W  33  34  Tete Jaune  52° 58' 23"N  Cache  119°25'54"W  Valemount  52° 47' 34»N 119° 15' 30"W  35  Lac le Jeune  50°18' 47"N 120° 38' 30"W  36  50° 07' 44"N  Canal Flats  116° 00' 30"W Na  Grand Forks' ' 1  Na  Nearest town Collected from A. americanum parasitizing Pinus banksiana Collected by Dr. Dick Smith, no GPS  36  BRITISH COLUMBIA  AAV,  X  Collection site  •  Colletotrichum gloeosporioides Caliciopsis arceuthobii  •  •  \  \ JNCEGEORGE  City V  Forest Region Boundary •  range of Arceuthobium americanum 0 100 200km 1  i  /-L  V"~  )  /  WKLIftMS LAKE  •  Ju ^  9 ~ c •  l  ]  ^r  1  \ \  V  i VANCOUVER ICTORIA  Figure 2-2. Geographic location of: A. Arceuthobium americanum collection sites, B . Sites where Colletotrichum gloeosporioides isolates were collected and, Caliciopsis arceuthobii was observed during this study.  37  C. Where  Colletotrichum gloeosporioides (Melanconiales, Barnett and Hunter, 1998) (anamorphic stage of Glomerella cingulatd) infection of Arceuthobium americanum resulted in brown to black necrotic regions at the shoot node, which eventually coalesced and ruptured through the shoot epidermal layer to expose black acervuli. Under moist conditions, masses of pink conidia were formed on the acervuli. Acervuli and conidia production were also observed on A. americanum fruit. The conidia were 12.5 - 2 0 x2.5 - 5 um, non-septate and straight. Typical melanized appressoria of 5 x 10 um formed when germ tubes from germinated conidia contacted the bottom of the petri plate. In culture on malt extract agar (MEA), the fungal colonies had a grey center with dark brown transitioning to golden brown at the colony margin and scattered black conidiomata. On PDA, the young mycelium was fluffy, white, transitioning to mottled grey / green. Abundant conidia were produced on both M E A and P D A . The symptoms and signs of disease, as well as the conidia and appressoria characteristics agree with descriptions in Parmeter et al. (1959) and Sutton (1980) and are shown in figure 2-3. A n A. americanum infection with typical C. gloeosporioides lesions, collected from Logan Lake, was deposited in the Forest Pathology Herbarium at the Pacific Forestry Centre (DAVFP 25861).  38  Figure 2-3. Colletotrichum gloeosporioides infection of Arceuthobium americanum and morphological characteristics. 1. Lesion on shoot, scale bar represents 1 mm, E = epidermis. 2. Acervuli exuding conidia (C), scale bar represents 1 mm. 3. Conidia (C) production on diseased fruit, scale bar represents 1 mm. 4. Conidia (C), scale bar represents 100 um. 5. Appressoria (A), scale bar represents 100 jam. 6. Culture on M E A .  39  40  Caliciopsis arceuthobii was identified based on the presence of clusters of shiny, black perithecia that were present on the stylar region of the female flowers (Figure 2-4).  Figure 2-4. Perithecia (P) of Caliciopsis arceuthobii infecting female flowers of Arceuthobium americanum.  Scale bar represents 1 mm.  2.3.2 Growth characteristics of Colletotrichum  gloeosporioides.  The temperature profile of mycelial growth, spore germination and germ tube elongation of 4 isolates of C. gloeosporioides was measured at temperatures ranging from 0°C to 35°C. Figure 2-5 shows the average growth of each isolate at each temperature after 12 days incubation, before the growth rate began to slow (Figure 2-6). Maximum mycelial growth of PFC-4277, PFC-4493 and PFC-4501 occurred at 20°C, while maximum growth of PFC-4278 occurred at 25°C. Although isolate PFC-4278 did not grow as rapidly at 20°C as the other isolates, growth of this isolate at 15°C, 20°C, and 25°C was very uniform. A l l isolates grew well at temperatures ranging from 15°C to 25°C and growth was dramatically reduced at 10°C and 30°C.  41  A:•»•-•  PFC-4277 PFC-4278 PFC-4493 PFC-4501  7/  \ \ V  -T5  —r 10  \  I-  -  15  20  25  30  35  Temperature (C) Figure 2-5. C. gloeosporioides colony diameter (mm) after 12 days incubation at temperatures ranging from 0°C to 35°C. Points plotted are the mean +/- standard error of the mean.  Plotting colony diameter growth over time at 20°C revealed that the increase in diameter was linear for the first 10 to 12 days, with a very slight decrease in rate over the remainder of the experiment, and that isolate PFC-4277 had the highest daily rate of growth (Figure 2-6).  42  80  Figure 2-6. Colony growth. Increase in colony diameter of C. gloeosporioides  incubated  at 20°C over 20 days. Mean +/- standard error of the mean.  Conidia germination of the four isolates at temperatures ranging from 0°C to 35°C after 18 hours incubation was highly variable between isolates (Figure 2-7). Isolate PFC-4277 had at least 95% conidia germination at temperatures ranging from 10°C to 30°C, with a maximum rate of 97% at 25°C, indicating that the conidia were able to germinate over a very broad range of temperatures. Conidia of isolate PFC-4501 also germinated over a broad range of temperatures but maximum percent germination was only 70%) at 15°C. Isolates PFC-4278 and PFC-4493 had poor conidial germination, with maximum germination of 54% and 42% respectively at 30°C. The selected isolates had maximum spore germination rates at temperatures that ranged from 15°C to 30°C.  43  100  0  5  10  15  20  25  30  35  Temperature (C) A •  PFC-4277 PFC-4278 PFC-4493 PFC-4501  Figure 2-7. Percent conidial germination of C. gloeosporioides isolates at temperatures ranging from 0°C to 35°C.  Germ tube length at temperatures ranging from 0°C to 35°C after 18 hours incubation was maximum at 25°C for all isolates and PFC-4277 had the maximum germ tube growth, with maximum germ tube length averaging 265 (am. As with colony diameter growth, isolate PFC-4278 did not have the maximum germ tube length, but this isolate had the least amount of change between 20°C and 25°C (Figure 2-8).  44  350  Temperature (C) Figure 2-8. Germ tube elongation at temperatures ranging from 0°C to 35°C after 18 hours incubation. Mean +/- standard error of the mean.  In all measured characteristics, the growth of C. gloeosporioides was inhibited at 0°C and 35°C and growth was very slow at 5°C and 30°C. Percent conidia germination was highly variable between isolates, while the growth characteristics were much more consistent. The growth characteristics of the four isolates were compared and utilized to determine which isolate should be selected as the lead biological control isolate. Isolate PFC-4277 had the maximum diameter growth at 20°C, the maximum hyphal growth rate at 20°C, at least 95% conidia germination at temperatures ranging from 5°C to 30°C and the maximum rate of germ tube elongation. Additionally, conidia production by PFC4277 was an order of magnitude higher than the other isolates. After conidia were harvested from the millet grain, PFC-4277 was found to produce 2.035 x 10 conidia per 8  45  ml, while PFC-4493 had the second highest conidia concentration at 1.725 x 10 conidia 7  per ml and PFC-4501 and PFC-4278 produced 1.55 x 10 and 1.1 x 10 conidia per ml 7  7  respectively.  2.3.3 Formulation Sodium alginate - kaolin clay formulation resulted in the formation of very uniform pellets of approximately 3 mm in diameter after drying. Following 108 days storage, C. gloeosporioides had emerged from 16/16 replicates of the 4°C stored formulation after 48 hours, while at 72 hours C. gloeosporioides had emerged from only 4/16 replicates of the lot stored at room temperature. When the 4°C stored formulation was ground to a powder using a mortar and pestle and placed on P D A , the fungus emerged from the powder within 24 hours. Powdered formulation was suspended in a corn oil emulsion and plated onto PDA. After 5 days incubation, all emulsions up to 25% oil had 100% fungal growth, while the 50% oil emulsion had growth from 7/12 spots, 75%) oil emulsion had 1/12 spots and 100% oil had no growth. The separation of the oil and water components of the emulsion occurred rapidly. The ground powder did not distribute well within the formulation, clogged the pipette tip easily and it settled out of solution very quickly. Due to the difficulty encountered suspending the powder, the rapid separation of the oil and water emulsion, as well as the negative effect of the oil on the fungus, the approach was abandoned. In the small scale greenhouse trial on A. tsugense, the ground formulation was sprinkled onto A. tsugense swellings and shoots that were soaked with a sucrose-gelatin solution and then hydrated by spraying more of the sucrose-gelatin solution on the treated areas.  2.3.4 Inoculation of Arceuthobium tsugense with sodium alginate formulated Colletotrichum gloeosporioides under greenhouse conditions. Two isolates of C. gloeosporioides were used: PFC-4277 isolated from A. americanum and PFC-2329 isolated from A. tsugense (Table 2-1). The fungus was applied after formulation in sodium alginate - kaolin clay, as well as a conidial suspension (PFC-4277 only). The control treatments, 2% sucrose and 0.5% gelatin solution and 2% sucrose and 0.5% gelatin solution plus sterile sodium alginate - kaolin 46  clay formulation, had no effect on the dwarf mistletoe. Thirteen days following inoculation, one replicate treated with the conidial suspension of PFC-4277 had two dead and two healthy shoots. One of the dead shoots from this replicate was sampled and C. gloeosporioides was isolated from the necrotic tissue. This was the only replicate of any treatment that became diseased as a result of C. gloeosporioides application. Application of the isolates of C. gloeosporioides formulated in sodium alginate - kaolin clay had no effect on A. tsugense. The powder remained on the dwarf mistletoe shoots for the duration of the experiment, but it is likely that the fungus never emerged from the formulation. The cut shoot treatment resulted in immediate necrosis at the cut ends of the shoots, but the tree showed symptoms of root damage 13 days after inoculation and was removed from the experiment, before the results of C. gloeosporioides infection on the emergence of new shoots could be assessed. The experiment was monitored for 55 days and was ended at this point because an outbreak of black vine weevils in the greenhouse compartment resulted in damage to 14 of the 15 remaining dwarf mistletoe plants; the aerial systems of 8 of the infections were completely destroyed.  2.4 Discussion 2.4.1 Collection of diseased A americanum and hyperparasite isolation The first step in the development of a biological control agent is the selection of a pathogen that causes disease on the target organism. Therefore, the first objective of this research was to survey the range of lodgepole pine within British Columbia and to isolate fungi that caused disease on A. americanum. The presence of Caliciopsis arceuthobii, Cylindrocarpon gillii and Colletotrichum gloeosporioides infecting Arceuthobium spp. in British Columbia have been reported (Wood, 1986) and these hyperparasites were sought in this study. Colletotrichum gloeosporioides was isolated from A. americanum at 24 of 36 sites visited (Table 2-2) and was distributed throughout the southern range of A. americanum in British Columbia (Figure 2-2, panel B). Caliciopsis arceuthobii was observed at 8 sites (Figure 2-2, panel C), however the fungus was not isolated in pure culture. The collection time or the slow rate of growth of C. arceuthobii were the likely factors that prevented isolation in pure culture. Parker (1970) isolated C. arceuthobii 47  from perithecia that were collected in October, November and April, and Knutson and Hutchins (1979) isolated it from December collections, while in this study, C. arceuthobii was collected in May, June, July and August, possibly after ascospores were released from the perithecia and before perithecia were present on newly infected female flowers. Perithecia were plated directly onto the glucose-asparagine media suggested by Parker (1970), however mycelial growth of C. arceuthobii was not recovered, possibly because C. arceuthobii grows very slowly on media (Knutson and Hutchins, 1979; Parker, 1970) and the fungus was overgrown by a Cladosporium sp. present on the perithecia. Cladosporium sp. has been reported to grow on the perithecia of C. arceuthobii after ascospore liberation from the perithecia (Kjuit, 1969) and it was isolated from perithecia in this study. Cylindrocarpon gillii was not recovered or observed during the survey. Cylindrocarpon gillii was first observed on A. americanum in Montana by Gill (1952) and three accessions of this fungus from British Columbia are stored in the forest pathology herbarium at the Pacific Forestry Centre in Victoria (DAVFP 15077, D A V F P 15081, D A V F P 24008). The herbarium samples were examined, but they were old and discoloured and it was difficult to differentiate them from herbarium samples of C. gloeosporioides. Microscopic examination of spores from the herbarium samples was not conducted. The range of C. gillii in British Columbia is much more restricted than C. gloeosporioides and C. arceuthobii (Wood, 1986), therefore, it is possible that had more sites been visited, C. gillii would have been observed on A. americanum in this study. It was decided to focus on C. gloeosporioides as a potential inundative biological control agent for A. americanum because it meets the requirements outlined by Wicker and Shaw (1968) and Colletotrichum spp. have been utilized as biological control agents in other pathosystems (Templeton, 1992). The range of C. gloeosporioides was found to coincide with the range of A. americanum that was surveyed in British Columbia and was readily isolated from diseased A. americanum. As the fungus is native to A. americanum in British Columbia, the risks associated with importing exotic pathogens as biological control agents are eliminated. Cultural characteristics that make C. gloeosporioides amenable to development as a biological control agent include rapid growth and abundant inoculum production in culture. The fungus was observed to infect and kill male and female shoots in all stages of development, as well as infect fruit, and it has been reported 48  to infect the endophytic system (Parmeter et al., 1959). Artificial inoculation by Parmeter et al. (1959) resulted in symptoms of disease 13 days after inoculation and girdling lesions 21 days after inoculation. In contrast, C. arceuthobii only infects female dwarf mistletoe flowers, grows very poorly in culture and the life cycle requires two seasons for completion (Knutson and Hutchins, 1979).  2.4.2 Growth characteristics of Colletotrichum gloeosporioides. The growth characteristics of 4 isolates of C. gloeosporioides were investigated for the purpose of lead isolate selection for future biological control work. The isolates originated from three different, geographic locations, representing different areas of British Columbia. Conidial production in culture was quantified, colony diameter growth rate was measured, conidial germination and germ tube elongation at temperatures ranging from 0°C to 35°C. Isolate PFC-4277 was selected as the lead isolate because it had the highest conidia production in culture, the fastest mycelial growth and highest conidia germination rates. Although isolate PFC-4278 had little variation in mycelial growth and germ tube elongation at temperatures ranging from 15°C to 25°C and 20°C and 25°C, respectively, this isolate was not selected as the lead isolate because the percent conidia germination was low. The percent conidia germination of different isolates at different temperatures described in figure 2-8 was highly variable. When germination was quantified, all plates were removed from treatment conditions at the same time and placed in a refrigerator at 4°C, which had the effect of greatly reducing the rate of growth, but the last plates to be assessed were in the refrigerator for approximately 5 hours. Percent conidia germination over time was not measured, and it is probable that after a longer incubation time, isolates PFC-4278 and PFC-4493 would have approached the level of isolate PFC-4277. Conidia germination decreases with increasing conidia concentration (Morin et al., 1996), possibly as a result of inhibitory compounds in the conidial matrix (Mondai and Parberry, 1992). The concentration of conidia that was utilized in this experiment was 1 x 10 , which was the 5  concentration at which Morin et al. (1996) achieved maximum germination, therefore, the variability of germination observed between isolates is not likely a result of self inhibition due to over crowding. The variability of conidial germination observed in this 49  study was corroborated in a study of C. gloeosporioides isolates that were collected from mango leaves in the Philippines (Estrada et al. 2000). The authors recorded 100% conidia germination at 20°C, 25 °C and 30°C for one isolate after 15 hours, while another isolate reached 100%) germination at the three temperatures after 30 hours. In both isolates, spore germination occurred most rapidly at 25°C and 30°C (Estrada et al., 2000). Speed of conidial germination is one of the most important characteristics of a biological control agent. As the length of time between application and conidial germination increases, the probability of successful establishment decreases because abiotic factors such as relative humidity and ultraviolet light damage will decrease the viability of the conidia. The formulation process is designed to reduce the effects of abiotic factors to increase the probability of establishment; however, rapid conidia germination is also an important component of the infection process. The growth characteristics measured provided an indication of which isolate may be the best isolate for field inoculation based on assumptions relating to the relationship between conidia production, germination and fungal establishment. Virulence was not assessed directly because no experimental units of A. americanum were available for testing under controlled conditions. The isolate selected for formulation may not be the most virulent of the selected isolates, but without the ability to directly assess virulence on A. americanum, it is impossible to determine if the most virulent isolate was selected.  2.4.3 Formulation Formulation of the biological control agent is a process whereby the active ingredient is mixed with inert carriers to enhance long term storage without loss of viability and biological activity (Boyette et al., 1996). It is an important step in the development of a biological control strategy because the formulation provides moisture to reduce or eliminate the dew period requirement (Boyett, 1994), allows application with conventional spray systems (Egley and Boyette, 1995), gives protection against U V irradiation and provides nutrition to the fungal propagules (Neumann and Boland, 1999), thus enhancing the efficacy of the biological control agent Formulation of C. gloeosporioides using the sodium alginate — kaolin clay method resulted in the formation of pellets that were easy to handle and provided stable long term 50  storage at 4°C. Grinding the pellets with a mortar and pestle likely caused a reduction in inoculum viability as a result of cell wall shearing and heat generation, but the reduction was not quantified. The fungus did emerge from ground pellets, indicating that some of the formulated conidia remained viable. Inhibition by the corn oil emulsion was found to increase with increasing corn oil concentration and it was difficult to work with; therefore, the formulation application method was modified for the greenhouse trial. A solution composed of 2% sucrose and 0.5% gelatin (Trujillo et al., 1994) was used to hydrate the formulation, provide a readily available carbon source to the fungus and to allow it to adhere it to the target dwarf mistletoe.  2.4.4 Inoculation of Arceuthobium tsugense with Colletotrichum gloeosporioides under greenhouse conditions. A small scale trial under greenhouse conditions was initiated to test the ability of sodium-alginate kaolin clay formulated C. gloeosporioides to infect A. tsugense infecting T. heterophylla. Arceuthobium tsugense was not the primary target organism of this research, however, experimental units of A. americanum that could be inoculated under greenhouse conditions were not available. The greenhouse study was therefore established with the primary intention of testing an inoculation strategy prior to use with A. americanum under field conditions and to verify Koch's postulates. A n isolate of C. gloeosporioides that was collected from A. tsugense was included in the trial to avoid any host / parasite specializations that may exist. To test the susceptibility of A. tsugense to C. gloeosporioides collected from A. americanum an isolate that was collected from A. americanum was also included in the study. Infection of A. tsugense from the formulated C. gloeosporioides did not occur in the highly controlled environment of the greenhouse following 60 hours of near 100% humidity, implying that it is unlikely that the fungus would become established if applied in this manner under field conditions. Grinding the formulation did not result in 100% mortality of the fungus; therefore, it is likely that the fungus did not become established from the formulation because the moisture available to the formulated conidia was too low. A conidial suspension of C. gloeosporioides isolate PFC-4277, collected from A. americanum, did however become established on, and was re-isolated from, A. tsugense 51  suggesting that the fungus was able to cause disease on a range of species of Arceuthobium and that the environmental conditions available to the fungus following inoculation were satisfactory for establishment. The results of this experiment were also important because it was proven that it is possible for C. gloeosporioides isolated from A. americanum cause disease on A. tsugense, confirming the results of Scharpf (1964) and the lack of host specificity. The effect of the black vine weevils, as well as the failure of sodium alginate kaolin clay formulated C. gloeosporioides to infect A. tsugense, prevents assessment of C. gloeosporioides as a biological control agent for A. tsugense. Although the effect of the fungus could not be assessed, the results of the greenhouse trial were valuable because they pointed out the weakness of the formulation. This resulted in the initiation of the search for an alternative formulation method for the field trial on A. americanum.  52  Chapter 3 - Development of a biological control strategy for Arceuthobium americanum, Part II: Field trial 3.1 Introduction It was decided to focus on Colletotrichum gloeosporioides as an inundative biological control agent for Arceuthobium americanum. Prior to inoculation in the field, it is preferable to perform inoculation studies under controlled conditions. The small greenhouse trial described in Chapter 2 allowed testing of an inoculation technique but did not provide efficacy data. To generate experimental units of A. americanum for such inoculation, A. americanum seeds were collected and placed on lodgepole pine seedlings in the greenhouse. It is known that a minimum incubation time of 2 years is required before aerial shoots are produced (Hawksworth and Wiens, 1996), and unfortunately, no experimental units were ready in time for this study. Due to the lack of experimental units for greenhouse testing, the study proceeded to field inoculation of A. americanum with a C. gloeosporioides isolate that was selected based on growth characteristics rather than a direct assessment of virulence. The infection process of C. gloeosporioides is initiated when conidia, produced on acervuli, are released during rain and dew (Wastie, 1972) and disease severity increases with increasing rain (Carrington et al., 2001). Conidia are encapsulated by a conidial matrix composed of exopolysaccharides and glycoproteins (Mondai and Parbery, 1992), that serves to prevent conidial germination before dispersal and to maintain germinability during periods of environmental stress (Louis and Cooke, 1985). Following splash dispersal (Yang et al., 1990), conidia must adhere to the plant surface. The exact mechanism of conidial adhesion is unknown, but it is not likely that specific adhesives are released from the conidia (Bailey et al., 1992). Pathogenesis is initiated by the formation of melanized appressoria, which penetrate the host epidermis without restriction to a specific host structure (Makowski and Mortensen, 1998). The dew period required for successful establishment on the host is directly correlated with the timing of germination, appressoria formation and penetration (Makowski and Mortensen, 1998; Makowski, 1993). Colletotrichum gloeosporioides has a two-stage pathogenesis process, termed intracellular hemibiotrophy. The initial phase of infection is symptomless and 53  occurs after penetration of the epidermal layer. Infection vesicles are formed in infected epidermal cells, but the cytoplasm of the epidermal cell is invaginated around the vesicle. Large diameter primary hyphae emerge from the infection vesicle and enter adjacent epidermal and cortical cells. The primary hyphae are constricted as they pass through the cell and the infected cell retains its ultrastructure and membrane function for 24 to 48 hours following penetration. After 2 to 3 days, the cytoplasm of the cell has degenerated and only membrane debris are present. The primary hyphae proceed from cell to cell and there is a slow transition to cell death (Bailey et al., 1992). On some hosts, C. gloeosporioides forms latent infections, whereby conidia germinate, form appressoria and penetrate the epidermal layer and form infection hyphae that remain latent within the upper cell layers. When the host plant becomes stressed through ripening, wounding or senescence, the membrane permeability of the host cells is changed and soluable nutrients are released that may trigger the end of latency (Cerkauskas, 1988). Following the initial biotrophic phase, thin secondary hyphae are formed that cause degradation and death of cells by intracellular and intramural growth during the necrotrophic phase of the pathogen. The necrotrophic phase is responsible for the formation of anthracnose symptoms and the fungus is able to grow throughtout host tissues causing extensive cellular destruction through the production of polygalacturonases and pectin lyase that degrade cell walls. Colletotrichum gloeosporioides also produces low molecular weight phytotoxins, however, the exact role of these phytotoxins in the pathogenesis process is unknown (Bailey et al., 1992). Colletotrichum gloeosporioides is widespread on many species of Arceuthobium in the western United States and Canada (Hawksworth et al., 1977). Initially described on Arcethobium abietinum by Parmeter et al., (1959), the fungus has been suggested as a biological control agent for dwarf mistletoes (Parmeter et al., 1959, Wicker, 1967; Knutson, 1978), however, the only study in which dwarf mistletoe was inoculated with C. gloeosporioides was by Parmeter et al., (1959). Other studies have surveyed infected dwarf mistletoe stands and estimated the natural control rate of C. gleoesporioides. Wicker (1967) indicated that C. gloeosporioides provided a significant level of natural control of A. campylopodum, while Muir (1977) found that C. gloeosporioides did not provide sufficient control of A. americanum. Although C. gloeosporioides can be 54  destructive, the overall effect of the fungus was judged to be minor (Hawksworth, 1972), likely due to the fact that the fungus is a native pathogen on a native host (Hawksworth et al., 1977). The conclusion that C. gloeosporioides would not provide adequate biological control of dwarf mistletoe was drawn prior to the recent advent of formulation technology and inundative biological control. The objective of this study was to assess the potential of C. gloeosporioides as an inundative biological control agent for A. americanum under field conditions by observing symptom development and quantifying the reduction in fruit production following fungal application.  3.2 Materials and Methods 3.2.1 'Stabilize' formulation The sodium alginate - kaolin clay formulation was found to be unsatisfactory for inoculation of dwarf mistletoe, therefore, the 'Stabileze' method developed by Quimby et al. (1999) was utilized to encapsulate C. gloeosporioides. Prior to formulation, C. gloeosporioides conidia of isolate PFC-4277 were harvested from millet grain by washing with sterile distilled water using the same technique as was employed to collect conidia for the germination study (Chapter 2). The harvested conidia were quantified using the hemacytometer and the volume of the conidial suspension was adjusted with sterile distilled water to 1.0 x 10 conidia per ml. The conidia were then formulated as 7  follows: 16.656 g Waterlock® B-204 (Grain Processing Corp., Muscatine, IA) was mixed with 16.7 ml of corn oil and then heated on 'high' in a microwave oven for 2 minutes. Once the mix had cooled to room temperature, 66.625 ml of conidial suspension was added and 66.625 g sucrose and 23.32 g Hi-Sil® 233 (PPG Industries Inc., Pittsburgh, PA) were slowly added and mixed into the conidia / Waterlock® B-204 / corn oil solution. The resulting powder was spread onto foil covered trays and allowed to air dry. A sterile control formulation was also made by substituting sterile distilled water for the conidial suspension. The dried formulation was weighed and stored at 4°C until required. The formulation was prepared for inoculation by placing 5 g of the dried formulation into 500 ml of sterile distilled water, resulting in a final concentration of approximately 53,000 conidia per ml, stirred for 30 minutes and then placed in a hand  55  atomizer. To inoculate A. americanum the formulation was sprayed onto the aerial shoots and swelling to run off.  3.2.2 Site description The field site was located in the Lillooet District of the Kamloops Forest Region. The site was approximately 2 hectares in size and was classified as the dry cool variant of the Interior Douglas Fir zone (IDF dkl) following the biogeoclimatic ecosystem classification system employed by the British Columbia Ministry of Forests (Lloyd et al., 1990). The overstory was composed of A. americanum infected lodgepole pine of 80 100 years and the understory was composed of A americanum infected lodgepole pine of approximately 30 years. A l l of the A. americanum infections included in the experiment were located in the lower crown of the understory trees. A Hobo data logger (Onset, Bourne, M A ) was installed near the center of the plot to record temperature and relative humidity (%) at 15 minute intervals from 1 week prior to inoculation day until 6 weeks following inoculation.  3.2.3 Experimental design Arceuthobium americanum infections were selected for inclusion in the field trial if they met the following criteria: A. americanum shoots were disease free, with well defined localized swellings (ie. no systemic infections) and a single A. americanum infection on the host branch. Male and female A. americanum infections were selected so that C. gloeosporioides symptom development could be monitored on as large a sample population as could be located within the inoculation site. Dwarf mistletoe age cannot be determined without destructive sampling, therefore, it is likely that a range of A. americanum ages was represented in the sample population. A total of 255 individual A. americanum infections and 7 treatments were included in the field trial (Table 3-1). There were 45 replicates of treatments 1, 2, 3, 4 and 5 and 5 replicates of treatments 6 and 7. Treatments 6 and 7 were included as observational controls and were not included in the statistical analysis. The number of male and female dwarf mistletoe infections was equalized between treatments, with treatments 1, 2 and 3 having 16 male and 29 female infections per treatment, treatment 4 56  having 14 male and 31 female infections and treatment 5 having 15 male and 30 female infections. The number of female infections per treatment was equalized so that the effect of C. gloeosporioides on fruit production could be assessed. Multiple A. americanum infections on the same host tree received the same treatment to avoid translocation effects and treatments 1,2 and 3 were each applied to A. americanum on 31 trees, treatment 4 was applied to 30 trees and treatment 5 was applied to 29 trees. The effect of relative humidity on the establishment of C. gloeosporioides on A. americanum was tested by randomly enclosing 22 replicates from each treatment within a clear polyethylene bag for 48 hours following inoculation. After application, P D A plates were sprayed with treatment 1 and the control 'Stabileze' used in treatment 4 to verify viability of C. gloeosporioides and sterility, respectively.  Table 3-1. Treatment description and number of replicates per treatment in the field trial designed to test the efficacy of C. gloeosporioides as a biological control agent for A. americanum. Treatment # 1  Treatment description  # of replicates  Isolate PFC-4277 formulated in 'Stabileze' sprayed on A. americanum  45  shoots to run off (two applications). 2  Isolate PFC-4277 formulated in 'Stabileze' applied directly to cut ends  45  of A. americanum shoots cut 5 mm from the bark. 3  Isolate PFC-4277 applied as a mycelial plug to artificially wounded  45  lodgepole pine bark in the center of the A. americanum swelling. 4  Formulation control: 'Stabileze' formulated with sterile distilled water  45  in place of fungal conidia and sprayed to run off. 5  Check: The A. americanum infection was not treated.  45  6  Cut shoot control: A. americanum shoots were cut and not treated.  5  7  Mycelial plug control: sterile media was applied to artificially wounded  5  lodgepole pine bark in the center of the A americanum swelling.  Treatments 3 and 7 were applied as follows: 1. The bark was thoroughly wiped with a 95% ethanol soaked cheesecloth to reduce surface contamination, 2. The cutting 57  end of a #4 cork borer was dipped in 95% ethanol and a hole was cut in the bark of the lodgepole pine branch and the bark removed to expose the cambium, 3. A plug of C. gloeosporioides mycelium and P D A (treatment 3, mycelium side down) or sterile P D A (treatment 7) was placed in the wound, 4. The wound was wrapped with Parafilm M (American National Can, Chicago, IL) to prevent colonization of the P D A by airborne contaminants. Treatments 2 and 3 were designed to inoculate the endophytic system of A. americanum. Dwarf mistletoe shoots are directly connected to the endophytic system and by inoculating the ends of cut shoots with C. gloeosporioides, a conduit was provided for the fungus to enter the endophytic system. Inoculation of the bark with C. gloeosporioides during treatment 3 application placed the fungus in close proximity to the endophytic system, possibly allowing fungal colonization. These treatments were included in the experimental design to observe the effect of C. gloeosporioides on new shoot production as well as to provide samples for destructive sampling that are required in Chapter 4.  3.2.4 Pre-treatment assessment Prior to inoculation, the following data was recorded for every A. americanum infection: swelling diameter, swelling length, distance between extreme shoots, number of shoots, number of buds (shoots less than 5 mm in height), number of fruit, maximum shoot length, sex, and vigour rating (Table 3-2, Figure 3-1).  Table 3-2. Vigour rating classes developed to classify individual A. americanum infections. Vigour rating  Description  0  Dead  1  Small infection with 1 or 2 shoots and few fruit (if female).  2  Small infection with few shoots and modest fruit (if female).  3  Average infection with approximately 10 shoots and fruit (if female).  4  Large infection with many shoots and fruit (if female)  5  Huge infection with many long shoots and large numbers of fruit (if female). 58  Figure 3-1. Typical A. americanum infections of each vigour rating. 1. Vigour rating 1. 2. Vigour rating 2. 3. Vigour rating 3. 4. Vigour rating 4. 5. Vigour rating 5.  3.2.5 Treatment application dates Treatments 1, 2, 4 and 5 were applied on July 23, 2000. The weather during inoculation day was cool and overcast with a maximum temperature of 22°C at 3:19 P M , which coincided with the minimum relative humidity of 46.6%. The weather remained cool with high relative humidity for the week following inoculation (Appendix 1,2). Treatment 6 was applied on July 25, 2000 and treatments 3 and 7 were applied on July 59  31, 2000. Treatment 1, but not the controls, was reapplied on August 14,2000, three weeks after the initial inoculation, as a precaution to provide adequate inoculum.  3.2.6 Treatment assessment Following treatment, data were recorded at regular intervals. The disease rating scale developed for the greenhouse trial (Chapter 2) was utilized to assess disease progression on the treated dwarf mistletoe infections. Disease rating and vigour rating of all treatments were assessed at 2 days, 4 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 2 months, 8 months and 1 year following inoculation from the date of application of treatments 1,2,4 and 5. Five weeks following inoculation, A. americanum swelling diameter and length, distance between extreme shoots, number of shoots, number of buds, number of fruit and maximum shoot were recorded as well as disease rating and vigour rating. One year following inoculation, the same measurements and counts were performed as were taken at 5 weeks, as well as the number of female flowers. A photographic record was collected to document the treatment effect on selected replicates of the different treatments.  3.2.7 Sampling at one year One year after inoculation, a sample of 10 randomly selected (via a random number generator) replicates was collected from each of treatments 1,2, 3,4 and 5 and 1 replicate of treatment 6 and 7. The samples were collected and returned to the laboratory where each sample was examined thoroughly for signs and symptoms of disease. If the dwarf mistletoe shoots had any symptoms of disease, even symptoms that were not characteristic of C. gloeosporioides, the shoots were surface sterilized and placed on PDA. Hyphae that emerged from the diseased shoots were subcultured and C. gloeosporioides was identified based on cultural characteristics and spore morphology. A plate trapped antigen ELISA kit that was specific for Colletotrichum spp. (Adgen Identikit B, Adgen Agrifood Diagnostics, U K ) was used to confirm the genus of these isolates. The manufacture's protocol was followed with one exception: 2 ml of coating buffer was introduced directly onto the mycelial mat in the petri plate, the culture was then scraped with a sterile loop and the buffer and mycelial fragments were incubated at 60  4°C for 1 hour followed by centrifugation at 10,000 xg for 5 minutes then 0.10 ml of the supernatant was placed in an ELISA well. The ELISA plate was incubated at 4°C overnight and then the wells were emptied and washed with the supplied wash buffer. The supplied blocking buffer was then added and the plates incubated at 37°C for one hour. Following incubation, the wells were emptied and washed and then the probe antibody (probe specificity proprietary information, Adgen Agrifood Diagnostics) was added and the plate incubated at 37°C for one hour. After washing, the conjugate was added to the wells and incubated at 37°C for one hour. A thorough wash preceded the addition of the substrate and following substrate addition, the plate was incubated in the dark at room temperature. A colour change from yellow to blue after the addition of the substrate indicated a positive reaction. The colour change was quantified by absorbance at 650 nm using a spectrophotometer that was blanked with the negative control.  3.2.8 Statistical analysis Analysis of variance on ranks was used to determine i f significant differences existed between treatments 1, 2, 3,4 and 5 and Dunn's method was used to reveal which treatments were significantly different. Treatment 2 results at one year were excluded from all analyses, except the number of buds and disease rating, because the treatment process, rather than C. gloeosporioides, caused the change in the assessed variable. The paired observation t-test or the signed rank test was utilized to determine i f significant changes in replicates of each treatment occurred over the course of the experiment by comparing the pretreatment condition to 1 year following treatment application. Chisquare analysis was used to study the relationship between disease rating and bagging the dwarf mistletoe infections with clear polyethylene bags for 48 hours post inoculation. Two by two contingency tables were constructed to compare the presence or absence of C. gloeosporioides and the presence or absence of the plastic bag. A l l statistical analyses were performed using SigmaStat version 2.03 (SPSS Inc., Chicago, IL).  61  3.3 Results 3.3.1 'Stabileze' formulation The formulation technique utilized in this field trial was the 'Stabileze' method developed by Quimby et al. (1999). This method was simple to perform and resulted in aggregates that ranged in size, with a maximum size of 5 mm. When placed on P D A , the aggregates rapidly became hydrated and C. gloeosporioides hyphae emerged within 24 hours. When suspended in water as a 1% solution, the formulation dissolved fully and was easily sprayed through a hand atomizer without clogging.  3.3.2 Establishment of C. gloeosporioides Four days following inoculation, 5 replicates of treatment 1 were showing disease, as were 3 replicates of treatment 4 and 2 replicates of treatment 5. One month following inoculation, one sample from treatment 1 was collected and C. gloeosporioides was recovered from the diseased tissue. Initially, brown necrotic regions were observed, which enlarged over time and girdled the shoot, resulting in death of the distal portion of the shoot. The formation of acervuli following inoculation was not observed. The result of successful establishment on A. americanum inoculated with C. gloeosporioides via treatment 1 is documented in figure 3-2. When treatment 1 was applied to the A. americanum shoots, many of the replicates that had symptoms of C. gloeosporioides disease were only showing symptoms on 1 or 2 shoots. This suggests that when the fungus became established, infection was not uniform. When the experiment was assessed one year after inoculation, 14 replicates of treatment 1 had less disease than they had at 2 months, while 7 had increased. The decrease in disease is likely due to abscission of the dead shoots.  62  Figure 3-2. Infection of A. americanum by C. gloeosporioides as a result of application of treatment 1, 'Stabileze' formulated C. gloeosporioides sprayed to run-off (DR = disease rating, V R = vigour rating). This replicate was not enclosed with a polyethylene bag. A . Pre-inoculation, DR = 0, V R = 5. B . One month postinoculation, DR = 1, V R = 5. C, Two months post-inoculation, D R = 2, V R = 5. D. One year post-inoculation, DR = 5, V R = 0.  3.3.3 Cut shoot inoculation - Treatment 2 A l l replicates that were subjected to treatment 2, 'Stabileze' formulated C. gloeosporioides applied to the cut shoots, had necrosis occurring at the cut ends of the shoots 4 days after treatment, while the controls (treatment 6) were dry and desiccated at the cut ends. Three weeks following application, it was noted that many of the inoculated shoots were being abscised at the basal cup. It was also noted that some buds that were not cut during treatment, because they were too small, had started to grow, resulting in  63  increased vigour rating. One year after inoculation, many of the replicates were producing buds, suggesting that they were recovering from treatment.  3.3.4 Dead replicates one year following inoculation. A l l A. americanum infections that were selected for inclusion in the field trial were healthy and not showing signs or symptoms of disease prior to inoculation and they were located on healthy lodgepole pine branches. One year following inoculation however, several of the replicates were lost from the experiment because the infected branch was dead as a result of self-pruning by the lodgepole pine host, therefore resulting in A. americanum death. Other replicates were lost from the experiment as a result of squirrel damage to the localized infection and some tags had fallen off of the host branch. Dwarf mistletoe infections that were killed as a result of host branch death or squirrel damage, or from which the tag had fallen off, were removed from the experiment and not included in the statistical analysis. The number of replicates remaining in the experiment one year after inoculation is recorded in table 3-3.  Table 3-3. Number of living dwarf A americanum infections prior to treatment and one year following treatment application. Treatment  Female Pre  Male  1 -year  Pre  Total 1-year  Pre  1-year  1  29  26  16  13  45  39  2  29  26  16  14  45  40  3  29  22  16  16  45  38  4  31  27  14  14  45  41  5  30  25  15  14  45  39  3.3.5 Effect of C. gloeosporioides on A. americanum fruit production Prior to treatment, there was no significant difference in the number of fruit present between treatments. In 2001, A N O V A on ranks indicated that the difference between treatments 1 and 3 and the controls was not significant, but the fruit crop was lower on the treated infections than the controls. The signed rank test indicated that the 64  number of fruit present on treatments 1, 4 and 5 were not significantly reduced from 2000 to 2001, but the number of fruit present on treatment 3 was (Figure 3-3). Variability in the number of fruit between replicates of the same treatment was high prior to treatment and in the year following treatment (Table 3-4, Figure 3-3). The number of fruit present on a single A. americanum  infection in 2001 was independent of the number of fruit  present in 2000: ie. replicate 2095 (treatment 1) had 44 shoots and 973 fruit prior to treatment and 0 shoots and 0 fruit in 2001 while replicate 2031 (treatment 1) had 17 shoots and 38 fruit prior to treatment and 27 shoots and 566 fruit in 2001. These two replicates are indicative of the amount of variation present in A. americanum  from year to  year, as well as the variation that was observed following application of treatment 1.  140  LTJ  1  2  A  0  CO  +"  -  ZZ.  100 80  o  60  •7> 40  20  -I  \s s i Pre treatment I I 1-year post  I  m  14  Trt 1  Trt 2  Trt 3  'A  Trt 4  Trt 5  Treatment Figure 3-3. Average number of fruit per female A. americanum  infection of each  treatment prior to treatment application in 2000 and one year following application in 2001. Treatment 2 excluded from 2001 A N O V A analysis. Mean +/- standard error of the mean.  65  Table 3-4. Distribution of the number of fruit present on A. americanum infections in 2000 and 2001. Treatment  Number of replicates with number of fruit in indicated range 1-10  0  11 - 1 0 0  101 - 1000  Pre  Post  Pre  Post  Pre  Post  Pre  Post  1  6  9  2  2  15  11  7  4  2  12  31  5  3  11  0  10  0  3  9  12  5  7  11  9  11  2  4  13  18  3  1  15  8  6  6  5  7  10  7  5  12  8  7  4  3.3.6 Number of female flowers The number of female flowers present on each infection was counted one year following inoculation to provide an estimate of the number of fruit that will be present two years after inoculation. A N O V A on ranks found no significant difference between treatments. As with fruit production, flower production was highly variable amongst replicates of the same treatment.  100  LU  if) 80 c CO  1 cn  J3>  co E  60-  40  *—  . CD  o  CD  20  Trt 1  Trt 2  Trt 3  Trt 4  Trt 5  Treatment Figure 3-4. Average number of female flowers present one year after inoculation. Treatment 2 excluded from A N O V A . Mean +/- standard error of the mean. 66  3.3.7 Effect of C. gloeosporioides on A. americanum shoot height Analysis of maximum shoot height prior to treatment application by A N O V A on ranks indicated that there was no significant difference between the maximum shoot height of A americanum infections of the different treatments. One year after treatment application, A N O V A indicated that there was no significant difference between treatments. When paired observation t-tests were conducted to compare the maximum shoot length of individual infections of each treatment in 2000 and in 2001, all treatments resulted in a significant decrease in maximum shoot height (Figure 3-5).  30  UJ 00  i  Pre treatment i 1-year post  60  1i  S £  5  40  o  E  20  Trt 1  Trt 2  Trt 3  Trt 4  Trt 5  Treatment  Figure 3-5. Average maximum shoot height (mm) of A. americanum swellings prior to treatment application in 2000 and one year following application in 2001. Treatment 2 excluded from 2001 A N O V A . Mean +/- standard error of the mean.  3.3.8 Effect of C. gloeosporioides on the number of A americanum shoots. A N O V A on ranks indicated that there was no significant difference in the number of A. americanum shoots present on each infection prior to treatment or one year after treatment. The number of female shoots was decreased in 2001 from the initial number in all treatments and the signed rank test indicated that treatments 2, 3, and 4 had a significant reduction (Figure 3-6).  67  14 i/^/i Pre treatment i i 1-year post  I + c:  10 H 8 -  t5 o  SZ  6 -  O  i  42  Trt  1  Trt 2  Trt 3  Treatment  1 Trt 4  Trt 5  Figure 3-6. Average number of shoots per A americanum infection prior to treatment application in 2000 and following inoculation in 2001. A N O V A in 2001.  Treatment 2 excluded from  Mean +/- standard error of the mean.  3.3.9 Effect of C. gloeosporioides on A. americanum bud production A N O V A on ranks indicated that the number of buds present was not significantly different between treatments prior to treatment application or one year later. The number of buds one year following treatment was not significantly different from the number present at the time of treatment for treatments 1, 3,4 or 5 as determined by the signed rank test. Treatment 2 however had a significant increase in the number of buds produced following treatment application, suggesting that A. americanum responded to the physical damage to the shoots by inducing new shoot production (Figure 3-7).  68  8 \/SA  Pre treatment 3 1-year post  Trt 1  Trt 2  Trt 3  Trt 4  Trt 5  Treatment Figure 3-7. Average number of buds (shoots <5mm high) present on A americanum infections prior to treatment in 2000 and one year following treatment in 2001. Treatment 2 included in A N O V A in 2001. Mean +/- standard error of the mean.  3.3.10 Analysis of disease rating In addition to quantifying the change in the physical characteristics of A. americanum, response to C. gloeosporioides infection was quantified by estimation of fungal colonization of A. americanum shoots. Two months following inoculation, A N O V A on ranks indicated that treatments 1 and 2 had significantly higher disease ratings than treatments 3,4 or 5 (Figure 3-8); however, one year following inoculation, the disease rating of each treatment was not significantly different between treatments (Figure 3-8). Unlike the other measures, the disease rating of A. americanum swellings subjected to treatment 2 was a result of fungal colonization of the cut dwarf mistletoe shoots and the associated necrosis, not an artifact of the treatment. Other than treatment 2, the disease rating was low following treatment and most of the treated A. americanum infections had disease ratings of 0 or 1 two months (Table 2-7), and one year (Table 2-8), following treatment application.  69  5 i/  2-months post 1-year post  co ro CD C  I  < D C O ro Q C Oj  Q  2  0  -I  T  1  Trt 1  Trt  2  Trt 3  Trt 4  Trt  5  Treatment Figure 3-8. Average disease rating of A. americanum infections two months and one year following inoculation. Mean +/- standard error of the mean.  Table 3-5. Number of A. americanum infections of each disease rating (DR) for all treatments two months after inoculation. Treatment  DR = 0  DR = 1  DR = 2  DR = 3  DR = 4  DR = 5  1  18  17  7  1  2  0  2  7  17  0  1  3  13  3  38  4  0  0  0  1  4  36  7  1  0  0  1  5  38  4  1  0  1  0  70  Table 3-6. Number of A. americanum infections of each disease rating (DR) for all treatments one year after inoculation. Treatment  DR = 0  DR= 1  DR = 2  DR = 3  DR = 4  DR = 5  1  26  6  3  0  1  2  2  21  8  2  0  0  9  3  20  6  4  1  2  5  4  28  7  0  0  1  6  5  23  11  0  0  1  2  3.3.11 Vigour rating analysis Vigour rating, a measure of the vitality of A. americanum, was assessed at the same time as disease rating. Prior to treatment application and at one year following inoculation, A N O V A on ranks indicated that there was no significant difference in vigour rating among treatments. When the signed rank test was used to compare individual dwarf mistletoe infections, it was found that all treatments had significantly decreased vigour ratings one year following inoculation (Figure 3-9).  71  5 I / A Pre treatment  Trt 1  Trt 2  Trt 3  Trt 4  Trt 5  Treatment Figure 3-9. Average vigour rating of A. americanum prior to treatment application and at one year following application. Treatment 2 excluded from A N O V A in 2001. Mean +/- standard error of the mean.  3.3.12 Effect of increased humidity The effect of artificially raising the humidity by enclosing 22 replicates of each treatment within a plastic bag for 48 hours after treatment application was analyzed using Chi-square analysis of the data from two months after inoculation when there was a significant difference in disease rating between treatments 1 and 2 and the controls. Two chi-square tests were conducted: the first included all replicates of treatments 1 through 5 (X = 3.537, df = 1) while the second included only replicates of treatment 1 (x = 2.681, df = 1). The results of both chi-square analyses indicated that the presence of C. gloeosporioides was not dependent upon the presence of the plastic bag.  3.3.13 Sampling in 2001 One year after inoculation, a random sample of 10 replicates of each treatment was collected from the field trial. The samples were returned to the lab where they were 72  screened for the presence of C. gloeosporioides. Of the 10 random samples collected from treatment 1, shoots of 8 were cultured, but C. gloeosporioides was not recovered. The basal cups and individual tissues of 2 shoots were randomly selected and cultured from all 10 samples of treatment 2 and C. gloeosporioides was not recovered. The branch tissue of treatment 3 was sectioned longitudinally and one half of the infection was cut at 3 to 5 mm intervals and placed on PDA. Of the 10 samples collected, C. gloeosporioides was recovered from 2 samples, but the fungus was restricted to the wound. ELISA confirmed the identity of the genus of this isolate as Colletotrichum. Three samples from treatment 4 and four samples from treatment 5 were cultured and C. gloeosporioides was not recovered.  3.4 Discussion Arceuthobium americanum was inoculated with C. gloeosporioides under field conditions to investigate the potential of the fungus as an inundative biological control agent of A. americanum. The trial was installed in July 2000 and assessed at regular intervals for a year following inoculation to quantify the effect of the fungus on A. americanum. Field inoculation of dwarf mistletoe with C. gloeosporioides was conducted in the past (Parmeter et al., 1959), but no trials of the scale conducted in this study have been established. The results of the trial suggest that the fungus did not have a significant impact on A. americanum and that future fruit production would not be significantly reduced as determined by flower production one year after inoculation. On some replicates C. gloeosporioides did become established, as shown in figure 3-1, and caused a large decrease in the reproductive ability of A. americanum. The fungus was isolated from the diseased tissue but the results were highly variable and a significant reduction in fruit production, compared to the controls, was not observed. One year following inoculation, there was little evidence of C gloeosporioides on the inoculated swellings, likely because dead shoots were shed. The shedding of dead shoots may have prevented secondary inoculum production that would have caused further disease and increased efficacy. There are several factors that likely contributed to the results that were observed in this field trial, including environmental conditions, inoculum concentration and isolate 73  selection. The maximum temperature immediately following inoculation (Appendix 1) was slightly below the optimum spore germination temperature of 25 °C, but growth of the fungus should not have been inhibited by temperature. Humidity was also relatively high immediately following inoculation (Appendix 2). Length and dew period temperature are critical factors in the establishment of C. gloeosporioides (Luo and TeBeest, 1999). Optimum temperature and dew period requirements have been established for Colletotrichum gloeosporioides f. sp. malvae infecting round-leaved mallow (Malvapusilla Smith) and velvetleaf (Abutilon theophrasti Medic). Makowski (1993) found that a minimum 20 hour dew period at 20°C or 25°C and 48 hours at 15°C was required to achieve 80% mortality on round-leaved mallow and 48 hours at 25°C on velvetleaf when conidia suspended in water were utilized. To establish 100% infection of northern jointvetch (Aeschynomene virginica (L.) B.S.P.), a minimum dew period of 12 hours at 28°C is required by C. gloeosporioides f. sp. aeschynomene (Luo and TeBeest, 1999). When field bindweed (Convolvulus arvensis L.) was inoculated with Phomopsis convolvulus Ormeno, 18 hours of dew resulted in much better infection than short interrupted dew periods (Morin et al., 1990). In this study, C. gloeosporioides was formulated in a solution that provided increased humidity, and the relative humidity was high following treatment, but the number of degree hours required for successful infection and the dew period requirement may not have been satisfied. The Cascade dry cool variant of the interior Douglas-fir zone (IDF dk2), where this experiment was conducted, is characterized by a warm, dry climatic regime with a long growing season and is commonly subjected to moisture deficits (Lloyd et al., 1990). The environmental conditions that occurred during the inoculation period are likely as close to optimum for C. gloeosporioides colonization as can be achieved at the time of year and location where the inoculation was conducted. Formulation of C. gloeosporioides was conducted to compensate for the dry conditions, but it is possible that the environmental conditions that characterize this zone limit C. gloeosporioides establishment and that the establishment of the fungus following inoculation may be higher in moister regions. Spring or fall inoculation in the IDF dk2 may result in greater infection success than was observed in this trial due to extended dew periods, however, low night temperatures may become limiting. 74  Inoculum concentration is another factor that may have affected C. gloeosporioides establishment in this study. The 'Stabileze' formulation proved to be a very good system for delivering C. gloeosporioides to A. americanum; however, the final inoculum concentration of the fungus was approximately 5.3 x 10 conidia per ml. This 4  concentration of inoculum is below the concentration that causes inhibition (Morin et al., 1990), therefore establishment was not limited by a high concentration of conidia. Luo and TeBeest (1999) used an inoculum concentration of 1.0 x 10 in their experiments 5  with C. gloeosporioides f. sp. aeschynomene and Makowski (1993) found the best control of round-leaved mallow and velvetleaf occurred with inoculum concentrations of 2 x 10  6  and 4 x 10 spores per ml, respectively. Morin et al. (1990) suggest that high inoculum 6  concentrations of 1.0 x 10 conidia per m be utilized to achieve satisfactory control 9  2  under sub-optimal moisture conditions; however, as conidia concentration increases disease does not increase due to inhibition of conidia germination (Makowski 1993). Unfortunately, experimental units of A. americanum that could be placed under controlled environmental conditions were not available. This prevented the detailed studies necessary to determine the exact temperature and moisture requirements, or optimum conidia concentration in the formulation, required for C. gloeosporioides to become established on A. americanum. The absence of experimental units under controlled conditions also prohibited study of the variation in virulence of C. gloeosporioides isolates and selection of the best isolate for field inoculation was made based on growth and sporulation characteristics in culture, not virulence assays. The results of successful establishment on a few replicates suggest that C. gloeosporioides has the potential to interfere with the life cycle of A. americanum and thereby reduce spread and intensification of the dwarf mistletoe. Before a successful inundative biological control strategy can be utilized, several obstacles must be overcome. Further work, in a controlled environment, is required to determine the optimum conditions required for successful infection of A. americanum by C. gloeosporioides so that the formulation and application aspects of the system can be modified. It is possible that the environmental conditions of the interior Douglas fir zone are limiting and that inoculation of A. americanum in a different biogeoclimatic zone would have resulted in greater establishment of C. gloeosporioides. If the requirements 75  for successful establishment can be defined and further trials suggest that C. gloeosporioides has potential as a biological control agent, additional challenges, including techniques for inoculation of A. americanum infections high in the crown, must be overcome before a successful inundative biological control strategy for A. americanum can be utilized by the forest industry.  76  Chapter 4 - Cultural and histopathological examination of the infection of the endophytic system of Arceuthobium americanum by Colletotrich um gloeosporioides.  4.1 Introduction Although the aerial shoots are the most obvious part of the dwarf mistletoe plant, the importance of the portion of the dwarf mistletoe growing within the host tissue, the endophytic system, cannot be overlooked. The endophytic system serves as the interface between the host and parasite. Mineral nutrients, carbon compounds and water are absorbed from the host (Alosi and Calvin, 1984), and new shoots arise from the cortical strands (Kuijt, 1955). If a fungal hyperparasite infects the endophytic system, not only will absorption of nutrients be affected, but new shoot production will be as well. From an inundative biological control standpoint, reduction of new shoot production would increase the interval between biocontrol treatment because the period of time between application and new fruit production would be extended. If the hyperparasite were able to kill the endophytic system, new shoot production, and therefore fruit production, would be eliminated. This chapter examines the role of Colletotrichum gloeosporioides in the endophytic system of Arceuthobium americanum. The aerial shoots have direct continuity with the endophytic system of the dwarf mistletoe. The endophytic system is composed of longitudinally and tangentially oriented strands and radially oriented sinkers (Alosi and Calvin, 1984). The radially oriented sinkers become embedded in the host xylem as the vascular cambium divides to add xylem tissue (Hunt et al. 1996). As the sinker penetrates the vascular cambium, the cambial cells remain undamaged and ray formation is stimulated. The ray gradually becomes embedded as new xylem is produced by the cambium (Kuijt, 1955). This occurs as fusiform initials are converted to ray initials adjacent to the parasite cells, resulting in the formation of a radially-oriented sinker that is surrounded by host ray cells (Alosi and Calvin, 1984b). This structure has been termed an "infected ray" by Srivastava and Esau (1961), which is an appropriate term as the development of the sinker occurs in close association with the host. As the sinker becomes embedded in the xylem tissue, meristematic activity must occur so that continuity with the cortical strands 77  is maintained. Exactly where within the sinker this meristematic activity occurs has been debated. Cohen (1954) suggests that sinkers are initiated by meristematic parenchyma cells within the cortical strand and that growth in length of the sinkers occurs by cell division at the base of the sinker where it joins the strand. Srivastava and Esau (1961) suggest that the parenchyma cells that cross the host cambium are meristematic because they are spindle-shaped and shorter and have denser cytoplasm than sinker cells in the xylem, as well as the presence of protoplasts and groupings of derivatives of single cells. Alosi and Carol (1984b) have observed distinct intercalary meristems juxtaposed to the host cambium, but they have also observed cellular arrangement within the neck of sinkers that suggests meristematic activity. It has been observed that many sinkers have no, or discontinuous, xylem and that no direct interspecific tracheary element connections exist. Water and mineral uptake by the mistletoe occurs via continuity of host and parasite cellulosic cell walls and through specialized half-bordered pits that are adjacent to sinker cells. It is hypothesized that water flows into the sinker and eventually to xylem in the cortical strand, where there is low resistance to movement and negative water potential created by transpiration in the aerial shoots results in continual water supply (Alosi and Carol, 1984). The aerial shoots and sinkers are connected by the cortical strands, which are located within the host phelloderm, cortex and phloem (Hunt et al., 1996). The strands are predominantly longitudinal and they cross and anastomose, as indicated by the presence of several steles within a single strand (Cohen, 1954). The sinkers are usually connected to the cortical strand by parenchyma cells, as xylem is typically not present in the sinker. Cortical strands have a central xylem core that is surrounded by a sheath of parenchymous cells (Hunt et al., 1996). Extension of the cortical strand occurs by an apical cell that divides transversly to create a row of segments, which divide longitudinally. These cells then undergo anticlinal and periclinal divisions to create longer strands of increased diameter. The periclinal divisions result in an inner core of small cells that are surrounded by larger cells. The small cells are meristematic and differentiate into xylem tracheary elements (Bhandari and Nanda, 1970). There is no phloem in the cortical strand (Cohen, 1954). The production of new shoots from the cortical strand occurs by the production of buds that appear as knob-like meristematic 78  protuberances on the outer cortical strand. As the bud develops, the basal cells divide and develop an intercalary stalk with tiered cells. When the bud is pushed to the surface, a cork cambium is formed by the host to produce a periderm that protects the cortical parenchyma cells of the host (Cohen, 1954). As outlined above, the aerial shoots are directly connected to the endophytic system within the host tissue and new shoots arise from the cortical strands. The connectivity between the aerial shoots and the endophytic system provides a conduit through which C. gloeosporioides may be able to infect the endophytic system of A. americanum. Colletotrichum gloeosporioides infection is initiated when a conidia, aided by the conidial matrix, is adhered to susceptible plant tissue. A melanized appressorium is produced at the terminal end of the germ tube after conidial germination, from which arises a penetration peg that penetrates the cuticle. After penetration of the cuticle, C. gloeosporioides hyphae ramify through the tissue during the symptomless biotrophic phase of infection and then secondary hyphae are formed that cause necrosis during the necrotic stage of infection (Bailey et al., 1992). If the endophytic system did become diseased as a result of C. gloeosporioides travelling from the shoots into the endophytic system, production of new shoots may be affected because new shoots arise from the cortical strands, which would effect the treatment periodicity of a biological control agent. The role of C. gloeosporioides within the endophytic system of Arceuthobium campylopodum Engelm. f. abietinum (Engelm.) Gill parasitizing red fir (Abies magnifica Murr) was investigated by Parmeter et al. (1959). The authors noted that when placed on water agar, superficial acervuli with setae developed in the region of the cortical strands of the endophytic system. The authors sectioned the fir bark and wood and were able to observe mycelium in the endophytic system. Diseased dwarf mistletoe infections were collected and C. gloeosporioides was isolated from the woody tissue of 12 of the 27 samples. In a second study, C. gloeosporioides was observed and isolated from the woody tissue of 5 of 5 diseased dwarf mistletoe swellings. From these observations, it was concluded that C. gloeosporioides was able to infect the endophytic system of A. campylopodum f. abietinum (Parmeter et al., 1959). Wicker and Shaw (1968) collected C. gloeosporioides from A. americanum, A. campylopodum, A. laricis and A. douglasii 79  and observed C. gloeosporioides hyphae penetrating the "interior cortical region, but never the vascular elements", but it is unclear whether the fungus was observed in the endophytic system of all these species. From a biological control standpoint, the results reported by Parmeter et al. (1959) and the observations of Wicker and Shaw (1968) are encouraging. They concluded that the endophytic system was colonized by C. gloeosporioides. However, many details were omitted. It is not stated precisely where within the endophytic system the fungus was observed and the pathological effects of the fungus in the endophytic system were not described. The objectives of this study were to determine i f C. gloeosporioides was able to infect the endophytic system of A. americanum and to describe the pathological effects of infection.  4.2 Materials and Methods 4.2.1 A. americanum naturally infected with C. gloeosporioides Diseased and healthy localized A. americanum infections were collected in the summer of 2000 from Logan Lake and Lytton, B C . Dwarf mistletoe infections that were showing extensive signs of disease, and were located on otherwise healthy branches, were split longitudinally and one half was surface sterilized with 95% ethanol and 10% sodium hypochlorite and the other half was preserved in formalin acetic acid (FAA) (Johansen, 1940). Samples were aseptically removed from the surface sterilized dwarf mistletoe infections and they were carefully dissected into small chips to isolate tissue from xylem, cambium, phloem, phellum, and the basal cups. The individual tissue types were then plated on potato dextrose agar (PDA, Difco), incubated at 20°C and observed at regular intervals. Fixation and isolation was conducted immediately after collection from the field. If C. gloeosporioides was isolated from any of the tissues sampled, the sample was selected for further microscopic investigation. Shoot sections with signs of disease were also surface sterilized and plated out to isolate C. gloeosporioides and preserved in F A A so that later microscopic examination could be conducted to observe C. gloeosporioides damage to the aerial tissues. Healthy A. americanum swellings were collected and processed in the same manner as the diseased samples to verify that the  80  sample was free of C. gloeosporioides and to provide uninfected checks for comparison with diseased material.  4.2.2 Artificial infection of A. americanum with C. gloeosporioides Two of the treatments applied in the field trial (Chapter 3) were designed to introduce C. gloeosporioides into the endophytic system of A. americanum. Treatment 2 involved placing 'Stabileze' formulated C. gloeosporioides directly onto the cut ends of shoots that were cut approximately 5 mm from the branch. Treatment 3 was applied by artificially wounding the dwarf mistletoe infection with a sterile cork borer, applying a mycelial plug of C. gloeosporioides to the cambium and wrapping the wound with Parafilm M . Inoculation of cut dwarf mistletoe shoots with sterile formulation and inoculation of an artificial wound with sterile P D A were controls for these treatments. In the summer of 2001, approximately one year after inoculation, 10 of the 45 replicates of each treatment were randomly selected and harvested to determine i f C. gloeosporioides had colonized the endophytic system of A. americanum. To compare the inoculated treatments with the controls, 1 of the 5 replicates of each control treatment was also collected. At the laboratory, the sample dwarf mistletoe infections of each treatment were examined closely under a dissecting microscope to identify signs and symptoms of C. gloeosporioides infection. After observation, the infections were sectioned longitudinally and one half of the infection was fixed in F A A and preserved in 70% ethanol and the other half was cultured. Basal cups of two shoots were randomly selected on the infections subjected to treatment 2, and its control, and the basal cups as well as all tissue layers below the basal cup were aseptically removed from the infection. The tissue layers were then separated and plated independently on P D A to determine how deeply C. gloeosporioides was able to penetrate the endophytic system. Dwarf mistletoe infections of treatment 3 were cut at 3 to 5 mm intervals, centered on the middle of the inoculation wound, and the pieces were plated on P D A to determine the distance that C. gloeosporioides had traveled from the wound. The control for treatment 3 was processed in the same manner as the inoculated swellings. The P D A plates were incubated in the dark at 20°C and checked regularly for the presence of C. gloeosporioides. 81  4.2.3 Paraffin embedding Samples from which C. gloeosporioides was isolated, check samples that were not infected and C. gloeosporioides infected A. americanum shoots were prepared for microscopic examination. The samples were fixed in F A A for 48 hours and then transferred to 70% ethanol for long term storage. After the presence of C. gloeosporioides was confirmed by culturing (or confirmed to be absent in the case of the check), three small pie shaped pieces were cut from the diseased swelling tissue. Three pieces were removed from each sample to allow radial, tangential and longitudinal sections to be cut. The samples were then taken through an alcohol dehydration process to remove water from the samples and then embedded in paraffin wax (Paraplast+, Oxford Labware, St. Louis, MO). The pieces were dehydrated by transferring from 70% ethanol to 70% T B E A (TBEA = 75% 2-methylpropan-2-ol tert-butanol, 25% absolute ethanol) overnight, then 70% T B E A to 85% T B E A for the day, followed by 85% T B E A to 95% T B E A overnight, then 95% T B E A to 100% T B E A for the day and 100% T B A (2methylpropan-2-ol tert-butanol) overnight. The paraffin embedding procedure was initiated by transferring from 100% T B A to TBA:paraffin oil (Sigma, St. Louis, MO) 1:1 for 8 hours, then pouring the pieces and liquid TBA:paraffin solution onto solidified Paraplast+ and placing the dish in a 60°C oven to melt. Once melted, the pieces dropped into the Paraplast+ and the liquid was poured out of the dish and pure Paraplast+ was poured in. The dish was then placed in the oven at 60°C under vacuum. Over a period of five days, the Paraplast+ was changed three times per day and the vacuum was gradually increased until 762 mm Hg was reached and bubbles stopped emerging from the pieces. Once the Paraplast+ was fully infused into the sample piece, the piece was embedded by placing the sample, with the side to be sectioned down, into molten Paraplast+ and allowing the Paraplast+ to solidify. After embedding, the pieces were mounted on a microtome object holder and the wax was removed down to the surface of the sample. The sample was then placed in a solution of molliflex (BDH, Toronto, ON):Ethylene glycol (FisherBrand, Pittsburgh, PA) 1:1 at room temperature for five to seven days to soften the sample prior to sectioning. After softening, 10 micron thick sections were cut with a rotary microtome (American Optical, model 820). The samples were returned to the softening solution when the 82  samples became too hard to section and were again sectioned after a further five to seven days in the softening solution. This cycle was repeated until adequate sections of the sample piece had been made. The sections were mounted on slides by floating a ribbon of sections upon a puddle of 4% formalin that was laid over a slide that had been pretreated with Haupts adhesive (Johansen, 1940). The formalin was evaporated and the sections bound to the slide by placing the slide on a hotplate at 50°C. Once the sample ribbons were bound to the slide, they were ready for staining.  4.2.4 Staining Three staining methods (safranin - picro=analine blue, Johansen's safranin and rhodamine B - methyl green; Appendix 3) were tested to determine which method provided the best differentiation between fungal and plant material. Safranin Picro=analine blue proved to be superior and it was used for all the tissue staining in this chapter.  4.2.5 Observation and photomicrography After staining, Permount (Fisher Chemical Co., Fair Lawn, NJ) was used to adhere a coverslip to the stained sections. Once the permount had set, slides were examined under the light microscope and observed at magnifications up to 400x using a Nikon Optiphot-2 microscope fitted with a phase contrast adapter or a Zeiss Photomicroscope-II fitted with a polarizing filter (Zeiss 47 36 00). Both microscopes had camera attachments and photomicrographs were taken using Kodak Elitechrome 160T tungsten film (Eastman Kodak Co., Rochester, N Y ) .  4.3 Results 4.3.1 Culturing and field observation of C. gloeosporioides inoculated A americanum. 4.3.1.1 A. americanum naturally infected with C. gloeosporioides Arceuthobium americanum infections that were located on otherwise healthy branches, yet had no shoots and appeared to be diseased, were collected and returned to 83  the lab where the woody tissues were dissected, aseptically removed and cultured. Colletotrichum gloeosporioides was isolated from the basal cup region of 8 of the 17 A. americanum infections collected. The fungus was never isolated from the inner bark tissues or xylem. Other fungi, including Cladosporium sp. and Sclerophoma pithyophila were commonly isolated from the outer bark tissues, but not the inner bark or xylem. Culturing the woody tissues from healthy A. americanum swellings resulted in isolation of Cladosporium sp. from 1 swelling. Colletotrichum gloeosporioides was not isolated from the woody tissues of healthy A. americanum swellings. To investigate the role of C. gloeosporioides as an endophyte, healthy A. americanum shoots adjacent to diseased shoots, and healthy portions of diseased A. americanum shoots, were surface sterilized and plated. Colletotrichum gloeosporioides was only isolated from the diseased portions of the A. americanum shoots, not the healthy tissues, suggesting that it does not act as an endophyte.  4.3.1.2 Artificial infection of A americanum with C. gloeosporioides Inoculation of the cut A. americanum shoots with 'Stabileze' formulated C. gloeosporioides resulted in necrosis that began at the cut surface and traveled down the shoot. One month after inoculation 100% shoot abscission had occurred on 27 of 45 replicates of treatment 2, while 1 of 5 replicates of the control had 0 shoots. One year after inoculation, 5 infections that were subjected to treatment 2 were dead as a result of the host branch death. Of the 40 remaining replicates, 24 had shoots and buds, 8 had buds but no shoots, 3 had shoots but no buds and 5 had neither shoots nor buds. Coincidentally, 2 of the 5 replicates with no shoots or buds (2183 and 2135) were collected during the random sampling and cultured. Shoots and buds were present on all of the control replicates at 1-year. Culturing the individual tissue types directly below the basal cups of 2 shoots of each of the 10 randomly selected replicates that were subjected to treatment 2 did not result in the recovery of any C. gloeosporioides isolates. Other fungi, including Sclerophoma pithyophila and Cladosporium sp. were recovered and every replicate had at least one fungus present. Plating the individual tissue types of the control for treatment  84  2 resulted in isolation of Sclerophoma pithyophila but not Cladosporium sp. or C. gloeosporioides (Table 4-1). Treatment 3 was an alternative method of inoculating the endophytic system with C. gloeosporioides. Immediately after wounding, the wound filled with copious quantities of pitch. Every replicate of treatment 3 that was cultured had the same fungal species as treatment 2 and Colletotrichum gloeosporioides was recovered from the inoculation site of 2 replicates. Both Sclerophoma pithyophila and Cladosporium sp. colonized the control for treatment 3 (Table 4-1).  85  Table 4-1. Recovery of fungi in culture from A. americanum subjected to treatment 2 and 3 during the field trial. A total of 10 replicates of the treatment and 1 of the control were randomly selected. Treatment  Treatment 2  Control for treatment 2  Treatment 3  Control for treatment 3  # of replicates  % infection  infected  of sample  Sclerophoma pithyophila  7  70  Cladosporium sp.  1  10  Colletotrichum gloeosporioides  0  0  Other  4  40  Sclerophoma pithyophila  1  100  Cladosporium sp.  0  0  Colletotrichum gloeosporioides  0  0  Other  0  0  Sclerophoma pithyophila  10  100  Cladosporium sp.  2  20  Colletotrichum gloeosporioides  2  20  Other  4  40  Sclerophoma pithyophila  1  100  Cladosporium sp.  1  100  Colletotrichum gloeosporioides  0  0  Other  0  0  Fungus  4.3.2 Histopathological examination 4.3.2.1 C. gloeosporioides infected A. americanum shoots Longitudinal and cross sections of diseased A. americanum shoots revealed that the epidermal layer of the shoot was blistered open to allow conidia to be released from acervuli. The acervuli did not contain setae and the acervulus was integrated into the mesophyll cells of the shoot and hyphae of C. gloeosporioides were observed to penetrate the shoot to the xylem. The mesophyll cells of the infected portion of the shoot were collapsed and unorganized, and intercellular and intracellular hyphae were observed. The boundary between healthy and necrotic shoot tissue was readily observed (Figure 4-1). 86  Figure 4-1. Colletotrichum gloeosporioides infected Arceuthobium americanum shoots. Safranin - Picro=analine stain. Scale bars represent 100 urn. 1. Acervulus (A) of C. gloeosporioides producing condia (C). Longitudinal section. 2. Acervulus of C. gloeosporioides. Cross section. Note: hyphae present in the center of the shoot at the xylem core. Epidermis (E), xylem (X). 3. Acervulus of C. gloeosporioides prior to breaking through the epidermal layer. Acervuli is integrated with host mesophyll cells. Hypha (H). 4. Longitudinal section of diseased shoot with intercellular hyphae (H) of C. gloeosporioides. Cells are disrupted and not organized. 5. Longitudinal section of healthy A. americanum shoot tissue with healthy, ordered mesophyll cells. Epidermis (E). 6. Diseased and healthy A. americanum tissue, showing the extent of necrosis. Acervuli (A) are present on the upper and lower shoot surfaces, close to the node (node is not in micrograph). Epidermis (E).  87  88  4.3.2.2 C. gloeosporioides infected A. americanum parasitizing P. contorta var. latifolia branch tissue Hyphae with the same morphology and staining characteristics of C. gloeosporioides in diseased shoots were observed in the dead outer bark tissues and associated with basal cups of A. americanum. A second hyphal type that was observed appeared to be melanized and it penetrated into the dead outer bark, and was present on the bark surface. Small circular cells were observed in association with this hyphal type, suggesting that the fungus was sporulating on the bark surface (Figure 4-2). The endophytic system of A. americanum was readily observed in the lodgepole pine xylem and bark tissues; however, no fungal hyphae were associated with these tissues. Xylem cells were observed in all parts of the endophytic system, including the sinkers, however, the xylem tissue observed in the sinkers are lodgepole pine ray tracheids as determined by the prominent and complex "dentate" cell wall thickenings (Koch, 1996). The xylem tissue appeared to be discontinuous and scattered throughout the sinker, rather than arrayed into a xylem column. Xylem tissue arrangement observed in longitudinal and cross sections of the cortical strands was also unorganized, however, it appeared that continuous xylem columns were present (Figure 4-3).  89  Figure 4-2. Transverse sections of healthy and C. gloeosporioides infected A. americanum tissues on and in lodgepole pine bark. Scale bars represents 100 um. 1. Spores (S) of melanized hyphae, on the surface of the bark? 2. Melanized hyphae (MH) on bark surface. 3. Melanized hyphae outside of bark tissue. 4. Melanized hyphae (MH) penetrating into the outer bark tissue. 5. Putative intercellular C. gloeosporioides hyphae (Cg H) present near bark surface. 6. C. gloeosporioides (Cg H) and melanized hyphae (MH) present in the outer bark tissue of lodgepole pine. 7. A. americanum shoot base with C. gloeosporioides hyphae present between shoot base and bark (arrow). 8. C. gloeosporioides hyphae (Cg H) in region shown in plate G.  90  91  Figure 4-3. Xylem cells in various endophytic tissues of Arceuthobium americanum. Pair of micrographs in transmitted light and birefringence to show xylem cells. Scale bar represents 100 um. 1,2. Long section of sinker in host xylem. Micrograph is oriented horizontally. X=host xylem, BP=borderd pit, RT=ray trachied. 3, 4. Cross section of sinker crossing the vascular cambium. S=sinker, VC=vascular cambium, P=phloem, X=xylem. 5,6. Cross section of a radial cortical strand in a longitudinal section of bark. X=A. americanum xylem. 7, 8. Longitudinal section of a cortical strand in a longitudinal section of bark. X=A. americanum xylem.  92  93  4.3.2.3 C. gloeosporioides inoculated A. americanum. The control and the two infections from which C. gloeosporioides  was recovered  after inoculation of the cut shoots were embedded in paraffin, sectioned and stained. No fungal hyphae were observed in the endophytic system or any of the host tissues; however, hyphae were observed on the bark surface. The hyphae appeared melanized and did not match the C. gloeosporioides  hyphae observed under acervuli in the shoots or  in the bark of the naturally infected A. americanum  infections that were described above.  4.4 Discussion Infection of the endophytic system of Arceuthobium parasitizing red fir by C. gloeosporioides  A. campylopodum,  f.  abietinum  was observed by Parmeter et al. (1959) and  Wicker and Shaw (1968) observed C. gloeosporioides of A. americanum,  campylopodum  A. laricis  infection of the endophytic system  and A. douglasii  (assumed because it is  not clearly stated exactly which species were colonized). Infection of the endophytic system of A. americanum  by C. gloeosporioides  during biological control treatment  would likely result in a decrease in the frequency of biological control application required to manage dwarf mistletoe because new shoot production would be reduced. Investigation of the role of C. gloeosporioides  in the endophytic system of A.  was undertaken to provide a better understanding of the role of the fungus in  americanum  this pathosystem.  4.4.1 Culturing The results of culturing the woody tissues of naturally infected A. infections indicated that C. gloeosporioides  was restricted to the basal cup and dead outer  bark tissues. Inoculation of cut A. americanum  shoots with C. gloeosporioides  result in infection of the endophytic system. Other fungi, including pithyophila  and Cladosporium  A. americanum  americanum  did not  Sclerophoma  sp. were commonly isolated from the outer dead bark of  swellings that were subjected to treatment 2. Field observation of the  response of cut A. americanum  shoots to inoculation suggests that it is likely that the  shoots were abscised from the dwarf mistletoe infection prior to C. reaching the endophytic system via the inoculated shoots. 94  gloeosporioides  Colletotrichum gloeosporioides was recovered from the woody tissue of lodgepole pine one year following C. gloeosporioides inoculation of an artificial wound in the lodgepole pine bark at the center of localized A. americanum infection, but the fungus was restricted to the inoculation point. Copious pitch flooding into the wound was likely inhibitory to C. gloeosporioides through the creation of an anerobic environment, or toxicity or both, thereby compartmentalizing the fungus and preventing further spread.  4.4.2 Histopathology  Conidia production from acervuli on the diseased shoots was copious, but no setea were observed in association with the acervuli on A. americanum shoots. Setae were not observed on C. gloeosporioides infected A. campylopodum f. abietinum shoots by Parmeter et al. (1959); however, setae production was recorded on C. gloeosporioides infected A. campylopodum shoots (Wicker, 1967) and shoots of A americanum, A. laricis and A. douglasii (Wicker and Shaw, 1968). It is possible that the differences in setae production were due to different races of C. gloeosporioides on the different Arceuthobium species. The presence of intracellular hyphae and hyphal penetration to the shoot xylem (Figure 4-1, panel B) observed in this study may represent a later stage in the infection process than was observed by Wicker and Shaw (1968) who found only intercellular hyphae that were limited to the exterior of the shoot cortex. In this study, destruction of A. americanum mesophyll cells in the aerial shoot as a result of the necrotic phase of C. gloeosporioides infection was clearly evident in the infected tissues. Colletotrichum gloeosporioides was not recovered from, or observed in, the endophytic system of A. americanum or the woody tissue of lodgepole pine; it was restricted to the aerial portion of A. americanum and the basal cup region of the swelling. These results are contrary to the observations of Parmeter et al. (1959) and Wicker and Shaw (1968) and may be because a different dwarf mistletoe / host association was studied here, or differences in the behaviour of C. gloeosporioides due to genetic variation of the fungus. It is also possible that if the sample size of this study was increased, C. gloeosporioides may have been observed in the endophytic system of A. americanum. 95  4.4.3 Potential C. gloeosporioides exclusion mechanism A combination of factors of the host and pathogen may be responsible for the fact that C. gloeosporioides was not observed to infect the endophytic system in this study. In order for the endophytic system to become diseased, the fungal hyphae must travel from the infection initiation point into the endophytic system. Continuity of the shoot with the endophytic system was observed in the histopathology portion of the study, providing a route for C. gloeosporioides to infect the endophytic system. During the necrotic phase of the disease cycle, diseased tissue containing hyphae was clearly distinguished from the healthy tissue, and fungal hyphae were not observed in healthy tissue. Necrosis was observed traveling down the shoot from the inoculation point to the shoot base, however, the diseased shoots were abscised from the dwarf mistletoe swelling and shed as the infection process progressed. This suggests that A. americanum prevented infection of the endophytic system by shedding diseased shoots. Infection of the endophytic system is not required for completion of the life cycle of C. gloeosporioides; conidia infect the aerial shoots and new conidia are produced in acervuli located on the shoots. Pectin lyase and polygalacturonase are produced during the infection process (Bailey et al., 1992; Senaratna et al., 1991), which would likely result in degradation of the parenchymous cells of the endophytic system but C. gloeosporioides is able to complete its life cycle in the shoot tissue, obviating the need for parasitism of the endophytic system.  4.4.4 Conclusion Colletotrichum gloeosporioides has not been reported to infect lodgepole pine and culturing of diseased A. americanum swellings resulted in isolation of the fungus from diseased portions of the shoots and the basal cup region only; however, several different fungi were isolated and observed on the dead outer bark tissue of P. contorta var. latifolia. The previous reports of C. gloeosporioides in the endophytic system are vague and refer to C. gloeosporioides exclusively (Parmeter et al., 1959; Wicker and Shaw, 1968). The results of this study indicate that many different fungi are associated with the dead outer bark tissues of P. contorta var. latifolia infected by A. americanum. The observations of this research suggest that infection of A. americanum with C. 96  gloeosporioides resulted in a decrease in the number of shoots on the dwarf mistletoe infection through shoot abscission, but that it had no effect on the production of new shoots because the endophytic system was not infected.  4.4.5 Future research The evidence presented in this research suggests that C. gloeosporioides does not infect the endophytic system of A. americanum. To further test this hypothesis, future research involving the use of isolates of C. gloeosporioides that have been genetically modified to express the green fluorescent protein of the jelly fish Aequorea victoria under the control of a constitutive promoter and in a controlled environment could be undertaken. Green fluorescent protein has revolutionized cellular biology (Heath, 2000; Lorang, 2001) and has been used to monitor gene expression (ie. Dumas et al., 1999) and the infection process (ie. Maor et al., 1998, Sexton and Howlett, 2001). A n efficient transformation protocol for the transformation of Colletotrichum gloeosporioides f. sp. aeschynomene has been reported (Robinson and Sharon, 1999) providing a method that can be utilized to transform an isolate of C. gloeosporioides collected from dwarf mistletoe. By utilizing an isolate of C. gloeosporioides that expresses green fluorescent protein under a controlled environment, time course experiments of the infection process can be conducted to follow hyphae in the infected shoot to unequivocally understand the extent of fungal colonization.  97  Colletotrichum gloeosporioides on Arceuthobium americanum in the crown of lodgepole pine  Chapter 5: Incidence of  5.1 Introduction  This study was conducted to observe the natural infection of americanum  by Colletotrichum  Pinus contorta  var. latifolia.  gloeosporioides  Arceuthobium  in the canopy of A. americanum  infected  The results of the survey may predict the efficacy of  inundative biological control of A. americanum  using C. gloeosporioides  positions, based on the distribution of naturally infected A.  at all crown  americanum.  The interactions between the forest canopy and the atmosphere are very complex. Water vapour is released to the atmosphere through transpiration, and transpiration rates depend upon biological and structural attributes that influence local concentrations in the canopy, such as sunlight, temperature, turbulence, humidity and leaf physiological condition (Rose, 1996). It is expected that gradients of sunlight, relative humidity, wind speed and leaf wetness exist in the crown of lodgepole pine. The top and south facing portions of the crown are exposed to greater solar radiation and lower relative humidity than the lower and north facing portions of the crown. Similarly, the branch tip is exposed to greater solar radiation than the region of the branch located near the main stem which is more sheltered and likely have higher relative humidity than the branch tip. Conidia of C. gloeosporioides  are released from acervuli during rain and dew, and  relative humidity is important during the infection process because conidia are rapidly inactivated by ultraviolet light and desiccation (Wastie, 1972). Free water is not required for conidia germination; however, germination is enhanced in the presence of free water (Wastie, 1972). Given the moisture requirements necessary for successful disease development by C. gloeosporioides, infected A. americanum  it is likely that a gradient of C.  exists within the canopy of A. americanum  gloeosporioides  infected lodgepole  pine. The hypothesis explored in this study is that successful establishment of C. gloeosporioides  on A. americanum  occurs on infections that are shaded and exposed to  increased relative humidity. Three tests of this hypothesis were addressed in this study: 1. Infection of A. americanum  by C. gloeosporioides 98  is highest in the lower portion of  the crown, 2. Infection of A. americanum by C. gloeosporioides is highest in the north facing side of the crown, and 3. A. americanum infections located at the branch tip have lower C. gloeosporioides infection than sheltered A. americanum infections located near the main stem, all versus the null hypothesis of no significant difference.  5.2 Materials and Methods 5.2.1 Stand and tree selection The stand selected for the study was located at 50° 29' 15"N latitude 121° 36' 19"W longitude and 1174m elevation in the Thompson dry cool variant of the interior Douglas-fir biogeoclimatic zone (Lloyd et al. 1990). The criteria for stand selection were: pure lodgepole pine, high dwarf mistletoe rating (DMR) (Hawksworth, 1977), C. gloeosporioides infection of the dwarf mistletoe present in the lower crown, mature trees with high live crown ratio and dwarf mistletoe infection throughout the crown. Six trees distributed throughout the selected stand were located and analyzed.  5.2.2 Data collection 5.2.2.1 Trees Diameter at breast height (DBH) was measured and the dwarf mistletoe rating was calculated based on Hawksworth's (1977) six-class system where 0 is no dwarf mistletoe infection and 6 is severe infection. The cardinal directions (aspect) were marked on the stem of each tree and the tree was then felled and the stem marked at 1-meter intervals. Tree height was recorded by measuring the length of the stem on the ground. A disk was cut from the stump for later aging of the tree.  5.2.2.2 Arceuthobium americanum Starting at the base of the tree, each branch was checked for the presence of A. americanum. If A. americanum was present on the branch, the height of the branch from the ground, aspect of the branch (quadrant centered on the cardinal direction), the distance of the A. americanum infection from the stem, total branch length, A. americanum sex, and the presence or absence of C. gloeosporioides on A. americanum shoots was recorded. Colletotrichum gloeosporioides was identified by the presence of 99  characteristic lesions on A. americanum shoots or fruit (Hawksworth and Weins, 1996). When systemic witches' brooms were encountered, they were treated as a single infection and all but infection location on the branch and branch length, were recorded.  5.2.3 Statistical Analysis The vertical pattern of C. gloeosporioides infection was assessed by dividing the crown of each tree into thirds and calculating the percent infection of A. americanum by C. gloeosporioides in each third. In an alternative approach, the presence of C. gloeosporioides at different locations in the canopy was calculated by dividing the crown of the tallest tree into thirds of 0-5m, 6-10m and 1 l-16m. The location of each dwarf mistletoe infection on every tree was then assigned to one of these height classes. The aspect of C. gloeosporioides in the crown was analyzed by stratifying the data based on aspect and calculating the percent infection of A. americanum by C. gloeosporioides. Horizontal position within the crown was estimated by computing the ratio of infection location to total branch length. Analysis of variance was used to compare percent infection of A. americanum by C. gloeosporioides at different crown and canopy heights and aspect positions. If the assumptions of normality and equal variance were not met, Kruskal-Wallis one-way analysis of variance on ranks was used; otherwise, two-way analysis of variance was used to compare the sampled trees and the treatment effect. The Chi-square test of independence was used to analyze the relationship between the presence of C. gloeosporioides and A. americanum sex and the presence of C. gloeosporioides and A. americanum horizontal location on the branch. Horizontal location on the branch was a ratio between 0 and 1 and intervals of 0.1 were used to calculate the chi-square statistic. A l l statistical analyses were computed using the computer package SigmaStat 2.03 (SPSS Inc, Chicago, IL).  5.3 Results 5.3.1 Individual trees Dwarf mistletoe infection of the host tree was highly variable. A l l trees were heavily infected, having an average D M R of 5.5 (Table 5-1), but the actual number of 100  dwarf mistletoe infections ranged from 28 to 323, averaging 126 ± 44 (mean ± standard error of the mean) (Table 5-1). The range of dwarf mistletoe infection of the host tree was broad; however, infection of A. americanum by C. gloeosporioides was very consistent, averaging 23.0 ± 1.1 % (Table 5-1).  Table 5-1. Characteristics of individual trees surveyed in this study. Tree  Height  DBH  Age  (m)  (cm)  (years)  1  12.6  20.4  117  2  8.6  17.4  3  15.4  4  # DM  # D M with  %  infections  Colletotrichum  infection  6  127  26  20.5  103  5  28  6  21.4  21.5  104  5  156  36  23.1  9.7  18.5  138  6  72  17  23.6  5  14.5  19.6  75  5  50  14  28.0  6  12.3  18.6  104  6  323  68  21.1  Mean  12.2  19.3  107  5.5  126  28  23.0  SE  1.1  0.6  8.4  0.2  44  9  1.1  f  DMR  Standard error of the mean  101  5.3.2 Crown position The presence of C. gloeosporioides infected A. americanum at different vertical locations within the host tree crown is shown in table 5-2. Kruskal-Wallis one-way A N O V A on ranks was used to analyze this data because the assumption of normality was not met. There was no significant difference in the presence of C. gloeosporioides between the trees (P=0.835); therefore, the crown thirds were pooled and crown position was compared. Kruskal-Wallis one-way A N O V A on ranks indicated that there were no significant differences between crown position and the presence of C. gloeosporioides (P=0.194). These results are illustrated graphically in figure 5-1.  Table 5-2. Infection of A. americanum by C. gloeosporioides at different tree crown thirds. Tree  Bottom Third  Middle Third  # D M #Inf % I n f  # D M #Inf % I n f  Top Third # DM  All  # Inf % I n f  1  5  1  20.0  42  11  26.2  80  14  17.5  20.5  2  1  1  100  7  5  71.4  20  0  0  21.4  3  46  4  8.7  80  27  33.8  30  5  16.7  23.1  4  24  4  16.7  38  9  23.7  10  4  40  23.6  5  10  2  20.0  24  7  29.2  16  5  31.3  28.0  6  127  23  18.1  180  41  22.8  16  4  25.0  21.0  21.8  22.9  Mean  30.6  34.5  102  Bottom  Middle Tree crown thirds  Figure 5-1. Percent infection of A. americanum by C. gloeosporioides in different tree crown thirds. Mean +/- standard error of the mean.  5.3.3 Canopy position The canopy of the stand was divided into thirds based on the height of the tallest tree sampled and the A. americanum infections assigned to thirds by their height above ground. Percent infection of A. americanum by C. gloeosporioides at different stand canopy positions was analyzed by two-way A N O V A because the assumptions of normality and equal variance were met. The two-way A N O V A results indicated that there was no significant difference in infection by C. gloeosporioides between trees (P=0.939) or canopy position (P=0.423) (Appendix 4). Canopy position data was pooled and is shown graphically in figure 5-2. Figure 5-3 illustrates graphically the distribution of A. americanum within the canopy. As can be seen in figure 5-3, 91% of the total A. americanum in the sampled trees was located in the bottom two-thirds of the canopy and C. gloeosporioides was present in all canopy positions. 103  50 45 40 35 U >> -o <x>  30 25  4—*  <_) ,£ _  20  "IHHRBHHI  15 10  Bottom  Middle  Top  Stand canopy position Figure 5-2. Percent infection of A americanum by C. gloeosporioides at different locations in the stand canopy. Mean +/- standard error of the mean.  104  100  £  No C. gloeo^orioides With C. gtoeo^orioides  80 4  60  6  6  40 A  20  n  0  2 4 Third 1  8 Third 2  10  J  In In -fl In  n  12  14 Third 3  16  Canopy position (m) Figure 5-3. Distribution of A. americanum  within the canopy. Numbers above bars  represent the number of trees having crowns within the interval.  5.3.4 C a n o p y aspect  Two-way A N O V A indicated no significant difference between percent A. americanum  infection by C. gloeosporioides  between trees (P=0.591) or aspect (P=0.914)  (Appendix 4). Percent infection of A. americanum pooled and is shown graphically in figure 5-4.  105  at different aspect locations was  CO  cu o o  & o o  o  £ 8 £ UJ  o <B North  East  South  West  Aspect Figure 5-4. Percent infection of A. americanum by C. gloeosporioides at different aspects. Mean +/- standard error of the mean  5.3.5 A. americanum sex and the presence of C. gloeosporioides The relationship between the presence of C. gloeosporioides and sex was analyzed using the chi-square test of independence and it was found that sex and the presence of C. gloeosporioides did not depend upon A. americanum sex (x with 1 degree 2  of freedom = 0.578; P=0.447).  5.3.6 A. americanum horizontal canopy position The location of A. americanum infection on the infected branch was calculated using the ratio of infection location over branch length. The frequency distribution of infected and uninfected A. americanum at different branch positions is presented in figure 5-5. Calculation of the chi-square statistic based on the intervals 0-0.6, 0.61-0.8, 0.81106  0.9, and 0.91-1.0 indicated that the presence of C. gloeosporioides could not be shown to be dependent upon horizontal position on the branch (x with 3 degrees of freedom = 2  2.484; P=0.478).  180 160  £ 140 A  i i No C. gloeotyorioides • B With C. gloeotyorioides  =3  CZ  <0  .o  CD  £  120  n  100  TO  < y—  o i_  CD -Q  E  80 60 A 40 A 20 A i  0.0  0.1  J ]  i  0.2  i l li 0.3  1 i 0.4  r  0.5  I 0.6  1 1 T r  0.7  0.8  -  0.9  "r  1.0  1.1  Ratio Figure 5-5. Distribution of C. gloeosporioides infected and uninfected A. americanum on the host branch. Values close to 0 represent infections close to the main stem; values close to 1 represent infections close to the branch tip.  5.4 Discussion The survey described here was conducted to observe the natural distribution of C. gloeosporioides infection of A. americanum within the crown of Pinus contorta var. latifolia. The distribution of C. gloeosporioides within the canopy was assessed to determine if there were any factors, such as solar radiation, that would limit the efficacy of an inundative biological control agent. The survey was relatively limited in scope, as only six trees within one stand were surveyed and it is possible that the results observed  107  here would not be observed in a stand with different structural characteristics given the complex relationships between the canopy and the atmosphere. The occurrence of C. gloeosporioides infection of dwarf mistletoe has been surveyed on Arceuthobium abietinum Englm. Ex Munz f. sp. magnificae Hawksworth and Weins (syn. Arceuthobium campylopodum Engelm. f. abietinum (Englem.) Gill) (Parmeter et al., 1959), Arceuthobium campylopodum Englm in Gray (Wicker, 1967) and Arceuthobium americanum (Muir, 1977). The percent infection of dwarf mistletoe by C. gloeosporioides observed in this study was lower than previously reported, averaging 22.9%. Muir (1977) observed 75% infection of plants at one site and 35.5% infection at another site. Wicker (1967) observed C. gloeosporioides infection of 67% of A. campylopodum on 75% of P. ponderosa trees sampled, while 100% of P. contorta var. latifolia trees surveyed in this study were parasitized by A. americanum that was infected by C. gloeosporioides. Parmeter (1959) observed 27.7% C. gloeosporioides infection of A. abietinum f. sp. magnificae plants. The variation in percent infection may relate to the ability of the fungus to infect different Arceuthobium hosts, alternatively, it could be due to different environmental conditions that affect inoculum and symptom production at the time of sampling or environmental differences between locations that affect C. gloeosporioides disease development. The frequency of C. gloeosporioides was not significantly different between trees, crown thirds or canopy thirds; compared to the vertical distribution described by Parmeter et al (1959) who observed uniform infection of the dwarf mistletoe in the upper % of the crown and a decrease in the lower A of the crown. The observation of no l  significant difference between crown or canopy thirds suggests that no gradients of relative humidity or solar radiation exist in the canopy of the stand that was sampled, or that if gradients were formed, they did not limit C. gloeosporioides infection. The stand that was sampled had been spaced and pruned from 1667 stems per hectare to 1208 stems per hectare 6 years prior to this study, thus the stand was relatively open. This stand was selected because the trees had a high live crown ratio, which was a necessary component of the study. It was desirable to sample trees that had foliage at all canopy positions because understory trees or overstory trees do not represent all canopy positions. The openness of the stand selected however may have prevented the formation of a relative 108  humidity gradient within the crown because the lower crown was not sheltered from the wind, as would be the case i f stand density were higher. Light penetration to the lower portion of the crown was also increased. The environmental conditions at the different canopy positions are likely to be representative of the conditions present in the upper canopy of a dense stand. The observation of C. gloeosporioides infection in the top third of the canopy is important from a biological control standpoint because it indicates that the A. americanum infections in the upper crown, which are the most important for A. americanum spread, are as susceptible to C. gloeosporioides infection as A. americanum in the lower crown. This finding also suggests that the results of the field trial of chapter 2 may also be applicable to upper crown A. americanum infections and open stands. In the future, if it is desirable to create a model to describe the infection of A. americanum by C. gloeosporioides, the model may be based on either crown or canopy position, as the trend was the same in both analyses. Aspect of C. gloeosporioides infected A. americanum and branch location were not related to infection of A. americanum by C. gloeosporioides. This suggests that solar radiation did not affect disease development. Shading of the crown by other trees in the stand may have decreased the amplitude of the gradient. The effect of aspect may also be reduced by the fact that the south facing side of one tree and the north facing side of an adjacent tree occupy the same space and may be exposed to very similar conditions. A n alternative explanation for the lack of a gradient of C. gloeosporioides infection of A. americanum is that C. gloeosporioides establishment likely occurs on cloudy days with high relative humidity, in which case, strong gradients of light do not exist. Dwarf mistletoe sex and the presence of C. gloeosporioides were independent, suggesting that pistillate and staminate A. americanum plants are equally susceptible to infection by C. gloeosporioides, as was observed by Wicker (1967). This is not surprising as C. gloeosporioides does not specifically parasitize one A. americanum tissue type, unlike Caliciopsis arceuthobii, which only parasitizes female flowers (Kuijt, 1969). This survey was conducted to observe any trends present in natural infection of A. americanum by C. gloeosporioides that might suggest reduced efficacy of an inundative biological control agent in certain crown locations. A l l tests failed to reject the null 109  hypothesis; results of this survey indicated that there were no significant differences between crown and canopy thirds, aspect within the crown or branch position and infection of A. americanum by C. gloeosporioides. This suggests that fungal inoculum is not limiting at different crown positions, or that it is equally limited at all positions, and environmental conditions were suitable for disease development at all crown positions. These results imply that fungal inoculum applied during biological control treatment, applied under appropriate climatic conditions, has an equal probability of causing disease on A. americanum, regardless of crown position.  110  Chapter 6 - Infection of  Arceuthobium americanum by Caliciops arceuthobii  6.1 Introduction Caliciopsis arceuthobii (ex. Wallrothiella arceuthobii) is an ascomycete fungus that parasitizes the female flowers of the spring flowering dwarf mistletoes Arceuthobium americanum, A. douglasii, A. pusillum, and A. vaginatum subsp. cryptopodium (Hawksworth and Wiens, 1996). Ascospores mature in perithecia in March and April and are liberated during flowering. It is hypothesized that ascospores are then transported to other flowers by insects, where they germinate and infect the female flower, preventing fruit development; therefore, pollination and disease initiation occur simultaneously. Perithecia are visible in the fall of the year of infection, but asci and ascospore development is delayed until the following spring, resulting in a life cycle that requires one year (Kuijt, 1969). Weir (1915) suggested using C. arceuthobii as a biological control agent and was able to infect A. americanum by binding diseased A. douglasii to a branch infected by A. americanum. Utilization of C. arceuthobii as a biological control agent for A. douglasii was investigated and inoculation with crushed perithecia resulted in 17% infection of 318 fruits (Knutson and Hutchins, 1979). The effect of C. arceuthobii on A. americanum under natural conditions was investigated in this study. A population of C. arceuthobii infected A. americanum was located and the percent fruit reduction caused by C. arceuthobii and the spread of C. arceuthobii to healthy A. americanum was quantified. The effect of the fungus on fruit production by A. americanum and the potential of this fungus as a biological control agent for A. americanum are discussed.  6.2 Materials and Methods 6.2.1 Stand characteristics The stand selected for this study was located at 52° 02' 54"N, 121° 48' 45"W, 1023 meters, within the Knife Creek Block of the Alex Fraser Research Forest, near 150 Mile House, British Columbia. The site was classified as the dk3 variant of Interior Douglas-Fir zone (IDFdk3) under the biogeoclimatic ecosystem classification system 111  (Steen and Coupe, 1997). Individual female A. americanum swellings were located on lodgepole pine trees that averaged 7.4 cm diameter at breast height and approximately 8 m in height. Prior to spacing to 1500 stems per hectare in 1990, the stand was assessed at 4699 coniferous stems per hectare (93% lodgepole pine, 3% Douglas-fir and 2% interior spruce). This stand was selected because C. arceuthobii was present and because it was located within the University of British Columbia research forest.  6.2.2 Arceuthobium americanum selection In 1998, 30 female A. americanum infections were randomly selected and tagged. Caliciopsis arceuthobii was present on the flowers of 22 of the 30 selected infections. Every female A. americanum infection selected bore a crop of fruit when the trial was initiated. Arceuthobium americanum infections were located on the branches and stems of the host trees and all infections observed were in the lower crown.  6.2.3 Data collection and analysis The initial data were recorded May 15, 1998 and assessment occurred July 28, 1999 and August 17 of 2000 and 2001. At every assessment, the number of fruit and number of flowers bearing C. arceuthobii perithecia were counted. During the 2001 assessment, the number of healthy fertilized flowers was counted to estimate fruit production in 2002. The percent reduction in fruit production caused by C. arceuthobii infection in the first year of the study was calculated assuming that the fruit present in May of 1998 were produced on flowers that escaped C. arceuthobii infection in 1997 and that the perithecia observed were on the same cohort of flowers that were producing fruit in 1998. The percent reduction in fruit production was therefore calculated according to the formula:  %=  a  xl00  .(<* + fi).  where a - number of flowers with perithecia in May 1998, /? = number of fruit present in May 1998. 112  The percent reduction in fruit production in 1999 could not be calculated because the 1998 cohort of C. arceuthobii perithecia was not recorded . The percent fruit 1  reduction for 2000 and 2001 was quantified assuming that fruit present in year x are a result of flowers that escaped infection in year x-1 and that C. arceuthobii observed in year x will cause a reduction in fruit production in year x+1 using the formula:  a  %= l(<*  xlOO  + z)_  where a = number of flowers with perithecia in year x-1, x = number of fruit present at year x.  The percent reduction in fruit production for 2002 was predicted based on the number of C. arceuthobii infected flowers and the number of C. arceuthobii free flowers using the formula:  %=  a xlOO (a + S)  where a = number of flowers with perithecia in 2001, d = number of flowers that escaped C. arceuthobii infection in 2001.  To clarify the relationship between perithecia production by C. arceuthobii and fruit production on A americanum, lifecycle drawings are presented in Appendix 5. To test the hypothesis that C. arceuthobii infected A. americanum is significantly more likely to have C. arceuthobii infection in the following year than A. americanum without C. arceuthobii, versus the null hypothesis of random infection, the Fisher exact test was calculated using SigmaStat 2.03. Two by two contingency tables for 1999 and  1  The road to the field site was closed due to heavy rain when I attempted to record data from June 25-27,  1999; therefore, the 1998 cohort of C. arceuthobii perithecia was not assessed.  113  2000, and 2000 and 2001 were constructed with infected in year JC and not infected in year x versus infected in year x+1 and not infected in year x+1. Only A. americanum infections that were alive in both years and had shoots in both years were included in the analysis. Only the years 1999 vs. 2000 and 2000 vs. 2001 were plotted because the C. arceuthobii perithecia observed in 1998 represent the 1997 cohort. Due to the timing of field data collection, the 1998 cohort of C. arceuthobii perithecia were not recorded (Appendix 5).  6.3 Results  6.3.1 Caliciopsis arceuthobii infection of Arceuthobium americanum Infection of A. americanum by C. arceuthobii was obvious in August of the year of infection. In Figure 6-1, taken in August 2001, fruit, C. arceuthobii infected and uninfected flowers of A americanum are visible. Arrows 1 and 2 of Figure 6-1 point to C. arceuthobii infected and uninfected flowers, respectively, that were produced in 2001, while arrow 3 points to a fruit that has developed as a result of escaping C. arceuthobii infection in 2000. Arrow 4 of Figure 6-1 points to an A. americanum flower that was infected by C. arceuthobii in 2000 and is dying.  114  Figure 6-1. Arceuthobium americanum infected by Caliciopsis arceuthobii. Photo taken August 17,2001 at the Knife Creek block of the Alex Fraser Research Forest, near 150 Mile House, British Columbia. 1. Caliciopsis arceuthobii perithecia present on A. americanum flower infected in 2001. 2. Arceuthobium americanum flower that escaped infection in 2001. 3. Maturing fruit of A americanum that escaped infection in 2000. 4. Arceuthobium americanum flower that was infected by C. arceuthobii in 2000.  6.3.2 Fruit production on A. americanum The average percent fruit reduction caused by C. arceuthobii was found to range from a low of 46% in 1998, to a high of 72% in 2000. The predicted percent fruit reduction caused by C. arceuthobii in 2002 is 39% (Table 6-1). Variation in the amount 115  of fruit reduction on C. arceuthobii infected A americanum was high, ranging from 1% to 100%. The average fruit reduction per year, caused by C. arceuthobii, over the course of the experiment, not including the predicted fruit reduction, was 58%).  Table 6-1. Average reduction in fruit production caused by Caliciopsis arceuthobii infection of Arceuthobium americanum. Year  Average % reduction  Standard error of the mean  1998  46  7.4  1999  Cannot calculate  2000  72  10.8  2001  53  13.1  39  11.0  2002  t  Predicted  Fruit production on individual A. americanum infections was found to be variable throughout the course of the experiment; large crops of fruit were not sustained on single infections over the course of the experiment and different infections had maximum fruit production in different years. Fruit production averaged 58, 23, 11 and 20 fruit per A. americanum infection in 1998,1999, 2000 and 2001 respectively. When the number of C. arceuthobii infected flowers at year x was plotted against the number of C. arceuthobii infected flowers at year x + 1 (Figure 6-2), no relationship was observed and there was no significant linear regression (P=0.271). This finding implies that the inoculum load present in year x does not affect infection of A. americanum the following year.  116  ~ +  400 -|  • O  x  CO CD >;  cn  1999 vs 2000 2000 vs 2001  300 A  i  CD  o> 200 CD  CD  100  H 8  .  oH —r-  0  100  200  300  400  Number of infected flowers (yearx) Figure 6-2. Scatter to compare the number of C. arceuthobii infected A. americanum flowers at year x with the number of infected flowers at year x+1. In the 1999 vs 2000 plot, 6 data points are located at the origin, while in the plot of 2000 vs 2001,2 data points are at the origin.  6.3.3 Movement in the stand Using the Fisher exact test to test the hypothesis that having C. arceuthobii in one year significantly predisposed the A. americanum infection to having C. arceuthobii in the following year failed to reject the null hypothesis for both pairs of successive years. Infection of A. americanum by C. arceuthobii was not significantly different than expected from random occurrence. Initially, eight of the A. americanum infections selected in this experiment were not infected by C. arceuthobii. One year later one replicate was dead and two were infected by C. arceuthobii. Of the two replicates that became diseased, one showed no sign of disease in 2000 or 2001 and one had perithecia in 1999 and 2000 and was dead in 117  2001. One replicate had no sign of C. arceuthobii in 1998 or 1999 and was dead in 2000 and one replicate had no sign of C. arceuthobii in 1998,1999, or 2000 and was dead in 2001. One replicate that had no C. arceuthobii in 1998, 1999, or 2000 bore perithecia in 2001. Two of the replicates remained free of C. arceuthobii throughout the experiment. Of the 22 replicates that had perithecia at the start of the experiment, six were dead in 1999. Six replicates had perithecia present every year for four years, while one replicate bore perithecia in 1998 only. Four replicates had C. arceuthobii perithecia in 1998 and 2001 only, three replicates had perithecia in 1998,1999 and 2001, one replicate had perithecia in 1998, 1999 and 2000, and one replicate had perithecia in 1998, 2000 and 2001. The raw data are presented in Appendix 6.  6.4 Discussion From a biological control perspective, the survey suggests that C. arceuthobii can reduce the spread and intensification of A. americanum by a significant amount through interfering with the production of new seed. Although only one stand was selected, the mode of action of this hyperparasite suggests that wherever it is present, it will cause a reduction in seed production. One of the attributes of a biological control agent for dwarf mistletoe, as defined by Wicker and Shaw (1968), is that the biological control agent must have an efficient mode of restricting development of the target disease. Clearly, C. arceuthobii meets this requirement by infecting the female flowers. Following the development of C. arceuthobii within the population of A. americanum surveyed indicated that it persists over many years on the host, affecting many seed crops, and that it can readily move from diseased individuals to healthy individuals. Although these traits are not specifically outlined by Wicker and Shaw (1968), they are important components of a biological control strategy. Spread to A. americanum infections that escaped initial biological control application is likely to occur as A. americanum that was C. arceuthobii free in one year was occasionally observed to be infected by C. arceuthobii in the following year. For C. arceuthobii to be maintained in the stand, A. americanum must also be maintained, and as the density of A. americanum increases, it is expected that disease caused by C. arceuthobii will also increase.  118  Infection of A. americanum by C. arceuthobii in one year was not related to the presence of C. arceuthobii in the preceding year. This suggests that i f insects vector the fungus, as suggested by Kuijt (1969), insects travel readily between infected and healthy A. americanum flowers and that the ascospores remain viable for a relatively long period of time. If the fungus is dispersed by splash dispersal or wind, it would be expected that A. americanum infections with many perithecia would have a high infection rate in the following year, however, the scatter plot in figure 6-2 does not support this. Although pathogenesis process of C. arceuthobii results in a natural level of control of A. americanum, some of the features of the Caliciopsis I Arceuthobium I lodgepole pine pathosystem described in Chapter 2 suggest that Caliciopsis may not be a good candidate for a biological control agent. The range of C. arceuthobii was the same as C. gloeosporioides, but C arceuthobii was observed in 8 sites and C. gloeosporioides was isolated from 21. When perithecia were collected from A americanum, C. arceuthobii was not successfully isolated, proving that it is difficult to culture. Caliciopsis arceuthobii has been cultured, but it grows slowly (Parker, 1970; Knutson and Hutchins, 1979), which is a possible reason why it was not isolated during the work conducted for this thesis. Cladosporium sp. is commonly associated with the perithecia of C. arceuthobii (Kuijt, 1969) and that fungus was commonly isolated in the survey portion of Chapter 2. The difficulty of culturing C. arceuthobii, the slow growth in culture, and the fact that no perithecia, and thus ascospores, have been produced in culture (Knutson and Hutchins, 1979), suggest that inoculum production necessary for biological control application would be difficult. The asexual stage of C arceuthobii is unknown, but it is possible that i f an asexual stage exists, conidia production in culture could provide inoculum for biological control studies. Inoculation studies that have been carried out with C. arceuthobii have used crushed perithecia in water (Knutson and Hutchins, 1979) and diseased A douglasii placed in close proximity to A americanum (Weir, 1915), but neither of these is practical at a commercial scale. Timing of application in the field must be synchronized with the flowering period of A americanum to result in successful establishment. The stigmata of A americanum is infected by C. arceuthobii, which is hypothesized to be inoculated by an insect vector. This may be difficult to replicate during biological control treatment application. 119  Caliciopsis arceuthobii has several characteristics that suggest that it would be an excellent biological control agent for Arceuthobium americanum. Unfortunately, there are several technical difficulties with culturing and inoculum preparation, which are major constraints in the development of inundative biological control agents (Auld and Morin, 1995), that must be overcome before this fungus can be utilized as a biological control agent for Arceuthobium americanum.  120  Chapter 1 - Arceuthobium  americanum response to shoot removal  7.1 Introduction Biological control of dwarf mistletoes differs from biological control of weeds because death of the aerial shoots may not ultimately result in death of the dwarf mistletoe infection. The endophytic system within the host branch can survive to produce a new crop of shoots after the shoots are killed (Hawksworth, 1972). If a biological control agent were able to infect the endophytic system and prevent the production of new shoots, re-application of the biological control agent would not be required, unless secondary inoculum production caused a reduction in new shoot production. If however, the biological control agent does not infect the endophytic system, the production of new shoots and new fruit is likely to occur. The production of a new crop of seeds after biological control application would necessitate reapplication of the biological control agent prior to the new seeds being cast if the canopy of the trees being protected has not grown above the treated dwarf mistletoe infections. The most efficient timing of biological control treatment would be during the spring, one year after pollination of the female flowers, immediately prior to fruit dispersal when environmental conditions for C. gloeosporioides establishment are favourable. This scenario is similar to the effect of Ethephon, an ethylene releasing compound that causes rapid dwarf mistletoe shoot abscission but does not affect the endophytic system, resulting in renewed fruit production following treatment (Nicholls, 1988). Application of an inundative biological control agent to dwarf mistletoe infections may result in uneven distribution of disease on the aerial shoots, allowing some shoots to escape infection and continue fruit production. The result of this scenario is dwarf mistletoe infections that have new shoots developing at the same time that fruit are maturing on shoots that escaped inoculation. It is not known if the dwarf mistletoe responds to partial shoot removal by increasing the production of new shoots or by increasing fruit production on existing shoots or i f it modifies growth characteristics at all. The first objective of this research was to observe how Arceuthobium americanum responds to biological control through mimicking the effect of a biological 121  control agent using manual shoot removal. The specific hypotheses examined were: 1) Shoot removal induces change in the growth rate of the endophytic system of A. americanum, 2) As damage to the aerial system of A. americanum increases, bud (=new shoot) production increases, 3) As damage to the aerial system of A. americanum increases, fruit production increases on the remaining shoots, 4) When the top portion of the shoot is damaged, A. americanum responds by increased fruit production on the existing shoots rather than increasing new shoot production, and 5) the average number of shoots, maximum shoot length and number of fruit remains constant over years when A. americanum is not damaged. The second objective of this research was to estimate the treatment periodicity necessary to prevent A. americanum seed production through mimicking the action of a biological control agent that was assumed to remove all of the aerial shoots but had no effect on the endophytic system of the dwarf mistletoe.  7.2 Materials and Methods To investigate the effects of partial A. americanum infection and the effects of killing the aerial system, but not the endophytic system of A. americanum, field trials were established that mimicked the action of the biological control agent by manually removing shoots.  122  7.2.1 Partial shoot removal experiment. The partial shoot removal experiment was designed to observe how the dwarf mistletoe plant responded following manual shoot removal that mimicked uneven establishment of a biological control agent. The experiment was established near Lytton (50° 27.324' N 121° 35.704' W 1166 m), British Columbia in the dry cool variant of the Interior Douglas-Fir zone (IDF dkl). Dwarf mistletoe infections that were selected were healthy individual A. americanum infections that were located on lodgepole pine trees approximately 30 years of age, averaging 7 cm diameter at breast height and 8 m in height. The overstory was composed of A. americanum infected lodgepole pine 8 0 - 1 0 0 years of age. Data collected prior to treatment included: A. americanum swelling diameter (measured at the point of maximum diameter), A. americanum swelling length, maximum shoot length, number of shoots and number of fruit (female). Arceuthobium americanum swelling diameter, swelling length and maximum shoot height were measured using digital calipers. The rate of growth of the endophytic system of A. americanum cannot be measured directly; therefore, in these experiments, the rate of A. americanum swelling diameter and length change was utilized as an index of endophytic system growth. The experiment was initiated in July 1999, prior to seed dispersal and assessed prior to seed dispersal in August one and two years after treatment application. One and two years following treatment, the number of buds (defined as shoots <5 mm in length) was counted and at 2 years the number of pollinated female flowers was counted as a predictor of fruit production three years after treatment. Pollinated female flowers could be distinguished from unpollinated flowers based on size. The treatments and number of replicates for each sex are outlined in table 7-1. The number of shoots was counted prior to treatment and then the designated percent reduction in shoot number was accomplished by breaking the shoots off at the basal cup. When treatment 4 was applied, scissors were used to trim each shoot at the midpoint. The number of fruit present immediately following treatment was not counted. The number of female replicates was variable because 7 infections that were assumed to be male due to the lack of fruit were actually female infections that were not yet producing fruit. These female infections were added to the female replicates and more male infections were located and treated to bring the 123  number of male replicates to 10 per treatment. When the maximum shoot length was measured after treatment application, the maximum bud length was utilized for the maximum shoot length of the replicate if no shoots were present because buds are immature shoots.  Table 7-1. Treatments applied at the partial shoot experiment and number of replicates. Treatment  Description  Male (n=)  Female (n=)  1  25% of aerial shoots removed.  10  11  2  50%) of aerial shoots removed.  10  11  3  75%) of aerial shoots removed.  10  14  4  A l l shoots trimmed to half original height.  10  11  5  No shoots removed.  10  10  6  A l l shoots removed.  10  10  7.2.2 Statistical analysis Analysis of variance (ANOVA) was used if the assumptions of a normal distribution and equal variance were met. If either assumption was not met, analysis of variance on ranks was used to detect differences due to treatment one and two years after treatment. If differences were detected, Tukey's test or Dunn's test was used to determine which treatments were different for normal or non-normal distributions respectively. Male and female infections were analyzed separately so that the effect of treatment on fruit production could be assessed. Variables that were significantly different as a result of treatment effect were plotted and trends were observed by calculating means. A l l statistical calculations were conducted using the software package SigmaStat 2.03 (SPSS Inc., Chicago, IL).  7.2.3 Total shoot removal In a separate experiment, the effect of shoot removal from A. americanum by a biological control agent was estimated by manually removing all of the shoots from A. americanum infections and comparing them with non-damaged controls. This experiment was conducted to estimate the time to new fruit production following 124  biological control treatment that removed all shoots. The A. americanum infections selected were all localized infections located on small lodgepole pine trees. Each treated infection was paired with a control, from which no shoots were removed. Although the primary variable of interest was the number of female fruit, other variables that related to the growth of dwarf mistletoe were also recorded; therefore, male and female infections were selected. The experiment was replicated near Canal Flats (50° 7.659' N 116° 0.405' W 1081 m) and Lytton (50° 29.310' N 121° 36.200' W 1291 m), British Columbia. Prior to treatment, the number of shoots, maximum shoot height, number of fruit (female), A. americanum swelling diameter and length were recorded. Twenty female infections were selected at both sites, while twenty and ten male infections were selected at Canal Flats and Lytton, respectively. Infections of the same sex were paired based on the number of shoots and one of the infections was randomly selected, by tossing a coin, to have all shoots removed. The experiment was established at both locations in August 1998 and was assessed in August of 1999, 2000 and 2001, prior to seed dispersal. Data collected at the assessments was the same as the pretreatment data and included the number of buds in 2000 and 2001, and the number of pollinated female flowers in 2001. Male and female infections, as well as sites, were analyzed independently. Analysis of variance and analysis of variance on ranks (if the assumptions of a normal distribution or equal variance were not met) was used to detect any differences between treatments using the statistical package SigmaStat (SPSS Inc., Chicago, IL).  125  7.3 Results 7.3.1 Partial shoot removal experiment At the 1-year assessment, no replicates were dead, but at the 2-year assessment of the experiment, 13 replicates were found to be dead, 11 of which were female (Table 72). Death of the dwarf mistletoe was not a result of treatment application; rather, it was the result of death of the host branch.  Table 7-2. Mortality two years after treatment. Treatment (% removed)  Female  Male  1 (25%)  2  1  2(50%)  3  1  3 (75%)  1  0  4 (cut)  2  0  5 (0%)  2  0  6(100%)  1  0  7.3.1.1 Effects of treatment on growth of the endophytic system Prior to treatment application, there were no significant differences between the swelling diameter or length of A. americanum infections assigned to each treatment. Throughout the course of the experiment, A. americanum swelling diameter and length were not significantly different between treatments; treatment application had no effect on the growth of the endophytic system. Swelling diameter and length increased throughout the course of the experiment (Figure 7-1).  126  2.0  20  1.8 1.6  • —  Swelling diameter Swelling length  LTJ  15 co  1.4 1.2 10 1.0 0.8 -\ 0.6 0.4 0.2 Pre  1-year  2- yea rs  Assessment time  Figure 7-1. Change in A. americanum swelling diameter and length over the course of the partial shoot removal experiment. Treatments pooled. Mean +/- standard error of the mean.  7.3.1.2 New shoot production  Following treatment application, the production of buds was quantified as a measure of the production of new shoots. There was no significant difference between treatments with respect to bud production on male or female A. americanum swellings one or two years after treatment due to high variability (Figure 7-2). Treatment 6,100% shoot removal, resulted in the production of the most buds two years following treatment, but it was not significantly different from the other treatments. The buds present at each assessment are representative of bud production during that year, there was no carry over of buds from 1-year to 2-years because the growth rate of the buds was fast enough that buds were classified as shoots in the following year.  127  12  1-year i 2-years  EZZ3  i  Trt 1  Trt 2  Trt 3  Trt 4  Trt 5  Trt 6  Treatment  Figure 7-2. Bud production one and two years after treatment. Male and female infections pooled. Mean number of buds per A. americanum infection +/- standard error of the mean.  The number of female shoots was not significantly different prior to, or following, treatment over the course of the experiment. The maximum shoot length of female A. americanum subjected to treatment 6 was significantly less (p=0.009) than treatment 1, but not the other treatments and there was no difference at 2-years. The number of male shoots were also not significantly different prior to treatment application or two years later, but one year after treatment, A. americanum subjected to treatment 2 had significantly more (p=0.002) shoots than A. americanum subjected to treatment 6. The maximum shoot length of male infections was not significantly different prior to treatment, or after two years, but at 1-year, treatments 2 and 3 had significantly longer (p=<0.001) shoots than infections treated with treatment 6.  128  7.3.1.3 Fruit production Average fruit production on A. americanum swellings of each treatment at 0-, 1and 2-years following treatment application is shown in table 7-3. Fruit production one year following treatment application was significantly different (p=0.009) between treatments 1 and 6,25% and 100% shoot removal respectively, but not significantly different between treatments at 2-years. Fruit produced in the year after treatment were a result of pollinated flowers that were present at the time of treatment application, while fruit produced in the second year were a result of flowers produced in the year after treatment. Generally, the total number of fruit present declined throughout the experiment (Table 7-3), and the average number of fruit per shoot declined as well (Figure 7-3).  Table 7-3. Partial shoot removal experiment. Average number of fruit present on female A. americanum infections of each treatment at every assessment and average number of flowers present at the final assessment. Treatment (% removed)  Pre treatment  1-year  2-years  Flowers, 2-years  1 (25%)  60  34  7  40  2 (50%)  105  7  6  64  3 (75%)  54  26  3  58  4 (cut)  66  9  10  118  5 (0%)  100  45  21  18  6 (100%)  204  0  0  13  129  25  UJ  CO  20  ro E 15  I  ts o  CO  .•!=!  10  E  0  i  Trt 1  Trt 2  Trt 3  Trt 4  Trt 5  Trt 6  Treatment Figure 7-3. Average number of fruit per shoot over the course of the experiment for every treatment. Mean +/- standard error of the mean. When the number of fertilized female flowers was counted as a predictor of fruit production three years after treatment, there was no significant difference between A. americanum infections treated with the different treatments. Flower production two years following treatment application suggests that the fruit crop three years after treatment will be larger than the previous years for all treatments except treatment 5, which appears to be declining (Table 7-3). As with fruit production, flower production was highly variable amongst replicates of the same treatment; some A. americanum replicates had many flowers while others had zero.  7.3.1.4 Cut shoots The cut shoot treatment was conducted to determine how A. americanum responded to dieback of the top portion of the shoot. When the shoots were cut, the cut segment died back to the proximal node and abscised at the node, while the remainder of the shoot appeared unaffected. Fruit production on the unaffected portions of the shoots continued and fruit were produced one year following treatment application.  130  7.3.1.5 Control The control for this experiment was treatment 5, zero shoot removal. Over the course of the experiment, the number of shoots, maximum shoot length and number of fruit declined, with the greatest decrease over the period of the first year, suggesting there was a general decline in the stand (Figure 7-4). The number of fruit was highly variable amongst replicates of the female control. The swelling diameter and length increased over the course of the experiment as it did with A. americanum infections of all treatments.  250  §  2 0  +  a.  •s  150  A  100  50  A  1-year  2-years  Assessment  Figure 7-4. Observation of control A. americanum infections. Number of shoots and maximum shoot length of male and female infections pooled. Mean +/- standard error of the mean for all plots.  7.3.2 Total shoot removal The effect of a biological control agent that removed all of the aerial shoots of A. americanum, but did not affect the endophytic system, was mimicked by manually removing all shoots and buds from A. americanum swellings and comparing the treated swellings with untreated controls. Results are summarized in table 7-4. Arceuthobium americanum mortality occurred during the course of the experiment, through a combination of squirrel damage, normal self-pruning and disease 131  by Colletotrichum gloeosporioides. During the assessment period, C. gloeosporioides was isolated from the shoots of 3 female and 4 male dwarf mistletoe infections at Canal Flats; all but 1 female were replicates of the control. Mortality was lower at Lytton, resulting in the death of 4 female infections; 3 of which were located on branches that died after treatment and 1 that was on a branch that was broken off. Although mortality occurred during the experiment, it was spread out over the treatments and when the results were pooled, Chi square indicated that mortality was independent of treatment.  Table 7-4. Average response of each variable measured up to 3 years following treatment application at both experimental locations. A l l measurements are recorded in centimeters. Trt = treatment, Ck = control. Location  Sex  Variable  Pre Trt  Canal  1-year Ck  2-years  Trt  Ck  Trt  3-years  Ck  Trt  Ck  Female Diameter  1.37  1.7  1.27  1.81  1.38  1.55  1.41  1.61  Length  3.70  4.45  4.67  4.94  8.11  7.37  9.83  9.15  # shoots*  20  18  4  7  6  10  7  13  Max shoot  4.6  4.85  0.6  2.32  1.4  2.07  2.20  1.97  #buds  _J  ~  ~  —  8  7  2  5  # fruit  54  16  0  20  0  0  4  6  # flowers  ~  —  ~  ~  ~  ~  50  69  Mortality  0  0  0  1  3  2  3  2  Diameter  1.39  1.47  1.14  1.59  1.19  2.11  1.26  1.97  Length  3.90  3.55  3.94  4.57  6.62  7.01  8.76  9.22  # shoots*  15  17  12  26  5  9  2  12  Max shoot  6.15  6.16  1.04  6.17  1.23  3.27  1.58  2.48  #buds  —  —  ~  ~  5  9  6  12  Mortality  0  0  1  3  1  3  2  6  §  §  e  132  §  §  §  Location  Lytton  Sex  Variable  Pre  1-year  2-years  Trt  Ck  Trt  Ck  Trt  Diameter  1.07  1.29  0.96  1.28  1.12  1.47  Length  2.58  3.55  3.33  3.99  6.37  9.26  # shoots*  14  12  0  18  8  6  Max shoot  3.86  3.94  0  3.83  1.60  2.51  #buds  ~  ~  ~  ~  4  # fruit  60  50  0  65  # flowers  ~  ~  —  Mortality  0  0  Diameter  0.98  Length  3-years  Ck  Trt  Ck  Female 1.06  1.55  6.52  10.54  10  14  1.86  2.02  13  9  18  2  5  2  5  —  —  —  39  82  0  0  3  1  3  1  1.00  1.02  1.06  0.93  1.63  1.02  1.04  3.60  3.10  4.40  3.50  7.14  9.49  8.37  9.10  # shoots*  13  14  0  10  12  4  18  9  Max shoot  4.06  4.72  0  5.70  0.92  3.05  2.54  2.93  #buds  —  ~  ~  ~  5  7  13  13  Mortality  0  0  0  0  0  0  0  0  §  §  §  §  §  §  §  §  Male  §  ' One year following treatment application, buds were not distinguished from shoots, therefore, the total number of shoots at 1-year includes buds. §  treatment is significantly different from the control by A N O V A with a = 0.05.  * Data not recorded.  7.3.2.1 Fruit production The time to reestablishment of fruit production, and fruit production on the controls, was quantified by counting the number of fruit at each assessment. Prior to treatment, the A. americanum infections designated to be treated had significantly (p=0.043) more fruit than the controls at Canal Flats, even though treatment and control were randomly assigned. This occurred because infections were paired based on the number of shoots, not the number of fruit. Following treatment application, there was no 133  significant difference in fruit production between the treatment and control A. americanum infections at every assessment at Canal Flats. At Lytton, there was no significant difference between A. americanum infections designated as treatment or controls prior to treatment application, but one year after treatment, the number of fruit at Lytton was significantly (p=0.004) higher on the controls than the treated infections. Two and three years after treatment, there was no significant difference in the number of fruit between the treatment and control. There was no significant difference between the treatment and control two or three years after treatment at Canal Flats or Lytton because fruit production on the control A. americanum infections decreased dramatically, not because fruit production on the treated infections increased. Prior to treatment all female infections at Canal Flats bore fruit, while at 1 year, 4 of 10 control infections bore fruit, but the difference between the treatment and control was not significant, and at 2 years only 1 fruit was present on 1 of 8 control infections. Three years after treatment, 2 of 8 control infections and 2 of 7 treated infections bore fruit. At Lytton, fruit production was greatly reduced two years after treatment. A l l control infections bore fruit in the year following application, while 4 of 9 control infections at 2 years and 2 of 9 control infections at 3 years bore fruit. Of the treated infections at Lytton, none produced fruit the year following shoot removal, while 1 of 7 infections produced consecutive fruit crops at 2 and 3 years after treatment. The production of consecutive crops of A. americanum fruit on control infections at Canal Flats and Lytton are recorded in table 7-5. One control infection at Canal Flats bore fruit prior to treatment and then again three years after treatment. After 2 consecutive crops of fruit, two infections died at Canal Flats and one infection died at Lytton.  134  Table 7-5. Number o f control replicates of A. americanum i n each category to record consecutive crops o f fruit at Canal Flats and Lytton. Site  1 crop  2 crops  3 crops  4 crops  Discontinuous  Canal Flats  5  3  0  1  1  Lytton  1  5  3  1  0  The number o f pollinated female flowers was counted three years after treatment as a measure o f potential fruit production four years following treatment. A t Canal Flats, 3 o f 8 controls and 4 o f 7 treated infections had pollinated female flowers and at Lytton, 5 o f 9 controls and 5 o f 7 treated infections had pollinated female flowers. The number o f flowers was not significantly different between treatments, and the average number o f flowers on each infection suggests that fruit production w i l l be high four years after treatment.  7.3.2.2 Endophytic system growth Growth o f the endophytic system was not significantly different between treatments at either site or for either sex. Treatment application did not affect endophytic system growth, the average diameter and length o f the dwarf mistletoe swelling increased throughout the course o f the experiment at both sites. The average increase i n swelling diameter and length over the assessment period was 1 m m and 5.6 cm, respectively, at both sites.  7.3.2.3 Shoot production The number o f shoots per swelling was not significantly different prior to treatment application. After treatment application, there was no significant difference i n the number o f male or female shoots at any time post treatment at Canal Flats or male shoots at Lytton. The number o f female shoots was significantly greater on the controls compared to the treated infections one year after treatment at the Lytton site (p=0.004), i n years 2 and 3, there were no significant differences. A decrease i n the number o f shoots on the control, coupled with an increase i n the number o f shoots on treated A. americanum swellings, accounted for the finding o f no significant difference. 135  One year after treatment, buds were not distinguished from shoots; therefore, the total number of shoots present at the 1-year assessment included buds and buds cannot be separated from the number of shoots. The production of new shoots was quantified two and three years following treatment by counting the number of buds and it was found that there was no significant difference between treatments, sexes or sites. The female maximum shoot length of control infections at Canal Flats was significantly longer than the treated infections at 1-year (p=0.043). The male maximum shoot length of controls was significantly longer than treated infections at 1-year (p=<0.001) and 2-years (p=0.023). Three years after treatment, there were no significant differences in maximum shoot length, as was the case prior to treatment. The maximum shoot length of female control infections at Lytton was significantly longer than the treated infections at 1-year (p= <0.001) and 2-years (p=<0.001), while the male controls were significantly longer than the treated infections at 1-year (p=<0.001). 7.4 Discussion  The partial shoot removal experiment was designed to mimic the effect of a biological control treatment that did not result in one hundred percent shoot death. The total shoot removal experiment was conducted to estimate the time to new fruit production following biological control treatment that killed all of the aerial shoots but not the endophytic system. Mortality and high variability between replicates of the same treatment affected the results of the experiments; however, general conclusions can be drawn from the response of the dwarf mistletoe to treatment. 7.4.1 Endophytic system growth  No decrease in the growth rate of the endophytic system, as estimated by change in the length and diameter of the A. americanum swelling, was noted following treatment application. It was hypothesized that endophytic system growth rate would slow because the nutrient sink generated by A. americanum (Clark and Bonga, 1970) would be reduced following removal of the aerial shoots; however, the null hypothesis of no significant difference was not rejected. To maintain connectivity between the sinkers in the xylem and the cortical strands in the phloem, meristematic activity must occur within the sinker 136  tissue as new xylem and phloem are laid down by the vascular cambium (Hunt et al., 1996). The finding of no change suggests that the growth of the endophytic system may be regulated or influenced by the growth of the host branch.  7.4.2 New shoot production The production of buds was not found to be significantly different between treatments following treatment application in the partial shoot removal or the total shoot removal experiments. The production of buds occurs via meristematic activity on the surface of the outer cortical strand, which ultimately results in emergence of the bud from the bark (Cohen, 1954). New shoots are produced at the margin of the endophytic system as the endophytic system expands, suggesting that the production of buds may be regulated by expansion of the endophytic system. Although the production of new buds was not significantly different between treatments in these studies, bud production was found to be significantly increased following treatment 2 application in the field trial that was described in Chapter 3. The variability of individual replicates observed in these studies was high and with increased sample size, significant differences may have been noted, but the trend observed suggests that A. americanum does increase new shoot production following treatment.  7.4.3 Fruit production Fruit production one year after treatment application was a result of immature fruit present on the shoots at the time of treatment, while fruit production in the following years was a result of flowers that were produced on the remaining shoots. There was a sharp decrease in fruit production in both treatments and controls in both experiments two years after treatment. The number of flowers present in the second year suggested that the seed crop will be higher in the following season, assuming each pollinated flower will produce a viable seed. The number of pollinated flowers present in the year following treatment was not counted; therefore, it is impossible to determine if all flowers counted in the second year will develop to maturity. It is unlikely that every pollinated flower counted will survive to produce a viable seed; of the 93% of A. tsugense shoots that bore flowers, only 43% produced fruit prior to dying (Smith, 1977). Variability 137  amongst replicates of the same treatments was high with respect to fruit and flower production. The general decline in the number of fruit present, as well as the variability between replicates of the same treatment, prevent a definitive conclusion with respect to changes in fruit production following treatment application.  7.4.4 Response of shoots to dieback from the tip Trimming the shoot at the midpoint mimicked the effect of a biological control agent that resulted in death of the top portion of the shoot. When the shoot was cut, the segment died back to the proximal node and fell off. Shoot development carried on normally and flowers that were present on the laterals developed into fruit. A n increase in the production of lateral branches on the shoots below the cut was not quantified; therefore, it is impossible to determine if A. americanum responded by increasing lateral branches. The production of new buds was no different from the other treatments, and fruit production declined in the following years as it did with all other treatments.  7.4.5 Shoot characteristics over time The controls of both experiments were utilized to test the hypothesis that A americanum infection characteristics do not change over time. Although the number of shoots remained constant, the maximum shoot height and number of fruit decreased. This suggests that A. americanum infections declined over time. Observation of A. americanum in the field suggests that the first shoots that are produced by the infection are vigorous and produce large crops of fruit. Individual shoots cannot be aged; however, under controlled conditions, individual shoots could be followed and their life history could be determined, providing an understanding of the number of consecutive crops of fruit they could bear. The health of the A. americanum infection is directly related to the health of the individual branch upon which it is located. As lodgepole pine increases in height, shade increases in the lower canopy. As the lower canopy becomes shaded, the branches die, effectively raising the living crown as the tree grows in height (Koch, 1996). Mortality of the host branch over the term of the experiment occurred in all experiments that were conducted in this study. The decline of the host branch associated with increased shading 138  and natural pruning of lodgepole pine has likely resulted in the decrease in A. americanum vigour over the course of the experiment.  7.4.6 Biological control treatment periodicity The total shoot removal experiment was established so that the time to new shoot production following biological control that removed all shoots, without damaging the endophytic system could be estimated. At Lytton, fruit were observed on shoots that had 100% shoot removal two years earlier. Seed development requires 16 months in A. americanum. For fruit to be produced two years after treatment, a shoot would need to grow and set flowers in the year following treatment application. The treatment was applied in August of 1998; it is highly doubtful that shoots grew and produced flowers that were pollinated in the spring of 1999. It is probable that the observation of fruit two years following treatment was the result of a shoot or bud that was accidentally missed during treatment application. This is supported by the fact that no fruit were observed on the 100% shoot removal treatment of the partial shoot removal experiment or at Canal Flats. Flowers were recorded two years after treatment on replicates of the 100% shoot removal treatment in the partial shoot removal experiment, suggesting that seed will be shed in the fall of the third year after treatment. Fruit were observed three years after shoot removal at Canal Flats and Lytton. When A. americanum was treated with ethephon, an ethylene releasing compound that causes shoot abscission, shoots bearing fruit were noted for 3% of infections two years after treatment, increasing to 52% of infections after 5-years (Nicholls, 1988). These data suggest that the recovery of A. americanum following shoot abscission is variable, perhaps as a result of host branch vigour and light availability, but that biological control reapplication may be required as soon as three years following the initial treatment. Consecutive crops of fruit on individual shoots of A. americanum have been observed for up to five years (Hawksworth and Wiens, 1996) and multiple crops of seeds over successive years have been observed on A. tsugense and A laricis (Smith 1977). In this study, individual shoots were not followed, but consecutive crops of fruit were observed on some infections for at least four years at Canal Flats and Lytton. If a biological control agent were able to remove the aerial shoots of A. americanum, seed 139  dispersal onto the regenerating stand would be significantly reduced for a period of at least 3 years and the age class distribution of shoots would be interrupted.  7.4.7 Conclusions Mortality, high variability and declining host branch vigour affected the results of this study. The impact of partial shoot removal on fruit production could not be assessed satisfactorily. Growth of the endophytic system did not change as a result of treatment, indicating that this characteristic may be closely related to growth of the host branch. Bud production did increase following total shoot removal in these experiments, and in the field trial in Chapter 3; however, the difference was not significant. Treatment application was designed to mimic the mode of action of a biological control agent; however, physically cutting the aerial shoots may induce a different host response than disease caused by C. gloeosporioides. Without inoculating A. americanum with C. gloeosporioides, it is difficult to determine exactly how the dwarf mistletoe responds to infection, but the wounds applied to A. americanum may give a minimum response time. Alternative methods of determining the maximum treatment interval include treatment with Ethephon and closely observing the development of individual shoots over time. From a biological control perspective, the results indicate that all shoots must be removed to cause a significant reduction in seed production, and that the maximum period of time between biological control applications necessary to achieve effective dwarf mistletoe control would be 3 years.  140  Chapter 8 - Conclusions Research in this study was focused toward the development of an inundative biological control strategy for lodgepole pine dwarf mistletoe. Previous studies into biological control of dwarf mistletoes were directed at establishing a self-regulating population of the biological control agent under the classical biological control paradigm. The development of an inundative biological control approach may provide an alternative dwarf mistletoe management tool to minimize establishment of dwarf mistletoe in regenerating stands. The initial stage of the study was directed at collecting diseased A. americanum for the isolation of fungi that may be potential inundative biological control agents. Colletotrichum gloeosporioides was collected throughout the region surveyed and Caliciopsis arceuthobii was observed but not isolated. Other fungi, including Cladosporium sp. and Schlerophoma pithyiophita were also isolated. Lodgepole pine were inoculated with A. americanum under greenhouse conditions to serve as experimental units for testing under controlled conditions, however, A. americanum infections had not emerged in time for any portions of the research conducted in this study. The lack of experimental units to test fungi isolated from diseased A. americanum that were collected in the survey resulted in the selection of C. gloeosporioides as the candidate organism because it met at least three of the six criteria of a dwarf mistletoe biological control agent as set out by Wicker and Shaw (1968). The distribution of the fungus coincided with the range of A. americanum that was surveyed, it produced abundant inoculum in culture and the field, and it has an efficient mode of curtailing A. americanum development. Further study of the infection process and an assessment of virulence are likely to confirm that C. gloeosporioides meets these criteria as well. Additionally, C. gloeosporioides was readily cultured and inoculum was easily generated in culture. Although C. arceuthobii has an efficient mode of reducing A. americanum spread and intensification, it was not selected because it was not cultured and inoculum has not been generated in culture in other studies. Without experimental units under controlled conditions, isolate selection was made based on the growth and sporulation characteristics of four different isolates. The isolate that produced the most conidia in culture and had the best growth over a wide range of temperatures was selected. Initially 141  a sodium alginate - kaolin clay formulation was tested in the greenhouse on experimental units of A. tsugense and this formulation was found to be unsatisfactory. The selected isolate was then formulated in 'Stabileze' and inoculated onto A. americanum under field conditions near Lytton, British Columbia. The results of successful establishment on a small number of replicates indicated that when C. gloeosporioides becomes established, it is able to cause extensive damage to A. americanum. A successful inundative biological control agent for A. americanum must interfere with seed production to prevent spread to uninfected trees and intensification in infected trees. There was no significant reduction in seed production as a result of inoculation with C. gloeosporioides in the field trial. This may be due to low inoculum concentration in the formulation, low virulence of the selected isolate, or the environmental conditions at the field site may have been limiting. It has been reported that C. gloeosporioides has been observed in the endophytic system of A. americanum. From a biological control perspective, this would likely result in increased time between treatment application because new shoot production would be inhibited. To study the relationship between C. gloeosporioides and A. americanum, naturally diseased and inoculated A. americanum swellings were cultured and examined microscopically. In this study, it was found that C. gloeosporioides was restricted to the basal cup region of A. americanum and did not infect the endophytic system. This may be indicative of a difference between this pathosystem and the other pathosystems that have been investigated. The natural distribution of C. gloeosporioides in a stand of A. americanum infected lodgepole pine was assessed to predict establishment success of C gloeosporioides at different crown positions following inoculation. It was found that A. americanum at all crown positions was equally susceptible to C. gloeosporioides indicating that if the inoculum contacts the dwarf mistletoe, probability of establishment is the same at all canopy positions. The study designed to follow Caliciopsis arceuthobii infection of A. americanum indicated that the fungus caused a significant reduction in fruit production and that it was able to infect A. americanum that was disease free in previous years. The difficulties of  142  inoculum production and field inoculation require extensive research before this fungus can be used as an inundative biological control agent for A. americanum. To study the response of A. americanum to biological control treatment and to determine the treatment interval required to prevent A. americanum infection of a regenerating stand, two separate experiments were conducted. The rate of endophytic system growth was not related to the amount of damage that was inflicted upon the aerial system, suggesting that endophytic system growth may be regulated by the host. There was not a significant increase in bud production following total shoot removal, however, the trend observed indicates that bud production may be increased following shoot removal. The time to fruit production following total shoot removal was three years, suggesting that the maximum treatment interval necessary to prevent seed dispersal, assuming no damage to the endophytic system, is three years. Future studies should be conducted on A. americanum so that fruit production over the lifespan of the dwarf mistletoe is better understood. It was obvious that A. americanum fruit production on individual infections varied from year to year. If the source of this variation can be determined, the biological control treatment periodicity may be extended. The results of these studies suggest that C. gloeosporioides has potential as an inundative biological control agent for A. americanum due to the damage that it causes when it becomes successfully established and that A. americanum at all crown positions was found to be susceptible to infection. Several challenges must be overcome before a successful inundative biological control strategy is developed. The optimum conditions for fungal establishment were not assessed because no experimental units were ready for these studies. Successful establishment of A. americanum on the lodgepole pine that were inoculated at the start of this study is now becoming evident, therefore, experimental units that can be placed under controlled conditions to study the infection process of C. gloeosporioides are now available. These experimental units can also be used for the selection of the most virulent isolate of C. gloeosporioides, as well as assessing the pathogenecity of other fungi that were collected during the collection phase. If the necessary conditions required for C. gloeosporioides establishment can be determined in the greenhouse, field inoculation can be timed to coincide with as close to optimum conditions as are available. If inoculation conditions can be optimized, frequent  143  reapplication is required, as demonstrated by the shoot removal experiment. The role of secondary inoculum production should also be studied, as secondary inoculum may serve to intensify C. gloeosporioides infection as well as increase the period of time necessary for preventing seed production. Another challenge that must be overcome is inoculation of A. americanum in the upper crown. Upper crown infections are the most important from a spread and intensification standpoint, and for biological control to be successful they must be inoculated. When applied to the upper crown by helicopter, Ethepon was ineffective in controlling dwarf mistletoe (Baker et al., 1989; Robbins et al., 1989), suggesting that considerable study will be required to develop an application system that effectively inoculates A. americanum in the upper crown. The primary objective of this research was to investigate the possibility of developing an inundative biological control strategy for A. americanum. Focusing the study on C. gloeosporioides provided important insight into what is required for a successful biological control strategy for A. americanum. The data collected in this study suggests that considerable research relating to conditions necessary for establishment of the fungus, improved efficacy and field inoculation techniques are required to develop C. gloeosporioides into a successful biological control agent. If these challenges to development cannot be overcome, then the use of C. gloeosporioides as an inundative biological control agent for A. americanum is unlikely, but further study should be conducted before this approach is abandoned. If the results of future research suggest that the C. gloeosporioides provides adequate control of A. americanum, future study of the genetic stability of C. gloeosporioides, as well as host range studies will need to be completed before the fungus can used commercially as an inundative biological control agent for A. americanum.  144  Literature Cited Alosi, M.C., and Calvin, C L . 1984. The ultrastructure of dwarf mistletoe (Arceuthobium spp.) sinker cells in the region of the host secondary vasculature. Canadian Journal of Botany 63: 889-898. Alosi, M.C., and Calvin, C L . 1984b. The anatomy and morphology of the endophytic system of Arceuthobium spp. In Biology of dwarf mistletoes: proceedings of the symposium. Edited by F.W. Hawksworth and R.F. Scharpf. U.S. Dep. Agric. For. Serv. Rocky Mount. Stn., Gen. Tech. Rep. RM-111. pp. 40-52. Anonymous. 1995. Dwarf mistletoe management guidebook. Forest Practices Code of British Columbia. Province of British Columbia. Ministry of Forests. 20 p. Auld, B.A., and Morin, L . 1995. Constraints in the development of bioherbicides. Weed Technology 9: 638-652. Baranyay, J.A. 1968. Squirrel feeding on dwarf mistletoe infections. Bi-Monthly Progress Report. Ottawa, Canada Department of Forestry 24(5): 41-42. Baranyay, J.A., and Smith, R.B. 1972. Dwarf mistletoes in British Columbia and recommendations for their control. Canadian Forest Service, Pacific Forest Research Centre. Report BC-X-72. 18 p. Bailey, J.A., O'Connell, R.J., Pring, R.J., and Nash, C. 1992. Infection strategies of Colletotrichum species. Pages 88-120 in: Colletotrichum: Biology, Pathology and Control. J.A. Bailey and M.J. Jeger, eds. C A B International, Wallingford, UK. Bailey, J.A., Nash, C , Morgan, L.W., O'Connell, R.J., and TeBeest, D.O. 1996. Molecular taxonomy of Colletotrichum species causing anthracnose on the Malvaceae. Phytopathology 86: 1076-1083. Barnett, H.L., and Hunter, B.B. 1998. Illustrated genera of imperfect fungi, 4 edition. APS Press, St. Paul, M N . 218 p. th  Bhandari, N . N . , and Nanda, K . 1970. The endophytic system of Arceuthobium minutissimum, the Indian dwarf mistletoe. Annals of Botany 34: 517-525. Biring, B.S., Comeau, P.G., and Boateng, J.O. 1996. Effectiveness of forest vegetation control methods in British Columbia. Canadian Forest Service and British Columbia Forest Service, F R D A Handbook 011. 51 p. Boyette, C.D. 1994. Unrefined corn oil improves the mycoherbicidal activity of Colletotrichum truncatum for hemp sesbania (Sesbania exaltata) control. Weed Technology 8: 526-529. 145  Boyette, C D . , Quimby, P.C., Jr., Bryson, C.T., Egley, G.H., and Fulgham, F.E. 1993. Biological control of hemp sesbania (Sesbania exaltata) under field conditions with Colletotrichum truncatum formulated in an invert emulsion. Weed Science 41: 497-500.  Boyette, C D . , Quimby, P.C.Jr., Caesar, A.J., Birdsall, J.L., Connick, W.J.Jr., Daigle, D.J., Jackson, M . A . , Egley, G.H., and Abbas, H.K. 1996. Adjuvants, formulations, and spraying systems for improvement of mycoherbicides. Weed Technology 10: 637-644. Broshot, N.E., and Tinnin, R.O. 1986. The effect of dwarf mistletoe on starch concentration in the twigs and needles of lodgepole pine. Canadian Journal of Forest Research 16: 658-660. Carrington, M.E., Roberts, P.D., Urs, N.V.R.R., McGovern, R.J., Seijo, T.E., and Mullahey, J.J. 2001. Premature fruit drop in saw palmettos caused by Colletotrichum gloeosporioides. Plant Disease 85: 122-125. Cerkauskas, R.F. 1988. Latent colonization by Colletotrichum spp.: Epidemiological considerations and implications for mycoherbicides. Canadian Journal of Plant Pathology 10: 297-310. Chapman, B., and Xiao, G. 2000. Inoculation of stumps with Hypholoma fasciculare as a possible means to control armillaria root disease. Canadian Journal of Botany 78: 129-134. Clark, J., and Bonga, J.M. 1970. Photosynthesis and respiration in black spruce (Picea mariana) parasitized by eastern dwarf mistletoe (Arceuthobium pusillum). Canadian Journal of Botany 48: 2029-2031. Cohen, L.I. 1954. The anatomy of the endophytic system of the dwarf mistletoe, Arceuthobium campylopodum. American Journal of Botany 41: 840-847. Dobie, J., and Britneff, A . A . 1975. Lumber grades and volumes from lodgepole pine infected with dwarf mistletoe. Wood and Fiber 7: 104-109. Dowding, S.E. 1931. Wallrothiella arceuthobii, a parasite of the Jack-pine mistletoe. Canadian Journal of Research 5: 219-230. Dumas, B., Centis, S., Sarrazin, N . , and Esquerre-Tugaye, M . 1999. Use of green fluorescent protein to detect expression of an endopolygalacturonase gene of Colletotrichum lindemuthianum during bean infection. Applied and Environmental Microbiology 65: 1769-1771.  146  Egley, G.H. and Boyette, C.D. 1995. Water-corn oil emulsion enhances conidia germination and mycoherbicidal activity of Colletotrichum truncatum. Weed Science 43: 312-317. Ellis, D.E. 1939. A fungus disease of Arceuthobium. Phytopathology 29: 995-996. Ellis, D.E. 1946. Anthracnose of dwarf mistletoe caused by a new species of Septogloeum. Journal of the Elisha Mitchell Science Society 62: 25-50. Estrada, A . B . , Dodd, J.C., and Jeffries, P. 2000. Effect of humidity and temperature on conidial germination and appressorium development of two Philippine isolates of the mango anthracnose pathogen Colletotrichum gloeosporioides. Plant Pathology 49: 608-618. Geils, B.W., and Mathiasen, R.L. 1990. Intensification of dwarf mistletoe on southwestern Douglas-fir. Forest Science 36: 955-969. Gilbert, J.A. 1984. The biology of dwarf mistletoes (Arceuthobium spp.) in Manitoba. M.Sc. Thesis, Department of Botany, University of Manitoba. 138p. Gill, L.S. 1935. Arceuthobium in the United States. Connecticut Academy of Arts and Science Transactions 32: 111-245. Gill, L.S. 1952. A new host for Septogloeum gillii. Plant Disease Reporter 36: 300. Hadfield, J.S., Mathiasen, R.L., and Hawksworth, F.G. 2000. Forest insect and disease leaflet 54, Douglas-fir dwarf mistletoe. United States Department of Agriculture Forest Service. lOp. Harper, G.J., Comeau, P.G., Hintz, W., Wall, R.E., Prasad, R., and Becker, E . M . 1999. Chondrostereum purpureum as a biological control agent in forest vegetation management. II. Efficacy on sitka alder and aspen in western Canada. Canadian Journal of Forest Research 29: 852-858. Hasan, S. and Ayers, P.G. 1990. Tansley review no. 23. The control of weeds through fungi: principles and prospects. New Phytologist 115: 201-222. Hawksworth, F.G. 1972. Biological control of the mistletoes. In: Nordin, V.J., comp. Biological control of forest diseases. Ottawa, Canada. The Canadian Forestry Service, p. 83-92. Hawksworth, F.G. 1977. The 6-class dwarf mistletoe rating system. Gen. Tech. Rep. RM-48. Fort Collins, CO: U.S. Department of Agriculture, Forest Service, Rocky Mountain Forest and Range Experiment Station. 7p. Hawksworth, F.G., Dixon, C.S., and Krebill, R.G. 1983. Peridermium bethelii: a rust 147  associated with lodgepole pine dwarf mistletoe. Plant Disease 67: 729-733. Hawksworth, F. G. and Johnson, D.W. 1989. Biology and management of dwarf mistletoe in lodgepole pine in the Rocky Mountains. Gen. Tech. Rep. RM-169. Fort Collins, CO: U.S. Department of Agriculture, Forest Service, Rocky Mountain Forest and Range Experiment Station. 38p. Hawksworth, F.G., Wicker, E.F., and Scharpf, R.F. 1977. Fungal parasites of dwarf mistletoes. Gen. Tech. Rep. RM-36. Fort Collins, CO: U.S. Department of Agriculture, Forest Service, Rocky Mountain Forest and Range Experiment Station. 14p. Hawksworth, F.G., and Wiens, D. 1972. Biology and classification of dwarf mistletoes (Arceuthobium). Agriculture Handbook 401. Washington, D C : U.S. Department of Agriculture, Forest Service. 234p. Hawksworth, F.G., and Weins, D. 1996. Dwarf mistletoes: biology, pathology, and systematics. Agricultural Handbook 709. Washington, D C : U.S. Department of Agriculture, Forest Service. 41 Op. Heath, M . C . 2000. Advances in imaging the cell biology of plant-microbe interactions. Annual Review of Phytopathology 38: 443-459. Hinds, T.E., Hawksworth, F.G., and McGinnies, W.J. 1963. Seed discharge in Arceuthobium: A photographic study. Science 140: 1236-1238. Hinds, T.E., and Hawksworth, F.G. 1965. Seed dispersal velocity in four dwarf mistletoes. Science 148: 517-519. Hintz, W.E., Becker, E . M . , and Shamoun, S.F. 2001. Development of genetic markers for risk assessment of biological control agents. Canadian Journal of Plant Pathology 23: 13-18. Holdenrieder, O., and Greig, B.J.W. 1998. Biological methods of control. In Heterobasidion annosum Biology, Ecology, Impact and Control. Edited by S. Woodward, J. Steinlid, R., Karjalainen and A . Huttermann. Wallingford, Oxon. C A B International, pp. 235-258. Hunt, R.S., Owens, J.N., and Smith, R.B. 1996. Penetration of western hemlock, Tsuga heterophylla, by the dwarf mistletoe Arceuthobium tsugense, and development of the parasite cortical system. Canadian Journal of Plant Pathology 18: 342-346. Johansen, D.A. 1940. Plant Microtechnique. Ed. 1, McGraw-Hill, New York. Knutson, D . M . , and Hutchins, A.S. 1979. Wallrothiella arceuthobii infecting Arceuthobium douglasii: culture and field inoculation. Mycologia71: 821-828. 148  Koch, P. 1996. Lodgepole Pine in North America. Wood Products Society, Madison, WL 1096p. Kope, H.H., Shamoun, S.F., and Oleskevich, C. 1997. First report of Colletotrichum gloeosporioides on Arceuthobium tsugense subsp. tsugense in Canada. Plant Disease 81: 1095. Kuijt, J. 1955. Dwarf mistletoes. The Botanical Review 21: 569-619. Kuijt, J. 1960. Morphological aspects of parasitism in the dwarf mistletoes (Arceuthobium). Publications in Botany 30. Berkeley, C A : University of California: 337-436. Kuijt, J. 1969. Observations on Wallrothiella arceuthobii, a fungus parasite of dwarf mistletoes. Canadian Journal of Botany 47: 1359-1365. Lennox, C.L., Morris, M.J., and Wood, A.R. 2000. Stumpout™ - Commercial production of a fungal inoculant to prevent re-growth of cut wattle stumps in South Africa. In Program and Abstracts of X International Symposium on Biocontrol of Weeds. July 4 - 14, 1999. Bozeman, Montana, U.S.A. Edited by N . Rush. Montana State University, Montana, U.S.A. p. 69. Leonard, O.A., and Hull, R J . 1965. Translocation relationships in and between mistletoes and their hosts. Hilgardia37: 115-153. Livingston, W.H., Brenner, M . L . , and Blanchette, R.A. 1984. Altered concentrations of abscisic acid, indole-3-acetic acid, and zeatin riboside associated with eastern dwarf mistletoe infections on black spruce. In Biology of dwarf mistletoes: proceedings of the symposium. Edited by F.W. Hawksworth and R.F. Scharpf. U.S. Dep. Agric. For. Serv., Rocky Mount. Stn., Gen. Tech. Rep. RM-111. pp. 53-61. Lloyd, D., Angrove, K., Hope, G., and Thompson, C. 1990. A guide to site identification and interpretation for the Kamloops forest region, Part 1. British Columbia Ministry of Forests, Victoria, B.C., Land Management Handbook 23. p. 103-108. Lorang, J.M., Tuori, R.P., Martinez, J.P., Sawyer, T.L., Redman, R.S., Rollins, J.A., Wolpert, T.J., Johnson, K . B . , Rodriguez, R.J., Dickman, M . B . , and Ciuffetti, L . M . 2001. Green fluorescent protein is lighting up fungal biology. Applied and Environmental Microbiology 67: 1987-1994. Louis, I., and Cooke, R.C. 1985. Conidial matrix and spore germination in some plant pathogens. Transactions of the British Mycological Society 84: 661-667.  149  Luo, Y . , and TeBeest, D.O. 1999. Effect of temperature and dew period on infection of northern jointvetch by wild-type and mutant strains of Colletotrichum gloeosporioides f. sp. aeschynomene. Biological Control 14: 1-6. Makowski, R . M . 1993. Effect of inoculum concentration, temperature, dew period, and plant growth stage on disease of round-leaved mallow and velvetleaf by Colletotrichum gloeosporioides f. sp. malvae. Phytopathology 83: 1229-1234. Makowski, R . M . , and Mortensen, K . 1998. Latent infections and penetration of the bioherbicide agent Colletotrichum gloeosporioides f. sp. malvae in non-target field crops under controlled environmental conditions. Mycological Research 102: 1545-1552. Manion, P.D. 1981. Tree Disease Concepts. Prentice-Hall, Inc., Englewood Cliffs, NJ. 399p. Maor, R., Puyesky, M . , Horwitz, B.A., and Sharon, A . 1998. Use of green fluorescent protein (GFP) for studying development and fungal-plant interaction in Cochliobolus heterostrophus. Mycological Research 102: 491-496. Mark, W.R., Hawksworth, F.G., and Oshima, N . 1976. Resin disease: a new disease of lodgepole pine dwarf mistletoe. Canadian Journal of Forest Research 6: 415-424. Mathiasen, R.L. 1996. Dwarf mistletoes in forest canopies. Northwest Science 70 (Special Issue): 61-71. Mathre, D.E., Cook, R.J., and Callan, N.W. 1999. From discovery to use. Traversing the world of commercializing biocontrol agents for plant disease control. Plant Disease 83: 972-983. Mercer, P.C. 1988. Biological control of decay fungi in wood. In Biocontrol of Plant Diseases, Vol. I. Edited by Mukerji, K . G . , and Garg, K . L . C R C Press Inc, Boca Raton, Florida, pp. 177-198. Mielke, J.L. 1959. Infection experiments with Septogloeum gillii, a fungus parasitic on dwarf mistletoe. Journal of Forestry 57: 25-26. Mondai, A . H . , and Parbery, D.G. 1992. The spore matrix and germination in Colletotrichum musae. Mycological Research 96: 592-596. Mordue, J.E.M. 1971. Glomerella cingulata. C.M.I. Descriptions of Pathogenic Fungi and Bacteria. No. 315. Morin, L., Watson, A . K . , and Reeleder, R.D. 1990. Effect of dew, inoculum density, and spray additives on infection of field bindweed by Phomopsis convolvulus. Canadian Journal of Plant Pathology 12: 48-56. 150  Morin, L., Derby, J.L., and Kokko, E.G. 1996. Infection process of Colletotrichum gloeosporioides f. sp. malvae on Malvaceae weeds. M y cological Research 100: 165-172. Mortensen, K . 1986. Biological control of weeds with plant pathogens. Canadian Journal of Plant Pathology 8: 229-231. Mortensen, K . 1988. The potential of an endemic fungus, Colletotrichum gloeosporioides, for biological control of round-leaved mallow (Malva pusilld) and velvetleaf (Abutilon theophrasti). Weed Science 36: 473-478. Mortensen, K . 1998. Biological contol of weeds using microorganisms. In PlantMicrobe Interactions and Biological Control. Edited by G.J. Boland and L.D. Kuykendall. Marcel Dekker, Inc. New York. pp. 223-247. Muir, J.A. 1967. Occurrence of Colletotrichum gloeosporioides on dwarf mistletoe (Arceuthobium americanum) in western Canada. Plant Disease Reporter 51: 798-799. Muir, J.A. 1970. Dwarf mistletoe spread in young lodgepole pine stands in relation to density of infection sources. Bi-monthly Research Note 26. Canada Department of Fisheries and Forestry, Forestry Service: 49. Muir, J.A. 1973. Cylindrocarpon gillii, a new combination for Septogloeum gillii on dwarf mistletoe. Canadian Journal of Botany 51: 1997-1998. Muir, J.A. 1977. Effects of the fungal hyperparasite C. gloeosporioides of dwarf mistletoe (Arceuthobium americanum) on young lodgepole pine. Canadian Journal of Forest Research 7: 579-583. Neumann, S., and Boland, G.J. 1999. Influence of selected adjuvants on disease severity by Phoma herbarum on dandelion (Taraxacum officinale). Weed Technology 13: 675-679. Nevill, R., and Winston, D. 1994. Forest health research needs survey. Forestry Canada. Pacific Forestry Centre. B C F R A C Report No. 3. F R D A Report 212. Nicholls, T. H . 1988. Ethephon tests for lodgepole pine dwarf mistletoe in Colorado. Proceedings of the 36 Western International Forest Disease Work Conference, Sept. 19-23, 1988, Park City, Utah, p. 34-37. th  Norman, D.J., and Trujillo, E.E. 1995. Development of Colletotrichum gloeosporioides f. sp. Clidemiae and Septoria passiflorae into two mycoherbicides with extended viability. Plant Disease 79: 1029-1032.  151  Oleskevich, C , Shamoun, S.F., Vesonder, R.F., and Punja, Z.K. 1998. Evaluation of Fusarium avenaceum and other fungi for potential as biological control agents of invasive Rubus species in British Columbia. Canadian Journal of Plant Pathology 20: 12-18. Parker, A . K . 1970. Growth of Wallrothiella arceuthobii on artificial media. Canadian Journal of Botany 48: 837-838. Parks, C.G., Bull, E X . , Tinnin, R.O., Shepherd, J.F., and Blumton, A . K . 1999. Wildlife use of dwarf mistletoe brooms in Douglas-fir in northeast Oregon. Western Journal of Applied Forestry 14: 100-105. Parmeter, J.R. Jr. 1978. Forest stand dynamics and ecological factors in relation to dwarf mistletoe spread, impact and control. In: Scharpf, R.F., and Parmeter, J.R. Jr. tech. Cords. Proceedings of the symposium on dwarf mistletoe control through forest management, April 11 - 13, 1978, Berkeley, C A . General Technical Report PSW-31. Berkley, C A . U.S. Department of Agriculture, Forest Service, Pacific Southwest Forest and Range Experiment Station: 16-30. Parmeter, J.R. Jr., Hood, J.R., and Scharpf, R.F. 1959. Colletotrichum blight of dwarf mistletoe. Phytopathology 49: 812-815. Pearce, R.B. 1984. Staining fungal hyphae in wood. Transactions of the British Mycological Society 82: 564-567. Peterson, R.S. 1966. Cronartium mycelium parasitizing gymnosperm and angiosperm tissues simultaneously. Mycologia 58: 474-477. Piirto, D.D., Crews, D.L., and Troxell, H.E. 1974. The effects of dwarf mistletoe on the wood properties of lodgepole pine. Wood and Fiber 6: 26-35. Pitt, D.G., Dumas, M.T., Wall, R.E., Thompson, D.G., Lanteigne, L., Hintz, W., Sampson, G., and Wagner, R.G. 1999. Chondrostereum purpureum as a biological control agent in forest vegetation management. I. Efficacy on speckled alder, red maple, and aspen in eastern Canada. Canadian Journal of Forest Research 29: 841-851. Prusky, D., Freeman, S., and Dickman, M . B . eds. 2000. Colletotrichum Host Specificity, Pathology, and Host - Pathogen Interaction. APS Press, St. Paul, M N . 393p. Quimby, P.C., Zidack, N.K., Boyette, C D . , and Grey, W.E. 1999. A simple method for stabilizing and granulating fungi. Biocontrol Science and Technology 9: 5-8. Robinson, M . , and Sharon, A . 1999. Transformation of the bioherbicide Colletotrichum gloeosporioides f. sp. aeschynomene by electroporation of germinated conidia. 152  Current Genetics 36: 98-104. Rose, C L . 1996. Forest canopy - atmosphere interactions. Northwest Science 70 (Special Issue): 7-14. Roth, L.F. 1974. Resistance of ponderosa pine to dwarf mistletoe. Silvae Genetica 23: 116-120. Sands, D . C , Ford, E.J., and Miller, R.V. 1990. Genetic manipulation of broad hostrange fungi for biological control of weeds. Weed Technology 4: 471-474. Scharpf, R.F. 1964. Cultural variation and pathogenicity of the Colletotrichum blight fungus of dwarf mistletoe. Phytopathology 54: 905-906. Schneider, H . 1981. Pathological and anatomy and mycology. In Staining Procedures, 4 ed. Edited by G. Clark. Williams and Wilkins, Baltimore, p. 372-373. th  Senaratna, L . K . , Wijesundera, R.L.C., and Liyanage, A . de S. 1991. Morphological and physiological characters of two isolates of Colletotrichum gloeosporioides from rubber (Hevea brasiliensis). Mycological Research 95: 1085-1089. Sexton, A . G . , and Howlett, B.J. 2001. Green fluorescent protein as a reporter in the Brassica-Leptosphaeria maculans interaction. Physiological and Molecular Plant Pathology 58: 13-21. Shamoun, S.F., Ramsfield, T.D., Shrimpton, G., and Hintz, W.E. 1996. Development of Chondrostereum purpureum as a mycoherbicide for red alder (Alnus rubra) in utility-rights-of-way. In Proceedings of the 1996 Expert Committee on weeds national meeting, Victoria, British Columbia, December 9 - 1 2 , 1 9 9 6 . Edited by P. Comeau and G. Harper, B.C. Ministry of Forests, Research Branch, Victoria, B.C., Canada, p. 199. Shamoun, S.F., Countess, R.E., Vogelgsang, S., and Oleskevich, C. 2000. The mycobiota of salal (Gaultheria shallon) collected on Vancouver Island and the exploitation of fungal pathogens for biological control. Canadian Journal of Plant Pathology 22: 192. Shaw, C.G. Ill, and Loopstra, E . M . 1991. Development of dwarf mistletoe infections on inoculated western hemlock trees in southeast Alaska. Northwest Science 65: 48-52. Shen, S., Goodwin, P.H., and Hsiang, T. 2001. Infection of Nicotiana species by the anthracnose fungus, Colletotrichum orbiculare. European Journal of Plant Pathology 107: 767-773. Smith, R.B. 1977. Overstory spread and intensification of hemlock dwarf mistletoe. 153  Canadian Journal of Forest Research 7: 632-640. Smith, R.B., and Wass, E.F. 1979. Infection trials with three dwarf mistletoe species within and beyond their known ranges in British Columbia. Canadian Journal of Plant Pathology 1: 47-57. Smith, R.B., Wass, E.F., and Meagher, M.D. 1993. Evidence of resistance to hemlock dwarf mistletoe (Arceuthobium tsugense) in western hemlock (Tsuga heterophylla) clones. European Journal of Forest Pathology 23: 163-170. Sproule, A . 1996. Branch age in jack pine at the time of dwarf mistletoe infection. The Forestry Chronicle 72: 307. Srivastava, L . M . , and Essau, K . 1961. Relation of dwarf mistletoe (Arceuthobium) to the xylem tissue of conifers. I. Anatomy of parasite sinkers and their connection with host xylem. American Journal of Botany 48: 159-167. Steen, O.A., and Coupe, R.A. 1997. Field guide to forest site identification and interpretation for the Cariboo Forest Region. British Columbia Ministry of Forests, Victoira, B.C., Land Management Handbook 39. Stojanovic, S. 1989. Proudavanje Sphaeropsis visci (Sallm) Sacc. I Colletotrichum gloeosporioides (Sacc.) Penz. - parazita bele imele. Zastita bilja 40 (4), br. 190: 493-503. Sutton, B.C. 1980. The Coelomycetes. Fungi Imperfecti with Pycnidia, Acervuli and Stromata. Commonwealth Mycological Institute, Kew, U K . 696p. Templeton, G.E. 1982. Biological herbicides: Discover, development, deployment. Weed Science 30: 430-433. Templeton, G.E. 1992. Use of Colletotrichum strains as mycoherbicides. In Colletotrichum Biology, Pathology and Control. Edited by J.A. Bailey and M.J. Jeger. C.A.B. International. Wallingford, Oxon, U K . p. 358-380. Trujillo, E.E., Norman, D.J., and Killgore, E . M . 1994. Septoria leaf spot, a potential biological control for banana poka vine in forests of Hawaii. Plant Disease 78: 883-885. van der Kamp, B.J., and Blenis, P.V. 1996. A model of hyperparasite regulation of the gall rust - lodgepole pine pathosystem. Canadian Journal of Forest Research 26: 1256-1265. Van Sickle, G.A., and Wegwitz, E. 1978. Silvicultural control of dwarf mistletoe in young lodgepole pine stands in Alberta and British Columbia. Environment Canada. Forestry Service. Pacific Forest Research Centre Information Report 154  BC-X-180. l i p . Wall, R.E. 1991. Pathological effects of Chondrostereum purpureum in inoculated yellow birch and beech. Canadian Journal of Plant Pathology 13: 81 -87. Walker, H.L., and Connick, W.J. Jr. 1983. Sodium alginate for production and formulation of mycoherbicides. Weed Science 31: 333-338. Wastie, R.L. 1972. Secondary leaf fall of Hevea brasiliensis: meteorological and other factors affecting infection by Colletotrichum gloeosporioides. Annals of Applied Biology 72: 283-293. Watson, A . K . , and Wall, R.E. 1995. Mycoherbicides: Their role in vegetation management in Canadian forests. In Recent progress in forest biotechnology in Canada, Edited by Charest, P.J., and Duchesne, L.C. Petawawa National Forestry Institute, Canadian Forest Service, Information Report PI-X-120. pp. 74-82. Watson, A . K . , Gressel, J., Sharon, A., and Dinoor, A . 2000. Colletotrichum strains for weed control. In Colletotrichum host specificity, pathology and host-pathogen interaction. Edited by D. Prusky, S. Freeman, M . B . Dickman. A P S Press, St. Paul, Minnesota, pp. 245-265. Wicker, E.F. 1967. Appraisal of biological control of Arceuthobium campylopodum f. campylopodum by Colletotrichum gloeosporioides. Plant Disease Reporter 51: 311-313. Wicker, E.F., and Shaw, C.G. 1968. Fungal parasites of dwarf mistletoes. Mycologia 60: 372-383. Weir, J.R. 1915. Wallrothiella arceuthobii. Journal of Agricultural Research 4: 369378. Wilcox, W.W., Pong, W.Y., and Parmeter, J.R. 1973. Effects of mistletoe and other defects on lumber quality in white fir. Wood and Fiber 4: 272-277. Wilson, C L . 1969. Use of plant pathogens in weed control. Annual Review of Phytopathology 7: 411-434. Winder, R.S., and Watson, A . K . 1994. A potential microbial control for fireweed (Epilobium angustifolium). Phytoprotection 75: 19-33. Wood, C. 1986. Distribution maps of common tree diseases in British Columbia. Pacific Forestry Centre, Victoria, B C . Information Report BC-X-281. 68p. Yang, X . , Madden, L . V . , Wilson, L.L., and Ellis, M . A . 1990. Effects of surface topography and rain intensity on splash dispersal of Colletotrichum acutatum. 155  Phytopathology 80: 1115-1120.  156  Appendix 1 - Temperature on inoculation day (July 23, 2000) and immediately following. 4035-  Inoculation  30 i  o 0) l_ =1 rt  <u  2520"  E »• •= ™5 •  07/17 2000  — i  07/19  1 07/21  1  1 07/23  07/25  Date  1 07/27  1 07/29  1— 07/31  Figure appendix 1-1. Temperature on inoculation day and following. Near Lytton, British Columbia. Maximum temperature on inoculation day was 22°C at 3:19 PM. 403530-  o 15  25-  20-  E  0 07/30  2000  1 08/01  1 08/03  1 08/05  Date  08/07  08/09  Figure appendix 1-2. Temperature at inoculation site from July 30 to August 9,2001. Near Lytton, British Columbia. 157  Appendix 2 - Relative humidity on inoculation day and immediately following 110100-  9080-  H  70H  E  H  =>  B0  0)  50 H  JS  40-  a: 30201007/17 2000  07/19  07/21  07/23  07/25  Date  07/27  07/29  07/31  Figure appendix 2-1. Relative humidity on inoculation day and immediately following. Minimum relative humidity at inoculation was 46.6% at 3:19 P M . Near Lytton, British Columbia.  07/30 2000  08/01  i  08/03  r  Date  08/05  08/07  08/09  Figure appendix 2-2. Relative humidity at inoculation site from July 30 to August 9, 2001. Near Lytton, British Columbia. 158  Appendix 3 - Histological staining methods Safranin - Picro=Analine blue (Schneider, 1981)  Hemo de (FisherBrand, Pittsburgh, PA)  15 min  Hemo de  5 min  Absolute ethanol / Hemo de  2 min  Absolute ethanol  2 min  Celloidin  Dip  95% ethanol  2 min  70% ethanol  2 min  50% ethanol  2 min  30% ethanol  2 min  18% ethanol  2 min  Double distilled H 0  5 min  1% Safranin  1 min  Tap H 0  1 + min  Picro+Aniline Blue  Simmer  18% ethanol  2 min  30% ethanol  2 min  50% ethanol  2 min  70% ethanol  2 min  95% ethanol  2 min  Absolute ethanol  2 min  Absolute ethanol / Hemo de  2 min  Clearing Solution "  5 min  Hemo de  5 min  Hemode  15 min  2  2  1  Permount and cover slip  Clearing solution = 50% clove oil, 25% absolute ethanol, 25% xylene 159  Johansen's Safranin (Johansen, 1940)  Hemode  15 min  Hemo de  5 min  Absolute ethanol / Hemo de  2 min  Absolute ethanol  2 min  Celloidin  Dip  95% ethanol  2 min  70% ethanol  2 min  50% ethanol  2 min  Johansen's Safranin in 95% ethanol  20 min  50% ethanol with trace HC1  10 sec  Picro=Analine Blue in 95% ethanol  20 min  100% ethanol  10 sec  Clearing solution  5 min  Hemo de  5 min  Hemode  15 min  Permount and cover slip  160  Rhodamine B - Methyl Green (Modified from Pearce, 1984)  Hemode  15 min  Hemo de  5 min  Absolute ethanol / Hemo de  2 min  Absolute ethanol  2 min  Celloidin  Dip  95% ethanol  2 min  70%) ethanol  2 min  50%) ethanol  2 min  30%) ethanol  2 min  18% ethanol  2 min  Double distilled H2O  5 min  1% Rhodamine-B  20 min  Tap H2O  Rinse  0.15% Methyl Green in 0.2M  5 min  Phosphate Buffer pH 8.0 Destain*  5 min  Clearing Solution  5 min  Hemo de  5 min  Hemode  15 min  Permount and cover slip  *Destain: 40% d H 0 , 50% methanol, 10% acetic acid 2  161  Appendix 4 - ANOVA tables for canopy study, Chapter 6 Table appendix 4-1. Two-way analysis of variance to compare trees and canopy thirds. Source  DF  SS  MS  F  P  Tree  5  548.206  109.641  0.233  0.939  Canopy third  2  882.243  441.122  0.938  0.423  Residual  10  4701.338  470.134  Total  17  6131.787  360.693  Table appendix 4-2. Two-way analysis of variance to compare trees and canopy aspect. Source  DF  SS  MS  F  P  Tree  5  728.977  145.795  0.763  0.591  Canopy aspect  3  97.945  32.648  0.171  0.914  Residual  15  2867.849  191.190  Total  23  3694.771  160.642  162  Appendix 5 - Diagrammatic representation of the lifecycle of Caliciopsis arceuthobii and fruit production by Arceuthobium americanum The diagrams below represent the lifecycle of C. arceuthobii and A. americanum and can be utilized to aid in the understanding of how individual cohorts of C. arceuthobii perithecia and A. americanum fruit are related in time. Data collection represented by an " A " . Caliciopsis perithecia are visible all year. The C. arceuthobii diagram was constructed based on the life cycle information in Kuijt (1969) and the A. americanum diagram was based on information presented in Hawksworth and Wiens (1996). A complete circuit around the diagram represents one year.  January February  December  November  March  October  April Ascospores releases infection initiation  2001  May  September  Empty perithecia visible  Immature perithecia present  August A = assessment date  Figure appendix 5-1. Timing of ascospore release and new perithecia production by Caliciopsis arceuthobii infecting Arceuthobium americanum. 163  January December  February  November  March  October Seed dispersal \2001 2002  September  August A = assessment date  July  June  Figure appendix 5-2. Fruit production on Arceuthobium americanum.  164  Appendix 6 - Presence of  Caliciopsis arceuthobii on Arceuthobi americanum  Table appendix 6-1. Number of Caliciopsis arceuthobii infected Arceuthobium americanum flowers observed at the Knife Creek Block of the Alex Fraser Research Forest, near 150 Mile House, British Columbia.  Replicate  Number of flowers with C. arceuthobii perithecia 1998  1999  2000  2001  1  8  0  0  17  2  5  35  35  3  3  17  5  0  2  4  1  0  0  4  5  32  0  0  2  6  0  0  0  Dead  7  0  0  0  1  8  0  39  0  0  9  4  Dead  Dead  Dead  10  0  0  Dead  Dead  11  0  0  0  0  12  180  103  149  0  13  27  30  11  27  14  125  Dead  Dead  Dead  15  36  Dead  Dead  Dead  16  39  206  22  23  17  400  0  0  44  18  80  Dead  Dead  Dead  19  169  0  0*  0  20  250  63  4  5  21  0  Dead  Dead  Dead  165  Replicate  Number of flowers with C. arceuthobii perithecia 1998  1999  2000  2001  22  150  0  63  111  23  0  2  8  Dead  24  28  1  56  12  25  154  Dead  Dead  Dead  26  300  56  423  73  27  100  12  0  3  28  51  168  0  2  29  87  Dead  Dead  Dead  30  0  0  0*  0*  * Indicates that there were no shoots present on the A. americanum infection. These replicates were not included in the Fisher exact test calculations.  166  

Cite

Citation Scheme:

        

Citations by CSL (citeproc-js)

Usage Statistics

Share

Embed

Customize your widget with the following options, then copy and paste the code below into the HTML of your page to embed this item in your website.
                        
                            <div id="ubcOpenCollectionsWidgetDisplay">
                            <script id="ubcOpenCollectionsWidget"
                            src="{[{embed.src}]}"
                            data-item="{[{embed.item}]}"
                            data-collection="{[{embed.collection}]}"
                            data-metadata="{[{embed.showMetadata}]}"
                            data-width="{[{embed.width}]}"
                            async >
                            </script>
                            </div>
                        
                    
IIIF logo Our image viewer uses the IIIF 2.0 standard. To load this item in other compatible viewers, use this url:
http://iiif.library.ubc.ca/presentation/dsp.831.1-0090452/manifest

Comment

Related Items