Open Collections

UBC Theses and Dissertations

UBC Theses Logo

UBC Theses and Dissertations

A Microbiological control of Melampsora medusae Thum. rust on Psuedotsuga menziesii (Mirb.) Franco seedlings McBride, Richard Phillips 1965

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

Item Metadata

Download

Media
831-UBC_1965_A6_7 M15.pdf [ 2.44MB ]
Metadata
JSON: 831-1.0104758.json
JSON-LD: 831-1.0104758-ld.json
RDF/XML (Pretty): 831-1.0104758-rdf.xml
RDF/JSON: 831-1.0104758-rdf.json
Turtle: 831-1.0104758-turtle.txt
N-Triples: 831-1.0104758-rdf-ntriples.txt
Original Record: 831-1.0104758-source.json
Full Text
831-1.0104758-fulltext.txt
Citation
831-1.0104758.ris

Full Text

A MICROBIOLOGICAL CONTROL OF MELAMPSORA MEDUSAE THUM. RUST ON PSEUDOTSUGA MENZIESII(Mirb.)Franco SEEDLINGS by RICHARD P. McBRIDE B . S c , University of B r i t i s h Columbia, 1964  A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in the Department of Bio]ogy and Botany  We accept this thesis as conforming to required standard  THE UNIVERSITY OF BRITISH COLUMBIA October, 1965  the  In p r e s e n t i n g t h i s  fulfilment  of  the requirements f o r an advanced degree at the U n i v e r s i t y  of  British  Columbia,  available mission  for  thesis  I agree that  in p a r t i a l  the L i b r a r y s h a l l  r e f e r e n c e and study.  make i t  I f u r t h e r agree that  f o r e x t e n s i v e copying o f t h i s  thesis  for  freely per-  scholarly  purposes may be granted by the Head o f my Department o r by his  representatives,,  cation of this  thesis  It  i s understood that copying o r p u b l i -  for financial  without my w r i t t e n  permission.  Department o f  < p f  The U n i v e r s i t y o f B r i t i s h Vancouver 8, Canada Date  /''  7  (  y  Columbia  gain s h a l l  not be allowed  ABSTRACT Saprophytic fungi and bacteria were recovered from healthy foliage of Douglas f i r (Pseudotsuga menziesii (Mirb.) Franco). The populations of microorganisms on foliage were variable and changed through the growing season. Three species of the bacteria isolated from Douglas f i r foliage were used in attempts to control the development of the rust, Melampsora medusae Thum.. on Douglas f i r seedlings in the greenhouse.  Effective  control was obtained when Bac? 1 lus cereus Frankland and Frankland and B_. mvcoides Flugge were applied to the seedlings in pure cultures of nutrient broth.  The most effective control was obtained from the application  of a mixture of these two species and a third unidentified species of Baci1lus in nutrient broth.  Less effective control resulted from the ap-  plication of bacteria suspended in water and c e l l free f i l t r a t e s of the bacterial cultures. The numbers of bacteria on the foliage of Douglas f i r were estimated using a modification of the s o i l dilution and bacterial plate count technique.  The bacterial populations on foliage of Douglas f i r in the  greenhouse and in the f i e l d were increased by the application of bacteria in nutrient broth and s t e r i l e nutrient broth.  • • • I II  CONTENTS Page INTRODUCTION  I  LITERATURE REVIEW  2  Leaf microfloras  2  Biological control of disease using foliage saprophytes  3  METHODS 1. Recovery of leaf microfloras 2.  6  The pathogen: c o l l e c t i o n , storage and germination of teliospores  6  3. The host  7  h.  7  The disease  5. Preparation of treatments 6. Estimation of bacterial populations  9 11  RESULTS Observations on foliage microfloras of Douglas f i r  13  Inoculation experiments  13  Field study  23  DISCUSSION  27  SUMMARY  31  LITERATURE CITED  32  APPENDIX  35  TABLES  The effect of water, nutrient broth, c e l l free f i l t r a t e and bacterial treatments on the development of Mel ampsora medusae on the foliage of Douglas f i r seedlings The effect of nutrient broth, c e l l free f i l t r a t e , and bacterial treatments on the development of Melampsora medusae and the number of bacteria on the foliage of Douglas f i r seedlings The effect of pure cultures in nutrient broth of Bacil lus cereus. B_. mycoides. Baci 1 lus sp., and of a mixture of these bacteria on the development of Melampsora medusae on the foliage of Douglas f i r seedl ings ,  FIGURES Page Figure 1.  Pycnial droplets of Melampsora medusae on a Douglas f i r seedl ing  8  Figure 2.  Aecia of Melampsora medusae on a Douglas f i r seedling  10  Figure 3.  The method of inoculations. From left to right: aspen leaves bearing germinating teliospores placed on a wire screen above the pot of seedlings; moist cotton placed over the aspen leaves; polyethylene bag placed over the pot  10  A comparison of the percent seedlings diseased in the f i r s t inoculation experiment with Melampsora medusae  16  A comparison of the percent seedlings diseased in the second inoculation experiment with Melampsora medusae  19  A comparison of the effects of pure cultures of three species of bacteria and a mixture of the cultures on the development of Melampsora medusae  2k  The effect of bacterial and water treatments on the number of bacteria recovered from Douglas f i r foliage in the f i e l d  26  Figure 4.  Figure 5.  Figure 6.  Figure 7.  vi  ACKNOWLEDGEMENTS Grateful acknowledgement is given to the National Research Council, Ottawa, for providing a Bursary in 1964-65 in support of this study. The writer expresses his appreciation to Dr. J . E . Bier, Professor of Forest Pathology, Department of Botany, University of B r i t i s h Columbia, for his guidance and encouragement throughout this study, and to Dr. J . Basaraba of Acadia University, Nova Scota, for the identification of two bacterial isolates.  Miss P. Ackland and Miss G.D. Pentland are kindly  thanked for their helpful suggestions and advice in writing this thesis.  1.  INTRODUCTION Frequently immature plant tissues are highly susceptible to paras i t i c microorganisms that are unable to infect more mature tissues of the same plant (9).  Presumably this susceptibility could be eliminated only  by chemical control methods because natural resistance was thought to depend on the development of anatomical and physiological defenses.  Bier  (2,3,4,5) and others (6,7,15,16,21,22,23,24) have shown that aerial portions of plants are complex biological communities comprised of numerous saprophytic bacteria and fungi as well as the host plant tissues.  Further-  more, Bier (2,3,4,5) has demonstrated that the interactional phenomena related to these saprophytic microorganisms may provide a natural form of defense against infection and/or disease development.  It has been report-  ed by Ruinen (24) and Last (15) that immature leaves had considerably lower populations of saprophytes than mature leaves.  Thus, i f the relatively  low microorganism populations of immature tissues could be supplemented by the application of saprophytes, perhaps natural resistance could be improved.  The present study was undertaken to investigate this concept in regard  to the development of the rust, Melampsora medusae Thum., on the immature foliage of Douglas f i r (Pseudotsuga menziesii (Mirb.) Franco). The aecial stage of M. medusae occurs in May and June on immature needles of Douglas f i r .  The uredial and t e l i a l stages develop on leaves  of trembling aspen (Populus tremuloides Michx.) from June until autumn defoliation.  Molnar and Sivak (18) have investigated the l i f e history of the  rust pathogen.  Z i l l e r (29,30) has presented detailed studies on the host  range and nomenclature of the rust fungus.  2  LITERATURE REVIEW Leaf microfloras The existence of non-paras i t i c microfloras on aerial plant structures has been known since the studies of Pasteur (21,22).  These studies  were concerned with the distribution of yeasts on fruits and suggested that physiological changes of the ripening fruit influenced the number of yeast microorganisms occurring on its surface.  Burri (6) and Duggeli (7)  isolated bacteria from the leaves of a wide range of plant species and established the fact that bacterial microfloras were ubiquitous on plant h  leaves.  q  Their bacterial counts varied from 10 to 10- bacteria per gram 7  fresh weight of leaf. Last (15), after studying the seasonal incidence of the yeast, Sporobolomyces. on wheat and barley leaves, concluded that the two main factors influencing the numbers of yeast c e l l s were the age of the leaf and atmospheric humidity. The results of his investigation showed that the yeast population remained low through the f i r s t half of the leaf's l i f e and then increased rapidly. Ruinen (24) studied the populations of microorganisms on leaves in Indonesia and Surinam. She found distinct differences in the species composition of the microfloras on (a) leaves of different plant species; (b) leaves of different ages; (c) leaves at different heights; and (d) leaves with different exposures to rain and to sunlight.  Furthermore, the results  of her studies indicated a definite succession in the populations during growth and senescence of the leaf.  The succession began with bacteria,  some of which were present on even the youngest leaves.  After this juven-  3.  i l e stage the most rapid increase in the population began.  This increase  coincided with the most active period of photosynthesis and transpiration of the host and resulted in a mixture of filamentous fungi, yeasts, and bacteria.  The bacteria at this time were the major component.  The decline  of leaf a c t i v i t y correlated with a change to a predominantly fungal microflora. The succession in the microflora apparently reflects a change in the quantity and quality of nutrients on the leaf surface.  Last (15)  found that the populations of Sporobolomyces were significantly higher on f e r t i l i z e d plants than on unfertilized plants.  Tukey et al_. (25) reported  that large quantities of nutrients were leached out of the leaf into water films on the leaf surface, with more nutrients being leached from f e r t i l ized plants than from unfertilized plants.  The quantities lost in this  manner increased when the leaf's a c t i v i t y began to decline at the start of leaf senescence.  Thus, the amount of nutrients on the leaf and size  of the microflora appear to be closely connected with the physiological condition of the host plant. Biological control of disease using foliage saprophytes The p o s s i b i l i t y that foliage saprophytes influence the development of parasitic microorganisms on plants has received only scant attention from plant pathologists.  In 1910, when discussing epiphyllous bacteria,  Potter (23) raised the question, "Are these bacteria at a l l concerned in the problem of immunity?"  F i f t y - f i v e years later, in a review of the  research on non-parasitic leaf microfloras, Last and Deighton (16) ask, "Do saprophytes play an important part in controlling the incidence of disease?"  As the s i m i l a r i t y of the two questions suggests, the knowledge  k  of this aspect of plant pathology has not developed rapidly! The fact that certain leaf saprophytes produce antifungal substances in v i t r o has been demonstrated by many workers (8,13,14, and others). These studies on a r t i f i c i a l media at constant humidity and temperature reveal l i t t l e about the natural interactions between saprophytes.  Only  in a limited number of cases has the effect of saprophytes on parasites been studied on host material. Bamberg (1) used saprophytes in an attempt to control the infection of corn plants by the smut, Ustilago zeae.  Bacteria were isolated from  corn plants which had failed to become infected after inoculation with virulent strains of the smut.  Bamberg (1) and Johnson (11) showed that  some of the bacteria were antagonistic to Ustilago zeae in a r t i f i c i a l culture.  The antagonistic bacteria when applied to the corn plants s i g -  nificantly reduced infection by the smut.  The reduced infection was evid-  ent when the bacteria were applied 3 days before, simultaneously with, or 3 days after the smut inoculum.  Bamberg (1) also demonstrated that c e l l  free f i l t r a t e s of the bacterial cultures were ineffective in controlling the disease. Wood (27,28) and Newhook (19,20) studied the antagonism of some s o i l microorganisms toward Botrytis cinerea Pers. on lettuce leaves.  Their  studies were initiated after i t was observed that lettuce seedlings grown in s o i l depressions were resistant to the disease.  It was suggested that  this resistance was a result of rapid colonization of the plants in the depressions by s o i l saprophytes.  Several bacteria, actinomycetes and fungi  antagonistic to B. cinerea were isolated from the s o i l .  Control of the  disease was achieved when the lettuce leaves were inoculated with the sapro-  5.  phytes before or simultaneously with B_. cinerea.  The best control was ob-  tained by spraying the plants with suspensions of the antagonistic saprophytes in a 1% glucose solution. The effect of bark and leaf microfloras on certain tree pathogens has been studied by Bier (2,3,4,5). The results of his work have indicated the protective value of the natural microflora.  The application of micro-  flora suspensions to watered greenwood cuttings of black cottonwood and willow prevented the development of Hypoxylon canker.  The microflora sus-  pensions were not effective in preventing canker development when the bark turgor of the cuttings was lowered. When the natural microflora was reduced by surface-sterilizing the cuttings, cankers developed at a l l turgor levels subsequent to inoculation with the pathogen. Bier (3) has also studied the microfloras of poplar leaves and their effect on the development of the rust, Melampsora occidental is Jacks, Microflora suspensions were found to inhibit germination of uredospores in vitro.  They also prevented the development of the rust on leaves of poplar  cuttings growing in the greenhouse.  When microflora suspensions were applied  to poplar leaves in the f i e l d the development of the rust was significantly reduced.  Furthermore, Bier (3) showed that the microflora composition of  poplar leaves was altered for several months following the application of microflora suspensions.  6  METHODS 1.  Recovery of leaf microfloras The healthy foliage of Douglas f i r to be sampled for microorganisms  was collected in the f i e l d , placed in s t e r i l e test tubes, and taken immediately to the laboratory.  Five grams of foliage were placed in a ster-  i l e metal-capped flask containing 100 ml. of s t e r i l e d i s t i l l e d water and shaken on a reciprocating shaker for s i x hours.  Two types of agar media  were used to culture the microorganisms, 5% malt extract and 2% agar, and 0.8% nutrient broth and 2% agar.  Ten ml. portions of the microflora sus-  pensions were added to cool agar media (44.5*C.) which were poured into petri dishes.  Filamentous fungi, yeasts and a few species of bacteria  were recovered in the malt extract medium. Bacteria were the only organisms recovered in the nutrient broth medium. 2.  The pathogen: c o l l e c t i o n , storage and germination of teliospores Leaves bearing t e l i a were collected in May of 1964 and 1965 from  the forest floor beneath aspen groves that had been heavily infected with M. medusae the summer prior to the c o l l e c t i o n s , Teliospores from two locations, Botanie Valley near Lytton, B.C. and Williams Lake, B . C . , were used in inoculation experiments.  The infected leaves were air-dried and stored  in kraft paper bags at room temperature until required for inoculations. Teliospore germination was induced by soaking the aspen leaves for one hour in d i s t i l l e d water at room temperature.  The leaves were then  placed in moist petri dishes in constant temperature chambers until the teliospores germinated.  Teliospore germination was tested at temperatures  7.  from 5*C. to 35°C.  The optimum temperature for germination was 10*C. for  the Botanie Valley material and 20*C. for the Williams Lake material. The distinct colour change from the black of the teliospores to the golden brown of the promycelia and basidiospores f a c i l i t a t e d a determination of the amount of germination. Unlike the teliospores used by Molnar and Sivak (18), those used in this study retained their capacity to germinate for only a few months. Even when the spores had been stored at 0*C. the germination was considered to be too low by the end of September to be used in inoculation studies. The period required for the teliospores to germinate at the optimum temperature increased from ten hours in June to 48 hours in September.  The increas-  ing period for germination was accompanied by a decrease in the amount of germination. 3.  The host The Douglas f i r seedlings for the inoculation experiments were grown  in 5 1/2" diameter pots from seed collected in 1959 at Duncan, B.C. The seed was s t r a t i f i e d before planting to obtain even germination and uniformly aged seedlings. to the rust. fected.  One month old seedlings proved to be the most susceptible  At this age both the cotyledons and the epicotyl became i n -  The rust was never observed to develop on foliage over five weeks  old. 4.  The disease Inoculation of Douglas f i r seedlings with basidiospores was carried  out using the technique described by Z i l l e r (30).  This technique is i l l u s -  trated in Figure 1 and consisted of the following steps:  Figure 1.  The method of inoculation: from left to right; aspen leaves bearing germinating teliospores placed on wire screens above the pot of seedlings, moist cotton placed on the aspen leaves, polyethylene bag placed over the pot.  9  (f) nets on wire frames were placed above the pots of Douglas f i r seedlings, ( i i ) the aspen leaves with germinating teliospores were placed ( t e l i a facing down) on the nets and moist absorbent cotton was placed over the leaves, ( i i i ) a s t e r i l e hand atomizer was employed to spray the seedlings with s t e r i l e d i s t i l l e d water, (iv) polyethylene bags were placed over the pots, (v) after four days of inoculation the leaves, nets, cotton and bags were removed. The intensity of disease was assessed by the number of pycnia which developed on the host.  The pycnia (Fig. 2) could be counted easily because  the pycnial droplets remained distinct even in heavy infections.  The aeeia  (Fig. 3) were more d i f f i c u l t to count because in heavy infections the i n dividual aecia did not remain distinct but merged with adjacent aecia. Frequently, mortality occurred in heavily infected seedlings before aecia were able to develop.  The number of aecia may be a less direct measure of  infection and disease development than pycnia because f e r t i l i z a t i o n must take place  before their development.  For these reasons the number of  pycnia was considered to be the most useful c r i t e r i o n of disease intensity. The number of pycnia was assessed in two ways; the percentage of seedlings on which pycnia developed and the average number of pycnia per diseased seedling calculated when the total number of pycnia was at a maximum. 5.  Preparation of treatments The bacteria were grown in liquid cultures of 0.8% Difco Nutrient  Broth for a period of s i x days on a reciprocating shaker at room tempera-  F i g u r e 3.  A e c i a of Melampsora medusae on a Douglas f i r 1 ing.  seed-  11.  ture.  The cell free controls were prepared by filtering one half of the  bacterial suspension through a Millipore f i l t e r with 0.45H pore size. When more than one species were applied in a treatment the bacteria were cultured individually in nutrient broth and mixed immediately prior to Millipore filtering and/or application to the seedlings. In experiments where bacteria were applied as water suspensions, the bacteria were cultured as described above. The cultures were then centrifuged at approximately 3000 r.p.m.  When the bacteria had precipit-  ated ,the nutrient solution was decanted and the bacteria resuspended in sterile d i s t i l l e d water. This water suspension was then centrifuged and the procedure repeated three times.  The final suspension was applied to  the seedlings. A small sterilized atomizer was employed to apply the material in all experiments. seedling.  Approximately five ml. of material were applied per  The seedlings treated with suspensions were incubated for 2k  hours before inoculation with rust spores was started;'. Freeze-dried samples of the three bacteria (Bacillus cereus Frankland and Frankland, B,. mycoides Flugge, and Baci1lus sp.) used in the inoculation experiments are deposited at the Forest Pathology Laboratory, U.B.C.  B_. cereus and JB. mycoides were  identified by Dr. J . Basaraba of Acadia University, Nova Scotia.  The Baci1 -  lus sp-. was tentatively identified by the author as a strain of B_. megaterium de Bary. 6.  Estimation of bacterial populations Field Experiment The bacterial populations of foliage of Douglas f i r trees were estim-  ated using a modification of the soil dilution technique (12). To ensure  12.  that samples contained approximately the same total surface area of f o l iage,on1y the d i s t a l 1.5 cm. of the needles were used.  Thirty such needle  portions were placed in a s t e r i l e test tube containing 10 ml. s t e r i l e d i s t i l l e d water that was shaken for 15 minutes on a wrist action shaker. The bacterial population of the water was then estimated using the s o i l dilution and plate count method (12). medium used in the plate counts.  Difco nutrient agar was the culture  This medium was selective for bacteria  and therefore the fungal components of the microflora were not studied in this experiment.  The foliage samples were always taken at 8:00 a.m.  Greenhouse Experiments The experimental procedures were the same as those used for the f i e l d experiment except for the size of the foliage sample.  Because of  the uniform size of the needles and the small amount of foliage on the seedlings, five whole needles were used in each sample . 1  The foliage sampl-  ing was made 48 hours after the nets, aspen leaves, cotton and bags used in the inoculations were removed.  13  RESULTS Observations on foliage microfloras of Douglas f i r The foliage of several Douglas f i r trees on the University of B.C. Endowment Lands was sampled to determine the types of microorganisms present'.  The foliage sampled ranged from three year old needles to those en-  closed by the bud scales.  Microorganisms were recovered from a l l samples.  The microorganisms most frequently isolated were: fungi:  Aureobasidium pullulans (de Bary) Arnaud, Epicoccum sp. and an unidentified pink yeast  bacteria:  Bac i i lus cereus. ]J. mycoides. Bac i 1 lus sp. and an unidentified yellow pigmented bacterium.  The microfloras of Douglas f i r needles were found to vary widely in the numbers and the types of microorganisms. After sampling over an entire growing season the following trends became evident: 1.  Young needles (1-2 months old) appeared to have microfloras more  restricted in numbers and species, and bacteria appeared to form the major portion of these microfloras. 2,  Mixtures of fungi and bacteria were present on the older needles  (3 months to 3 years old) and there appeared to be an increase in the incidence of fungi compared to the incidence of bacteria as the age of the needles increased. Inoculation experiments The bacteria, Bacil lus cereus. B_. mycoides and Bac? 11 us sp., were chosen for use in the biological control experiments because they appeared  14.  to be among the f i r s t colonizers of immature Douglas f i r foliage. The f i r s t inoculation experiment included the following treatments: (i) s t e r i l e d i s t i l l e d water, ( i i ) Bacillus cereus. B_. mycoides and Bacillus sp. in s t e r i l e d i s t i l l e d water, ( i i i ) s t e r i l e nutrient broth, (iv) Baci1lus cereus. B. mycoides and Bacillus sp. in nutrient broth, (v) a c e l l free f i l t r a t e of ( i v ) . Teliospores from the Williams Lake collection of aspen leaves were used for the inoculation. Four pots, each containing 18, two-month-old Douglas f i r seedlings, were used in each experiment. The results of the f i r s t inoculation experiment are summarized in Table 1.  Compared to the s t e r i l e d i s t i l l e d water control in which 75% of  the seedlings became diseased, a l l treatments resulted in a significant reduction (at the  ,1% level of probability) in the percentage of seedlings  diseased (Figure 4 ) . The application of bacteria in nutrient broth resulted in the most effective control of the rust with only 1.4% of the seedlings becoming d i s eased.  This treatment also had the greatest effect on the number of bact-  eria recovered from seedling foliage, the number increasing to 400 times that recovered from foliage sprayed with s t e r i l e d i s t i l l e d water. The applications of bacteria in water and of s t e r i l e nutrient broth provided less effective control of the rust disease with respectively 36% and 39% of the seedlings becoming diseased.  Four times as many bacteria  were recovered from foliage of seedlings treated with bacteria in water than that treated with s t e r i l e d i s t i l l e d water.  The application of s t e r i l e  TABLE 1 The effect of water, nutrient broth, c e l l free f i l t r a t e and bacterial treatments on the development of Melampsora medusae on the foliage of Douglas f i r seedlings  No. of seedlings  Average no. of bacteria per needle seven days after treatment  % of seedlings d iseased  Average no. of pycnia per diseased seedling  S t e r i l e water  72  1,500  75.0  89.6  Bacteria in water  72  6,500  36.1  95.1  S t e r i l e nutrient broth  72  64,000  38.9  96.4  Cell free filtrate  72  60,000  30.5  71.0  Bacteria in nutrient broth  72  328,000  1.4  46.0  Treatment  Figure k,  A comparison of the percent seedlings diseased in the f i r s t inoculation experiment.  17.  nutrient broth increased the number of bacteria recovered from the foliage to kO times that recovered from foliage treated with sterile distilled water. The cell free f i l t r a t e was not as effective as bacteria in nutrient broth but was more effective than sterile nutrient broth in controlling the rust disease.  The effect of the cell free f i l t r a t e on the number of  bacteria recovered from the foliage was similar to that resulting from the application of sterile nutrient broth* The effect of the nutritional and bacterial elements on the development of M, medusae was examined in a second experiment in which teliospores on aspen leaves from Botanie Valley were used as inoculum.  Four pots, each  containing 16, one-month-old Douglas f i r seedlings, were used in each treatment.  The treatments were: (i) sterile nutrient broth, Bacillus cereus, B_. mycoides. and Bacillus sp. in nutrient broth,  ( i i i ) cell free f i l t r a t e of ( i i ) (Mi 11ipore fiItered). The results of this experiment are summarized in Table 2. As in the f i r s t experiment, application of bacteria in nutrient broth provided the most effective control of the rust disease with only 12.5% of the seedlings in that treatment becoming diseased compared to 70% in the sterile nutrient broth treatment and 58% in the cell free f i l t r a t e treatment (Figure 5). The effect of the treatments on the numbers of bacteria recovered from the foliage seven days after the treatments were applied was of the same magnitude as in the previous experiment (Table 2).  Approximately eight times  as many bacteria were recovered from foliage treated with bacteria in nutrient broth as from foliage treated with the cell free f i l t r a t e or  TABLE 2 The effect of nutrient broth, c e l l free f i l t r a t e , and bacterial treatments on the development of Melampsora medusae and the number of bacteria on the foliage of Douglas f i r seedlings  Treatment  No. of seedlings  Average no. of bacteria per needle seven days after treatment  % of seedlings diseased  Average no. of pycnia per diseased seedling  S t e r i l e nutrient broth  64  65,500  70.3  138.6  Bacteria in nutrient broth  64  508,000  12.5  31.5  Cell free filtrate  64  60,500  57.8  130.5  Figure 5*  A comparison of the percent seedlings diseased in the second inoculation experiment.  20.  s t e r i l e nutrient broth. As mentioned previously the bacterial treatments in the preceding experiments were mixtures of three species of bacteria, Bacillus cereus. B, mycoides and Bacillus sp.  It was considered of interest to determine  if each of the species would be effective In controlling the rust when applied individually.  Therefore, the species of bacteria were applied  individually as pure cultures in nutrient broth.  A mixture of the three  species of bacteria similar to the bacterial treatments of the previous experiments was also applied. The control treatment in each case was a c e l l free f i l t r a t e of the bacterial treatment.  Thus the following treat-  ments were applied: (i) Baci11 us cereus in nutrient broth, ( i i ) c e l l free f i l t r a t e of ( i ) , ( i i i ) Bacillus mycoides in nutrient broth, (iv) c e l l free f i l t r a t e of ( i i i ) , (v) Bac?1lus sp. in nutrient broth, (vi) c e l l free f i l t r a t e of (v), ( v i i ) a mixture of ( i ) , ( i i i ) and (v), ( v i i i ) c e l l free f i l t r a t e of ( v i i ) . Teliospores on aspen leaves from the Botanie Valley collection were used for the inoculum in this experiment.  Three pots, eachicontaining 12,  three-week-old Douglas f i r seedlings, were used for each treatment. The results of this experiment are summarized in Table 3 . Compared to their c e l l free f i l t r a t e s the Baci1lus mycoides treatment reduced the percentage of diseased seedlings from 94.4% to 55.5%, the Baci1lus cereus treatment from 97.2% to 27.8%, and mixture of bacteria treatment from  TABLE 3 The effect of pure cultures in nutrient broth of Bac? 1 lus cereus. B_. mycoides. Baci11 us sp., and of a mixture of these bacteria on the development of Melampsora medusae on the foliage of Douglas f i r seedlings  Treatment  No. of seedlings  Average no. of bacteria per needle seven days after treatment  % of seedlings d iseased  Average no. of pycnia per diseased seedling  Bac i11 us cereus in nutrient  36  416,000  27.8  11.2  Cell free f i l t r a t e of B_. cereus culture  36  25,000  97.2  140.4  Bacillus mycoides in nutrient  36  400,000  55.5  53.9  Cell free f i l t r a t e of B_. mycoides culture  36  33,000  94.4  129.8  Bac ?11 us sp, in nutrient  36  250,000  69.4  75.2  Cell free f i l t r a t e of Bac ?11 us sp. culture  36  32,000  91.7  326.4 - continued  TABLE 3, cont'd  Treatment  B. cereus, P' Bac?1lus sp. and B_. mycoides in nutrient  No. of seedlings  Average no. of bacteria per needle seven days after treatment  % of seedlings diseased  36  420,000  5.5  36  26,000  Average no. of pycnia per diseased seedling  15.5  Cell free f i I t r a t e of B_. cereus. B. mycoides and Baci11 us sp.  cultures  Sk.k  217.7  23.  94.4% to 5.5% (Fig. 6). There was no significant difference in the percentages of diseased seedlings within the four different c e l l free f i l t rate treatments ( i i , i v , v i , v i i i ) .  The application of the unidentified  species, Bac i1lus s p . . d i d not significantly reduce the percentage of diseased seedlings. As shown before the treatments which were most effective in cont r o l l i n g the rust were also most effective in increasing the number of bacteria recovered from the seedling foliage. A l l seedlings in the inoculation experiments were grown in the greenhouse for s i x months after inoculation. During this time there were no signs of damage resulting from any of the spray treatments. The analyses of variance of the results of the preceding experiments are tabulated in Appendix 1. Field study The numbers of bacteria recovered from the foliage of seedlings used in the inoculation experiments indicated that the high bacterial population resulting from the spray treatments persisted for at least four weeks under greenhouse conditions.  Field experiments for the control of  M. medusae were impossible at this time because no area near the University had a history of chronic M. medusae infection.  It was considered of inter-  est, however, to determine i f the microflora of immature Douglas f i r foliage could be altered under f i e l d conditions. Buds of 8?year-old Douglas f i r s growing in a nursery at the Univers i t y of B r i t i s h Columbia were used in the experiment.  Buds which broke  dormancy at the same time and which had similar exposure to sun and rain were chosen for the experiment.  Three treatments were applied:  24.  "1  F i g u r e 6.  The Bacteria in Nutrient  Broth  |  |  The R e s p e c t i v e c e l l Free Filtrate  A comparison o f t h e e f f e c t s o f p u r e c u l t u r e s on t h r e e s p e c i e s o f b a c t e r i a and a m i x t u r e o f t h e t h r e e s p e c i e s on t h e development o f Melampsora medusae.  (i) sterile d i s t i l l e d water, (i i) Bac? 1 lus cereus, B_. mycoides and Baci 11 us sp. in sterile dist i l l e d water, ( i i i ) Bacillus cereus. B_. mycoides and Bacillus sp. in nutrient broth. The buds were treated as soon as the needles had broken free from the bud scales.  The number of bacteria recovered from the needles was  determined oneday before the treatments were applied and one, two, four, seven, twelve and fifteen days after the treatments were applied. During the f i r s t five days temperatures were moderate and the skies were generally overcast.  From the sixth day until the termination of the  experiment on the 15th day there was an increase in daytime temperatures and the hours of sunshine. Figure 7.  The results of this experiment are given in  The application of bacteria in nutrient broth resulted in a  greatly increased bacterial population for k to 6 days.  The application  of bacteria in water caused a much smaller increase which lasted for only 2 to 3 days.  Coinciding with the change to warm sunny weather the bacter-  ial populations of needles of a l l treatments, including the water control, declined.  The influence of weather conditions appeared to have an import-  ant influence on the microflora populations.  Further field studies in an  area with a history of chronic M. medusae infection are required to determine if the weather conditions suitable for infection are also suitable for the maintenance of a large microflora.  Figure 7. The effect of water and bacterial treatments on the number of bacteria recovered from Douglas f i r foliage in the f i e l d . ls> ON  27.  DISCUSSION Healthy needles of Douglas f i r proved to be complex biological communities consisting of the plant tissues and microfloras of saprophytic bacteria and fungi.  Immature needles had considerably fewer microorganisms  in their microfloras than mature foliage.  Control of the rust, M. medusae,  was obtained when the microfloras of immature needles were supplemented by suspensions of bacteria isolated from Douglas f i r foliage.  Thus, one  factor in the natural susceptibility of immature foliage to this rust may be the lack of sufficiently developed microfloras. Because common s o i l organisms such as Baci1lus cereus and Aureobasidium pullulans occurred as part of foliage microfloras, a microflora may be p a r t i a l l y dependent for its formation on dust-borne inoculum from the s o i l .  Consequently, the microflora composition and level of rust  resistance may in part be dependent on factors related to s o i l microbiology. The microfloras of needles of the same age and from the same tree were found to be variable and, therefore, genetically identical host material may have varying degrees of resistance to disease.  Part of the varia-  tion of microfloras may be a result of microclimatic factors.  A recent  study (26) has correlated the incidence of white pine b l i s t e r rust with microclimatic conditions.  In considering the effect of the microclimate,  attention should be given to both the development of the host's microflora and the development of the pathogen. Attempts to alter the microfloras of the needles of Douglas f i r seedlings in the greenhouse indicated that nutrition was B n important factor influencing the number of bacteria in the microfloras.  When s t e r i l e  28.  nutrient broth was applied to the foliage the number of bacteria increased to approximately hO times the number of bacteria on untreated foliage. The application of bacteria suspended in s t e r i l e d i s t i l l e d water to foliage increased the number of bacteria to only four times that on untreated needles.  The greatest increase in the number of bacteria on foliage was  attained when bacteria were applied in nutrient broth suspension. Attempts to alter microfloras of Douglas f i r foliage under f i e l d conditions indicated the possible importance of the influence of weather on microflora, A decline of the numbers of bacteria recovered from both control and treated foliage coincided with a change to hot dry weather. The influence of weather on the development of microfloras may account in part for variation in the severity of disease in nature. Inoculation experiments were carried out on Douglas f i r seedlings in the greenhouse to determine i f bacteria from the natural microfloras of Douglas f i r foliage would be effective in controlling the disease caused by M. medusae.  Significant levels of rust control were achieved by indiv-  idual applications of s t e r i l e nutrient broth, bacteria in water, three species of bacteria in nutrient broth, and c e l l free f i l t r a t e s of three species of bacteria in nutrient broth.  Two species of bacteria, Bacillus  cereus and B_. mycoides provided significant control of the rust when applied individually as pure cultures in nutrient broth.  The most effective control  was attained when three species of bacteria, Baci 1 lus cereus. B_. mycoides and Bac?11 us sp. were applied as a mixture in nutrient broth.  The degree  of rust control attained by a l l treatments appeared to be related to the effect the treatment had on the total number of bacteria on the foliage. The treatments which resulted in the greatest increase in the number of  29  bacteria on the foliage provided the most effective control of the rust, and the treatments which resulted in a relatively low number of bacteria on the foliage did not provide effective control.  Therefore, the bacteria  appeared to be an active agent in the control of the rust.  However, the  manner in which the bacteria controlled the rust is not known. The cell free filtrates of bacteria in nutrient broth cultures were more effective than sterile nutrient broth in controlling the rust. some bacterial metabolite may  This indicated that  have been inhibitory to the pathogen.  Several species of the genus Bac?11 us are known to produce antibiotics (17),  It is possible that interactional phenomena of the bacteria on the  foliage resulted in an inhibition of basidiospore germination similar to the inhibition of germination of uredospores of Melampsora occidentialis Jacks by microfloral organisms as reported by Bier (5).  However, the  control could also have occurred later in the infection process or perhaps after infection had taken place. The three Bac ?11 us species used in the inoculation experiments appeared to be suitable for the biological control of M. medusae. They occurred frequently on immature foliage of Douglas f i r and therefore have demonstrated their ability to become established on susceptible tissues. Resistant endospores are produced by these bacteria thus enabling them to persist over periods of unfavorable environmental conditions.  The applica-  tion of bacteria to susceptible foliage resulted in rust control in the greenhouse.  Further studies are required to investigate the feasibility  of this control method under field conditions. The use of organisms from the natural microflora of the host t i s sues to achieve one form of biological control of a plant disease could  30.  have advantages over chemical control methods.  One advantage would be in  avoiding the use of toxic sprays which could destroy beneficial as well as pathogenic microorganisms.  Because the organisms used in the control  occur naturally on the host there are no foreign substances introduced as there are in chemical control methods.  There is also the possibility that  the microorganisms and, therefore, the protection would become self-perpetuating.  The results of the present study indicate that no damage is done  to the plant by the altered microflora, whereas, toxic chemical sprays often result in damage to young plant tissues (10),  31  SUMMARY  The needles of Douglas f i r were found to have natural microfloras of saprophytic fungi and bacteria.  As the foliage matured the microfloras  increased in numbers of organisms present and changed in species composition.  Attempts to increase the size of the microflora a r t i f i c i a l l y were  successful and indicated the importance of n u t r i t i o n , inoculum, and weather as factors controlling the microflora s i z e .  The microflora of immature  needles included saprophytic bacteria which were effective in controlling the rust, Melampsora medusae.  The most effective control was obtained  when three species of bacteria, Bacillus cereus. B_. mycoides and Baci 1 lus sp. were applied as a mixed suspension in nutrient broth.  Two species,  B,. cereus and B. mycoides provided effective control of the rust when applied as pure cultures in nutrient broth.  Cell free f i l t r a t e s of bacterial  cultures provided a small degree of rust control indicating the possible production of a bacterial metabolite which inhibited rust development. S t e r i l e nutrient broth treatments resulted in partial control of the rust. Apparently this control was due to the increased number of bacteria in the microflora which followed the application of additional n u t r i t i o n . None of the treatments had any harmful effect on the seedlings.  32.  LITERATURE CITED 1.  Bamberg, R.H. 1930. Bacteria antibiotic to Ust i laoo zeae. path. 21: 881-890.  Phyto-  2.  Bier, J . E . 1963. Tissue saprophytes and the p o s s i b i l i t y of biological control of some tree diseases. For. Chronicle 39 (1): 81-84.  3.  Bier, J . E . 1965. Some effects of foliage saprophytes in the control of Melampsora leaf rust on black cottonwood. For. Chronicle 41 (3): 306-313.  4.  Bier, J . E . and Rowat, M.H. 1962. The relation of bark moisture to the development of canker diseases caused by native, facultative parasites, 7. Some effects of the saprophytes on the bark of poplar and willow on the incidence of Hypoxylon canker. Can. J . Botany 40: 61-69.  5.  Bier, J . E . and Rowat, M.H. 1963. phytes on Hypoxylon canker.  6.  B u r r i , R. 1903. Die Bakterienvegetation auf der Oberflache normal entwickelter Pflanzen. Z b l . Bakt. (2 Abt.) 10: 756-763.  7.  Duggeli, M. 1904. Die Bakterienflora gesunder Samen und daraus gezogener Keimpflanzen. Z b l . Bakt. (2 Abt.) 12: 602-614.  8.  French, Richard C , Novotny, J . F . , and Searles, R.B. 1964. ties of bacteria isolated from wheat stem rust spores. pathology 54 (8): 970-974.  9.  Gauman, E. 1950. Principles of plant infection. Co., New York.  Further effects of the bark saproFor. Science 9 (3): 263-270.  ProperrPhyto-  Hafner Publishing  10.  Horsfall, J . G . , Hervey, G.E.R., and Suit, R.F. 1939. Dwarfing of cucurbits, sprayed with bordeaux mixture. Jour. Agr. Res. 58: 423-443.  11.  Johnson, Delia E. 1931. The antibiosis of certain bacteria to smuts and some other fungi. Phytopathology 21: 843-863.  12.  Johnson, L . F . , C u r l , E . A . , Bond, J . H . , and Frigbourg, H.A. 1 9 5 9 . Methods for studying s o i l microflora-plant disease relationships. Burgess Publishing Co., Minneapolis, Minn., U.S.A.  13.  K r s t i c , M. 1954. Note on the susceptibility of the chestnut-blight fungus towards some microorganisms in cultures. Plant Dis. Rep. 38: 427-428.  33  14.  K r s t i c , M. 1956. Prospects of appl icat ion of biological control in forest pathology. Bot. Rev. 22: 38-44.  15.  Last, F.T. 1955. Seasonal incidence of Sporobolomyces on cereal leaves. Trans. B r i t , mycol. Soc. 38 (3): 221-239.  16.  Last, F . T . , and Deighton, F.C. 1965. The non-parasitic microflora on the surfaces of l i v i n g leaves. Trans. B r i t , mycol. Soc. 48 (1): 83-99.  17.  M i l l e r , Max V/. 1961. The Pfizer Handbook of Microbial Metabolites. McGraw-Hill Book Company, Inc.  18.  Molnar, A.C. and Sivak, B. 1964. Melampsora infection of pine in B r i t i s h Columbia. Can. J . Botany 42: 145-158.  19.  Newhook, F . J . 1951. Microbiological control of Botrytis cinerea Pers. I. The role of pH changes and bacterial antagonism. Ann. Appl. B i o l . 38: 169-184.  20.  Newhook, F . J . 1951. Microbiological control of Botrytis cinerea Pers. II. Antagonism by fungi and actinomycetes. Ann. Appl. B i o l . 38: 185-202.  21.  Pasteur, L .  I876.  Etude sur la biere.  Paris.  22.. Pasteur, L . 1878. Examen c r i t i q u e d'un escrit posthume de Claude Bernard sur la fermentation alcoolique. C. r . hebd. Seanc. Acad. S c i l , Paris 87: 813-819. 23.  Potter, M.C. 1910. Bacteria in their relation to plant pathology. Trans. B r i t , mycol. Soc. 3: 150-168.  24.  Ruinen, Jakoba 1961. The phyllosphere. i . An ecologically neglected m i l i e u . Plant and Soil 15 (2): 81-109.  25.  Tukey, H.B. J r . , Wittwer, S . H . , and Tukey, H.B. 1957. Leaching of carbohydrates from plant foliage as related to light intensity. Science 126: 120.  26.  Van Arsdel, E.P. 1965. Relationships between night breezes and b l i s t e r rust spread on Lake State white pines. U.S. Forest Service Research Note LS-60.  27.  Wood, R.K.S. 1951. The control of diseases of lettuce by the use of antagonistic organisms. I. The control of Botrytis cinerea Pers. Ann. Appl. B i o l . 38: 203-216.  28.  Wood, R.K.S. and Tveit, M. 1955. Control of plant diseases by use of antagonistic organisms. Bot. Rev. 21: 441-492,  34.  29.  Z i l l e r , W.G. 1955. Studies of western tree rusts. II. Melampsora occidental is and M. albertens i s . two needle rusts of Douglas f i r . Can. J . Botany 33: 177-188.  30.  Z i l l e r , W.G. 1965. Studies of western tree rusts. VI. The aecial host ranges of Melampsora albertens i s . M. medusae and M. o c cidental i s . Can. J . Botany 43: 217-230. ~  35  APPENDIX Summary tables of analyses of variance and Duncan's New Multiple Range Tests of the results of the inoculation experiments  A.  The effect of water, nutrient broth, c e l l free f i l t r a t e and bacterial treatments on the development of Melampsora medusae on the foliage of Douglas f i r . (i)  Source of variation  Analysis of Variance d.f.  Sum of squares  Mean square  Calculated F  Tabled F at 5%  Treatments Block Error Total (ii)  4 3 12 19  115,535.5 2,414.7 2,701.3 120,651.5  28,883.9 804.9 225.1  128.3 3.6*  1%  3 . 2 6 5.41 3.49 5.95  Duncan's New Multiple Range Test of treatment means (5% level)  S t e r i l e d i s t i 1 led water  S t e r i l e nutrient broth  Bacteria in water  3 8 J  2 i J  75.0  Cell free filtrate 20,1  Bacteria in nutrient broth 1.9  B.  The effect of nutrient broth, c e l l free f i l t r a t e and bacteria in nutrient broth treatments on the development of Melampsora medusae on the foliage of Douglas f i r .  36  (')  Analysis of Variance  Source of variation  d.f.  Sum of squares  Mean square  Calculated F  Tabled F at 5%  2 3 6 11  Treatments Block Error Total (i?)  105.2** .50  5.14 10.92 4.76 9.78  Duncan's New Multiple Range Test of treatment means (1% level)  S t e r i l e nutrient broth  Cell free filtrate  70.3  C.  2,090.8 9.9 19.9  4,181.6 29.8 119.2 4,330.6  1%  Bacteria in nutrient broth  57.8  12.5  The effect of pure cultures of Bac? 1 lus cereus. §_. mycoides. and Baci 1 lus sp. and a mixture of the three species on the development of Melampsora medusae. (i)  Analysis of Variance  Source of variation  d.f.  7 2 14 23  Treatments Block Error Total (ii)  22,254.3 1,406.9 2,903.3 26,564.5  Mean square  Calculated F  3,179.2 703.5 207.4  15.3** 3.4  Tabled F at 5%  1%  2.77 3.74  4.28 6.51  Duncan's New Multiple Range Test of treatment means (5% level)  Cell free f i l t r a t e of Baci1lus cereus  97.2  Sum of squares  Cell free Cell free f i l t r a t e of a f i l t r a t e of mixture of Baci11 us Baci1lus cereus. mycoides EJ. mycoides, and Baci1lus sp. 9ib4  94.4  Cell free f i l t r a t e of Baci11 us sp.  91.7  Bacillus sp. in nutrient broth  6^4  37.  Baci1lus mycoides in nutrient broth  55.5  Baci11 us cereus in nutrient broth 2Z£  Footnote * Significant at the 5% l e v e l . ** Significant at the 1% l e v e l .  A mixture of Baci11 us cereus. B. mycoides and Baci11 us sp„ in nutrient broth  5^1  

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.831.1-0104758/manifest

Comment

Related Items