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Incidence, etiology and epidemiology of stonefruit dieback in the Okanagan Valley Cujec, Thomas Peter 1988

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INCIDENCE, ETIOLOGY AND EPIDEMIOLOGY OF STONEFRUIT DIEBACK IN THE OKANAGAN VALLEY By THOMAS PETER CUJEC B.Sc, The University of Brit ish Columbia, 1985 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES (Department of Plant Science) We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA August, 1988 ® Thomas Peter Cujec, 1988 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of ' -Cft/v-J -4<AJ/ The University of British Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3 DE-6(3/81) i i ABSTRACT A C y t o s p o r a s p e c i e s i s o l a t e d f r o m i n f e c t e d t i s s u e s and s p o r u l a t i n g s t r o m a t a o n d i s e a s e d t r e e s c a u s e d t y p i c a l d i e b a c k s y m p t o m s when i n o c u l a t e d i n t o P r u n u s s p e c i e s and was i d e n t i f i e d as t h e p r i m a r y c a u s e o f s t o n e f r u i t d i e b a c k i n t h e O k a n a g a n . B a s e d on t h e m o r p h o l o g y o f t h e s t r o m a t a , s p o r e d i m e n s i o n s , and c o l o n y g r o w t h and c o l o r on m a l t e x t r a c t a g a r , t h e f u n g u s was i d e n t i f i e d a s C\ l e u c o s t o m a ( S a c c ) . A f t e r i n c l u d i n g t h e number o f t r e e s removed d u r i n g t h e w i n t e r o f 1985-86 and 1986-87 b e c a u s e o f C y t o s p o r a s p . , an a v e r a g e o f 1 4 . 8 % o f t h e t r e e s i n 17 s t o n e f r u i t o r c h a r d s were a f f e c t e d by d i e b a c k f r o m S e p t e m b e r 1985 t o S e p t e m b e r 1987. The i n c i d e n c e o f C y t o s p o r a s p . i n t h e i n d i v i d u a l b l o c k s r a n g e d f r o m 3.0-56.9%. In 11 o f t h e 17 o r c h a r d s s u r v e y e d i n 1986 and r e s u r v e y e d i n 1987, d i e b a c k symptoms were e v i d e n t on t r e e s w h i c h had b e e n s y m p t o m l e s s i n 1986. The p e r c e n t o f n e w l y i n f e c t e d t r e e s i n t h e s e 11 b l o c k s r a n g e d f r o m 0.4-8.8% and a v e r a g e d 2.9%. The m a j o r i t y o f s p o r u l a t i n g C y t o s p o r a s p . i n f e c t i o n s w e r e f o u n d on t h e s c a f f o l d l i m b s ( 6 9 % ) o r t r u n k s ( 2 8 % ) o f i n f e c t e d t r e e s . P r u n i n g wounds ( 6 5 % ) , r a t h e r t h a n w i n t e r i n j u r y ( 2 5 % ) , were t h e m a j o r i n f e c t i o n c o u r t s . F a l l a n d s p r i n g i n o c u l a t i o n s o f a s p o r e s u s p e n s i o n ( 1 0 3 s p o r e s / m l ) o f e i t h e r a p e a c h i s o l a t e ( P 8 - 1 9 ) t o p e a c h , o r a c h e r r y i s o l a t e ( C 9 - 2 3 ) t o c h e r r y r e v e a l e d t h a t i n t r a s p e c i e s s p r e a d o f t h e d i s e a s e c a n o c c u r a t a n y t i m e o f t h e y e a r . A l t h o u g h s p r i n g s p o r e i n o c u l a t i o n s o f t h e p e a c h i s o l a t e t o c h e r r y o r t h e c h e r r y i s o l a t e t o p e a c h r e s u l t e d i n s i g n i f i c a n t l y (P = 0.05) more i n f e c t i o n s t h a n t h e c o n t r o l t r e a t m e n t s , i d e n t i c a l f a l l i n o c u l a t i o n s d i d n o t . T h i s s u g g e s t s i i i that spread of Cytospora sp. between cherry and peach is most l ikely to occur in the spring. The effect of temperature on spore germination and mycelial growth of Cytospora sp. in vitro was isolate-dependent. The minimum lag period for Cytospora sp. spore germination occurred at 27° C. Spores germinated at temperatures as low as 10° C, and remained viable even after exposure to -18° C for 1 week. The temperature optima for the in vitro growth of most stonefruit isolates in this study was 20-23° C. Viable Cytospora sp. spores were washed from infected trees (10 5-10 6 spores/ml) and adjacent healthy trees (104 spores/ml) in mid-December and collected in funnel traps after the f i r s t rain the following spring (late Apr i l ) . Under Okanagan conditions, infection of fresh pruning wounds made in the spring can occur either by spores which overwintered on infected trees and were dispersed by spring rains, or by spores dispersed by f a l l rains to healthy trees on which they overwintered and infected following pruning. Benomyl (1 g a . i . /L ) , dichlone (1 g a . i . /L ) , f lus i lazole (0.01 g a . i . / L ) and ziram (5 g a . i . / L ) applied as water sprays did not s ign i f icant ly (P = 0.1) reduce the percent infection compared to the unprotected, inoculated controls. Of eight fungicide-pruning paste mixtures, only benomyl added to either Heal 'n ' Seal or linseed o i l s ignif icantly (P = 0.1) reduced the number of cankers which developed compared to the untreated control. iv TABLE OF CONTENTS Page ABSTRACT i i LIST OF TABLES vi LIST OF FIGURES v i i i ACKNOWLEDGEMENTS xi INTRODUCTION 1 LITERATURE REVIEW 1 Etiology 3 Variabi l i ty 6 Epidemiology 8 Control 14 MATERIALS AND METHODS 19 Survey of Diseased Orchards 19 Isolation Procedures 20 Isolations from Naturally Infected Trees 22 Fungus Identification 23 Inoculation Techniques 23 Pathogenicity and Virulence Trial • 25 Inoculation of Mature Cherry and Peach Trees 27 Susceptibil ity of Pruning Wounds to Infection 28 Leaf Scar Inoculations 29 Effect of Temperature on Mycelial Growth 30 Effect of Temperature on Spore Germination 30 Survival of Cytospora sp. Spores Following Exposure to Low Temperature 31 Spore Collection in Naturally Infected Orchards 32 A r t i f i c i a l Irrigation of Infected Limbs 34 Chemical Control Trial 35 RESULTS 38 Symptoms and Signs of Cytospora sp. Infection 38 Survey of Diseased Orchards 43 Incidence of Cytospora sp 45 Orientation, Location and Infection Courts of Cytospora sp. . 49 Isolation of Cytospora sp 54 Sample Description 54 Colony Description 58 Pathogenicity and Virulence of Cytospora sp. Isolates 58 Infection of Mature Cherry and Peach Trees by Two Cytospora sp. Isolates 63 A) Mycelial Inoculations 63 B) Spore Inoculations 65 V RESULTS (continued) Susceptibil ity of Pruning Wounds to Infection by Cytospora sp. Spores 69 Effect of Temperature on Spore Germination 71 Effect of Temperature on Mycelial Growth 71 Epidemiology of Cytospora sp 77 A) Spore Collection in Diseased Orchards 77 B) Experimental Irrigation of Infected Limbs 82 Chemical Control 82 DISCUSSION 89 SUMMARY 106 LITERATURE CITED 108 vi LIST OF TABLES Table 1. Characteristics which distinguish Cytospora sp. cincta from 5 C_^  leucostoma. Table 2. Background information on 30 stonefruit orchards in the 44 Okanagan and Similkameen Valleys of B.C. having a problem with dieback in 1986. Table 3. The incidence of Cytospora sp. dieback in 1986 and 1987 in 46 those stonefruit orchards having greater than 1% of their trees infected. Table 4. Inoculum potential and spread of Cytospora sp. from 1986 to 48 1987 in 17 stonefruit orchards having > 1% disease incidence in 1986 in Oliver and Osoyoos. Table 5. Orientation, location and apparent infection courts of 50 Cytospora sp. infections on diseased stonefruit trees in 18 orchards in 1986, and 17 orchards in 1987 located in the Oliver-Osoyoos region of the Okanagan Valley. Table 6. Location and infection courts of new Cytospora sp. 53 infections in 1987 on trees that had been symptomless in 1986. Table 7. Seasonal var iab i l i ty in the efficiency of Cytospora sp. 55 isolation on malt extract and potato dextrose agars from the margins of sporulating infections. Table 8. Pathogenicity of Cytospora sp. isolates obtained from 62 naturally infected apricot, cherry, and peach trees when inoculated to wounds on 1-year-old 'Springold' peach trees. X Table 9. Virulence of Cytospora sp. isolates obtained from naturally 64 infected apricot, cherry and peach trees when inoculated to wounds on 1-year-old 'Springold' peach t r e e s / Table 10. The effect of the season of inoculation on the percent 66 infection caused by representative cherry and peach isolates of Cytospora sp. when inoculated to limbs of mature 'Van' cherry and 'Fairhaven' peach t r e e s / Table 11. The percent infection caused by representative cherry and 67 peach isolates of Cytospora sp. when inoculated as separate spore suspensions to the limbs of mature 'Van' cherry and 'Fairhaven' peach trees in the f a l l and spr ing/ v i i Page Table 12. The effect of pruning wound age on the pathogenicity and 70 virulence of representative cherry (C9-23) and peach (P8-19) isolates of Cytospora sp. inoculated as spore suspensions to mature 'Van' cherry and 'Fairhaven' peach trees in the spring. Table 13. A comparison between the optimum and maximum temperatures 76 for the mycelial growth of 13 Cytospora spp. isolates obtained from naturally infected stonefruit and apple trees in the Okanagan and Similkameen Valleys of B.C. and authentic isolates of cincta and persoonii. Table 14. Effectiveness of post-inoculation fungicide treatments in 86 preventing infection of fresh pruning wounds by a r t i -f i c i a l l y applied Cytospora sp. spores/ Table 15. Influence of fungicide treatments in limiting Cytospora sp. 87 canker elongation on peach trees following a r t i f i c i a l inoculation of fresh pruning wounds with a spore suspension of Cytospora sp. x V I 1 1 LIST OF FIGURES Paoe Figure 1. Typ ica l symptoms of Cytospora sp. on natura l ly 39 infected stonefruit trees in the 01 iver-Osoyoos area of B.C. A, Gumming associated with Cytospora sp. infection. Note the "running" or steady progression of the gum along the infected limb. B, Death of an entire cherry tree due to Cytospora sp. infection. C, Sunken wood (arrow) infected with Cytospora sp. D, A sharp, dist inct boundary between healthy (top) and infected (bottom) wood. Figure 2. Typical signs of Cytospora sp. on naturally infected 41 stonefruit and apple trees in the 01iver-Osoyoos area of B.C. A, Stromata characteristic of Cytospora sp. on an infected cherry limb after the bark has been peeled away. B, A single, light-red spore horn on an infected peach soon after i t was exuded from the stroma, and C, blackish spore horns several months after they were exuded from the stromata. D, White ostioles of the stromata on an infected peach trunk after a l l the spores were exuded. E, Amber-coloured spore horns (arrow) characteristic of the Cytospora sp. species which infects apple, compare the colour of these with that of the spore horn on peach in B. Figure 3. Typical Cytospora sp. canker development on a peach 51 tree with the bark removed to show how the infection began through the unprotected pruning stub and spread down the branch into the scaffold limb. Figure 4. Free-hand sections of typical Cytospora sp. stromata 56 obtained from naturally infected stonefruits in the 01iver-Osoyoos area of B.C. In A, textura intr icata, note the arrow pointing to the loosely interconnected hyphae, in B, textura angularis; polyhedral cel l s with no intercel lular spaces. C, Convoluted chamber within a stromata. D, Vertical cross-section of a stromata, note the single ost iole, the central p i l l a r and the two chambers; one on each side of the p i l l a r . E, Typical Cytospora sp. spores viewed under o i l immersion. Figure 5. Colony morphology and color on MEA of a typical 59 isolate of Cytospora sp. (P8-19) isolated from a naturally infected stonefruit tree in Oliver B.C. A, A 6-day-old culture, note the "feathered" appearance of the culture at its edge. B, A 2-week-old o l ive-green culture and C, a 5-week-old almost black culture. Note the whitish, newly developed stromata at the centre of the culture in C. Page Figure 6. Inoculation of 1-year-old 'Springold' peach trees 61 using the "crushed bark" technique. A, Canker development 6 months after inoculation with a plug of mycel ia l from a t yp i c a l s tonefru i t i s o l a te of Cytospora sp., and B, with a plug of s ter i le malt extract agar. Figure 7. Effect of temperature on the lag phase for spore 72 germination of three Cytospora sp. isolates; apricot (A5-16), cherry (C9-23) and peach (P18-19) on malt extract agar. The lag phase was the time (hours) required for 90% spore germination. Dishes were checked 12, 18, 24, 36, 48, 60, 72 hours after inoculat ion. Each point is an average of three replicate dishes. The analyses of variance indicated a s i gn i f i cant temperature X i so late interact ion (P=0.05). Figure 8. Effect of temperature on the mycelial growth of six 73 Cytospora sp. isolates obtained from infected peach trees in the Okanagan Valley. Each point is a mean of duplicate measurements on each of four replicate dishes. Measurements were recorded after a 1-week incubation period of malt extract agar. Figure 9. Effect of temperature on the mycelial growth of an 74 apr icot (A5-16) and two cherry (C9-23, Cl-28) isolates of Cytospora sp. obtained from the Okanagan Valley and of IL^  cincta 106 (ATCC). Each point is a mean of dupl icate measurements on each of four replicate dishes. Measurements were recorded after a 1-week incubation period on malt extract agar. Figure 10. Effect of temperature on the mycelial growth of four 75 apple isolates of Cytospora sp. obtained from the Okanagan and Similkameen Valleys and of persoonii 105 (ATCC). Each point is a mean of duplicate measurements on each of four rep l i cate dishes. Measurements were recorded after a 1-week incubation period on malt extract agar. Figure 11. Concentration of waterborne Cytospora sp. conidia and 78 volume of rainwater collected during rainy periods in the summer and f a l l of 1986 by funnel-type spore traps placed beneath infected trees with sporulating stromata. Values on each collection date in A are means of three spore traps placed beneath three infected trees in a peach orchard in Oliver, and in B, of four traps placed beneath four infected trees in a peach orchard in Osoyoos, B.C. X Figure 12. Average temperatures and the concentrat ion of 79 waterborne Cytospora sp. conidia and volume of rainwater col lected during rainy periods in the spring and summer of 1987 by funnel-type spore traps placed beneath infected trees with sporulating stromata. Temperature values obtained from local temperature recording centres, are averages for a 7-day period immediately preceding each col lect ion date. Spore concentration and rainwater volumes for each collection date are means of four spore traps placed beneath four separate infected trees in a peach orchard in Oliver (A) and Osoyoos (B), B.C. N.D. = no data. Figure 13. Average temperature (••••) and rates of water (—) 83 and Cytospora sp. conidia collection (-) in funnel-type spore traps during a r t i f i c i a l i r r igat ion of infected peach limbs having sporulating stromata. Breaks in the lines represent drying intervals. (A) Average temperature during the irr igation interval was calculated from the temperature reading at the beginning and end of each interval. Temperatures were measured using a mercury thermometer placed in a Stevenson weather screen situated just beyond the i r r i g a ted area. (B) Rates of spore and water collection are averages of eight traps placed 10 cm beneath four peach limbs with sporulating infections (two traps/limb). xi ACKNOWLEDGEMENTS I am most grateful to my thesis supervisor, Dr. R.J. Copeman, for his essential guidance, encouragement and assistance throughout the course of this study and for generous financial support. Thanks are also due to the other members of my committee: Dr. P.L. Sholberg, Agriculture Canada, who read an earl ier draft of the thesis and offered many helpful suggestions, Dr. R.J. Bandoni, Department of Botany, and Dr. V.C. Runeckles, Department of Plant Science, who made numerous useful comments during the course of the project. Thanks to Dr. D. Bowden, Director of the Agriculture Canada Research Station in Summerland, for his permission to use the f a c i l i t i e s at the station and to a l l the station staff for their assistance. And f i n a l l y , a h e a r t f e l t thanks to my parents , f o r t h e i r encouragement, love, and support. 1 INTRODUCTION In British Columbia, Canada approximately 2280 ha (5700 acres) were planted to stonefruits in 1984. About half of this (952 ha = 2380 acres) was devoted to peach production. Another 744 ha (1860 acres) acres were planted to sweet cherry, and the remaining 584 ha (1460 acres) were devoted to apricot, plum, and prune production (8). In 1984, the cash value of the peach and cherry crops was approximately $4 mil l ion each. The apricot and prune crops were valued at $1 mill ion and $0.6 mil l ion each respect ive ly. In B r i t i sh Columbia (B.C.), peach and apricot production is situated mainly in the Oliver-Osoyoos and Penticton areas of the Okanagan Valley, but there are also some orchards north of Kelowna and in the Similkameen Valley. Cherry production occurs throughout both the Okanagan and Similkameen Valleys. LITERATURE REVIEW In the early 1980's, there was a dramatic increase in the incidence of a stonefruit dieback in the Oliver-Osoyoos region of the Okanagan Valley. Local extension off icers estimated that the problem was present in 50% of a l l stonefruit orchards in the region and was causing a 10% reduction in overall y ields. Some growers, however, were experiencing losses of up to 50% and a few affected stonefruit blocks were taken out of production because of the dieback problem. Since stromata exuding a l l an to id hyaline spores in reddish sporehorns were often present beneath the bark of infected tissue, i t was believed that the dieback problem in the Okanagan might be similar to the 2 disease known as Cytospora sp. dieback or perennial canker in other regions of North America. ' Although Cytospora sp. spp. are wound pathogens, typical ly infecting trees weakened by some other stress, they can also be saprophytes on dead wood. Therefore, the poss ibi l i ty that Cytospora sp. was a secondary organism infecting tissues previously k i l led by low winter temperatures or infected with Pseudomonas syrinqae pv. syringae (van Hall) could not be disregarded. Two fungi, Leucostoma cincta (Fries) Hohnel (= Valsa cincta (Fries)) and Leucostoma persoonii (Nitschke) Hohnel (= Valsa leucostoma (Fries)) are the causal organisms of perennial canker on stonefruits in other regions of North America. Their imperfect stages Cytospora sp. cincta (Saccardo) and Cytospora sp. leucostoma (Saccardo) respectively, are most commonly encountered in the f i e l d . These two organisms have been reported throughout the stonefruit growing regions of North America (48, 50), as well as in Europe (10, 46) and Japan (Togashi as cited by Defago (10)). The disease is considered a limiting factor of peach production in New York (45), Colorado (37), and Ontario (49) and of prune production in California (57) and Idaho (25). In 1933, V^ cincta and V^ leucostoma were f i r s t reported on peach in Ontario. Since then, both species have been reported on various Prunus species throughout Canada (9, 17). In B.C., V^ leucostoma was reported on nectarine in 1946, on peach on Vancouver Island in 1948, and on plum in 1957 (9). V_^  cincta has not been reported on stonefruits in B.C. In the United States, Valsa ambiens (Fries) and V^ leucostoma have been reported on domestic apple (Malus pumila) in the Pacific Northwest, North Central and New England states (48, 50). Unidentified Valsa sp. 3 have been reported on domestic apple in six Canadian provinces. In B.C., \L ambiens and V^ leucostoma were reported on apple on Vancouver Island in 1953 (9). The death of trees or limbs infected with Cytospora spp. can occur in two ways. In B.C., Idaho and California the disease is characterized by a rapid dieback of the infected tree or limb. Perennial canker-type symptoms are not observed on the diseased trees. In Ontario, Colorado and New York, the yearly alternation between fungal invasion of healthy tissue, and callus development by the host, results in the formation of concentric callus rings and a perennial canker-type symptom. Eventually (5-6 years) the expanding canker girdles the branch resulting in the dieback of the infected limb. In a l l regions, a d i s t inc t margin separates the diseased and healthy cambial tissue and stromata are formed beneath the bark of infected wood. Etiology cincta (= V^ cincta) and persoonii (= V^ leucostoma), the commonly used names for the perfect states of the perennial canker-causing fungi, are often used interchangeably in the l iterature (30). Originally, the genus Valsa was proposed by Fries in 1849 and included al l stromatic pyrenomycetes with hyaline and allantoid spores. In the following years, workers removed some of the organisms from the genus because of differences in perithecia l structure and development and placed them in separate genera. Eventually, only the species that are presently in the Leucostoma, Valsa, and Valsella genera remained in the original Valsa genus proposed by Fries. In 1917, von Hohnel proposed 4 that these subgenera be given the rank of genera and claimed that they could be distinguished on the basis of the presence or absence of a black basal zone (conceptable) beneath the perithecia and the number of spores in the asc i . Subsequent workers (30, 51) have adopted von Hdhnel's system to differentiate these genera. In 1973, Muller and von Arx (40) also used von Hdhnel's system of c lass i f icat ion to differentiate the Leucostoma, Valsa, and Valsella species. They considered the imperfect stages of these three genera to be species of Cytospora sp. D i f fe rent i a t ion of species within the genus Cytospora sp. is d i f f i c u l t . The s ize of the stromata or conidia are not useful taxonomical character i s t i c s because they are very similar for many species (29, 30, 55). Characteristics which can be used to distinguish C_^  cincta from C^ leucostoma are given in Table 1. Mycelial growth at various temperatures appears to be of l i t t l e use in distinguishing the two Cytospora sp. species. After testing six C_^  leucostoma isolates, Defago (10) concluded that the effect of temperature on mycelial growth was isolate-dependent. The temperature optima of i s o l a t e s obtained from prune (27° C), blackthorn (27° C) and an unidentified host in Japan (30° C) were higher than those of isolates from cherry (20° C), peach (20° C) and apricot (24° C). Mycelial growth occurred between 5 and 35° C. The growth rates and temperature optima for the f ive C_^  cincta isolates tested were more uniform than those of C^ leucostoma. Isolates of C^ cincta grew at temperatures ranging from -0.9 to 30° C with an optimum of 22-24° C. In Idaho, Helton and Konicek (23) studied the growth rates of four (\ cincta and two C_;_ leucostoma isolates on L i l l y and Barnett agar medium. Al l isolates grew at the lowest temperature 5 Table 1. Characterist ics which distinguish Cytospora cincta from C_. leucostoma Characteristic C. cincta C. leucostoma Reference Asexual fruct i f icat ions embedded in perithecial stromata not embedded in perithecial stromata 51 Spore horns light pinkish reddish-brown 51 Locule morphology convoluted, no central p i l l a r highly convoluted, with central p i l l a r 10 Colony color on Malt extract agar (MEA) at f i r s t white-grey then yellow-ish at f i r s t white, then olive-green to black 10, 29 Colony morphology on MEA star-form growth, even margins spiral growth form, irregular margins 10, 29 Aerial mycelium sparse to dense absent 10, 29 6 (3° C) tested, and were k i l led at 45° G. C\ leucostoma and C\ cincta had temperature optima of 20 and 30° C respectively. Isolates varied not only in their temperature optima but also in their relative growth rate at each of the temperatures tested. One isolate of (\ cincta obtained from an unspecified host in Michigan, and an isolate of C\ leucostoma obtained from 'French' prune in Cal i fornia, grew at temperatures of 0-36° C and 6-40° C with optima of 24 and 30° C respectively (1). Variability Differences in the requirements for optimal growth and colony growth patterns of Cytospora sp. isolates belonging to the same species, as well as, between monoascospore isolates obtained from the same ascus have been documented (25, 39). Helton and Konicek demonstrated that isolates of the same Cytospora sp. species required d i f f e r e n t hydrogen-ion concentrations (32), and carbon (31) and nitrogen (24) sources for optimal growth in v itro. Recent work (18) confirmed the var iab i l i ty between i so l a te s belonging to the same Cytospora sp. species by demonstrating that isolates of either cincta or persoonii, obtained from the same orchard could be segregated into several interaction groups based on vegetative incompatibility reactions (barrages) on oatmeal agar. Cytospora sp. isolates of the same species also d i f fer in their pa thogen ic i ty when inoculated to various hosts. In Idaho, 10 unidentified Cytospora sp. isolates obtained from various f ru i t tree and forest hosts, were inoculated to eight forest or ornamental species and 12 cultivated or native f ru i t species (19). Isolates obtained from the forest tree species were either nonpathogenic or only weakly virulent on 7 cu l t ivated stonefruits, while those from the tree f r u i t hosts were generally nonpathogenic on the forest species. None of the isolates tested were pathogenic on apple. One of the apple i so lates was pathogenic on a l l the cult ivated stonefruit variet ies tested, while another one was pathogenic only on 'Stanley' prunes. Cytospora sp. isolates obtained from 'J.H. Hale' peach and an ' I ta l ian ' prune were weakly virulent on 'Bing' cherry, while another isolate from ' I ta l ian ' prune and one from 'Pres ident ' plum were nonpathogenic on cherry. Although the cherry isolate was pathogenic on a l l the cultivated tree f ru i t hosts, i t was most virulent when inoculated to cherry. In West Germany (46) unidentified Cytospora sp. isolates obtained from apple, apricot, plum and two peach cult ivars, as well as, a cherry isolate of C\ leucostoma were cross-inoculated to the same series of hosts. The apple and apricot isolates were nonpathogenic on a l l of the hosts. None of the isolates were pathogenic on apple. Only the plum isolate was pathogenic on apricot. The C^ leucostoma isolate from cherry was pathogenic on cherry, plum and peach, however, the plum and two peach isolates were nonpathogenic on cherry. The plum and both peach isolates were pathogenic on plum and peach. Inherent va r i ab i l i t y in the virulence of Cytospora sp. isolates belonging to the same species has been reported. Differences in the virulence of the isolates ( 2 2 ) , as well as, a significant cult ivar x isolate interaction has been reported for both cincta (12) and leucostoma (59) on peach in Ontario and Colorado respectively, as well as, for C^ leucostoma on prune in California ( 1 ) . In addition to the inherent va r i ab i l i t y among isolates, ambient 8 temperatures and host act iv ity have also been implicated in influencing the virulence of Cytospora sp. isolates. Following inoculations to 6-month-old peach seedlings, C^ leucostoma was more virulent than C^ cincta at temperatures above 15° C, while at temperatures below 10° C the reverse was true (52). Subsequent inoculations to mature prune trees and 1-year-old peach twigs confirmed these results (1, 12). In Colorado, Jones and Luepschen (27) inoculated 3-year-old peach trees with mycel ia l plugs of C_^  leucostoma and recorded canker enlargement over the period of a year. The greatest canker enlargement occurred in the spring, when host activity was minimal; while the least occurred in the summer, the period of maximum stem growth. This occurred despite the fact that the ambient temperatures in the summer were approximately equal to those required for optimal growth of C_^  leucostoma in v i tro. They therefore concluded that the rate of canker enlargement was more c lose ly correlated with host a c t i v i t y than with ambient temperatures. Epidemiology Fungi belonging to the form order Sphaeropsidales, whose spores are exuded in spore horns, are better adapted to dispersal by rain splashing and wind-blown rain than by wind (33). Viable conidia were collected in funnel traps placed beneath infected trees throughout the year in Cal i fornia and Colorado following either natural rain or a r t i f i c i a l washing of infected limbs (2, 36). In addition, during rainstorms in Cal ifornia, conidia (concentration not specified) could be collected in traps placed 76 m away from infected trees. In Colorado (36), the 9 greatest number of conidia were collected in the summer, while in California the greatest number were collected in the f a l l . A regression equation accounting for 47% of the variation in the number of conidia trapped in California was produced. Time and temperature between rains, as well as the rate of r a in f a l l , had a significant effect on the number of conidia collected in Cal i fornia. Rohrbach and Luepschen (44) demonstrated that both a carbon source and a relat ive humidity of 100% are required for germination of C^ leucostoma spores. Spore germination occurred from 10-32° C. The minimum lag period (24 hours) before germination occurred at 27 and 32° C. At 21° C, maximum germination occurred after 36 hours. Exposure to 38° C for 13 days was lethal. Spores sprayed on petri dishes and incubated at -1° C for 13 days remained f u l l y viable. However, spores frozen in water (-23° C) for 13 days experienced a 50% reduction in v iab i l i t y . Although Cytospora sp. spp. are wound pathogens, different types of wounds are commonly ut i l ized as entry points for Cytospora sp. in various regions of North America. In Cal i fornia, sunburn injury on scaffold limbs is considered the major infection court for persoonii (57). In Idaho, (20, 22) low-temperature injury causing localized areas of dead tissue on the scaffold limbs and trunks of the trees is believed to be the most important entry point for the pathogen. After Helton (20) simulated localized winter injury on plum trees using dry ice, 77% of the injured scaffold limbs and 44% of the trunks became naturally infected with Cytospora sp. The percent infection increased i f cuts were made into the simulated winter-injured tissue using a knife. Nevertheless, not more than 1/3 of a l l infect ions ever developed into expanding 10 cankers. Isolations from tissues suspected of being infected with Cytospora sp. were not attempted. Due to the fact that minor low-temperature injury on stonefruits is common at northern latitudes such as Idaho, and because of the high percent of freeze-kil led tissue invaded by Cytospora spp., Helton reasoned that localized winter injury is the most important entry point for the pathogen in Idaho. In New York (28), winter injury was implicated as an important infection point in the early 1960's after expanding Cytospora sp. cankers developed on sweet cherry trees one year after they had been subjected to natural winter injury. Cytospora sp. was isolated from the expanding cankers. In Colorado (37) a survey of 2040 trees in 85 dif ferent peach orchards (24 trees/orchard) revealed that 57% of Cytospora sp. infections were associated with pruning wounds while nearly 40% were associated with winter injury. Nearly 50% of the diseased trees had at least one infected scaffold limb, 30% of the trees had a canker on the main trunk, while 42% of the trees had cankers on the fruit ing wood. The authors examined the trees " . . . for the presence of canker symptoms on the trunks, scaffold limbs, and smaller limbs of the f ru i t bearing wood They did not specify i f the presence of stromata was required as positive evidence of Cytospora sp. infection. No isolations were attempted from any of the diseased trees. After surveying 330 peach trees in an orchard in St. Catherine's, Ontario, Willison (54) concluded that 72% of a l l cankers were associated with the following types of wounds in approximately equal proportions: 1) oriental peach moth (Grapholitha molesta (Busck)), 2) pruning wounds 11 made flush with the surface of a larger branch, 3) pruning stubs and 4) dead bearing wood. The remaining cankers were associated with mechanical damage, damaged crotches, winter injury on the main trunk or pruning wounds made as a result of the removal of a previous canker. The susceptibil ity of pruning stubs to infection was emphasized by the fact that although the number of pruning wounds made flush with the subtending branch was much greater than the number of pruning stubs, the percent of cankers developing from the two wounds was approximately equal. W i l l i son (56) la ter postulated that the importance of various infection courts changed as the trees matured. In the f i r s t 2-3 years after planting, pruning wounds were the most important infection court because they were the most common type of injury made at the time. Later, as the trees developed a more extensive branching system, twig injuries caused by the oriental peach moth became the most important infection court. Dead twigs and f ru i t pedicels were associated with the majority of the cankers when the trees were 6 to 7 years old. Indirect evidence for the importance of pruning wounds as entry points was provided by the fact that trees pruned in October had more natural infections than trees pruned in the summer; when wound healing occurred much more rapidly. More recently, Tekauz and Patrick in Ontario reported (49) that peach nodal elements (buds and leaf scars) on 1-year-old twigs are important entry points for Cytospora spp. by demonstrating that their removal resulted in a decrease in the incidence of infection. Removal of the entire node (bud and leaf) resulted in much fewer natural infections than i f the leaves were allowed to f a l l of f na tu ra l l y , thereby 12 implicating peach nodes as infection courts. Removal of the buds or leaves alone resulted in more lesions/twig than i f the whole node was removed, thereby implicating both leaf scars and buds as infection courts. In a separate experiment, only 16% of the a r t i f i c i a l leaf scars made by cutting the leaf off with a scalpel at the point of natural leaf absc i s s ion , became infected a f te r being inoculated with a spore suspension (9 x l O 5 spores/ml) of cincta. Bud inoculations did not result in infection. Because twig remnants were frequently observed at the centre of established cankers, the researchers concluded that many of the perennial cankers found on peach scaffold limbs in Ontario orig inated from infections on 1-year-old twigs which later progressed to the subtending branch. The relat ively low percent of successful leaf scar infections, and the fa i lure to infect buds was explained on the basis of relat ive cu l t i va r res i s tance, premature a r t i f i c i a l leaf abscission and the inoculation of healthy, as opposed to low-temperature damaged buds. Dhanvantari (12) achieved 100% infection of leaf scars by gently pulling off leaves ready to f a l l and placing a drop of a spore suspension of either cincta or persoonii on the wound. Recently the importance of host response to wounding has been recognized because of its role in a plant's resistance to fungal and bacterial pathogens (47). The healing-over process in woody plants follows a set sequence: the cel l s in the injured area f i r s t undergo hypertrophy, t h i s is fo l lowed by c e l l wall 1 i g n i f i c a t i o n and suberization, immediately inside the new 1 ignosuberized layer phellogen 13 cel ls are then generated and these eventually differentiate into phellum cel ls (4). The sequence of phellum cel l production following inoculation with Cytospora sp. was similar to that observed in uninoculated wounds. However, the extent of the host response was different. Formation of the 1ignosuberized layer (3) and phellum ce l l s (58) was delayed in the inoculated wounds. In addition, the inoculated wounds had a thinner phellum layer than the control wounds. Mycelial wedges through the phellum cel l s were sometimes apparent, thereby allowing for the infection of new l iving tissue and continued expansion of the canker. In a recent study (5) a r t i f i c i a l l y made wounds of varying ages were inoculated with mycelium of C^ leucostoma. The formation of an adequate phellum layer 14 days after wounding was found to be responsible for resistance to Cytospora sp. infection. Lignification and formation of the 1ignosuberized boundary layer was correlated with a decrease in the rate of canker expansion. Using seven unidentified peach cultivars, Biggs (4) quantitatively compared the process of phellum regeneration following wounding of 5-year-old scaffold limbs in the months of June, July and August. The wounds were made using a cork borer. Wound age, and to a lesser extent the date of wounding, and peach cult ivar had significant effects on lignin and suberin deposition, as well as, phellum cel l production. Al l stages of the healing over process occurred more rapidly when wounds were made in July or August. Wounds made in August had s ignif icantly more phellum cel l s than those made in June or July. Earl ier work (53) based on measurements of callus widths suggested that a r t i f i c i a l wounds made in July healed-over more rapid ly than those made in June, August or 14 September. Wounds made in April or October healed-over the slowest. A positive correlation was found between the rate of wound healing and the reported f ie ld resistance of 13 peach cultivars to Cytospora spp. (53). Wounds made on 'Redhaven' peach healed-over at an average rate while those made on 'Early Elberta 1 and 'Veteran' peach healed-over relat ively slowly. In general, plants subjected to stress due to freezing, drought, transplanting, nutrient deficiencies, defoliation or insect damage are probably not as effective in forming new phellum cel ls after wounding as unstressed plants and are therefore more susceptible to infection by wound pathogens (47). In Ca l i f o rn i a (1), mycelial inoculations of "vigorous" trees resulted in smaller cankers than inoculations made to "non-vigorous" ones. Vigorous trees were successful in healing over the cankers within one year of inoculation while the "nonvigorous" ones were not. Recent work has shown that wounds made on peach trees which were adequately irrigated had a larger number of phellum cel ls than wounds made on trees subjected to water s tress (7). Formation of the lignosuberized boundary layer, however, was not influenced by i r r igat ion. Control Cytospora spp. are a serious problem in many peach growing regions of North America. As a result, numerous attempts have been made to screen different peach cultivars for Cytospora spp. resistance, with the aim of procuring resistant germplasm for use in future breeding programs. Despite extensive t r i a l s , however, a completely resistant cult ivar has 15 not yet been found (13, 35, 42). Out of 26 cult ivars, 'Veteran,' 'Redhaven,' and 'Fairhaven' were ranked fourth, eighth and twenty-fourth respectively in increasing susceptibi l ity to Cytospora sp. in New York (42). Susceptibil ity was based on the number of naturally occurring infections on the trees. In Colorado, there was l i t t l e variation in the canker lengths following a r t i f i c i a l inoculation of 10 peach cu l t ivars with mycelium of C^ leucostoma. Nevertheless, 'Redhaven' and 'Fairhaven' were ranked as moderately and highly susceptible respectively to the pathogen (38). The importance of f i e ld surveys in establishing relative cult ivar susceptibi l ity to Cytospora spp. was emphasized by Luepschen (35). He found no correlation between cult ivar susceptibi l ity, as determined by canker size following a r t i f i c i a l mycelial inoculations, and the natural incidence of infection on trees in the f i e l d . Field surveys have the advantage of ref lecting the occurrence of infection over a period of several years. This is an important point because recent results (13) demonstrating the significant effect of the year of inoculation and the year x c u l t i v a r i n te rac t i on on canker length following mycelial inoculations to peach cult ivars, suggest that non-genetic factors are important in canker development. At concentrations of 1-10 ppm, many chemicals can inhibit the growth and/or spore germination of Cytospora spp. in v i tro (11, 15, 43). However, the known phytotoxicity and/or mammalian toxicity of many of these compounds have prec luded t h e i r use in orchard t r i a l s . I n te re s t i ng l y , spores of C^ c inc ta and C_;_ 1 eucostoma responded differently to some fungicides (11). Ferbam, Kalo 100 (75% sulphur + 16 3.5% dichlone) and dodine inhibited spore germination of both Cytospora sp. species at 1-10 ppm. However, captan and dichlone inhibited the germination of C^ c incta spores at 50 ppm, while 100 and 500 ppm respectively were required to inhibit the germination of C_. leucostoma spores. Intensive research in other stonefruit growing areas of North America has fai led to identify an environmentally safe fungicide which is effective in eradicating established infections. In Idaho (26) large numbers of experimental chemicals were evaluated as protectants and/or eradicants against C_. cincta on peach. The chemical sprays were applied on separate trees at the beginning and end of June. Inoculations of peach limbs with mycelium of C^ cincta were made either 13 days prior to the appl icat ion of the sprays or three days following the sprays. Although some of the pre-inoculation treatments were successful in l i m i t i n g canker s i z e , a l l the pos t - inocu la t ion treatments were i n e f f e c t i v e . Because a p re - inocu la t ion spray of cycloheximide thiosemicarbazone (500 ppm) inhibited new infections, exhibited limited chemotherapeutic act ion, and was pers istent in the t rees, i t was considered a particularly promising chemical control agent and further tests were done with i t . Most of the other chemicals tested, including cycloheximide mixed with l inseed o i l , were phytotox ic at the concentrations required for adequate control. In a second experiment (21), a single spray of cycloheximide at 3200 ppm or repeated sprays at 800 ppm, were required to limit the elongation of established cankers. In Colorado (34), pre-inoculation sprays of benomyl alone (300 ppm), in combination with supreme o i l , or after sprays of the growth regulator 17 succ in ic ac id -2 ,2 -d imethy lhydraz i le (SADH), resulted in a 80-98% reduction in canker size compared to the untreated, mycelium-inoculated controls. Al l post-inoculation treatments were completely ineffective in reducing canker length. No information was given on the effectiveness of the pre-inoculation treatments in preventing infection. At present there are no fungicides registered against Cytospora spp. on stonefruits in Canada. Fall or spring sprays of ferbam or captafol on peach are recommended in Ontario primarily for peach leaf cur l , but also because of their apparent effectiveness in decreasing the incidence of leaf scar i n fec t i on s (6, 41). There is an obvious need for an environmentally safe protectant and/or eradicant fungicide which is effective against Cytospora spp. Currently the following cultural control measures designed to reduce the spread of Cytospora spp. are recommended in areas of North America where the disease is a problem (6, 45, 54). 1) Plant disease-free, winter-hardy stock. 2) Avoid wounds caused by rodents, insects or mechanical implements as these are potential infection courts. 3) Maintain trees in a vigorous, healthy condition, since trees stressed by drought, or nutrient deficiencies are more susceptible to infection because of their inabi l i ty to heal-over wounds rapidly. 4) Encourage a rapid rate of healing over by pruning as late in the spring as possible to take advantage of the warmer temperatures, and by making the cuts just beyond the ridge of thickened bark connecting the branch to the larger limb. 5) Apply a pruning paste to large cuts as soon as they are made. 6) Paint the trunks and scaffold limbs of the trees with a white latex paint to avoid southwest injury. 7) Avoid the development of narrow 18 crotch angles by training the trees properly. 8) Remove dead and/or infected wood from the orchards immediately, since they are a source of inoculum. Stonefruit dieback in the 01iver-Osoyoos area is causing serious economic losses to many growers and is continuing to spread unchecked within blocks. Since the problem is relat ively new in the region, very l i t t l e is known about its etiology and epidemiology. Research with the following objectives was undertaken: 1) To identify the causal organism(s) of stonefruit dieback; 2) To determine the in fect ion court(s) commonly used by the pathogen(s) under Okanagan conditions; 3) To determine the epidemiology of the disease under Okanagan conditions; 4) To evaluate the host speci f ic i ty of different strains in order to determine i f interspecies spread of the disease among stonefruits and to apple can occur; 5) To identify fungicides which might be effective as protectants against the disease. 19 MATERIALS AND METHODS Survey of Diseased Orchards In May and June of 1986, 30 stonefruit orchards reported to have a problem with limb dieback were v is i ted. The orchards were chosen at random from l i s t s of "problem" orchards supplied by extension off icers and packinghouse fieldmen. Because limb dieback was reported to be most widespread in the Oliver-Osoyoos area of the Okanagan Valley, the majority of the orchards examined were located in this region of the Valley. The orchardists were asked the following questions: 1) what cultivars were planted and i f there were any significant differences in cultivar susceptibi l i ty, 2) what the age of the majority of the trees was, 3) what year the disease became a serious problem and possible reasons for the increase in disease severity, 4) how much replanting had been done in the past year because of the disease, 5) whether recent leaf or soil nutrient analyses had revealed any nutrient deficiencies and 6) what control measures, i f any, had been t r i ed . An estimate of the incidence of dieback was made after a preliminary tour of the blocks with the orchardists. Blocks with > 1% of the trees exhibiting dieback symptoms were mapped and surveyed in deta i l . The position of every f i f t h tree in the block was mapped and the tree class i f ied as being either healthy or diseased. A tree was considered diseased i f one or more of the following c r i te r i a were met: 1) at least one of i ts limbs was dead, 2) leaf size was reduced, 3) profuse gumming was present, or 4) i f a discrete margin was present between healthy and sporulating infected tissue. The positions of any obviously diseased trees situated between 20 every f i f t h tree were also marked on the map. The following information was obtained for every diseased tree in the block: 1) orientation of the dead limb or canker (N, E, W, S), 2) the part of the tree infected (main trunk, scaffold limbs or bearing wood), 3) the type of wound associated with infection (the method of pruning or presence of a large pruning cut, winter injury on the trunk near the ground, mechanical injury or insect injury), and 4) the presence or absence of stromata beneath the bark and/or gummosis. Trees with old, callused-over cankers having no v is ib le effect on the vigor or productivity of the tree were not included in the survey results. The 18 stonefruit orchards having > 1% infection in 1986 were re-surveyed during the summer of 1987. The orchardists were asked the following questions: 1) how many new infections were evident since 1986, 2) how much replanting had been done in the past year due to the disease, and 3) i f any new control measures were being t r ied. Unlike 1986, each tree in the block was examined before being class i f ied as either healthy or d iseased. The c r i t e r i a used to c l a s s i f y the t rees , and the information obtained for diseased trees were the same as in 1986. Isolation Procedures A standard isolation procedure was used to isolate Cytospora spp. from the margins of infected tissue on both naturally infected and a r t i f i c i a l l y inoculated trees. Samples of tissue ( 3 x 1 cm) were taken from the margins of healthy and infected wood and submerged in a 5% commercial bleach solution (5% a . i . NaOCl) for 2 minutes. The tissue pieces were blotted dry with paper towels. Three chips of diseased 21 tissue (1 x 0.4 cm) were removed from the margin of healthy and diseased wood and placed on a 100 x 15 mm petri dish containing approximately 15 ml of either Difco malt extract agar (MEA) or Difco potato dextrose agar (PDA). This procedure was repeated a second time using a different piece of infected tissue obtained from the same tree. The dishes were placed in plastic bags and incubated on the laboratory bench at room temperature (25° C). After 1 week, they were checked for Cytospora spp. Plugs of mycelium were taken from suspected Cytospora spp. co lonies and transferred to individual MEA dishes. These dishes were checked 2-3 weeks later for the presence of dark, olive-green colonies characteristic of C_^  leucostoma. Recovery was considered successful i f (\ leucostoma was cultured from at least two of the six tissue samples plated on two repl icate dishes, or from three of the nine samples plated on three replicate dishes. Nonsporulating stromata on infected bark or small twigs were induced to sporulate by placing the samples in sealed plastic bags with wet paper towels for 1 week. Exuded spores were mixed in 5 ml of s ter i l i zed, d i s t i l l ed water. An aliquot (1-2 ml) of the spore suspension (1 x 105 spores/ml) was spread over dishes containing MEA with the aid of a flame-s te r i l i z ed , L-shaped glass rod. After 5-7 days incubation at room temperature (25° C), the dishes were checked for the presence of a dark, olive-green mat of mycelium, characteristic of C_. leucostoma. Leaves from diseased trees having symptoms similar to those caused by F\ syrinqae pv. syrinqae were sampled and indexed for the presence of the bacterium. Small pieces of leaf tissue were cut out aseptically from the margins of lesions and placed in 2-3 drops of s ter i l i zed, d i s t i l l ed 22 water for 5 minutes. The resulting suspension was streaked on nutrient sucrose agar (16). After 2-3 days incubation at 28° C, the colonies on the plates were compared to those of control P^  syringae pv. syringae strains. I s o l a t i o n s from N a t u r a l l y Infected Trees Samples were obtained from trees having dieback symptoms in a l l 30 stonefruit orchards visited in May and June 1986. Typically, four to seven trees were sampled at random in orchards having > 1% infection. Samples of diseased tissues taken from the advancing edge of both sporulating (stromata present) and nonsporulating (stromata absent) infections were obtained from a total of 100 trees. Similar samples were also taken from trees with sporulating Cytospora sp. infections in August, October and December 1986. During the summer of 1987, tissue samples obtained from margins of nonsporulating infections were collected from 54 trees exhibiting dieback symptoms. In May and June 1986, samples of infected tissue were plated on both MEA and PDA, however, on a l l the other dates that samples were col lected, only MEA was used as the isolation media. Spores exuded from stromata were mixed in s ter i l i zed, d i s t i l l e d water and dispersed on MEA. Whenever bacterial canker was suspected, both infected leaves and woody tissues were collected. Al l samples were identified according to orchard and tree number. Isolations were attempted within 24 hours after sampling using the methods previously described. Cytospora spp. isolates obtained in May and June 1986 were stored on PDA slants at 2-3° C. Al l isolates were identified according to the orchard from which they were obtained and 23 a s s i g n e d an i s o l a t e number ( O r c h a r d c o d e - N u m b e r ) . T h i s d e s i g n a t i o n was u s e d i n a l l f u t u r e r e f e r e n c e s t o t h e i s o l a t e s . Fungus I d e n t i f i c a t i o n The d i m e n s i o n s and m o r p h o l o g y o f t h e s t r o m a t a were d e t e r m i n e d f o r two c h e r r y and f o u r p e a c h s a m p l e s o f f r u i t i n g s t r u c t u r e s w i t h t h e a i d o f a d i s s e c t i n g m i c r o s c o p e . F r e e - h a n d s e c t i o n s o f t h e s t r o m a t a were made and e x a m i n e d u n d e r a compound m i c r o s c o p e t o d e t e r m i n e t h e a r r a n g e m e n t o f t h e h y p h a e i n t h e s t r o m a t a . T h e s p o r e h o r n s f r o m e a c h s a m p l e w e r e s u s p e n d e d i n w a t e r and t h e d i m e n s i o n s o f 50 s p o r e s m e a s u r e d u n d e r o i l i m m e r s i o n . C y t o s p o r a s p . i s o l a t e d f r o m t h r e e p e a c h , two c h e r r y and one a p r i c o t t r e e was i n c u b a t e d a t 25° C on 20 ml o f MEA f o r 1 m o n t h . The c u l t u r e s were e x a m i n e d a t 2-3 d a y i n t e r v a l s f o r t h e f i r s t 10 d a y s and t h e n a t w e e k l y i n t e r v a l s a f t e r w a r d s . T h e c o l o r and g r o w t h p a t t e r n s o f t h e d e v e l o p i n g c u l t u r e s , a s w e l l a s t h e p r e s e n c e o r a b s e n c e o f a e r i a l m y c e l i u m a n d / o r s t r o m a t a were n o t e d . I n o c u l a t i o n Techniques A l l i n o c u l a t i o n s u s i n g p l u g s o f m y c e l i u m o r s p o r e s u s p e n s i o n s were made u s i n g a s t a n d a r d " c r u s h e d b a r k " i n o c u l a t i o n p r o c e d u r e (28, 35, 38). The a r e a t o be i n o c u l a t e d ( 5 x 2 cm) was swabbed w i t h a c o t t o n wad s o a k e d i n 95% e t h a n o l . A f t e r t h e e t h a n o l had e v a p o r a t e d , a wound ( 2 x 1 cm) was made by s t r i k i n g a b o l t ( d i a m e t e r = 5 mm) p l a c e d a g a i n s t t h e s u r f a c e o f t h e b a r k w i t h a hammer u n t i l t h e b a r k was b r o k e n and t h e x y l e m e x p o s e d . T h e b o l t was f l a m e d b e f o r e m a k i n g e a c h new w o u n d . P l u g s o f MEA c o n t a i n i n g a c t i v e l y g r o w i n g m y c e l i u m o b t a i n e d f r o m 7 t o 1 0-day-old 24 cultures of Cytospora sp. were placed on the wounds using a flame-s t e r i l i z e d spatula. Wounds inoculated with plugs of mycelium were covered with black plastic tape. Spore suspensions were applied to the wounds using a ca l ibrated p ipette. Wounds inoculated with spore suspensions were l e f t uncovered to simulate natural condi t ions. Regardless of the source of inoculum, a l l wounds were inoculated immediately after being made. A twig inoculation technique was developed to obtain spores of Cytospora sp. whenever they were required. Two-year-old twigs, 15-20 cm in length and approximately 1 cm in diameter, were obtained from mature 'Springold' peach trees and inoculated with plugs of mycelium using the "crushed-bark" technique (one inoculat ion/twig). The wounds were approximately 1 x 0.5 cm in size. Immediately following inoculation, a l l the twigs inoculated with the same isolate were placed upright in a 500 ml beaker containing 50 ml of d i s t i l l ed water and incubated at 25° C. The water was changed at weekly intervals for 6 weeks. Stromata and spore horns were evident on infected twigs 4-5 weeks after inoculation. The infected twigs, with the spore horns s t i l l attached to the stromata, were stored at room temperature until required. Approximately 5 days before the spores were needed, spore horns were dissolved in s ter i l i zed, d i s t i l l ed water and a few drops of the spore suspension (1 x 105 spores/ml) spread on MEA. Spore v i a b i l i t y was checked after 24 hours incubation at 25° C using a compound microscope and always found to be > 95%. Spore suspensions in water were made as required and spore concentrations ca l i b ra ted with the aid of a hemocytometer. 25 Pathogenicity and Virulence Trial At the end of Apri l 1987, 1-year-old 'Springold' and 2-year-old 'Redhaven' peach trees on S iber ian C rootstocks and 1-year-old 'Starkcrimson' apple trees on MM 111 rootstocks were interplanted among 1-year-old 'Mcintosh' and 'Jonagold' apple trees in Field 20 of the Canada Agriculture Research Station at Summerland, B.C. (CARS). The trees were planted 0.6 m apart within the rows which were 3.6 m apart. The so i l was a sandy-loam type. The 'Redhaven' trees were 2 cm in diameter at the graft union, while the other trees were approximately 1 cm in diameter. A l l t rees were pruned according to standard hort icu l tura l practices immediately after planting. Irr igat ion was supplied with a system of overhead sprinklers. Gramoxone was applied in bands along the tree rows in the last week of May, and again' in early July, to control emerged weeds. No pesticides were applied to the trees at any other times of the year. In the second week of May 1987, three apricot, six cherry and 23 peach isolates of Cytospora sp. which had been obtained in infected stonefruit orchards in 1986 were inoculated to the 'Springold' peach trees. At the time of inoculation the leaves on the trees had expanded to 50% of their fu l l size. The "crushed-bark" technique was used to inoculate plugs of mycelium from each isolate to the main trunks of four replicate trees at a point approximately 0.5 m above the ground. The isolates were assigned to the trees in a random manner. In the last week of October 1987, the isolation procedure previously described was used to confirm Cytospora sp. infection on the inoculated trees. The pathogenicity of the isolates was compared by assigning dead 26 trees a disease index of 90, trees with expanding cankers an index of 60, and a value of 10 to uninfected trees. The virulence of the isolates was determined by averaging the canker length and width measurements. Canker length was determined by measuring the distance from the center of the inoculated wound to the apex of the vis ible canker. Width values were determined by measuring the circumference of the canker immediately above the tape used to cover the wound. Oead trees were given a width value equal to the circumference of the tree above the tape and a canker length of 60 mm. A value of 60 mm was chosen because i t represented a canker length longer than any of the cankers found on the l iving trees. Trees with no cankers were assigned a canker size value of 10 mm. In addition to the individual disease indices and canker lengths determined for each isolate, a mean disease index and a mean canker length was calculated for al l those isolates obtained from the same stonefruit host. Six apple, six peach and one apricot isolate of Cytospora spp. were inoculated to 'Redhaven' peach and 'Starkcrimson' apple trees on May 10 1987. At the time of inoculation, the 'Redhaven' trees were at the bud-swell stage while the apple trees were in f u l l leaf. The apple trees were inoculated pn the main trunk, while the peach trees were inoculated near the base of one of the scaffold limbs. Inoculations were made to the peach limbs since their diameters were approximately equal to those of the main trunks of the apple trees. Inoculations were made using the "crushed-bark" technique. Each isolate was assigned to four replicate trees at random. 27 Inoculation of Mature Cherry and Peach Trees In October 1986 and April 1987, f ive mature 'Fairhaven' peach trees and two mature 'Van' cherry trees situated in two separate orchard blocks at CARS were inoculated with two strains of Cytospora sp. The peach trees had not been pruned during the winter of 1986-87, but some of the f ru i t was removed in the summer of 1987 to avoid limb breakage. No fungicides or insecticides were applied to the peach trees while the experiment was in progress. In June 1986 and 1987, the two cherry trees were sprayed with diazinon to control the cherry mealybug. At the end of June 1987, iprodione was accidentally sprayed on one of the cherry trees. The experimental trees were not situated near any natural sources of inoculum. Inoculations were made on October 18-19, 1986, when the trees were approximately 10-20% defoliated, and on April 26-27, 1987 immediately after the trees had leafed out. The experiment was arranged as a completely randomized design. Each treatment was assigned to eight replicate limbs in a random manner. Inoculated cherry limbs were 2-5 cm in diameter, while those of peach were 2-3 cm. In both the f a l l and spring, there was no prec ip i ta t ion for at least 1 week after the inoculations had been made. One peach (P8-19) and one cherry (C9-23) isolate of Cytospora sp. were used as separate sources of inoculum. Each.isolate was inoculated to both cherry and peach in the following ways: 1) separate spore suspensions of 5 x 103, 5 x 105 and 5 x 107 spores/ml applied to wounded bark; 2) a spore suspension of 5 x 105 spores/ml applied to unwounded bark; and 3) a plug of mycelium applied to wounded bark. S ter i l ized, 28 d i s t i l l e d water , and plugs of MEA were app l i ed to wounded t i s s u e s and served as the two con t r o l t reatments . Spores were obta ined us ing the twig i n o c u l a t i o n technique p rev i ou s l y de s c r i bed . A l l wounds i nocu l a ted with plugs of mycelium or spore suspensions were made us ing the " c ru shed -bark" techn ique. Wounds on the cherry l imbs were 2-3 x 1-2 cm i n s i z e , w h i l e those on peach were 1-2 x 1-1.5 cm. The a p p r o p r i a t e spore suspension (0.5 ml) was g radua l l y p i p e t t e d to the wound to avoid r u n - o f f from the i n o c u l a t i o n s i t e . A handheld atomizer was used to spray a spore suspension (5 x 10 5 spores/ml) on unwounded cherry and peach l imbs . A la rge p l a s t i c bag was p laced beneath the l imbs and held up r i gh t on both s ides i n order to e l i m i n a t e s p r a y d r i f t t o a d j a c e n t b r a n c h e s . A rea s o f the l imb approximately 5 cm i n length were sprayed u n t i l thoroughly wet. Black tape was wrapped around the limb at the outer e x t r e m i t i e s of the sprayed areas so that they could be i d e n t i f i e d at a l a t e r date . The i nocu l a ted t r ee s were examined p e r i o d i c a l l y throughout the experiment to ensure prompt removal of any l imbs with s po r u l a t i n g i n f e c t i o n s . In mid-October 1987, the i s o l a t i o n procedure p r e v i ou s l y descr ibed was used to conf i rm Cytospora sp. i n f e c t i o n . S u s c e p t i b i l i t y of Pruning Wounds t o I n f e c t i o n Pruning wounds vary ing from 1.5-2.0 cm and 1.0-1.5 cm i n diameter on two mature ' V an ' and f i v e mature ' F a i r h a v e n ' t ree s r e s p e c t i v e l y were inocu la ted w i th two i s o l a t e s (P8-19, C9-23) of Cytospora sp. at the end of A p r i l 1987. Ha l f of these pruning wounds were i nocu la ted immediately a f t e r being made w i th a spore suspension of Cytospora sp. The remainder 29 were inoculated 1 week later. A 0.5 ml aliquot of 4 x 105 spores/ml was slowly pipetted to each pruning cut to allow for adequate absorption of the suspension by the tissue. Ster i l ized, d i s t i l l ed water applied to fresh pruning wounds served as the controls. The pruning wounds were le f t uncovered. Each isolate-pruning age treatment combination was rep l i cated eight times on both the cherry and peach trees. The treatments were assigned to the limbs of the individual trees at random. In October 1987 samples from the diseased margins of the pruning cuts were brought back to the lab and the standard isolation procedure used to confirm Cytospora sp. infection. Canker length was measured from the pruning cut to the furthest extremity of the discolored cambial tissue. Mean percent infection and mean canker lengths were analysed by separate analyses of variance and compared by using Fisher's protected LSD (P = 0.05). Leaf Scar Inoculations In mid-October 1986, a r t i f i c i a l leaf scars were made by gently pulling off the leaves of 1-year-old potted 'Valiant* peach trees. The fresh leaf scars were immediately inoculated with a spore suspension of Cytospora sp. (1 x 106 spores/ml) obtained from either a peach (P8-19) or cherry (C9-23) isolate. Spores were obtained using the twig inoculation technique previously described. The spore suspensions from each isolate were applied in two ways. In the f i r s t method, a pipette was used to place one drop (0.1 ml) of a spore suspension on a l l the leaf scars of the tree individually, while in the second method an atomizer was used to spray the spore suspension on the twigs of the entire tree to the point 30 of run-off. The control treatments consisted of s ter i l i zed, d i s t i l l ed water applied similarly. Each treatment was replicated on f ive separate trees. The trees were examined for bud infection and/or twig dieback in April 1987. Effect of Temperature on Mycelial Growth The optimum temperatures for the mycelial growth of 13 Cytospora spp. isolates obtained from stonefruit and apple trees in B.C. were compared to those of known ATCC isolates of L^ cincta and L_. per soon i i . Plugs of agar (diameter = 5 mm), containing actively growing mycelium from each 8-day-old Cytospora spp. culture, were inverted in the center of four replicate petri dishes containing 20 ml of MEA,. The dishes were bagged and incubated in the dark at 1, 5, 10, 15, 20, 23, 25, 27, 30 or 35° C for 1 week. After 1 week, colony diameters on each dish were determined by averaging two diameter measurements obtained at right angles to one another and a mean diameter for each isolate calculated. Mean colony diameters were analysed by the analysis of variance. Mean colony diameters of isolates having the same optimum temperature were compared using Fisher's protected LSD (P = 0.05). Effect of Temperature on Spore Germination Separate spore suspensions (1 x 106 spores/ml) of one apricot (A5-16), one cherry (C9-23) and one peach (P8-19) isolate of Cytospora sp. were prepared in s ter i l i zed, d i s t i l l ed water. Spores of each isolate were incubated at 1, 5, 10, 15, 20, 25, 27, 30 and 35° C on three rep l icate dishes containing 15 ml of MEA. A pipette was used to aseptical ly place 0.1 ml of the appropriate spore suspension at the 31 center of each petri dish. The suspensions were dispersed over the surface of the agar using a f lame-steri l ized, L-shaped glass rod. The dishes were examined under a compound microscope after 12, 18, 24, 36, 48, 60 and 72 hours incubation. The percent of viable spores was determined by randomly counting a total of 100 spores from at least f ive d i f ferent microscope f i e l d s on each dish. A spore was considered germinated i f i ts germ tube was at least three times longer than the length of an ungerminated spore. Dishes with more than 90% germination were discarded because formation of mycelial mats from the hyphae of the germinated spores made i t impossible to observe individual, ungerminated spores. One week after the commencement of the experiment, dishes in which no spore germination occurred were transferred to 25° C and spore v iab i l i ty checked 24 hours later. Survival of Cytospora sp. Spores Following Exposure to Low Temperature The v iab i l i ty of Cytospora sp. spores from isolates A5-16, C9-23 and P8-19 were determined following exposure to low temperatures in v i t ro. Peach twigs 2 cm long were cut longitudinal ly and sprayed with the appropriate spore suspension (5 x 105 spores/ml) until thoroughly wet using an atomizer. After spraying, the twigs were placed in a laminar airflow hood to dry. Dried twigs were placed in empty petri dishes with the cut side down to prevent ro l l ing over. After acclimatization at 0° C for 5 hours, the dishes were transferred to -18° C for 1, 3, 5 or 7 days. Each isolate was sprayed on a total of 16 individual twigs. Two twigs sprayed with the same isolate were placed in each petri dish for a total of 8 dishes/isolate. On each sampling date, two petri dishes of each 32 isolate were removed from the -18° C incubator, subjected to 0° C for 5 hours, and then transferred to room temperature (25° C). Three hours later, an atomizer was used to spray approximately 1 ml of s ter i l i zed, d i s t i l led water on the twigs in order to wash off the spores. The water used to wash twigs original ly sprayed with spores of the same isolate was combined and dispersed on three replicate dishes of MEA. The dishes were incubated at 25° C and spore v iab i l i ty checked 24 hours later using a compound microscope. Spore Collection in Naturally Infected Orchards Funnel-type spore traps were used to monitor the dispersal of Cytospora sp. spores in naturally infected stonefruit orchards. The traps were placed beneath limbs with sporulating infections and beneath healthy limbs of healthy trees 1.8 to 6 m away from the source of inoculum. The traps consisted of a plastic funnel (12 cm diameter), connected to a 500 ml col lection bottle by a 6 mm plast ic tube inserted through a hole in the cap. The traps were attached to the limbs of the trees using grape t r e l l i s wire. Depending on which was more convenient, the bottles were either attached to another limb of the same tree or were placed on the ground beside the trunk. Irrigation in the orchards was supplied by solid set, ground sprinklers. The funnels were placed above the reach of the irr igat ion water. On July 4 1986, 18 spore traps were placed in two peach orchards infected with Cytospora sp. One of the peach orchards was situated in Oliver while the other one was in Osoyoos. The volume of rainwater in the traps was measured at approximately 7-day intervals until freeze-up 33 on November 12, 1986. A sample of the rainwater from each of the traps was retained and the concentration of spores (spores/ml) estimated with a hemocytometer. On April 20 1987, 26 spore traps were placed in two peach blocks infected with Cytospora sp. Spore traps had to be placed under different trees in 1987 in the peach block in Osoyoos because the infected trees used in 1986 had been removed during the winter. The peach orchard monitored in Oliver in 1987 was different from the one monitored the previous year. As in 1986, the traps were placed beneath diseased and surrounding healthy trees. The concentration of spores (spores/ml) and the amount of rainwater collected in the traps was determined on a weekly basis unti l October 30 1987. At monthly intervals in 1986 and 1987, 1 ml aliquots of spore suspensions (1 x 105 spores/ml) col lected beneath infected trees were dispersed on separate MEA dishes. After a 24-hour incubation period at 25° C, the number of viable spores was counted under a compound microscope and the percent v iab i l i t y calculated. In 1987, daily maximum and minimum temperatures in the monitored orchards were estimated from the temperatures recorded at the nearest Environment Canada meteorological stations. The temperature recording station at the Oliver Sewage Treatment Plant was located 2-3 km from the peach orchard monitored in Oliver in 1987, while the Osoyoos recording station was 1 km west of the infected peach orchard monitored in that area. Temperature data were not obtained from either temperature recording station during the summer of 1986 because of a labor dispute. At the end of December 1986 an atomizer was used to spray d i s t i l l ed water on the limbs of healthy peach trees 1.8-6 m from trees with 34 s p o r u l a t i n g i n f e c t i o n s . A 1 - f o o t s e c t i o n o f a s i n g l e l i m b on e a c h t r e e was s p r a y e d t o r u n - o f f w i t h a p p r o x i m a t e l y 30 ml o f w a t e r . The r u n - o f f w a t e r was c o l l e c t e d by a f u n n e l a t t a c h e d t o a c o l l e c t i o n b o t t l e . In t o t a l , 23 h e a l t h y l i m b s , on 23 t r e e s a d j a c e n t t o f i v e t r e e s w i t h s p o r u l a t i n g i n f e c t i o n s i n two d i f f e r e n t peach b l o c k s , were s p r a y e d . The c o n c e n t r a t i o n o f s p o r e s i n t h e c o l l e c t e d w a t e r was d e t e r m i n e d w i t h t h e a i d o f a hemocytometer and t h e p e r c e n t o f v i a b l e s p o r e s d e t e r m i n e d a f t e r s p r e a d i n g t h e s p o r e s u s p e n s i o n s on MEA. In a d d i t i o n , s p o r e h o r n s f r o m i n f e c t e d t r e e s were mixed i n 10 ml o f s t e r i l i z e d , d i s t i l l e d w a t e r . One ml o f t h e r e s u l t i n g s p o r e s u s p e n s i o n (1 x 1 0 5 s p o r e s / m l ) was s p r e a d on MEA and t h e p e r c e n t o f v i a b l e s p o r e s d e t e r m i n e d a f t e r a 2 4 - h o u r i n c u b a t i o n p e r i o d a t 25° C. A r t i f i c i a l Irrigation of Infected Limbs F o u r p e a c h l i m b s w i t h s p o r u l a t i n g C y t o s p o r a s p . i n f e c t i o n s were i r r i g a t e d f o r a t o t a l o f 50 h o u r s u s i n g a s i n g l e o v e r h e a d s p r i n k l e r . The i n f e c t e d l i m b s were suspended h o r i z o n t a l l y 60 cm above t h e g round by two w i r e s a t t a c h e d t o p o l e s hammered i n t o t h e g round a t o p p o s i t e ends o f t h e l i m b . Two s p o r e t r a p s , i d e n t i c a l t o t h o s e used f o r s p o r e t r a p p i n g i n n a t u r a l l y i n f e c t e d o r c h a r d s , were p l a c e d 10 cm b e n e a t h each s p o r u l a t i n g peach l i m b . The t r a p s were h e l d u p r i g h t by n a i l i n g t h e f u n n e l t o wooden s t i c k s . The f o u r i n f e c t e d l i m b s were p o s i t i o n e d e q u i d i s t a n t l y f r o m one a n o t h e r i n a c i r c u l a r m a n n e r a p p r o x i m a t e l y 5 m f r o m t h e o v e r h e a d s p r i n k l e r . The e x p e r i m e n t was c o n d u c t e d i n an open f i e l d , w i t h no n a t u r a l s o u r c e s o f C y t o s p o r a s p . i n o c u l u m n e a r b y . No n a t u r a l r a i n f a l l o c c u r r e d d u r i n g t h e e x p e r i m e n t . 35 The average temperature during each i r r i g a t i on interva l was calculated from temperature readings at the beginning and end of each interval. Temperatures were measured using a mercury thermometer placed in a Stevenson weather screen situated just beyond the irrigated area. The average volume of water and concentration of spores collected in the eight bottles after each irr igation interval were described as ml/cm2/h and spores/cm2/h respectively. Chemical Control Tr ia l The e f f e c t i v e n e s s of benomyl, captan, c a p t a f o l , d i ch lone, f lus i lazo le, iprodione, tr i for ine and ziram in inhibiting the germination of Cytospora sp. spores in vitro was determined. A 0.1 ml aliquot of a spore suspension (1 x 105 spores/ml) of the peach isolate P8-19 was spread on 3 replicate dishes of MEA amended with 0.1, 1, 10, 100, or 1000 ppm active ingredient (a.i.) of each fungicide. The fungicides were added to the media as concentrated water suspensions immediately before pouring. The pH of the media varied from 4.9-5.2. The dishes were checked for germinated spores after incubation at 25° C for 48 hours. At the beginning of May 1987, pruning cuts (diameter = 1-2 cm) were made on the scaffold limbs of 4-year-old 'Fairhaven' peach trees located in a peach block at CARS. The leaves of the trees were fu l ly expanded at the time of pruning. Immediately after the cut was made, 0.5 ml of a spore suspension (8 x 105 spores/ml) obtained from the peach isolate (P8-19) was slowly pipetted to the wound to allow for complete absorption by the tissue. No fungicides, other than the ones used in the experiment, were sprayed on the trees during the course of the experiment. Al l other 36 normal horticultural practices, however, were maintained. One day after inoculation, benomyl, dichlone, f lus i lazole and ziram were applied to the wounds at rates of 1000, 1000, 10 and 5000 ppm a . i . respectively. Each fungicide was applied as a water spray, and in combination with either Heal 'n ' Seal (Farwell Products, Wenatchee WA) or double boiled l inseed o i l (Recochem Inc., Vancouver, B.C.). The fungicides were applied at the stated concentrations regardless of the method of application. Control treatments consisted of unamended Heal 'n ' Seal, linseed o i l or 2 ml of s t e r i l i zed , d i s t i l l e d water. An atomizer was used to apply approximately 5 ml of the fungicide-water suspension to each pruning wound. Adjacent pruning cuts were protected from spray dr i f t by passing the pruning stub through a hole made at the bottom of a large plastic bag. While the spraying was done, the bags were held upright, thereby enclosing the pruning cut on a l l sides except the top. A new bag was used for each of the four fungicide sprays. The desired concentration of fungicides in the paste treatments was obtained by di lut ing 1 ml of a concentrated fungicide suspension in 24 ml of either Heal 'n ' Seal or linseed o i l and then mixing the paste thoroughly. The fungicide-pruning paste treatments were applied d i rect ly to the pruning cuts, as well as on the sides of the limbs to a point 5 cm below the cut. The 2.5 cm nylon paintbrush used to apply the paste treatments was washed thoroughly before each new treatment was applied. The unamended Heal 'n 1 Seal and linseed o i l control treatments were applied prior to any of the fungicide-amended mixtures. Each treatment was applied to nine replicate pruning cuts chosen at random among the 28 peach trees used in the t r i a l . Visual observations on the response of 37 the host to the fungicide treatments were made in July. Five months after inoculation, the lengths of a l l the cankers were determined by measuring the distance from the pruning cut to the furthest extremity of the discolored cambial t i ssue. In order to confirm Cytospora sp. infection, tissue samples were taken from the discolored margins of a l l the pruning cuts and isolations attempted using the standard technique previously described. Mean percent infection (P = 0.1) and mean canker lengths (P = 0.05) were analysed by separate analyses of variance and compared by using Fisher's protected LSD. 38 RESULTS Symptoms and Signs of Cytospora sp. Infection The f i r s t symptom of Cytospora sp. infection on cherry and peach trees was the presence of gumming on infected trunks or scaffold limbs (Figure 1A). The gumming associated with Cytospora sp. infections differed from that caused by abiotic factors such as mechanical injury in that there was a steady progression or "running" of the gum up the tree, rather than a local ization in one area. Gumming was not observed on apricots. The death of an entire tree or single limb was the most obvious symptom of Cytospora sp. infection (Figure IB). Dieback was usually f i r s t seen in the spring after healthy trees had leafed out normally. It could, however, occur at any time during the summer. Infected wood was sometimes sunken (Figure 1C) and there was always a sharp, d i s t i nc t boundary between i t and healthy wood (Figure ID). Stromata (Figure 2A) f i r s t appeared as raised pimples beneath the bark and were found on infected trunks or scaffold limbs. In late spring and throughout the summer the ostioles broke through the bark and spore horns were exuded. At f i r s t the spore horns were a light red color, later they became dark red or purple (Figure 2B-C). White ostioles were vis ible after the stromata had exuded a l l of their spores (Figure 2D). Perithecia were not observed on any of the infected trees, nor were they found on infected limbs which had been piled for firewood. The symptoms of Cytospora sp. infection on apple were very similar to those found on stonefruits, except that gumming was never observed on 39 Figure 1. Typical symptoms of Cytospora sp. on naturally infected s tone f ru i t trees in the Oliver-Osoyoos area of B.C. A, Gumming associated with Cytospora sp. infection. Note the "running" or steady progression of the gum along the infected limb. B, Death of an entire cherry tree due to Cytospora sp. infection. C, Sunken wood (arrow) infected with Cytospora sp. D, A sharp dist inct margin between healthy (top) and infected (bottom) wood. 40 41 Figure 2. Typica l signs of Cytospora sp. on natural ly infected stonefruit and apple trees in the 01 iver-Osoyoos area of B.C. A, Stromata characteristic of Cytospora sp. on an infected cherry limb after the bark has been peeled away. B, A single, light-red spore horn on an infected peach soon after i t was exuded from the stroma, and C, blackish spore horns several months after they were exuded from the stromata. D, White ostioles of the stromata on an infected peach trunk after a l l the spores were exuded. E, Amber-coloured spore horns (arrow) characteristic of the Cytospora sp. species which infects apple, compare the colour of these with that of the spore horn on peach in B. 42 43 apple. In addition, the spore horns exuded from stromata on apple were a yellowish-amber color (Figure 2E) while those on stonefruits were reddish or purple. Survey of Diseased Orchards Trees in 27 of the 30 stonefruit orchards examined in 1986 were infected with Cytospora sp. (Table 2). Most of the orchardists stated that Cytospora sp. f i r s t became a serious problem during the 1982 growing season. Some, however, remarked that a small number of their trees had symptoms before 1980. None of the orchardists noticed any major differences in the susceptibi l ity of different apricot, cherry or peach cult ivars to Cytospora sp. infect ion. In orchards having different stonefruit species interplanted in the same block, Cytospora sp. was usually predominant on one species, however infection was not always limited exclusively to that host. Prune trees were variable in their susceptibil ity to Cytospora sp. infection. In one orchard, ' I ta l ian ' prunes growing near an infected peach block were infected with the same species of Cytospora sp. present on nearby peach trees. In two other orchards, however, the prune trees adjacent to infected cherry trees, or separating two infected peach blocks were not infected with Cytospora sp. Peach trees less than 4-years-old and greater than 15-years-old rarely appeared infected. Symptoms of infection on apricot and cherry trees were most common on 7 to 20-year-old t rees . Cytospora sp. was occasionally observed on newly planted 1 or 2-year-old trees. Whether infection originated in the nursery or after the trees were planted in the infected blocks was not determined. Recent leaf and soil nutrient TABLE 2. Background Information on 30 stonefruit orchards 1n the Okanagan and Slmilkameen Valleys of B.C. having a problem with dieback 1n 1986 Cytospora spp P. syringae Orchard Growers Location Type of Predominant Age of Present > IX code name stonefruit cult1var(s) y most trees Incidence Present Al DalIwhal 011ver Apricot P J 10 + + A2 Grewal Oliver Apricot T 15 + + + A3 Hahn Oliver Apricot T.w 20-30 - - -A4 M. Hearle Osoyoos Apricot T 15 + - -A5 Hart1nu1ck 01Iver Apricot WJ 10-20 + + -A6 Miller 011ver Apricot K J 15 - - -Cl Brummer Osoyoos Cherry S.V.L 15 + + + C? C1a Oliver Cherry B.L.V 10-15 - - -C3 Duarte Oliver Cherry L.V 10-15 + + -C4 Field 13 Summer-land Cherry Experimental Cultivars 15 + C5 Ganho Oliver Cherry U, some L 15 + - -C6 Machlal Oliver Cherry V,L 30 + + -C7 McCarty Oliver Cherry U 20 + - + C8 Pew Osoyoos Cherry U 10-15 + + -C9 Wells Oliver Cherry U 20-25 + + -PI Cachola A Oliver Peach F.R.GM 10-15 + + -PZ Cachola B Oliver Peach S.E.F.R 7-12 + + -P3 DeCosta Oliver Peach ER.F 7-10 + -P4 Gomes Osoyoos Peach R.F.S 10 + + -P5 E. Hearle Osoyoos Peach G.S.F 10 + + -P6 Hedge Oliver Peach & R-V.L Peach 10 + - + Cherry Cherry 20 P7 Kovac Oliver Peach R.E.F 10 + + -P8 Markus Oliver Peach F,R 15 + + -P9 Perelra A Oliver Peach GM.S.F 10-15 + + -PIO Perelra B Oliver Peach U 10 + - -P l l Relvas Oliver Peach F,R 5-10 + - -P12 Starling Oyama Peach F.ER 10-15 + - -P13 Swlntack Oliver Peach R.S 10 + + -P14 Vriends Keremeos Peach U 10 + - -PR Forty Oliver Prune I 15 + - -y Apricot cultivars: K = Kaledon, P « Perfecton, T = Tilton, W = Wenatchee Moorpark; Cherry cultivars: B - B1ng, L = Lambert, S = Stella, V = Van; Peach cultivars: E = Elberta, ER = Early Elberta, F «= Falrhaven, G •= Glohaven, GM = Golden Monashee, R = Redhaven, S « Springold; Prune cultlvar: I = Italian; U = Unknown. z + * present, - = absent. 45 analyses commissioned by the orchardists did not reveal any nutrient deficiencies. Two control measures sometimes used against Cytospora sp. were the painting of the trunks of trees with white latex paint, and the application of pruning pastes to very large cuts. Neither of these measures provided adequate control. Symptoms of syringae infection were observed on trees in two apricot and three cherry blocks, but the number of trees infected was always < 1%. On no occasion did symptoms of both syringae and Cytospora sp. infection appear on the same tree. Cytospora sp. was not observed in three of the 30 stonefruit orchards examined in 1986. Symptoms in two of the three orchards included dead trees and scaffold limbs while in the third orchard only dead fruit ing spurs were observed. Neither "running" gum nor stromata were observed on any of the trees in the three blocks. Since attempts to isolate Cytospora sp. from the margins of diseased tissues were unsuccessful, this organism is not believed to be the causal agent of limb dieback in these orchards. Incidence of Cytospora sp. In 1986, more than 1% of the trees in 18 of the 30 orchards examined were infected with Cytospora sp. (Table 2). In these 18 orchards the disease was evident on an average of 7% of the trees (Table 3). In nine orchards, a significant amount of replanting had been done during the winter of 1985-86 because of Cytospora sp. When the number of trees affected with the disease and the amount of replanting due to the disease were considered, an average of 11.7% of the trees were affected by Cytospora sp. In the individual blocks total disease incidence varied TABLE 3. The Incidence of Cytospora sp. dieback in 1986 and 1987 in those stonefruit orchards having greater than 1% of their trees infected Trees (%) 1986 1987 Affected" Orchard No. of trees Total 1 Total v Sept. 85 to code in block Infected Removeds affected Infected Removed5 affected Sept. 87 Al 225 46.2 10.7 56.9 46.2 0 46.2 56.9 A2 85 9.4 14.1 23.5 0 9.4 9.4 23.5 A5 72 6.9 0 6.9 13.9 0 13.9 13.9 Total 382 Adjusted Mean x 30.6 9.4 40.0 29.8 2 31.8 41.2 CI 6.9 11.8 18.7 4.9 2.8 7.7 19.5 C3 176 10.2 12.5 22.7 16.5 0 16.5 29 C6 54 5.6 0 5.6 5.6 0 5.6 5.6 C8 115 3.5 0 3.5 3.5 0 3.5 3.5 C9 224 3.6 0 3.6 3.6 0 3.6 3.6 Total 713 Adjusted Mean 5.8 5.4 12.2 6.9 0.6 7.5 12.9 PI 102 4.9 O 4.9 8.8 0 8.8 8.8 P2 500 2.6 3 5.6 1.6 1.4 3.0 6.0 P3 302 3.0 0 3.0 0 3.0 3.0 3.0 P4 420 6.0 4.5 10.5 14.3 0.9 15.2 19.7 P5 1265 4.1 0 4.1 6.5 0.6 7.1 7.1 P7 145 6.2 0 6.2 11.0 0 11.0 11.0* P8 210 5.7 12.4 18.1 13.8 0 13.8 26.2 P9 300 7.7 18.3 26 2 7.7 9.7 28 P l l 340 5.9 12.9 18.8 5 5 10 22.9 P13 332 6.9 0 6.9 - - - z Total 3916 Adjusted Mean 4.9 4.1 9.0 6.3 1.9 8.2 12.3 Total 5011 Overall Mean 7 4.7 11.7 8.4 1.7 10.1 14.8 s Trees removed during the winter of 1985-86 because of Cytospora sp. t Total affected = % Trees Infected 1986 + St Trees removed 1986. u Trees removed during the winter of 1986-87 because of Cytospora sp. v Total affected = % Trees Infected 1987 + % Trees removed 1987. w Affected Sept. 1985 to Sept. 1987 « % Total affected 1987 + % Removed 1986. x Means were adjusted by adding the actual number of trees Infected, removed or affected in each block and dividing by the total number of trees in the blocks for each stonefruit species and for a l l species combined. y Block taken out of production 1n August 1987 due to Cytospora sp. z A l l trees removed for reasons not associated with Cytospora sp. 47 from 3.0 to 56.9% of the trees. More than 10% of the trees were affected in four peach, two cherry and two apricot blocks. During the winter of 1986-87, one peach orchard was taken out of production for reasons unrelated to Cytospora sp. Cytospora sp. infection was evident on an average of 8.4% of the trees in the remaining 17 orchards in the Oliver-Osoyoos area when they were re-surveyed during the summer of 1987. When the removal of diseased trees during the winter of 1986-87 was taken into account, 10.1% of the trees in the 17 blocks were affected. Based on the number of trees removed during the winter of 1985-86 and the number of trees affected in 1987 for each individual block, a mean of 14.8% of the trees in the 17 orchards surveyed in 1986 and 1987 had been affected by the disease from September 1985 to September 1987. On this basis, more than 10% of the trees in f i ve peach, three apricot and two cherry blocks had been affected by Cytospora sp. in this two-year period. In August 1987, a 15-year-old peach block (P8) was taken out of production because of the inabi l i ty of the orchardist to stop the continued spread of the disease. "New infections," defined as trees having dieback symptoms in 1987 but which were symptomless in 1986, were present in eight of the nine peach, two of the f ive cherry, and one of the three apricot blocks re-surveyed in 1987 (Table 4). The percent of newly infected trees in the individual blocks ranged from 0.4 to 8.8%, with an overall average of 2.9%. More "new infections" occurred on peach (3.3%) than on apricot (1.0%) or cherry (1.7%). In the 17 orchards surveyed in 1986 and 1987 there was no correlation between the percent of "newly infected" trees in 1987 and either the percent of trees infected in 1986 (r s = 0.22) or the 48 TABLE 4. Inoculum potential and spread of Cytospora sp. from 1986 to 1987 in 17 stonefruit orchards having > 1% disease incidence in 1986 in Oliver and Osoyoos Trees (%)  Sporulating Total Newly Orchard Infected infections Removed affected affected code 1986 1986 1986 1986 1987 Al 46.2 9.4 10.7 56.9 0 A2 9.4 2.9 14.1 23.5 0 A5 6.9 1.4 0 6.9 5.6 Apricot Adjusted Meany 30.6 6.3 9.4 40.0 1.0 CI 6.9 2.8 11.8 18.7 0.7 C3 10.2 2.3 12.5 22.7 6.3 C6 5.6 1.9 0 5.6 0 C8 3.5 3.5 0 3.5 0 C9 3.6 2.7 0 3.6 0 Cherry Adjusted Mean 5.8 2.7 5.4 12.2 1.7 PI 4.9 2.0 0 4.9 3.9 P2 2.6 1.0 3.0 5.6 0.4 P3 3.0 1.3 0 3.0 0 P4 6.0 4.3 4.5 10.5 8.8 P5 4.1 1.1 0 4.1 2.8 P7 .2 2.1 0 6.2 2.8 P8 7 1.9 12.4 18.1 8.1 P9 7 7.0 18.3 26.0 2 P l l 5.9 3.2 12.9 18.8 3.8 P13 6.9 4.2 0 6.9 z Peach Adjusted Mean 4.9 2.5 4.1 9.0 3.3 Overal1 Adjusted Mean 7 2.8 4.7 11.7 2.9 y Means were adjusted by adding the actual number of trees infected, having sporulating infections, removed or affected and dividing by the total number of trees in the blocks for each stonefruit species and for a l l species combined. Total number of trees in each block as given in Table 3. z Block taken out of production during the winter of 1986-87, for reasons unrelated to Cytospora sp. 49 percent of trees with sporulating infections in 1986 ( r s = 0.06). Orientation, Location and Infection Courts of Cytospora sp. In 1986, as well as 1987, the orientat ions (N,E,W,S) of the i n fec t i ons were s imi lar (21.8 to 28.6%) i f results from a l l the stonefruit orchards were analysed together. In both years the adjusted means for the stonefruit species surveyed revealed that most of the infections were found on the scaffold limbs. However, some were also found on the trunks and twigs of diseased trees (Table 5). Pruning wounds (Figure 3) were associated with the majority of infections while winter injury, and to a much lesser extent mechanical or insect injury, were associated with the remaining infections. Some differences among the orientation of the infections were evident for each of the individual stonefruit species, however the predominant orientation of the infections varied depending on the year and species of stonefruit. In 1986, the percent of sporulating infections (stromata present) found on scaffold limbs ranged from 67.8% on peach to 75% on apricot and averaged 69.2%. While in 1987, i t ranged from 69.3% on peach to 72.7% on cherry and averaged 69.9%. In both years, approximately 10% of the sporulating infections on apricot trees appeared to have been init iated on small twigs, and later progressed on to the scaffold limbs. In 1986, the number of nonsporulating infections (stromata absent) found on the scaffold limbs of peach (61.1%) was almost double that found on the main trunks (38.7%). Apricot trees had s l ightly more infections on the scaffold limbs (52.7%) than on the trunks (47.3%), while cherry trees had an equal number in both locations. In 1987, the TABLE 5. Orientation, location and apparent infection courts of Cytospora sp. infections on diseased stonefruit trees In 18 orchards 1n 1986, and 17 orchards In 1987 located In the OHver-Osoyoos region of the Okanagan Valley Cytospora sp. Infection [%) Number of trees Orientation Location Infection courts Year and host Infected Stroma Stroma present absent North East West South Trunk" Scaffold limbs Twig Winter v Injury Pruning* injury Mech.* Injury Insect injury 1986 24 (21)y Apricot 117 - 16.6 33.3 29.2 Cherry 41 13 (32) - 7.7 30.8 30.8 Peach 191 96 (50) - 25 20.8 24 Total 349 133 Adjusted mean2 - 21.8 24.1 25.6 1987 Apricot 113 34 (30) - 17.6 29.4 26.5 Cherry 49 22 (45) - 9.1 22.7 36.4 Peach 226 127(56) - 28.3 22.8 20.5 Total 388 183 Adjusted mean - 24 24 23.5 1986 Apricot 117 - 93 (79) 29 25 23.6 Cherry 41 - 28 (68) 14.3 21.4 32.1 Peach 191 - 95 (50) 24 24 23 Total 349 216 Adjusted mean - 216 25 24 24.5 1987 Apricot 113 - 79 (70) 25.3 24.1 22.8 Cherry 49 - 27 (55) 29.6 25.9 18.5 Peach 226 - 99 (44) 30.3 25.3 24.2 Total 338 205 Adjusted mean - 28 25 23 20.8 30.8 30 12.5 30.8 32.3 75 69.2 67.8 12.5 25 46.2 22.9 75 53.8 67.7 8.3 1.0 28.6 28.5 69.2 2.3 25.6 67.8 6.0 .8 26.5 31.8 28.3 17.6 27.3 30.7 70.6 72.7 69.3 11.8 26.5 54.5 26.8 70.6 45.5 65.4 2.9 7.1 28.4 27.9 69.9 2.2 30.6 63.9 5.5 0 22.6 32.1 28 47.3 50 38.7 52.7 50 61.1 44.1 46.4 51.6 49.5 50 46.3 6.5 3.6 2.1 26.4 43.9 56 0 47.7 48.1 4.2 0 27.8 25.7 20.2 35.4 33.3 28.3 64.6 66.6 71.7 13.9 37 32.3 77.2 59.3 62.6 8.9 3.7 5.1 24 31.7 68.3 0 25.9 67.8 4.3 0 u Includes those infections which started on the main trunk and subsequently progressed up a main limb. v Includes those infections 1n which there was no obvious entry point. w Includes Infections through open pruning wounds, narrow crotch angles or pruning stubs. Includes Injury due to orchard machinery, as well as from wires and ropes used to t i e limbs, y Number in brackets 1s the percent. z Means were adjusted by adding the actual number of Infections for each orientation, location or Infection court and dividing by the total number of infections (with or without stroma, as appropriate) for each stonefruit species and for a l l species combined. Figure 3. Typical Cytospora sp. canker development on a peach tree with the bark removed to show how the infection began through the unprotected pruning stub and spread down the branch into the scaffold limb. 52 percent of nonsporulating infections found on the scaffold limbs of the three stonefruit hosts ranged from 71.7% on peach to 64.6% on apricot and averaged 68.3%. In 1986 and 1987, pruning injury was associated with 65 to 75% of a l l sporulating infections on apricot and peach trees. Pruning injury was associated with the majority (53.8%) of sporulating infections on cherry trees in 1986, but in 1987, winter injury was the most predominant infect ion court (54.5%). In 1986, the percent of nonsporulating infections associated with pruning injury was approximately (±5%) equal to that associated with winter injury for each of the three stonefruit species. In 1987, the percent of nonsporulating infections associated with pruning injury on the stonefruit hosts ranged from 77.2% on apricot to 59.3% on cherry and averaged 67.8%. An average of 70% of the "new infections" which occurred in 11 of the 17 orchards re-surveyed in 1987 were located on the scaffold limbs (Table 6). Although the percent of "new infections" located on the scaffold limbs varied from 25 to 100% and averaged 70%, in only two orchards were less than 60% of the "new infections" located on the scaffold limbs. In the 11 stonefruit orchards, the percent of "new infections" associated with pruning injury ranged from 25 to 100% and averaged 62%. Pruning injury was associated with 50 to 75% of a l l "new infections" in the eight peach blocks. Winter injury was associated with 55 and 75% of a l l the "new infections" in one cherry (C3) and one apricot block (A5) respectively. A pruning wound was associated with the one "new infection" in the remaining cherry orchard (CI). 53 TABLE 6. Location and infection courts of new Cytospora sp. infections in 1987 on trees that had been symptomless in 1986 Cytospora sp. infection (%) Locationy Infection court Orchard No. of newly Trunk Scaffold Winter Pruning code infected trees 1 imb injury injury A5 4 25 75 75 25 Cl 1 0 100 0 100 C3 11 36 64 55 45 PI 4 0 100 25 75 P2 2 50 50 50 50 P4 37 27 73 24 76 P5 36 36 64 44 55 P7 4 75 25 50 50 P8 17 24 76 35 65 P9 6 17 83 33 67 Pl l 13 31 69 38 62 Peach total 119 Adjusted mean2 30 70 35 65 Overall total 135 Adjusted mean 30 70 38 62 y Explanation of location and infection courts as in Table 5. z Means were adjusted by adding the actual number of infections for each location or infection court and dividing by the total number of infections for peach and for all stonefruit species combined. 54 Isolation of Cytospora sp. Cytospora sp. was the only organism consistently isolated from trees exhibiting dieback symptoms. In 1986, i t was isolated from 80 of the 100 samples of necrotic tissue obtained from diseased trees (80%). Malt extract agar was more reliable (93%) than PDA (65%) during isolation of Cytospora sp. from the margins of sporulating infections in the spring (Table 7). In December, Cytospora sp. was recovered from only three of the f ive trees (60%) known to be infected with Cytospora sp. During the summer of 1987, Cytospora sp. was isolated from 42 of the 54 samples (78%) of diseased tissue obtained from trees exhibiting dieback symptoms but having no stromata. Sample Description Cytospora sp. samples obtained from three peach and two cherry trees were described as follows. Sporocarps black, not erumpent as separate pycindial f ruct i f icat ions, but immersed just below outer periderm with ostiole exposed to outside (on cherry; 2.0-2.5 (x = 2.4) x 1.3-1.7 (x = 1.5) mm, on peach; 2.3-2.8 (x = 2.6) x 1.1-1.6 (x = 1.3) mm). Stromata orb icular when viewed from os t i o l a r s ide, flattened in side view, composed of compact hyphae (textura intr icata and textura angularis; Figure 4 A-B) and brown; chamber convoluted (Figure 4C), sometimes subdivided, typ ica l ly with a central column, with a single ost iole (Figure 4D). Paraphysis absent, conidiophores amphigenous within chambers, hyaline 14.0-16.0 (x = 14.5) pm long on peach, 14.0-16.5 (x = 15.5) pm long on cherry, cyl indr ic, mostly simple, sometimes branched, smooth, bearing conidia singely, apical ly. Conidiogenous cel l s TABLE 7. Seasonal variability in the efficiency of Cytospora sp. Isolation on malt extract and potato dextrose agars from the margins of sporulating infections Number % Time of Isolation Trees Dishes Recoveryy Amount of isolation media sampled inoculated contamination May-June MEAZ 40 80 93 Some PDA 40 80 65 A lot August MEA 6 12 83 None October MEA 5 10 80 Some December MEA 5 15 60 All Successful recovery = Cytospora sp. cultured from at least two of six samples of diseased tissue plated on two replicate dishes or from three of nine samples plated on three replicate dishes. Isolation media: MEA = malt extract agar, PDA = potato dextrose agar. 56 F i g u r e 4 . F r e e - h a n d s e c t i o n s o f t y p i c a l C y t o s p o r a s p . s t r o m a t a o b t a i n e d f r o m n a t u r a l l y i n f e c t e d s t o n e f r u i t s i n t h e O l i v e r - O s o y o o s a r e a o f B . C . I n A , t e x t u r a i n t r i c a t a , n o t e t h e a r r o w p o i n t i n g t o t h e l o o s e l y i n t e r c o n n e c t e d h y p h a e , i n B , t e x t u r a a n g u l a r i s ; p o l y h e d r a l c e l l s w i t h n o i n t e r c e l l u l a r s p a c e s . C , C o n v o l u t e d c h a m b e r w i t h i n a s t r o m a t a . D , V e r t i c a l c r o s s - s e c t i o n o f a s t r o m a t a , n o t e t h e s i n g l e o s t i o l e , c e n t r a l p i l l a r a n d t h e t w o c h a m b e r s , o n e o n e a c h s i d e o f t h e p i l l a r . E , T y p i c a l C y t o s p o r a s p . s p o r e s v i e w e d u n d e r o i l i m m e r s i o n . 57 58 integrated, cy l indr ica l , hyaline, smooth. Conidia (Figure 4E) hyaline 4.0-6.5 (x = 5.0) x 0.5-1.5 (x = 1.5) um on peach, 4.0-6.5 (x = 5.5) x 1.0-1.5 (x = 1.5) um on cherry, aseptate, thin-walled, smooth, al lantoid, sometimes rounded-oblong, in red to purple droplets or tendri ls. Colony Description Strains obtained from al l stonefruits were similar in culture. On MEA, colonies at f i r s t transparent whitish (Figure 5A), becoming l ight green, then olive-green (Figure 5B) and f ina l ly almost black at center but remaining whitish at margins (Figure 5C). The hyphae growing on and in the medium. Reverse, blackish with interspaced dense black clumps of mycelium. Expanding colonies with highly irregular margins, "feathered" owing to the large number of branches developing from both sides of the main hyphae. The main hyphae often grow in a curved manner from the center of the plate, producing a spiral arrangement. Mature colonies densely matted, with irregular margins. Cultures not forming stromata, are black at the center and yellowish-green marginally, their margins are more irregular and the mycelium a l i t t l e sparser. Aerial mycelium sparse or absent in a l l cultures. Mature stromata with white disc and drop of clear water resting on top, diameter 2-3mm. Pathogenicity and Virulence of Cytospora sp. Isolates A l l of the 32 Cytospora sp. isolates inoculated to 1-year-old 'Springold' peach trees were pathogenic (Figure 6 and Table 8). Twenty-two isolates were pathogenic on a l l four replicate trees, eight isolates were pathogenic on three of the four replicates, while two isolates were 59 Figure 5. Colony morphology and color on MEA of a typical isolate of Cytospora sp. (P8-19) isolated from a naturally infected stonefruit tree in Oliver, B.C. A, A 6-day-old culture, note the "feathered" appearance of the culture at i ts edge. B, A 2-week-old olive-green culture and C, a 5-week-old almost black culture. Note the whitish, newly developed stromata at the centre of the culture in C. Figure 6. Inoculation of 1-year-old 'Springold' peach trees using the "crushed bark" technique. A, Canker development 6 months after inoculation with a plug of mycelium from a typical stonefruit isolate of Cytospora sp. , and B, with a plug of sterile malt extract agar. TABLE 8. P a t h o g e n i c i t y of Cytospora sp. i s o l a t e s obtained from n a t u r a l l y Infected a p r i c o t , cherry, and peach trees when Inoculated to wounds on 1-year-old 'Springold' peach trees." Isolate code Y Disease index Pl-7 90.0 a z Pl-5, P3-26 82.5 ab P9-3, P8-18, P13-31 75.0 abc P5-10, P8-17, P8-19, C6-22, C9-23, Cl-28, Cl-29 67.5 abed P9-1, P9-2, P9-4, P5-11, P8-20, P3-24, Cl-27, P13-30, A2-32 60.0 bede P3-25, P5-8, P l l - 1 2 , Al-15 55.0 cde P2-6, P5-9, A5-16, C6-21 47.5 de Pl l - 1 3 , P l l - 1 4 35.0 ef Control 10.0 f Trees were wounded by s t r i k i n g a b o l t placed against the surface of the bark with a hammer u n t i l the bark was broken. The wounds were inocu-lated with plugs of a c t i v e l y growing mycelium and covered with p l a s t i c tape. Controls consisted of plugs of MEA. Le t t e r and number before dash = the orchard code as given i n Table 1; number following dash = Isolate number. Trees were assessed 6 months a f t e r i n o c u l a t i o n . Oead trees = 90, Infected t r e e s with expanding canker = 60, uninfected trees = 10. Indices are means of four r e p l i c a t e t r e e s , those followed by the same l e t t e r are not s i g n i f i c a n t l y d i f f e r e n t according to Fisher's protected LS0 (P = 0.05). 63 pathogenic on two replicates. Moreover, 11 isolates k i l led one out of the possible four trees, three isolates k i l led two trees, two isolates ki l led three trees, and one isolate k i l led a l l four replicate trees. The average disease indices for the six cherry (62.9) and 23 peach isolates (62.3) were not s ignif icantly different from each other, however, both indices were s ignif icantly greater than the average index (54.2) for the three apricot isolates (P = 0.05). The number of isolates having the same disease index followed a normal distr ibution. With the exception of orchard P3, i so lates obtained from the same orchard did not have s i gn i f i c an t l y d i f fe rent disease indices. There was no correlat ion between the disease index of the isolates and the percent of infected trees ( r s = 0.05) in the orchards from which the isolates were obtained. The average virulence of the six cherry isolates (30.6 mm) was not signif icantly different (P = 0.05) from that of either the 23 peach (32.9 mm) or the three apricot isolates (27.9 mm). The peach isolates were, however, s ignif icantly more virulent' than those from apricot. With the exception of orchard P3, isolates obtained from the same orchard did not di f fer s ignif icantly in their virulence (Table 9). Only one of the six apple isolates of Cytospora sp. was pathogenic on peach. Apple isolates of Cytospora sp. were nonpathogenic when inoculated back to their original host. None of the stonefruit isolates were pathogenic on apple. Infection of Mature Cherry and Peach Trees by Two Cytospora sp. Isolates A) Mycelial Inoculations Mycelial inoculations to the limbs of mature 'Van' and 'Fairhaven' TABLE 9. Virulence of Cytospora Isolates sp. obtained from naturally infected apricot, cherry and peach trees when inoculated to wounds on 1-year-old Springold' peach trees" Isolatey Canker Size (mm Pl-7 55.1 a z Pl-5 47.2 ab P3-26 46.9 ab P9-3 43.0 abc P13-31 41.9 abed P8-18 38.8 abede P8-17 38.7 bede C9-23 37.2 bedef P8-19 34.8 bedef C6-22 33.8 bedefg CI-29 33.1 bedefg P9-4 32.9 bedefg P5-10 32.3 bedefg P3-25 31.1 cdefg P13-30 31.0 cdefg Al-15 30.2 cdefg Cl-28 29.8 cdefg A2-32, P9-2 29.5 cdefg P8-20 29.1 cdefg P5-8 28.8 cdefg P9-1 27.9 cdefg P5-11 26.5 defg P3-24 25.9 efg Cl-27 25.3 efgh Pll-12 25.2 efgh P2-6, P5-9 24.6 efgh A5-16, C6-21 24.1 efgh Pll-13 21.9 fgh Pll-14 19.1 gh Control 10 h Method of inoculation was as described in Table 8. Letter and number before dash = the orchard code as given in Table 1; number following dash = isolate number. Canker size = the average of the length and width measurements, length = the distance from the center of the inoculated wound to the apex of the visible canker, width = circumference of the canker immediately above the tape. Dead trees = length of 60 mm and width = to circumference of the tree measured Immediately above the tape. Uninfected trees = canker size of 10 mm. Means of four replicates (one 1noculat1on/tree), followed by the same letter are not significantly different according to Fisher's protected LSD (P = 0.05). 65 trees showed that the representative cherry isolate (C9-23) was equally pathogenic on both cherry and peach during f a l l and spring inoculations (Table 10). S imi lar ly, there was no s ignif icant difference in the percent infection caused by the representative peach isolate (P8-19) when i t was inoculated to both hosts during the f a l l or spring. In April 1987, the typical symptoms and signs associated with Cytospora sp. dieback were observed on two cherry limbs and one peach limb which had been inoculated with plugs of mycelium the previous f a l l . The peach limb, and one of the cherry limbs, had been inoculated with the peach isolate while the other cherry limb had been inoculated with the cherry isolate. Three peach limbs which had been inoculated with the peach isolate in April 1987, developed dieback symptoms in June 1987. B) Spore Inoculations The percent infection resulting from the inoculation of cherry with the cherry isolate in the f a l l was not signif icantly different from that achieved with spring inoculations (Table 11). This observation held over the range of spore concentrations employed. Identical results were observed when the peach isolate was-inoculated back to its original host. Even inoculat ion with 103 spores/ml of either the peach or cherry isolates back to their original hosts resulted in signif icantly more infection than the control treatments. Regardless of the isolate, and time of inoculation, 107 spores/ml yielded a signif icantly higher percent infection than 103 spores/ml. At each of the spore concentrations used in the f a l l and spring, the cherry isolate caused a higher percent infection when inoculated to cherry than did the peach isolate when i t was inoculated to peach. These d i f f e rence s , however, were not TABLE 10. The effect of the season of Inoculation on the percent Infection caused by representative cherry and peach Isolates of Cytospora sp. when Inoculated to limbs of mature 'Van' cherry and 'Fairhaven' peach trees" Cytospora sp. infection (%) Cherry inoculated in Peach inoculated in Isolate Fall Y Sprlnq Fall Sprlnq Cherry 88 abz 100 a 100 a 100 a Peach 100 a 88 ab 100 a 100 a Control 0 c 0 c 0 c 0 c Plugs of mycelium were inoculated to wounds as described in Table 8. Fall = trees 10-20* defoliated, Spring = immediately after the trees had leafed out. % infection = (number of infections, as confirmed by the re-isolation of Cytospora sp./total of eight replicate inoculations) x 100. Percent values followed by the same letter are not significantly different according to Fisher's protected LSD (P = 0.05). Data in this Table and Table 11 were analysed together as one experiment. TABLE 11. The percent infection caused by representative cherry and peach isolates of Cytospora sp. when inoculated as separate spore suspensions to the limbs of mature 'Van' cherry and 'Fairhaven' peach trees in the f a l l and spring x Isolate Inoculum spores/ml Cytospora sp. infection (%) Cherry inoculated in^ Peach inoculated in Fall Spring Fall Spring Cherry °3 0 g o g o g lot 50 cde 50 cde 25 efg 63 bed 10= 88 ab 75 abc 63 bed 100 a 107 100 a 100 a 100 a 100 a Peach °? o g o g o g o g 10;? 13 fg 63 bed 38 def 38 def 10? 25 efg 63 bed 63 bed 63 bed 107 38 def 100 a 88 ab 75 abc All treatments were applied to wounds made by striking a flame-steril ized bolt placed against the surface of the bark with a hammer until the bark was broken and the xylem exposed. Prior to wounding, the area of the limb to be inoculated was swabbed with a cotton wad soaked in 95% ethanol. Fall = trees 10-20% defoliated, Spring = immediately after the trees had leafed out. % infection = (number of infections, as confirmed by the re-isolation of Cytospora sp./total of eight replicate inoculations) X 100. Percent values followed by the same letter are not significantly different according to Fisher's protected LSD (P = 0.05). 68 significant (P = 0.05). By contrast, spring inoculations of the peach isolate to cherry and the cherry isolate to peach resulted in a s ignif icantly higher percent infection than identical inoculations in the f a l l . With the peach i s o l a t e , th i s s i g n i f i c a n t increase was observed at a l l inoculum concentrations, whereas with the cherry isolate i t was observed only at inoculum concentrations of 103 and 105 spores/ml. Fall inoculation of cherry with the peach isolate at spore concentrations of 103 and 105 spores/ml did not result in a percent infection that was s ignif icantly different from the control treatments. Fall inoculation of the cherry isolate to peach at 103 spores/ml was also not s ignif icantly different from the control. Identical spring inoculations at spore concentrations of 103 spores/ml resulted in a s ignif icantly greater percent infection than the control treatments. Peach inoculated with the cherry isolate in the f a l l at 105 or 107 spores/ml had s ign i f icant ly higher percent in fect ion than cherry inoculated with the peach i so late. Similar comparisons of infections resulting from spring inoculations resulted in only spring inoculations of the cherry strain (105 spores/ml) to peach being s ignif icantly greater than the peach isolate to cherry. Only in one case were greater percent infections observed when the i so late was inoculated back to i ts host of or ig in rather than the alternate host. The peach isolate inoculated at 105 and 107 spores/ml in the f a l l caused a greater percent infection on peach than on cherry. No differences were observed in the spring inoculation of the peach isolate, nor in either f a l l or spring inoculations of the cherry isolate to either host. 69 Regardless of the i s o l a t e , host, or season of i nocu l a t i on , application of a spore suspension (5 x 105 spores/ml) to unwounded bark did not result in infection. Similarly, inoculations to fresh peach leaf scars made in October did not result in infection regardless of the method of spore application used. No Cytospora sp. was re-isolated from any of the control treatments. Susceptibil ity of Pruning Wounds to Infection by Cytospora sp. Spores Regardless of the i so la te , spore inoculations to fresh pruning wounds on cherry and peach always resulted in s i gn i f i c an t l y more infections and larger cankers than the respective control treatments (Table 12). Inoculation of 1-week-old peach wounds with the cherry i so late resulted in s i gn i f i c an t l y more in fect ion than the control treatments. However, inoculation of 1-week-old peach wounds with the peach isolate fai led to result in any infection. The percent infection resulting from inoculations to 1-week-old pruning wounds on cherry by either the cherry or peach isolates did not d i f fer significantly from the contro l t reatments . Regardless of the i s o l a t e - h o s t treatment combination, the lengths of the cankers on 1-week-old pruning wounds were not s ignif icantly different from the control treatments. There was no significant difference in the percent infection caused by the peach or cherry isolates when they were inoculated to fresh pruning wounds of e i ther host. However, the cherry i so late was s ign i f icant ly more virulent on cherry than the peach isolate was on peach. The cherry isolate also caused signif icantly more infection and longer cankers on fresh pruning wounds of cherry than i t did on fresh 70 TABLE 12. The effect of pruning wound age on the pathogenicity and virulence of representative cherry (C9-23) and peach (P8-19) isolates of Cytospora sp. inoculated as spore suspensions to mature 'Van' cherry and 'Fairhaven' peach trees 1n the spring Virulence Wound age canker leng Isolate Host when inoculated* 51! infection" (mm)y Cherry Cherry 0 (freshly made) 100 a 141.3 a Peach Cherry 0 88 ab 71 be Peach Peach 0 75 ab 102.3 b Cherry Peach 0 63 b 71 be Cherry Peach 1 week 63 b 21.5 de Cherry Cherry 1 week 25 c 20.8 de Peach Cherry 1 week 13 c 12.1 de Peach Peach 1 week 0 c 1.0 e Control z Cherry 0 0 c 1.0 e Control Peach 0 0 c 1.0 e Pruning wounds (diameter on peach = 1.0-1.5 cm, on cherry 1.5-2.0 cm) were made immediately after the trees had leafed out in the spring and were either inoculated Immediately after being made, or one week later, with 0.5 ml of a 2 x 105 spores/ml spore suspension from one of the Isolates. % Infection = (number of infections, as confirmed by the re- isolat ion of Cytospora sp./total of eight replicate Inoculations) x 100. Percent values followed by the same letter are not significantly different according to Fisher's protected LSD (P = 0.05). Canker length = distance from the pruning cut to the furthest extremity of the discolored t i ssue. No Infection = 1 mm. Mean of eight replicates followed by the same letter are not significantly different according to Fisher's protected LSD (P = 0.05). z Control = 0.5ml of s ter i l i zed, d i s t i l l ed water 71 wounds of peach. By comparison, there was no significant difference in the pathogenic i ty or virulence of the peach i so la te when i t was inoculated to fresh wounds of either cherry or peach. Effect of Temperature on Spore Germination Spore germination did not occur at 0, 5 or 35° C. Nevertheless, spores subjected to these temperatures for up to one week germinated within 24 hours after being placed at 25° C. More than 90% of the Cytospora sp. spores sprayed on peach twigs in vitro were viable after exposure to -18° C for one week. Over 90% germination occurred at temperatures ranging from 10 to 30° C (Figure 7). The e f fect of temperature on the length of the lag period before germination was isolate-dependent. A minimum lag period of 18 hours was observed for a l l isolates, but i t occurred at 27° C for the apricot isolate and at 25 and 27° C for the peach and cherry isolates. Effect of Temperature on Mycelial Growth The effect of temperature on the mycelial growth of the 15 Cytospora spp. isolates tested i_n v i tro was also isolate-dependent. Isolates varied not only in their temperature optima, but also in their relative growth response at other temperatures (Figures 8, 9, and 10). Even isolates P8-18, P8-19 and P8-20, which were obtained from the same peach orchard in Oliver, had different optimum temperatures (Table 13). None of the isolates grew at 1 or 5° C, f ive of the nine stonefruit isolates, and a l l four apple isolates obtained in B.C. grew at 35° C. Six of the nine isolates obtained from stonefruits in the Okanagan had a temperature optima of either 20 or 23° C, while two peach and one cherry 72 12 H 1 1 1 1 1 1 ' 1 0 10 20 30 40 Temperature (°C) Figure 7. Effect of temperature on the lag phase for spore germination of three Cytospora sp. i so lates: apricot (A5-16), cherry (C9-23) and peach (P8-19) on malt extract agar. The lag phase was the time (hours) required for 90% spore germination. Dishes were checked 12, 18, 24, 36, 48, 60 and 72 hours after inoculation. Each point is an average of three repl icate dishes. The analyses of variance indicated a s ignif icant temperature x isolate interaction (P = 0.05). 801 0 10 20 30 40 Temperature (°C) Figure 8. Effect of temperature on the mycelial growth of six Cytospora sp. isolates obtained from infected peach trees in the Okanagan Valley. Each point is a mean of duplicate measurements on each of four replicate dishes. Measurements were recorded after a 1-week incubation period on malt extract agar. 601 0 10 20 30 40 Temperature (°C) Figure 9. Effect of temperature on the mycelial growth of an apricot (A5-16) and two cherry (C9-23, Cl-28) isolates of Cytospora sp. obtained from the Okanagan Valley and of cincta 106 (ATCC). Each point is a mean of duplicate measurements on each of four rep l i ca te dishes. Measurements were recorded after a 1-week incubation period on malt extract agar. 0 10 20 30 40 Temperature (°C) Figure 10. Effect of temperature on the mycelial growth of four apple isolates of Cytospora sp. obtained from the Okanagan and Similkameen Val leys and of persoonii 105 (ATCC). Each point is a mean of duplicate measurements on each of four replicate dishes. Measurements were recorded after a 1-week incubation period on malt extract agar. 76 TABLE 1 3 . A c o m p a r i s o n b e t w e e n t h e o p t i m u m and max imum t e m p e r a t u r e s f o r t h e m y c e l i a l g r o w t h o f 13 C y t o s p o r a s p p . i s o l a t e s o b t a i n e d f r o m n a t u r a l l y i n f e c t e d s t o n e f r u i t and a p p l e t r e e s i n t h e Okanagan and S i m i l k a m e e n V a l l e y s o f B . C . and a u t h e n t i c i s o l a t e s o f c i n c t a and L^ p e r s o o n i i C o l o n y d i a m e t e r (mm) a t t e m p e r a t u r e (° C ) y I s o l a t e " O r i g i n a l L o c a t i o n 20 23 25 27 30 M a x . h o s t t e m p . C C) A 5 - 1 6 a p r i c o t O l i v e r 5 4 . 8 a z 30 P 8 - 1 9 p e a c h O l i v e r 4 5 . 6 b 30 P 9 - 4 p e a c h O l i v e r 4 5 . 4 b 30 P 1 2 - 3 4 p e a c h Oyama 3 5 . 5 c >35 P 8 - 1 8 p e a c h O l i v e r 46, . 3 a >35 C 9 - 2 3 c h e r r y O l i v e r 44, . 8 a 30 106 - - 7 . 6b 27 P 8 - 2 0 p e a c h O l i v e r 6 7 . 5a >35 P 3 - 2 6 p e a c h O l i v e r 5 4 . 8b >35 C l - 2 8 c h e r r y O s o yoo s 4 1 . ,1c >35 102 a p p l e Oyama 5 6 . 8 a >35 101 a p p l e V e r n o n 3 0 . 1 b >35 104 a p p l e Ke remeos 2 3 . 6 b >35 105 _ _ 56 . . l a >35 100 a p p l e V e r n o n 50 , . 6b >35 x I s o l a t e s 105 a n d 106 w e r e k n o w n i s o l a t e s o f L_^  p e r s o o n i i a n d L^ _ c i n c t a r e s p e c t i v e l y . y I s o l a t e s w e r e i n o c u l a t e d on MEA d i s h e s a t 1 , 5 , 1 0 , 1 5 , 2 0 , 2 3 , 2 5 , 2 7 , 30 o r 35° C f o r 1 w e e k . E a ch d i a m e t e r v a l u e i s a mean o f d u p l i c a t e m e a s u r e m e n t s on e a c h o f f o u r r e p l i c a t e p l a t e s . Max imum t e m p e r a t u r e = t e m p e r a t u r e a b o v e w h i c h no m y c e l i a l g r o w t h o c c u r r e d . z V a l u e s i n t h e same c o l u m n and f o l l o w e d by t h e same l e t t e r a r e n o t s i g n i f i c a n t l y d i f f e r e n t a c c o r d i n g t o F i s h e r ' s p r o t e c t e d LSD (P = 0 . 0 5 ) . 77 isolate had an optimum of 25° C (Table 13). The four apple isolates had optimum temperatures of 27 or 30° C. Sub-cultures of cincta and persoonii or ig ina l ly obtained from the ATCC by Dr. A.R. Biggs, had temperature optima of 23 and 30° C respectively. Epidemiology of Cytospora sp. A) Spore Collection in Diseased Orchards In 1986, current-season spore horns were observed on naturally infected trees at the end of June. Spore traps were placed beneath infected trees with sporulating stromata at the beginning of July and viable spores were collected following rainy periods from July 10 until freeze-up in mid-November (Figure 11). In both Oliver and Osoyoos, the concentrations of spores collected in July were comparable to those collected following rains in September and the f i r s t week of October. In both areas, there was an increase in the concentration of spores collected on November 12 compared to that collected on the two preceding collection dates. In 1987, current-season spore horns were not observed on infected trees' until the end of May. Nevertheless, viable spores were collected immediately following the f i r s t rainy period of 1987 which occurred on April 30 and May 1 (Figure 12). In Osoyoos, the concentrations of spores collected early in the spring (May) were lower than those collected in July. However, in Oliver, the concentrations of spores collected in May were comparable to those collected in July. A low concentration of spores was col lected in both Osoyoos and Oliver following the last rainfal l of the year (1987) during the f i r s t week of August. 78 o a 01 e o a co 6.0 -5.0 . 4.0 ~ 3.0 2.0 1.0 Spon— Rainwater I up—'-T— 160 - 140 - 120 - 100 - 80 - 60 _ 40 20 e a i c 'to CC 7/10 7/18 7/23 7/30 9/12 9/19 9/25 10/110/29 11/5 11/12 C o l l e c t i o n D a l e ( m o n t h / d a y ) B o Q. 0) o a co 3 - I 1 -Spores Rainwater II 7/10 7/18 7/23 7/30 9/12 9/19 9/25 10/110/29 11/5 11/12 - 160 - 140 - 120 - 100 - 80 _ 60 _ 40 20 a a 5 c C o l l e c t i o n D a t e ( m o n t h / d a y ) Figure 11. Concentration of waterborne Cytospora sp. conidia and volume of rainwater collected during rainy periods in the summer and f a l l of 1986 by funnel-type spore traps placed beneath infected trees with sporulating stromata. Values on each collection dfate in A are means of three spore traps placed beneath three infected trees in a peach orchard in Ol iver, and in B, of four traps placed beneath four infected trees in a peach orchard in Osoyoos, B.C. 79 Figure 12. Average temperatures and the concentration of waterborne Cytospora sp. conidia and volume of rainwater collected during rainy periods in the spring and summer of 1987 by funnel-type spore traps placed beneath infected trees with sporulating stromata. Temperature values obtained from local temperature recording centres, are averages for a 7-day period immediately preceding each collection date. Spore concentration and rainwater volumes for each collection date are means of four spore traps placed beneath four separate infected trees in a peach orchard in Oliver (A) and Osoyoos (B), B.C. N.D. = no data. 5/2 5/21 6/3 6/11 6/22 7/1 7/8 7/23 7/30 8/E Co l l ec t i on Date (month/day) VA spores ES] rainwater E "a 8. in i o x 0) a & 0 ) 6.0 5.0 4.0 3.0 " 2.0 -1.0 -n.d. n.d. E - 25 2 0 15 10 50 40 30 -20 10 5/21 6/3 6/11 6/22 7/1 7/8 7/23 7/30 8/6 C o l l e c t i o n Date (month/day) 81 In both years, the concentration of spores collected did not appear to be correlated with the volume of water in the traps. In some instances, increased spore counts from one trapping to the next reflected an increase in the amount of rainwater in the traps, while in other instances higher spore counts occurred in spite of a decrease in the volume of water collected compared to the previous collection date. In 1987, temperature had no apparent effect on the concentration of spores collected. In both Oliver and Osoyoos, the last week of June was the hottest week of the summer, and at the end of that week (7/1) the lowest concentration of spores was collected. However, the last week of July was the second hottest week of the summer and after that week (7/30), the greatest concentration of spores in 1987 was collected in Osoyoos, while in O l i ver an average concentration of spores was collected. On each spore co l lect ion date in 1986 and 1987, 0 to 5 x 104 spores/ml were detected in the rainwater collected by traps placed beneath healthy trees adjacent to trees with sporulating stromata. Spore concentrations similar to those obtained in traps placed beneath healthy trees in the summer and f a l l were collected when healthy limbs on trees adjacent to diseased ones were washed with d i s t i l l ed water in December 1986 (1 x 104 - 8 x 104 spores/ml). Spores collected beneath sporulating limbs at various times of the year when germinated on MEA were found to have at least 90% v iab i l i t y . Because of the large population of overwintering microflora, and the low concentration of Cytospora sp. spores collected, the exact percent of viable spores washed from healthy limbs in December 1986 was d i f f i c u l t to 82 determine. Nevertheless, the spores which could be observed were viable. Spores obtained from spore horns brought into the laboratory in December 1986 had > 90% v iab i l i t y . B) Experimental Irrigation of Infected Limbs During the a r t i f i c i a l irr igat ion of infected peach limbs, the rate of spore collection gradually decreased (Figure 13). The greatest number of spores (3 x 106 spores/cm2/h) were collected during the second 2-hour irrigation interval at the beginning of the experiment. At the end of the experiment, following 50 hours of intermittent i r r igat ion, 9 x 104 spores/cm2/h were collected. With the exception of the 2230-700 interval and the 700-930 irrigation interval on the third morning of the experiment, there was always a concomitant increase or decrease in the rates of spore and water collection from one irr igat ion interval to the next. However, the rate of spore col lect ion at the beginning of the experiment was generally greater than at similar rates of water collection near the end. Temperature had no apparent effect on spore col lect ion. On the f i r s t day of the experiment, a low concentration of spores was collected during the hottest part of that day, while on the second day, the highest concentration of spores for that day was collected during the hottest part of the day. Increasing or decreasing temperatures had no apparent effect on the relative rates of spore col lection. Chemical Control Germination of Cytospora sp. spores in vitro was inhibited at a minimum concentration of 0.1 ppm (a.i.) by captafol and f lus i lazo le , at 1 83 Figure 13. Average temperature (••••) and rates of water (—) and Cytospora sp. conidia collection (—) in funnel-type spore traps during a r t i f i c i a l i r r i g a t i o n of infected peach limbs having sporulat ing stromata. Breaks in the lines represent drying intervals. (A) Average temperature during the i r r i gat ion interval was calculated from the temperature reading at the beginning and end of each i n t e r v a l . Temperatures were measured using a mercury thermometer placed in a Stevenson weather screen situated just beyond the irrigated area. (B) Rates of spore and water collection are averages of eight traps placed 10 cm beneath four peach limbs with sporulating infections (two traps/limb). Spores ( x 10 spores/cnv/h) CD > Average Temperature (°C) u « o> CD o ro J> - ' - ' N U M M N U U c n c o o K } J > < n ' c o o r o Irrigation Water (ml/cm'/h) 85 ppm by iprodione, benomyl and dichlone and at 10 ppm by captan, t r i for ine and ziram. Benomyl, dichlone, f lus i lazole and ziram were applied to pruning wounds at concentrations 1000, 1000, 100 and 500 times greater than the concentration required for each respective fungicide to inhibit spore germination in v itro. Spray applications of the fungicides, as well as the boiled linseed o i l and Heal 'n ' Seal control treatments, were ineffective in preventing Cytospora sp. infections (P = 0.1) (Table 14). Although a higher percent infection occurred i f unamended Heal 'n ' Seal was applied to the wounds rather than linseed o i l , this difference was not s ignif icant. Of the four fungicides, only benomyl, when applied in combination with either Heal 'n ' Seal or linseed o i l , was effective in s ignif icantly reducing the incidence of Cytospora sp. infection compared to the untreated control. There was no significant difference in the effectiveness of benomyl when mixed with either Heal 'n ' Seal or linseed o i l . Because the partitioning of the sums of squares for the treatment effect indicated a significant effect of fungicide in preventing infection (P = 0.05), but not the method of app l i ca t i on nor the fungicide x method of appl icat ion interaction, the abi l i ty of each fungicide and the unamended carriers to prevent infection was averaged over the three methods of application and compared. On average, only benomyl (44%) resulted in s ignif icantly less infection (P = 0.05) than the mean of the unamended carriers (77%). The analys i s of variance on the canker lengths indicated a significant fungicide x method of application interaction (P = 0.05). Only benomyl, in combination with Heal 'n ' Seal or linseed o i l , resulted in significantly smaller cankers than the untreated controls (Table 15). TABLE 14. Effectiveness of post-inoculation fungicide treatments in preventing infection of fresh pruning wounds by a r t i f i c i a l l y applied Cytospora sp. sporesx Cytospora sp. infection (%) five months after the inoculated pruning wounds were treated with Carrier plus  Carr ier Carr ier alone Benomy1y Dichlone F l u s i l a ? o l e Ziram Heal 'n ' Seal 88 ab 2 33 c 55 be 100 a 88 ab Linseed o i l 66 abc 33 c 55 be 88 ab 55 be Water 77 ab 66 abc 66 abc 66 abc 66 abc Fungicide treatments were applied 24 hours after peach wounds (1-2 cm diameter) were inoculated with 4 x 105 spores of a peach isolate of Cytospora sp. (P18-19). Benomyl, dichlone, f lusi lazole and ziram were applied at rates of 1000, 1000, 10 and 5000 ppm active ingredient respectively. % Infection = (number of infections, confirmed by the re-isolation of Cytospora sp./total of nine replicate inoculations) X 100. Percent values followed by the same letter are not significantly different according to Fisher's protected LSD (P = 0.1). CO CTl TABLE 15. Influence of fungicide treatments in limiting Cytospora sp. canker elongation on peach trees following a r t i f i c i a l inoculation of fresh pruning wounds with a spore suspension of Cytospora sp. Canker length (mm) five months after the inoculated pruning wounds were treated with y Carrier plus  C a r r i e r C a r r i e r alone Benomvl ni chi one F1usila7ole Ziram Heal 'n ' Seal 62.3 bc z 10.2 d 106.4 a 83.5 ab 95.8 ab Linseed o i l 41.5 cd 9.8 d 29 cd 34.6 cd 36.3 cd Water 47.5 c 38.5 cd 40 cd 41 cd 40.5 cd Fungicides applied at the rates and according to the methods described in Table 14. Canker length measured from the pruning cut to the furthest elongation of the discolored cambial tissue. Unsuccessful infection - fai lure to re-isolate Cytospora sp. five months later = canker length of 1 mm. Means of nine replicates followed by the same letter are not signif icantly different according to Fisher's protected LSD (P = 0.05). 88 The H e a l 'n' S e a l and l i n s e e d o i l c o n t r o l t r e a t m e n t s w e r e i n e f f e c t i v e i n r e d u c i n g c a n k e r l e n g t h compared t o t h e u n t r e a t e d c o n t r o l . A l t h o u g h t h e unamended H e a l 'n' S e a l t r e a t m e n t r e s u l t e d i n l o n g e r c a n k e r s t h a n t h e unamended l i n s e e d o i l o r u n t r e a t e d c o n t r o l t r e a t m e n t s , t h e s e d i f f e r e n c e s were n o t s i g n i f i c a n t . A p p l i c a t i o n s o f d i c h l o n e , f l u s i l a z o l e o r z i r a m i n c o m b i n a t i o n w i t h H e a l 'n' S e a l r e s u l t e d i n s i g n i f i c a n t l y l o n g e r c a n k e r s t h a n i f t h e f u n g i c i d e s were a p p l i e d as w a t e r s p r a y s o r i n c o m b i n a t i o n w i t h l i n s e e d o i l . DISCUSSION 89 Stonefruit dieback was found to be widespread among a i l stonefruit cultivars in the Oliver-Osoyoos area. In 1986, trees exhibiting dieback symptoms typical of Cytospora sp. were found in 27 of the 30 stonefruit blocks surveyed and in 18 of these blocks, > 1% of the trees were affected. From 1986 to 1987, the problem continued to spread (2.9%) in most (11 out of 17) blocks. From September 1985 to September 1987 dieback affected an average of 14.8% of the trees in the 17 blocks surveyed in 1986 and 1987. The incidence in the individual blocks ranged from 56.9 to 3.0%, with losses occurring at a time when the trees were most productive, and were due not only to the removal of entire trees but also to the debil itating effect of repeated pruning of scaffold limbs. Some orchardists have incurred serious economic losses in the last 2 years and are considering removing entire blocks from production. The fact that many of the dead limbs had stromata exuding hyaline, allantoid spores in reddish spore horns suggested that a Cytospora sp. species might be responsible for the dieback of stonefruits. The o l ive-green color and growth patterns of cultures obtained from spore horns exuded from stromata were similar to those obtained from the margins of diseased tissues of the majority of trees (80%) with limb dieback. Inoculation of these cultures to 1-year-old peach trees and to the limbs of mature 'Van' cherry and 'Fairhaven' peach trees resulted in typical dieback symptoms and the development of stromata identical to those observed on naturally infected trees. No dieback symptoms occurred with the control treatments. Subsequent re-isolation of Cytospora sp. from 90 the inoculated trees and limbs f u l f i l l e d Koch's postulates and confirmed that a Cytospora sp. was the primary cause of stonefruit dieback in the Okanagan. Closer examination of the morphological characterist ics of the stromata found on infected stonefruits revealed a convoluted chamber, which was sometimes subdivided, but having a central column and a single o s t i o l e . These features correspond to those of C^ leucostoma as described elsewhere (10, 29, 51) and distinguish i t from C^ cincta, the other Cytospora sp. species reported to infect stonefruits. (9, 17) The size of the stromata on cherry (2.0-2.5 (x = 2.4) x 1.3-1.7 (x = 1.5) mm) and peach (2.3-2.8 (x = 2.6) x 1.1-1.6 (x = 1.3) mm) was similar to that found by Defago (2.86 ± .67 x 1.5 ± .36 mm) (10). Kastir (29) and Kern (30) reported stromata diameters of 1.8-3.0 mm and 1.0-1.5 mm respectively for C_^  leucostoma on stonefruits. Spore dimensions measured in this study (on peach 4.0-6.5 (x = 5.0) x 0.5-1.5 (x = 1.5) pm, on cherry: 4.0-6.5 (x = 5.5) x 1.0 - 1.5 (x = 1.5) um are in f a i r l y close agreement with those found by Kastir (29) (5.9 x 1.4 pm), Urban (51) (4.0-6.0 x 1.0-1.4 um) and Kern (30) (4.0 - 6.5 x 0.5 - 1.5 pm), but are generally larger than those reported for C_^  leucostoma by Defago (10) (5 ± 0.13 x 1.05 ± 0.05 um). The olive-green color of cultures of the fungus isolated from the margins of infected tissue and of spore horns exuded from stromata distinguish C^ leucostoma from other Cytospora spp. (Kastir (29), Spielman personal communication). Based on the morphology and size of the stromata, spore dimensions and cultural character i s t ics , the causal organism of stonefruit dieback in the Okanagan most closely resembles descriptions of C_^  leucostoma. Since the sexual stage (L^ 91 persooni i ) has not been found, conf i rmat ion of t h i s ten ta t i ve identif ication is impossible. Therefore, the organism wil l be referred to as Cytospora spp. The temperature optima for the Cytospora sp. isolates obtained from infected stonefruit trees in the Okanagan (23° C) was in close agreement with that found by Helton (20° C) (23) and Defago (20-24° C) (10) for isolates of C^ leucostoma. The difference between this temperature optimum (23° C) and that of persooni i (30° ) and the optimum (30° C)reported by Bertrand and English in California (1) might be due to inherent differences between isolates of the di f ferent populations. Differences among the temperature optima reported for persoonii in the literature might also be due to the mistaken identif ication of the fungus because of the apparent absence of the sexual stage in many regions. The temperature optima for the mycelial growth of C^ leucostoma in vitro should not be based on results obtained from one or two isolates given the significant temperature x isolate interaction found in this (Figures 8, 9 and 10) and other studies (10, 23). Mistaken identif ication of C^ cincta might also be responsible for the differences in temperature optima reported in the l iterature. The temperature optimum of cincta (23° C) reported in this study agrees closely with that of Defago (22-24° C) (10) and Bertrand and English (24° C) (1) but di f fers from that reported by Helton (30° C) (23). Stromata exuding hyaline, allantoid spores in reddish to purplish spore horns are the diagnostic sign of Cytospora sp. infection. However, they are not always associated with a l l dead limbs or trees. This can pose a problem in the correct diagnosis of the cause of dieback. 92 However, the high percent of Cytospora sp. recovered from the diseased margins of sporulating infections from early spring unt i l late f a l l indicates that Cytospora sp. can be diagnosed with confidence i f infected tissues are plated on MEA. As in B.C., Cytospora sp. infection in Idaho and California is characterized by the rapid dieback (2-3 years) of infected limbs. Perennial cankers are not observed on any of the diseased trees. In Colorado and Ontario, however, perennial cankers are present on infected trees, and infected limbs take 5-6 years to d ie. The absence of perennial cankers in B.C., Idaho and California suggest a more rapid invasion of vascular tissue by the fungus and/or a less effective host response in these regions than in Colorado or Ontario. The length of time between the in i t iat ion of infection and the appearance of dieback symptoms on naturally infected stonefruit trees in the Okanagan is unknown. However, some limb dieback symptoms were apparent 3-6 months following mycelial inoculations to mature cherry and peach trees in October 1986 and Apr i l 1987. S imi lar inocu la t ions using spore suspensions had not resulted in dieback symptoms as of 30 October 1987. As in B.C., C^ leucostoma has been identified as the causal organism of stonefruit dieback in Cal i fornia, Colorado and Ontario. However, unlike other regions (19, 46), isolates from apricot and peach trees in B.C. were pathogenic to peach and cherry respectively. In addition, B.C. isolates of C_^  leucostoma di f fer from those of Michigan, California and Ontar io in the pa t te rn of t h e i r p ro te in bands f o l l ow ing gel electrophoresis (Adams, personal communication). The differences in the pathogenicity and protein patterns of Cytospora sp. isolates from B.C. 93 suggest that they might be different from those found in other areas of North America. If the B.C. isolates are more pathogenic than the ones found in other areas, then this might explain the inabi l i ty of the host to part ia l ly wall-off infections and the absence of perennial cankers on infected trees in B.C. It is possible that a different species of Cytospora sp. is present in the Okanagan: one whose asexual stage closely resembles that of C_^  leucostoma, but whose sexual stage is different from persoonii. More research is needed to compare the pathogenicity of Cytospora sp. isolates from B.C. with those from other regions of North America. The presence or absence of perennial cankers in various regions of North America is not a r e f l e c t i on of d i f ferences in the re l a t i ve susceptibil ity of local cultivars to Cytospora sp. 'Redhaven', the major peach cul t ivar grown in B.C., is considered moderately resistant to infection in other peach growing areas of North America (38, 42, 53). The rapid dieback symptom observed in B.C. is probably not due to the fact that the major B.C. cultivars are more susceptible to infection than those commonly grown in Ontario or Colorado. Pruning wounds are the major infection court for Cytospora sp. in B.C. and Colorado. In Cal i fornia, Idaho and Ontario, the major infection courts are sunburn injury, mild low-temperature winter injury and leaf scars respectively. One might expect that exposure of a large amount of vascular tissue following wounding would result in a more rapid invasion of Cytospora sp. than i f very l i t t l e vascular tissue was exposed. However, differences in the infection courts and the accessibi l ity of vascular tissue does not appear to explain the presence or absence of 94 perennial cankers in various areas. In Colorado, perennial cankers are present on infected trees even though pruning wounds offer excellent accessibi l ity to the vascular tissue. In Idaho and California perennial cankers are not observed in spite of the inaccessibil ity of vascular tissue. The unusually cold temperatures experienced in the 01iver-Osoyoos area from January 5-7, 1982, might explain the sudden and simultaneous increase of Cytospora sp. in orchards throughout the region in the following years. During this period, temperatures were unseasonably cold with mean daily temperatures of -12 to -15° C, and a minimum temperature of -20° C on January 6. This temperature, (-20° C) was the second lowest recorded in Osoyoos in the last seven years. The lowest (-25° C) occurred on November 28, 1985. Although severe winter injury on peach trunks usually occurs at temperatures below -25° C, mild low-temperature injury can occur at higher temperatures. A tree's susceptibil ity to winter injury depends upon the hor t i cu l tura l pract ices followed the previous summer because they influence the winter hardiness of the tree. If the trees did indeed experience some mild low-temperature injury in January 1982, then the presence of a large number of potential infection courts would have provided an opportunity for isolates of the existing endemic population to spread and infect new trees. Subsequent sporulation of these infections would have resulted in an increase in the inoculum pressure within the orchards and rendered a l l types of wounds more prone to infect ion. The high inoculum pressure within the orchards and the necessity of annual spring pruning would have promoted the continued 95 spread of Cytospora sp. within the blocks. Infected nursery stock, or the emergence of a highly pathogenic mutant of Cytospora sp. from the endemic population, cannot explain the sudden and simultaneous increase of the disease in stonefruit blocks throughout the Oliver-Osoyoos area. If these explanations were va l id, then one would have expected a slow and gradual spread of Cytospora sp. from the orchards where infected nursery stock were planted or from the single orchard where the mutation occurred. It is not known whether choke cherry (Prunus virginiana L.), or pin cherry (Prunus pensylvanica L.) are infected by C_^  leucostoma in B.C. (9,17). C^ leucostoma has been reported on choke cherry in Ontario and Nova Scotia and a Cytospora sp. has been reported on pin cherry in Ontario. Because choke cherry is an alternate host for several diseases of Prunus spp., i t has been eradicated from land adjacent to the orchards. The hot summer temperatures in the southern Okanagan limit the amount of natural vegetation in the h i l l s adjacent to the orchards. Artemisia sp. and Pinus ponderosa, the most common native plants in the Oliver-Osoyoos area, are not reported to be hosts of C^ leucostoma (9,17). Valsa pini has been reported to infect ponderosa pine in B.C. (48). However, research in Idaho (19) suggests that Cytospora isolates obtained from native tree hosts are generally nonpathogenic on cultivated stonefruits. It is therefore unlikely that native wild plants in the Oliver-Osoyoos area act as reservoirs of Cytospora sp. inoculum for infection of stonefruits. The disease index (Table 8), might be as good an indicator of the virulence of the isolates as canker size. In this study, many of the 96 isolates did not d i f fer s ignif icantly in their virulence. A difference of 36 mm separated the most virulent isolate (Pl-7) from the least v i ru lent one (P l l -14) . However, i f dead trees and trees with no infections were excluded from the results, a difference of only 17.7 mm separated the largest canker from the smallest one. This suggests that there is l i t t l e va r i ab i l i t y in the abi l i ty of the isolates to cause canker elongation once they have i n i t i a ted in fec t ion . The high correlation between the disease index of the isolates and the size of the cankers they caused (r s = .917) supports the claim that the virulence of the isolates can be compared using a disease index scheme where a l l expanding cankers are given the same value. Regardless of the age of the peach trees, the cherry (C9-23) and peach (P8-19) i s o l a t e s did not d i f f e r s i g n i f i c a n t l y in t h e i r pathogenicity when plugs of mycelium were used as the source of inoculum. There was no significant difference in the pathogenicity of the peach and cherry isolates when they, were inoculated to 1-year-old peach trees. Similarly there was also no significant difference in the pathogenicity of the isolates i f they were inoculated to the limbs of mature peach trees in either the f a l l or spring. The cherry and peach isolates also did not generally d i f fer s ignif icantly in their pathogenicity when spore inoculum was used, however, one exception was noted. At each spore concentration there was no significant difference in the pathogenicity of the peach and cherry isolates i f they were inoculated to limbs of mature peach in the f a l l . In the spring, however, the cherry isolate inoculated at 105 spores/ml was more pathogenic on peach than the peach isolate was. At 103 or 107 spores/ml there was no s ignif icant difference in the 97 pathogenicity of the isolates. Similar host specif ic ity relationships were generally observed for the peach and cherry isolates at a l l the spore concentrations tested. The cherry isolate was as pathogenic on cherry as i t was on peach at each spore concentration in both fa l l and spring inoculations. In the spring, the peach isolate was as pathogenic on peach as i t was on cherry at each spore concentration. However, in the f a l l , the peach isolate was less pathogenic on cherry than on peach at 105 and 107 spores/ml. Host spec i f i c i t y has important implications in the tree-to-tree spread of Cytospora sp. within blocks planted entirely to one stonefruit species, as well as in the spread of the disease from one species of stonefruit to another. Results obtained from spore inoculations at 103 spores/ml are important in understanding intra and inter-species spread of the disease because this is the spore concentration commonly found on healthy trees surrounding diseased ones. Tree-to- t ree spread of Cytospora sp. within blocks planted entirely to either peach or cherry can occur equally well in both the f a l l and spring. Based on the percent infection caused by the isolates at 103 spores/ml, the disease is as l ikely to spread within infected cherry blocks as i t is in infected peach blocks in both the spring and f a l l . Since the percent infection caused by the f a l l inoculation of 103 spores/ml of the cherry isolate to peach or the peach isolate to cherry was not s i gn i f i c an t l y d i f ferent from the control treatments i t is unlikely that very much interspecies spread of the disease occurs in the f a l l . It can, however, occur in the spring. In the spring, sporulating cherry trees can serve as a source of inoculum for healthy cherry or 98 p e a c h t r e e s e q u a l l y w e l l . I n a d d i t i o n a h e a l t h y c h e r r y t r e e i s a s l i k e l y t o b e i n f e c t e d b y i n o c u l u m f r o m i n f e c t e d c h e r r y t r e e s a s i t i s f r o m i n f e c t e d p e a c h t r e e s . T h e s e s a m e p r i n c i p l e s a l s o a p p l y t o i n f e c t e d a n d h e a l t h y p e a c h t r e e s . I n t h e s p r i n g , C y t o s p o r a s p . i s a s l i k e l y t o s p r e a d f r o m c h e r r y t o p e a c h a s i t i s f r o m p e a c h t o c h e r r y . I n t e r s p e c i e s s p r e a d o f C y t o s p o r a s p . i s p r o m o t e d b y t h e s u s c e p t i b i l i t y o f c h e r r y a n d p e a c h t r e e s t o " f o r e i g n " i n o c u l u m i n t h e s p r i n g , a n d b y t h e f a c t t h a t p r u n i n g c u t s a r e a l s o m a d e a t t h i s t i m e o f t h e y e a r . T h e g r e a t e r s u s c e p t i b i l i t y o f t h e h o s t s i n t h e s p r i n g i s s u r p r i s i n g i n v i e w o f t h e i n c r e a s i n g r a t e o f h o s t a c t i v i t y w h i c h o c c u r s a t t h i s t i m e c o m p a r e d t o t h e d e c r e a s i n g r a t e w h i c h o c c u r s i n t h e f a l l . M o r e r e s e a r c h n e e d s t o b e d o n e o n t h e s u s c e p t i b i l i t y o f p r u n i n g w o u n d s t o " f o r e i g n " i n o c u l u m i n t h e s p r i n g b e c a u s e t h e y , a n d n o t " c r u s h e d - b a r k " w o u n d s , a r e t h e m a j o r i n f e c t i o n c o u r t s i n d i s e a s e d o r c h a r d s i n B . C . . C o n f l i c t i n g r e s u l t s c o n c e r n i n g t h e p a t h o g e n i c i t y o f t h e c h e r r y i s o l a t e ( 1 0 5 s p o r e s / m l ) w h e n i t w a s i n o c u l a t e d t o f r e s h p r u n i n g w o u n d s o f c h e r r y a n d p e a c h c o m p a r e d t o i d e n t i c a l i n o c u l a t i o n s t o " c r u s h e d - b a r k " w o u n d s s u g g e s t s t h a t t h e a b i l i t y o f t h e h o s t t o p r e v e n t i n f e c t i o n c a n d e p e n d o n t h e t y p e o f w o u n d t h a t i s i n o c u l a t e d . C y t o s p o r a s p . d o e s n o t s p r e a d f r o m i n f e c t e d a p p l e t r e e s t o h e a l t h y s t o n e f r u i t t r e e s o r v i c e v e r s a . I n n a t u r a l l y i n f e c t e d o r c h a r d s i n B . C . , t h e a m b e r - c o l o r e d s p o r e h o r n s f o u n d o n a p p l e t r e e s i n f e c t e d w i t h C y t o s p o r a s p . w e r e n o t f o u n d o n s t o n e f r u i t t r e e s , w h i l e t h e d a r k - r e d s p o r e h o r n s c h a r a c t e r i s t i c o f C ^ l e u c o s t o m a w e r e n e v e r o b s e r v e d o n a p p l e t r e e s . T h e h o s t s p e c i f i c i t y o f t h e i s o l a t e s w a s c o n f i r m e d i n t h i s s t u d y w h i c h d e m o n s t r a t e d t h a t i s o l a t e s o f C ^ l e u c o s t o m a c a n n o t i n f e c t a p p l e 99 trees, while apple isolates of Cytospora sp. generally cannot infect peach trees. The specif ic i ty of the apple and stonefruit isolates of Cytospora sp., as well as differences in stromata morphology and colony characteristics on MEA suggest that they are different species. In the f i e l d , Cytospora sp. infections were only observed on apple trees that had previously been exposed to severe and extensive injury due to cold winter temperatures or f i r e . This suggests that the Cytospora sp. species found on apple is either a secondary organism which colonizes t issue previously infected by another organism, or a weak pathogen requiring an extensive amount of dead tissue before i t can become established in the host. Perhaps the wounds made on the otherwise healthy and vigorous apple trees did not provide enough dead tissue to enable the apple isolates of Cytospora sp. to in i t iate infection. In the Okanagan, the majority of new Cytospora sp. infections on previously healthy trees occurs through pruning wounds. Since peach trees require much more pruning than apricot or cherry trees i t is not surprising that Cytospora sp. is spreading most frequently and to the greatest extent in peach blocks. Regardless of the inoculum pressure, successful infection requires a susceptible host. Since Cytospora sp. spores can only be dispersed by rain, infection can only occur i f i t rains while pruning wounds are susceptible to infection. The time and method of pruning, and tree vigor, are factors which influence the length of time that pruning wounds remain susceptible to Cytospora sp. infection in the spring. Since a l l orchardists claimed that they pruned their stonefruits in March, the timing of the pruning operation is not the reason for the differences in the percent of newly 100 infected trees among the orchards in 1987. However, the ab i l i ty of the orchardists to avoid making large pruning cuts, leaving pruning stubs, or allowing narrow crotch angles to develop can vary a great deal. Also the horticultural practices which allow for the establishment of healthy and vigorous trees capable of rapid healing-over following wounding, varies from orchard to orchard. The lack of a corre lat ion between the percent of sporulating infections in diseased orchards in 1986 and the percent of newly infected trees in 1987 is probably due to the large number of different orchard management practices which affect not only the number of infection courts which are established, but also influence a tree's susceptibi l ity to in fect ion by af fect ing i t s a b i l i t y to heal-over wounds. Because Cytospora sp. is a wound pathogen dispersed by rain, disease spread would be minimal in orchards where the majority of the pruning wounds had healed-over before the spring rains began. Trees having a slow rate of healing-over, however, would be prone to infection when the spring rains began even in blocks with low inoculum pressure. In this manner, Cytospora sp. might spread more rapidly in orchards having a low inoculum pressure but whose trees also had a slow rate of healing-over, than in blocks with a high inoculum pressure but whose trees could heal-over wounds rapidly. Spore trapping done in this study demonstrated that Cytospora sp. has a tremendous inoculum potential and that spores can be dispersed by wind-blown rain at any time of the year. Any wounds made on stonefruit trees in the 01iver-Osoyoos area from mid-March until mid-October would be susceptible to infection since mean daily temperatures during this 101 period are > 10° C. New infections could occur in diseased orchards by at least four different scenarios. Pruning wounds made in the spring could be infected by spores from overwintered spore horns which were dispersed to the wounds by spring rains. Alternatively, pruning wounds made in the spring could be infected by spores that were dispersed in the f a l l and overwintered on healthy trees which they infected following pruning. Spores, dispersed in the f a l l , could infect wounds made at that time. There is also the possibi l i ty that pruning wounds made in the spring might not have suff icient time to heal-over before current-season spores were exuded and dispersed. The observations made in this study suggest that some of these alternatives are more important than others. In the Okanagan, spores in the spore horns exuded from infected trees in the summer were s t i l l viable in December. In addition, viable spores were collected in traps placed beneath healthy trees adjacent to diseased ones in early spring before current-season spore horns were exuded. These facts support the hypothesis that overwintering spore horns can serve as a source of inoculum for infections in the spring. This is the f i r s t study to demonstrate that viable Cytospora sp. spores are present on healthy trees during dry periods of the year. The fact that Cytospora sp. spores remained viable after a 1-week exposure to-18° C i_n vitro supports the claim that in the Okanagan, Cytospora sp. spores can overwinter on adjacent healthy trees and in i t ia te infection when pruning cuts are made in the spring. Mechanical implements and leaf f a l l are the major causes of wounds made on stonefruit trees in the f a l l . Since a low percent of natural infections were associated with mechanical injury and infections through 102 leaf scars does not occur in the Okanagan, Cytospora sp. probably does not spread to a great extent in the f a l l . There are no reports in the l iterature concerning how long pruning wounds made in the spring remain susceptible to infection by Cytospora sp. spores. This study suggests that 1-week-old pruning wounds made in the spring are generally resistant to infection. However, since the experimental trees were pruned when they were in f u l l leaf, the greater physiological activity of the tree, and the higher ambient temperatures at the end of Apr i l , would have promoted a faster rate of healing-over than might normally be expected in commercial orchards where pruning was done when the trees were dormant in March. Nevertheless, based on results from this experiment and work done with plugs of C_^  leucostoma inoculated to "crushed bark" wounds in Ontario (5), i t is probable that pruning cuts made in commercial stonefruit orchards in B.C. are resistant to infection 3-4 weeks after being made. In the Okanagan, current-season spore horns were observed on naturally infected stonefruits 2-3 months after the pruning operation would normally have been completed. It is therefore unl ikely that current-season spore horns provide the inoculum for infection of pruning wounds made earl ier that same year. Pruning wounds are the primary infection court for Cytospora sp. in the Okanagan and therefore an effective control strategy must be geared towards protecting these wounds from infection. The fungicide t r i a l was a factorial experiment. A partitioning of the sums of squares for the main treatment effect i l lustrated a significant (P = 0.05) effect of fungicides in preventing Cytospora sp. i n fec t ion . The method of appl icat ion, and the fungicide x method of application interaction 103 f a c t o r s were no t s i g n i f i c a n t . C o m p a r i s o n o f t h e f o u r f u n g i c i d e p e r c e n t means w i t h t h e unamended c a r r i e r p e r c e n t i n f e c t i o n mean (77%) u s i n g F i s h e r ' s p r o t e c t e d LSD (P = 0 . 1 ) d e m o n s t r a t e d t h a t on a v e r a g e , o n l y benomyl (44%) s i g n i f i c a n t l y r e d u c e d t h e i n c i d e n c e o f i n f e c t i o n . Upon e x a m i n i n g t h e d a t a i t appeared t h a t i f benomyl was a p p l i e d as a s p r a y (66%) i t m i g h t no t be s i g n i f i c a n t l y more e f f e c t i v e i n r e d u c i n g i n f e c t i o n t h a n t h e unamended w a t e r s p r a y t r e a t m e n t (77%). T h i r d l y , i n o r d e r t o i l l u s t r a t e p r e c i s e l y w h i c h t r e a t m e n t s were most e f f e c t i v e i n p r e v e n t i n g i n f e c t i o n s , a n d t h e r e b y m a k e i t p o s s i b l e t o p r o v i d e s p e c i f i c r e c o m m e n d a t i o n s t o t h e o r c h a r d i s t s , a l l 15 means were compared u s i n g F i s h e r ' s p r o t e c t e d LSD. The e f f e c t o f t h e c a r r i e r s on t h e r e l e a s e o f t h e f u n g i c i d e s was unknown a t t h e b e g i n n i n g o f t h e e x p e r i m e n t . The e f f e c t i v e n e s s o f benomyl m i x e d w i t h H e a l ' n ' S e a l o r l i n s e e d o i l was p r o b a b l y b e c a u s e t h e s e c a r r i e r s a l l o w e d f o r t h e g r a d u a l r e l e a s e o f t h e f u n g i c i d e i n t o t h e l i m b as r e p o r t e d by F l e t c h e r and Shape ( 1 4 ) . S p r a y a p p l i c a t i o n s e v e n t u a l l y d r y o u t on t h e a p p l i e d s u r f a c e s and t h e n t h e f u n g i c i d e i s no l o n g e r a b l e t o p e n e t r a t e i n t o t h e l i m b s t o p r e v e n t i n f e c t i o n . In t h i s s t u d y , t h e h i g h c o n c e n t r a t i o n o f i n o c u l u m u s e d , and t h e 2 4 -hour d e l a y between i n o c u l a t i o n o f t h e p r u n i n g wounds and t h e a p p l i c a t i o n o f t h e f u n g i c i d e t r e a t m e n t s , f a v o r e d t h e i n i t i a l d e v e l o p m e n t o f t h e p a t h o g e n . In c o m m e r c i a l o r c h a r d s , a p p l i c a t i o n o f a benomyl and Hea l 1 n ' S e a l m i x t u r e t o a l l p r u n i n g wounds ( > 2 - 3 cm d i a m e t e r ) i m m e d i a t e l y a f t e r t h e y a r e made s h o u l d p r o v i d e e x c e l l e n t p r o t e c t i o n a g a i n s t C y t o s p o r a s p . s p o r e s d i s p e r s e d by e a r l y s p r i n g r a i n s . Use o f l i n s e e d o i l as a c a r r i e r i s not recommended because o f i t s p h y t o t o x i c e f f e c t s on t r e a t e d l i m b s . 104 Application of fungicide pastes to pruning cuts (> 2 cm diameter) immediately after they are made is a labor intensive process. Given the facts that Cytospora sp. spores can overwinter on healthy trees, and that orchardists in the Oliver-Osoyoos area normally apply dormant sprays of lime sulphur or ferbam in the spring to control Coryneum blight, the opportunity exists for integrated control. Although dormant sprays of lime sulphur or ferbam are effect ive against Coryneum, there are no reports in the l iterature concerning the effectiveness of these or other fungicides in reducing the population of overwintering Cytospora sp. spores. More research is needed to identify a broad spectrum fungicide which could be applied as a dormant spray and which would be effective in reducing or eradicating the overwintering inoculum of both Cytospora sp. and Coryneum. Intensive research in other peach growing areas of North America has f a i l ed to ident i fy a fungicide which is e f fect ive in eradicat ing established infections. As a result, current control strategies are based on preventing new infections from occurring. Since Cytospora sp. can infect any wounds made on the trees, and because spores are present throughout the year, any preventative control strategy must be integrated into a l l aspects of orchard management. The following f ive orchard management practices aimed at encouraging a rapid rate of wound healing, and reducing the presence of inoculum within the orchards, should be followed in order to reduce the incidence of new infections. F i r s t l y , trees should be maintained in a vigorous state. Trees stressed by insects, drought or nutrient def i c ienc ies are more susceptible to infect ion because of the i r i n a b i l i t y to heal-over wounds rap id ly . 105 Secondly, pruning should be done as late in the spring as possible to take advantage of the more rapid rate of wound healing which occurs at higher temperatures. Thirdly, in order to encourage a rapid rate of healing-over, branches should be cut just beyond the ridge of thickened bark which connects them to larger limbs. Pruning stubs should never be lef t . The pruning operation should be well planned every year so that large pruning cuts can be avoided, however, i f large cuts (> 2 cm) are necessary, they should be covered with a mixture of benomyl and Heal 'n ' Seal (1 g a.i./L) immediately. Fourthly, the trees should be trained properly so that wide crotch angles are developed. Tissues in narrow crotch angles are susceptible to winter injury or sp l i t t ing ; i f the branch is carrying a heavy load of f ru i t . And f i na l l y , sporulating infections on scaffold limbs or trees should be removed immediately and burned because they are a source of inoculum. Until resistant stonefruit cultivars and/or eradicant fungicides are identif ied, a successful control strategy must stress the prevention of new Cytospora sp. infections and be incorporated into a l l aspects of orchard management. Adherence to the integrated control program outlined above wi l l reduce the incidence of new Cytospora sp. infections and result in extended orchard l i f e , lower replant costs, and greater economic returns to the orchardists. 106 SUMMARY 1. Stonefruit dieback was widespread on a l l stonefruit cult ivars throughout the 01iver-Osoyoos area. 2. From September 1985 to September 1987 the number of trees affected by the disease ranged from 3.0-56.9% and averaged 14.8%. 3. New infections were evident on an average of 2.9% of the trees in 11 of the 17 orchards re-surveyed in 1987. 4. Cytospora leucostoma was tentat ive ly ident i f ied as the causal organism of stonefruit dieback in the Okanagan and was restricted to stonefruits. 5. In the Okanagan, the symptoms of the disease are different from those in Ontario or Colorado. 6. The majority of Cytospora sp. infections (66%) were found on the scaffold limbs and were associated with pruning wounds (65%). Leaf scar infections do not occur in the Okanagan. 7. Diagnosis of Cytospora sp. infection was shown to be possible anytime during the growing season i f margins of infected tissue were plated on MEA. 8. A new twig inoculation technique was developed for the production of Cytospora sp. spores. 9. Intraspecies spread of Cytospora sp. in cherry or peach blocks can occur in both the f a l l and spring. 10. Cytospora sp. is more l ikely to spread from cherry to peach or from peach to cherry in the spring than in the f a l l . 11. An unidentified Cytospora sp. species was found on apple trees in the Okanagan and Similkameen Valleys but did not infect stonefruits. 12. Although C^ leucostoma spores germinated most rapidly at 27° C, germination did occur at 10° C, and spores remained viable even after exposure to -18° C for 1 week. 13. The effect of temperature on mycelial growth i_n vitro was isolate-dependent, with optima for most isolates ranging from 20-23° C. 14. In the Okanagan, pruning wounds made in the spring can be infected by overwintered spores produced the previous summer which were dispersed to the wounds by spring rains, or by spores which were dispersed in the f a l l and overwintered on the trees which they 107 infected when the cut was made in the spring. Benomyl mixed with Heal 'n ' Seal or linseed o i l (1 g a.i./L) was effective in preventing Cytospora sp. infection i f the mixture was applied to pruning wounds within 24 hours after they had been inoculated with a suspension of Cytospora sp. spores. 108 LITERATURE CITED 1. Bertrand, P.F. and English, H. 1976. Virulence and seasonal activity of Cytospora leucostoma and C_^  cincta in French prune trees in California. Plant Dis. Rep. 60: 106-110. 2. Bertrand, P.F. and English, H. 1976. Release and dispersal of conidia and ascospores of Valsa leucostoma. Phytopathology 66: 987-991. 3. Biggs, A.R. 1984. Boundary-zone formation in peach bark in response to wounds and Cytospora leucostoma infection. Can. J . Bot. 62: 2814-2821. 4. Biggs, A.R. 1986. Phellogen regeneration in injured peach tree bark. Ann. Bot. 57: 463-470. 5. Biggs, A.R. 1986. Wound age and infection of peach bark by Cytospora leucostoma. Can. J . Bot. 64: 2319-2321. 6. Biggs, A.R. 1986. Peach canker. Pest management program for peach. Factsheet 86-034. Ontario Ministry of Agriculture and Food. Loose-leaf pub. n.p. 7. Biggs, A.R. and Cline, R.A. 1986. Influence of irr igat ion on wound response in peach bark. Can. J . Plant Pathol. 8: 405-408. 8. B r i t i s h Columbia M in i s t ry of Agr i cu l ture and Food. 1985. Agriculture stat ist ics prof i le 1984. Victor ia, B.C. 134 pp. 9. Connors, I.L. 1967. An annotated index of plant diseases in Canada. Can. Dep. Agric. Publ. 1251. Queen's Printer, Ottawa. 381 pp. 10. Defago, G. 1935. De quelques Valsees von Hohnel parasites des arbres a noyau deperissants. Beitr. Kryptogamenflora Schweiz. 8(3): 109 pp. 11. Dhanvantari, B.N. 1968. Effects of selected fungicides on germination of conidia of Cytospora cincta and C^ leucostoma in v i tro. Can. J . Plant Sci . 48: 401-408. 12. Dhanvantari, B.N. 1982. Relative importance of Leucostoma cincta and persooni i in perennial canker of peach in southwestern Ontario. Can. J . Plant Pathol. 4: 221-225. 13. Dhanvantari, B.N. and Dirks, V.A. 1983. An evaluation of peach cultivars and selection for resistance to Leucostoma cincta. Can. J . Plant Sci. 63: 307-310. 109 14. F le tcher , J . T . and Shape, K. 1978. Control of Didymel1 a  lycopersici on tomato stems with fungicidal paints. Plant Pathol. 27: 194-197. 15. French, W.J. and Helton, A.W. 1962. In vitro toxicity of forty-six compounds to a Cytospora isolate from Italian prune. Phytopathology 52: 810-814. 16. Garrett , G.M.E., Panagopoulos, C.G., and Crosse, J . E . 1966. Comparison of plant pathogenic pseudomonads from f ru i t trees. J . appl. Bact. 29: 342-356. 17. Ginns, J.H. 1986. Compendium of plant diseases and decay in Canada 1960-1980. Can. Dep. Agric. 1251. Queen's Printer, Ottawa. 416 pp. 18. Hammer, S.A. and Adams, G.C. 1987. Methods and media for determining mycelial reaction zones in L^ cincta and L^ persoonii. Phytopathology 77: 1721 (Abstr.). 19. Helton, A.W. 1961. First year effects of 10 selected Cytospora isolates on 20 f ru i t and forest species and varieties. Plant Dis. Rep. 45: 500-504. 20. Helton, A.W. 1961. Low temperature injury as a contributing factor in Cytospora invasion of plum trees. Plant Dis. Rep. 45: 591-597. 21. Helton, A.W. and Kochan, W.J. 1967. First and second year effects on Cytospora canker disease of ' I ta l ian ' prune trees sprayed with four concentrations of cycloheximide thiosemicarbazone. Plant Dis. Rep. 51: 655-658. 22. Helton, A.W. and Konicek, D.E. 1961. Effects of selected Cytospora i s o l a t e s from s tonef ru i t s on ce r ta in s tone f ru i t v a r i e t i e s . Phytopathology 51: 152-157. 23. Helton, A.W. and Konicek, D.E. 1962. An optimum environment for the c u l t u r i n g of Cytospora i s o l a t e s from s t o n e f r u i t . I. Temperature. Mycopath. and Mycol. Appl. 16: 18-26. 24. Helton, A.W. and Konicek, D.E. 1962. An optimum environment for the culturing of Cytospora isolates from stonefruit. III. Nitrogen sources. Mycopath. and Mycol. Appl. 16: 125-132. 25. Helton, A.W. and Moisey, J.A. 1955. Cytospora damage in Idaho prune orchards. Plant Dis. Rep. 39: 931-943. 26. Helton, A.W. and Rohrbach, K.G. 1967. Chemotherapy of Cytospora canker disease in peach trees. Phytopathology 57: 442-446. 110 27. Jones, A.C. and Luepschen, N.S. 1971. Seasonal development of Cytospora canker on peach in Colorado. Plant Dis. Rep. 55: 314-317. 28. Kable, P.F., F l iege l , P., and Parker, K.G. 1966. Cytospora canker on sweet cherry in New York State: Association with winter injury and pathogenicity to other species. Plant Dis. Rep. 51: 155-157. 29. Kastirr, V. and Ehrig, F. 1984. Morphologische Differenzierung von Cytospora - Arten an Apfelgeholzen. Arch. Phytopathol. v. Planzenshutz. 20: 453-467. 30. Kern, H. 1955. Taxonomic studies in the genus Leucostoma. Papers of the Michigan Academy of Science, Arts and Letters. 40: 9-22. 31. Konicek, D.E. and Helton, A.W., 1962. An optimum environment for the culturing of Cytospora isolates from stonefruits. II. Carbon sources. Mycopath. and Mycol. Appl. 16: 27-34. 32. Konicek, D.E. and Helton, A.W. 1962. An optimum environment for culturing of Cytospora isolates from stonefruits. IV. Hydrogen-ion concentration. Mycopath. and Mycol. Appl. 16: 243-248. 33. L i l l y , V.G. and Barnett, H.L. 1951. Physiology of fungi. 1st ed. McGraw-Hill, Toronto, pp. 464. 34. Luepschen, N.S. 1976. Use of benomyl sprays for suppressing Cytospora canker on a r t i f i c a l l y inoculated peach tree. Plant Dis. Rep. 60: 477-479. 35. Luepschen, N.S. 1981. Cr i te r i a for determining peach variety suscept ib i l i ty to Cytospora canker. Fruit Varieties Journal 35: 137-140. 36. Luepschen, N.S. and Rohrbach, K.G. 1969. Cytospora canker of peach trees: Spore ava i lab i l i ty and wound susceptibi l i ty. Plant Dis. Rep. 53: 869-872. 37. Luepschen, N.S., Hethington, J .E . , Stohl, F . J . , and Mowrer, K.F. 1979. Cytospora canker of peach trees in Colorado: Survey of incidence, canker location and apparent infection courts. Plant Dis. Rep. 63: 685-687. 38. Luepschen, N.S., Rohrbach, K.G., Jones, A . C , and Dickens, L.E. 1975. Susceptibil ity of peach cultivars to Cytospora canker under Colorado orchard conditions. HortScience 10: 76-77. 39. Lukezic, F.L., deVay, J . E . , and English, H. 1965. Comparative physiology and pathogenicity of Leucostoma persoonii and Rhobosticta  quercina. Phytopathology 55: 511-518. I l l 40. Muller, E., and von Arx, J.A. 1973 Pyrenomycetes: Meliolales, Coropophorales, Sphaerioles. Pages 87-132 In Ainsworth, G.C., Sparrow, F.K., and Sussan, A.S. (Eds.). The Fungi, Vol. 4A. Academic Press, New York, N.Y. 41. Northover, J . 1976. Protection of peach shoots against species of Leucostoma with benomyl and captafol. Phytopathology 66: 1125-1128. 42. Palmiter, D.H., and Hickey, K.D. 1970. Relative resistance of 26 peach cultivars to bacterial spot and Valsa canker. Plant Dis. Rep. 54: 395-399. 43. Rohrbach, K.G., and Helton, A.W. 1965. In vitro effects of f i f t y pesticidal substances on an isolate of Cytospora cincta from Italian prunes. Phytopathology 55: 382-386. 44. Rohrbach, K.G., and Luepschen, N.S. 1968. Environmental and nutritional factors affecting pycnidiospore germination of Cytospora  leucostoma. Phytopathology 58: 1134-1138. 45. Rosenberger, D.A. 1982. Biology and control of Cytospora fungi peach plantings. New York (Cornell) Agric. Exp. Stn. Mem. 92, 6 pp. 46. Schmidle, A., Krahmen, H., and Brenner, H. 1979. Ein beitrag zur taxonomischen abgrenzung von Leucostoma persoonii (Nits. Hohnel) und Leucostoma cincta (Fr.) Hohnel. Phytopath. Z. 96: 294-301. 47. Schoeneweiss, D.F. 1981. The role of environmental stress in diseases of woody plants. Plant Dis. Rep. 65: 308-314. 48. Shaw, C.G. 1973. Host fungus index for the Pacific Northwest. I. Hosts. Washington Agric. Extension Station Bulletin 765. 121 pp. 49. Tekauz, A., and Patrick, Z.A. 1974. The role of twig infections on the incidence of perennial canker of peach. Phytopathology 64: 683-688. 50. United States Department of Agriculture. 1960. Index of plant diseases in the United States. Agric. Handbook 165. Washington, D.C. 531 pp. 51. Urban, Z. 1958. Revise ceskoslovenskych zastupoo roduu Valsa, Leucostoma a Valsel la. Rozprovny Cesk. Akad. Ved. 68: 1-100. 52. Wensley, R.N. 1964. Occurrence and pathogenicity of Valsa (Cytospora) species and other fungi associated with peach canker in southern Ontario. Can. J . Bot. 42: 841-857. 53. Wensley, R.N. 1966. Rate of healing and its relationship to canker of peach. Can. J . Plant Sci. 46: 257-264. 112 54. Wi11ison, R.S. 1933. Peach canker investigations. I. Some notes on incidence, contributing factors and control measures. Sc i . Agric. 14: 32-47. 55. Will ison, R.S. 1936. Peach canker investigations. II. Infection studies. Can. J . Res. 14: 27-44. 56. Will ison, R.S. 1937. Peach canker investigations. III. Further notes on incidence, contributing factors, and related phenomena. Can. J . Res. 15: 324-339. 57. Wilson, E.E., and Ogawa, J.M. 1979. Fungal, bacterial, and certain nonparas i t ic diseases of f r u i t and nut crops in C a l i f o r n i a . Division of Agricultural Sciences. University of Cal i fornia. No. 4090. 58. W i s e n i e w s k i , M., Bog le , A . L . , and Wi l son , C L . 1984. Histopathology of canker development on peach trees a f t e r inoculation with Cytospora leucostoma. Can. J . Bot. 62: 2804-2813. 59. Wysong, D.S., and Dickens, L.E. 1962. Variation in virulence of Valsa leucostoma. Plant Dis. Rep. 46: 274-276. 

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