MILHIUILUEIMHUW”Hill"!!!HIHIHIHHIIDHII I 10494 8314 rum This is to certify that the thesis entitled CYTOSPORA CANKER OF COLORADO BLUE SPRUCE (PICEA PUNGENS): ETIOLOGY, SYMPTOMOLOGY, EPIDEMIOLOGY AND CONTROL presented by Lewis Kageche Kamiri has been accepted towards fulfillment of the requirements for Ph.D. degree in Plant Ethology jwpw Major professor Date M47 /2’, I770 0-7639 { fl-“\\\| r. “ ». ”I‘ll/I,” ‘ _, W: 25¢ per day per item RETURNING LIBRARY MATERIAL§z Place in book return to renew charge from circulation racer CYTOSPORA CANKER OF COLORADO BLUE SPRUCE (PICEA PUNGENS): ETIOLOGY, SYMPTOMOLOGY, EPIDEMIOLOGY AND CONTROL BV Lewis Kaqeche Kamiri A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Botany and Plant Pathology 1980 ABSTRACT CYTOSPORA CANKER OF COLORADO BLUE SPRUCE (PICEA PUNGENS): ETIOLOGY, SYMPTOMOLOGY, EPIDEMIOLOGY AND CONTROL Bv Lewis Kageche Kamiri Cytospora canker of Colorado blue spruce (31933 pungens Engelm.) caused by Valsa kunzei Fr. (imperfect stage Cytospora kunzei Sacc.) is the most destructive and easily recognized disease of blue spruce in Michigan. Blue spruce trees are rarely killed by the disease, but the continuous removal of cankered branches destroys the symmetrv and sub- sequently the aesthetic value of the tree. Development of disease svmptoms was studied on naturally-infected blue spruce. Resinosis and needle drOp began in spring and continued into late fall. Disease development was greatest during summer, as indicated by symptoms expression. Laboratory studies indicate that 27°C was the optimum temperature for conidia germination, germ tube elongation and mycelial growth. Maximum conidial germination occurred in 32 hours at 27°C. Histological observations of cankered branches showed that the pathogen invades host tissue intracellularlv. Hyphal activity was greatest in the cells Lewis Kageche Kamiri of the periderm, secondary phloem, cortex, the vascular cam- bium and its recent derivatives. Hyphae were seen easily in phloem resin canals. Xylem tracheids and xylem rays were invaded to a lesser extent. Trees subjected to water stress in the greenhouse following inoculation with monoascospore cultures developed significantly (p=.001) more cankered branches than did inoculated, non-drought-stressed trees. Inoculation of drought-stressed and non-drought-stressed trees with mono- conidium cultures did not cause infection. No infection occurred without wounding or through needles with either inoculum. In field studies conidia and ascospores were water— and air-borne. Conidia were trapped during periods of wet- ness throughout the season, with the highest number occurring in spring. Ascospores were common only in spring. The number of water- and air-borne conidia always exceeded the number of water- and air-borne ascospores. Both spore types exhibited diurnal-nocturnal periodicity. Dispersal of air- borne conidia and ascospores was correlated with mean daily temperature, hours of 100% relative humidity, hours of leaf wetness, and hours of rainfall (p=.001). Conidia and ascospores isolated throughout the year were viable. in yitrg evaluation of fungicides for control of Cytospora canker showed Arbotect 20-S (2-(4 thiazolyl) ben- zimidazole hypophosphite), Benlate 50% WP (Methyl l- (butylcarbamyl)-2-benzimidazole carbamate), Bravo 6F (Tetrachloroisophthalonitrile), and Topsin M 70% WP (Thiophanate methyl (dimethyl 1,2-phenylene) bis (iminocarbonothioyl) bis carbamate) to be effective in inhi- biting mycelial growth of Q; kunzei at 1000 ppm. In field studies involving 10 sprays over a 2 year period, both Benlate at 5.0 lb. and Topsin M at 3.57 lb. per 100 gallons of water resulted in 45.4% control of Cytospora canker on pruned, naturally-infected blue spruce trees. Arbotect 20-S appeared to be phytotoxic as a foliar protectant spray, and Bravo 6F was inconsistent in its performance. DEDICATION This dissertation is dedicated to mv father, the late Stephen Njuguna Karanja, who taught me as a child to love and appreciate plants; and to my mother, brothers and sisters. ii ACKNOWLEDGEMENTS A number of pepple provided me with valuable assistance as I conducted my research. I am grateful to Dr. Franklin F. Laemmlen, my major professor, for the guidance and encouragement he gave me during the course of this research and in the preparation of this manuscript. I wish to express appreciation to Drs. H. Davidson, J. H. Hart, and W. H. Weidlich for critical evaluation of this manuscript. I am grateful to Dr. W. H. Weidlich for his help with histological studies. This research was made possible by the management of Ella Sharp Park and Sharp Nursery who provided the necessary field plots. The following companies provided fungicides for field spray trials: Diamond Shamrock Corporation, DuPont Agrichemicals, Merck and Company, Inc., and Pennwalt Corporation. Primary financial support was provided by the Agricultural Experiment Station, MSU. Additional assistance came through a grant from the International Society of Arboriculture. Last, but not least, I am grateful to my family whose encouragement and support made it possible for me to complete my studies in the United States of America. I also thank Anne Mason for her friendship, understanding, and patience. iii TABLE OF CONTENTS Page DEDICATION . . . . . . . . . . . . . . . . . . . . . . ii ACKNOWLEDGMENT . . . . . . . . . . . . . . . . . . . . . iii LIST OF TABLES. . . . . . . . . . . . . . . . . . . . .viii LIST OF FIGURES . . . . . . . . . . . . . . . . . . . . . x GENERAL INTRODUCTION AND LITERATURE REVIEW . . . . . . 1 LITERATURE CITED 0 O O O O O O O O O O O O O O O O O O O 6 PART I. THE HOST, PATHOGEN, SYMPTOMS, AND HISTOLOGY. . . 7 INTRODUCTION 0 C O I O O O O 0 O O O O O O O O O O O O O 7 MATERIALS AND METIIODS O O O O O O O O O O O O O O O O O I 8 A. Pattern of Symptom Development in the Field . . . . . . . . . . . . . . . . . . . . . 8 B. Effect of Temperature on Conidia Germination . . . . . . . . . . . . . . . . 9 C. Effect of Exogenous Nutrients on Conidia Germination . . . . . . . . . . . . . . .10 D. Effect of Temperature on Mycelial Growth . . . .11 E. Histological Observation of Cytospora Canker . . . . . . . . . . . . . . . . . . . .11 RESULTS 0 O O O O O O O O I O O O O O O C O O I O O O O O 13 A. Pattern of Symptom Development in the Field . . . . . . . . . . . . . . . . . . . . .13 B. Effect of Temperature on Conidia Germination . . . . . . . . . . . . . .15 C. Effect of Exogenous Nutrients on Conidia Germination . . . . . . . . . . . . . . . . . .15 iv D. Effect of Temperature on Mycelial Growth . . E. Histological Observation of Cytospora canker O O O O O O O O C O I O O O O O O O O DISCUSS ION O O O O O O O O O O O O O O O O O O O O O 0 LITERATURE CITED 0 O O O O O O O O O O O O O O O O O 0 PART II. PLANT-WATER RELATIONS AND SITES OF VALSA INFECTION O O O O O O O O C O O O O O O O 0 INTRODUCTION 0 O O O O O O O O O O O I O O O I O O O 0 MATERIALS MD METHODS O O I O O O O O I I O O O O O O A. Effect of Drought Stress on Cytospora Canker Development . . . . . . . . . . . . . . . . B. Infection Through Wounds . . . . . . . . . . C. Infection Through Needles . . . . . . . . . RESULTS 0 ‘ O O O O I O O O O O O O O I O O O O O I O O A. Effect of Drought Stress on Cytospora Canker Development 0 O O O O O O O O I O O O O O B. Infection Through Wounds . . . . . . . . . . C. Infection Through Needles . . . . . . . . . DISCUSSION 0 O O O O O O O O O O O O O O O O O O O O 0 LITERATURE CITED 0 O O O O O O O O O O O O O O O O O 0 PART III. DISPERSAL OF ASCOSPORES AND CONIDIA OF VALSA KUNZEI O O O O O O O I O O C O O O O I O O I NTRODUC TI ON C O O O O O O O O O O O O C O O I O O O 0 MATERIALS AND METHODS O O O O O O O O O O O C O O O O A. Trapping of Rain Dispersed Conidia and Ascospores . . . . . . . . . . . . . . . B. Trapping of Air- borne Conidia and Ascospores C. Environmental Factors Affecting Conidia and Ascospores Numbers . . . . . . . . . . . . . Page 19 21 25 30 31 31 31 32 34 34 34 36 38 38 44 47 47 48 49 50 51 D. RESULTS A. B. C. D. DISCUSSION LITERATURE PART IV. Spore Isolation and Spore Viabilitv . . . . . Relative Populations of Water-borne Conidia and Ascospores . . . . . . . . . . . . . . . . . The Occurrence and Importence of Air-borne Conidia and Ascospores . . . . . . . . . . . Environmental Factors Affecting Conidia and Ascospore Numbers . . . . . . . . . . . . . . Spore Isolation and Spore Viabilitv . . . . . CITED 0 O O O O O O O O O O O O O O O O O O CONTROL OF CYTOSPORA CANKER . . . . . . . . . INTRODUCTION 0 O O O O O O I O O O O O O O O O O O O 0 MATERIALS AND METHODS O O O O O O O O O O O O O O O C O A. B. RESULTS A. B. Preliminarv in Vitro Screening for Fungicides Active Against Cytospora kunzei . . . . . Field Evaluation of Fungic1des for Control of Cytospora Canker . . . . . . . . . . . . . . Preliminary in Vitro Screening for Fungicides Active Against Cytospora kunzei . . . . . . Field Evaluation of Fungicides for Control of Cytospora Canker . . . . . . . . . . . . . . DISCUSSION 0 O O O O O O O O O O O O O O O O O O O O 0 LITERATURE CITED 0 O O O O O O O O O O O O O O O O O 0 APPENDIX A. B. Stepwise Multiple Regression Analvsis of the Parameters Affecting the Number of Air-borne Ascospores. 1978 . . . . . . . Stepwise Multiple Regression Analysis of the Parameters Affecting the Number of Air- -borne Conidia. 1978 . . . . . . . . . . . . . . vi . 52 O 60 . 70 Stepwise Multiple Regression Analvsis of the Parameters Affecting the Number of Air-borne Ascospores. Stepwise Multiple Regression Analysis of the Parameters Affecting the Number of Air- -borne Conidia. Total and Mean Number of Cankered Branches on Fungicide Treated Blue Spruce. 1978 and 1979 1979 1979 vii Page LIST OF TABLES Table Page PART I. THE HOST, PATHOGEN, SYMPTOMS AND HISTOLOGY 1. Pattern of Symptom Development on Colorado Blue Spruce in the Field. . . . . . . . . . . . l4 2. Mean Percent Germination and Germ Tube Length of Cytospora kunzei Conidia After Exposure to Low Temperatures. . . . . . . . . . . . . . . . 17 PART II. PLANT-WATER RELATIONS AND SITES OF VALSA INFECTION 1. Effect of Drought Stress and Wounding on CytoSpora Canker Development. 1978 . . . . . . 35 2. Effect of Drought Stress and Wounding on Cytospora Canker Development. 1979 . . . . . . 37 PART III. DISPERSAL OF ASCOSPORES AND CONIDIA OF VALSA KUNZEI 1. Number of Air-borne Ascospores and Conidia of Valsa kunzei Caught at Albion, Michigan in a Burkarfi Spore Trap. 1979 . . . . . . . . . . . 58 2. Number of Air-borne Ascospores and Conidia of Valsa kunzei Caught at Albion, Michigan in a Burkara Spore Trap. 1979 . . . . . . . . . . . 63 3. Pearson Correlation Coefficients of the Parameters Affecting the Number of Air-borne Spores Trapped. 1978 and 1979. . . . . . . . . 69 viii Table Page 4. Twigs with Pvcnidia and Viable Conidia/10 Twigs ‘ Sampled from Cvtospora Infected Blue Spruce at Baker's Natural Wood Lot, MSU. 1977. . . . . . 71 5. Twigs with Pycnidia and Viable Conidia/10 Twigs Sampled from Cvtospora Infected Blue Spruce at Baker's Natural Wood Lot, MSU. 1978. . . . . . 73 6. Twigs with Fruiting Structures and Viable Spores/10 Twigs Sampled from CvtOSpora Infected Norwav Spruce at Albion, Michigan. 1978. . . . . . . . . . . . . . . . . . . . . . 74 7. Twigs with Fruiting Structures and Viable Spores/10 Twigs Sampled from Cytospora Infected Norwav Spruce at Albion, Michigan. 1979. . . . . . . . . . . . . . . . . . . . . . 75 PART IV. CONTROL OF CYTOSPORA CANKER 1. Mean Number of Cankered Branches Showing Symptoms on Fungicide Treated Colorado Blue Spruce From July to November. . . . . . . . . . 88 2. Fungicides, Disease Indexes and Percent Disease Control of Cytospora Canker. 1978 and 1979 O O O O I O I O O O O O O O O O O 90 ix Figure LIST OF FIGURES Page PART I. THE HOST, PATHOGEN, SYMPTOMS, AND HISTOLOGY Germination of Cytospora kunzei Conidia on Double Strength medium E O O O O O O O O O O O O O O O O Effect of Low Temperature Exposure on Germination of Cytospora kunzei Conidia . . . . . . . . . . . Effect of Temperature on Mvcelial Growth of Valsa kunzei Cultured on Medium B . . . . . . Cvtospora hvphae in the Phloem Resin Canals of Infected Colorado Blue Spruce Twigs . . . . . . . vahae of Cytospora Canker Fungus in the Phelloderm of Infected Colorado Blue Spruce Twigs . . . . . Cytospora Hyphae in the leem of Infected Colorado Blue Spruce TWiqS O O O O O O I O O O O O O I O O Cvtospora Hyphae in the Xylem of Infected Colorado Blue Spruce Twigs . . . . . . . . . . . . . . . . Phloem Resin Canals of Non-infected Colorado Blue Spruce Twigs . . . . . . . . . . . . . . . . . . PART III. DISPERSAL OF ASCOSPORES AND CONIDIA OF VALSA KUNZEI Number of Water-borne Conidia of Cytospora kunzei Caught from Cankered Colorado Blue Spruce Branches MSU, Michigan. 1978 . . . . . . . . . Number of Water-borne Conidia of Cytospora kunzei Caught from Cankered Colorado Blue Spruce Branches MSU, Michigan. 1979 . . . . . . . . . . 16 18 20 22 23 23 24 53 54 Figure 3. Number of Water-borne Ascospores and Conidia of Valsa kunzei Caught from Cankered Norway Spruce Branches. Albion, Michigan. 1978 . . Number of Water-borne Ascospores and Conidia of Valsa kunzei Caught from Cankered Norway Spruce Branches. Albion, Michigan. 1979 . . Number of Air-borne Cytospgra kunzei Conidia Trapped/Hour. Albion, Michigan. 1978 . . . Number of Air—borne Valsa kunzei Ascospores Trapped/Hour. Albion, Michigan. 1978 . . . Number of Air-borne Cytospora kunzei Conidia Trapped/Hour. Albion, Michigan. 1979 . . . Number of Air-borne Valsa kunzei Ascospores Trapped/Hour. Albion, Michigan. 1979 . . . PART IV. CONTROL OF CYTOSPORA CANKER Effect of Various Fungicides on Mycelial Growth of Cxtospora kunzei . . . . . . . . . . . . . xi Page GENERAL INTRODUCTION AND LITERATURE REVIEW GENERAL INTRODUCTION AND LITERATURE REVIEW Colorado blue spruce (Picea pungens Engelm.) is one of the most commonly planted and highly valued ornamental, shelterbelt, and Christmas trees in the north temperate zones. It is used little for timber production (3). Cvtospora canker caused by Valsa kunzei Fr. (imperfect stage Cytospora kunzei Sacc.) is the most destructive and easilv recognized disease of Colorado blue spruce in Michigan. Blue spruce trees are rarely killed by the disease, but the continuous removal of cankered branches destrovs the sym- metry and subsequently the aesthetic value of the tree. However, tree mortality has-been reported on Douglas-fir (Pseudotsuga menziesii (Mirb.) Franco) (5,12) and Engelmann spruce (Picea engelmannii Parry)(4) infected with Cvtospora canker. In 1933, Gilgut and Bovd (2) first reported the occurrence and symptoms of Cytospora canker on blue spruce in Massachusetts. They noted that the disease attacks small branches as well as large limbs, but not the trunk. The disease usually starts on a lower limb and spreads upward and laterally. Gummosis is pronounced on diseased branches. There is no hypertrophy or atrophy on infected branches. No organism other than Cytospora was isolated from the cankers. 2 The old belief of horticulturalists was that the slow dying of the lower limbs was a natural process. Gilgut, in 1936 (1), confirmed Cytospora kunzei Sacc. as the causal agent by artificially wound—inoculating healthy Norway spruce (gigs; 'abigg (L.) Karst.) with a suspension of spores. No perfect stage of the fungus was observed on spruce. In 1937, Waterman (10) reviewed the distribution and host range of g; kunzei. Marsden, in 1948 (7), reported isolation from cankers of Norway and Colorado blue spruce of a Valsa that was identified by Wehmeyer as Valsa kunzei Fr. The pathogeni- city of the Valsa was confirmed by inoculating Norway spruce trees in the greenhouse. In 1960, Hawksworth and Hinds (4) reported the disease for the first time on Engelmann spruce (Picea engelmannii Parry) near Kenosha Pass, Pike National Forest, Colorado. They observed fruiting bodies of both the perfect and imperfect stages of the fungus on cankered branches in June. Trunk and branch infections were common on small trees about 6 inches in diameter, while only branch cankers were noted on larger trees. Jorgensen and Cafley, in 1961 (6), recorded the occurrence of Cytospora canker on white and Norway spruce in southern Ontario, Canada. Their study on tree growth in relation to the develOpment of stem cankers revealed that trees had been predisposed to attack as indicated by the sudden decrease in diameter growth. The predisposing factor 3 was believed to be drought, which weakens trees with a poorly develOped root system. Ouellette, et al. (8) observed Cvtospora canker in all plantations of white and Norway spruce in Quebec and in some natural stands of indigenous spruce species in 1965. The development of cankers was more pronounced in some years than others. They found no clear- cut correlation between rainfall, temperature and disease intensity. Wright, in 1957 (12), and Hinds and Stewart, in 1965 (5), studied pitch-girdle canker on Douglas—fir in Colorado and found it to be caused by Cytospora kunzei Sacc. Wright stated that diseased trees have a poorly developed root system due to the site conditions and that any pronounced deficiency in precipitation causes a rapid reduction in growth, which subsequently leads to infection and killing by g; kunzei. In 1955, Waterman (11) compared species of Cytospora and Valsa commonly found on cankers of various species of spruce and other coniferous hosts. There was considerable similarity in the development of the perithecial stromata on the various hosts. The fungus on all coniferous hosts corresponded with Valsa kunzei Fr. Slight differences, however, indicated that three types of stromata development were involved. These differences were the basis for distinguishing three varieties of the species Valsa kunzei Fr.: namely, Valsa kunzei var. kunzei var. nov. typical, Valsa kunzei var. piceae var. nov. and Valsa kunzei var. superficialis conb. nov. When nursery trees were inoculated with isolates of Valsa kunzei representing each of the varieties, infections were obtained with only the V; kunzei var. piceae on Picea species (g; pungens, P; ables, 2. 25132- talis and P; glauca) and Pseudotsuga menziesii. Pycnidial stromata were variable on any one host. Morphological characters of the Cytospora stage were not sufficiently distinctive to indicate that more than one species was involved. Data concerning spore liberation and natural infection periods are lacking. Control measures available at the present time are not satisfactory. Strong (9) was the first to give a good description of the symptoms of Cytospora canker and recommended the following control measures: (1) removal of cankered branches; (2) immediate burning of the affected branches after removal; (3) continuous surveillance for the appearance of dving branches; and (4) fertilization and watering of trees to improve vigor when cankers have been found and removed. Waterman (11) failed to control the disease by spraying cankered blue spruce trees with Bordeaux mixture and by cutting out infected bark. There is no other chemical control available at the present time. The purpose of this investigation was to study the etiology and epidemiology of Cytospora canker of Colorado blue spruce and possible measures of control. The 5 information obtained from this study should contribute to an understanding of the disease cycle and provide practical information for homeowners and nurservmen. 10. 11. 12. 6 LITERATURE CITED Gilgut, C. J. 1936. Cvtospora canker of spruces. National Shade Tree Conf. Proc. 12:113-119. Gilgut, C. J. and O. C. Bovd. 1933. Cvtospora canker of Picea spp. Phytopathologv. 23:11. Hanover, J. W. 1975. Genetics of blue spruce. USDA For. Serv. Res. Pap. WG-28. 12 p. Hawksworth, F. G. and T. E. Hinds. 1960. Cvtospora canker of Englemann spruce in Colorado. Plant Disease Reporter. 44:72. Hinds, T. E. and J. L. Stewart. 1965. Cytospora canker recurrence on Douglas-fir in Colorado. Plant Disease Reporter. 49:481-482. Jorgensen, E. and J. D. Cafley. 1961. Branch and stem cankers of white and Norwav spruce in Ontario. Forestry Chronicle. 37:394-400. Marsden, D. H. 1948. PhytOpathological notes. A Valsa associated with Cytospora canker of spruces. Pytopathology. 38:307-308. Ouellette, G. B., J. M. Conway and G. Bard. 1965. Frequence et intensite du chancre Cvtosporeen dans les plantations depinette du Quebec (In French, English Abstract). Forestry Chronicle 41:444-453. Strong, F. C. 1953. Spruce branch canker. National Shade Tree Conf. Proc. 29:30-35. Waterman, A. M. 1937. Cytospora canker of spruce. Plant Disease Reporter. 21:55. Waterman, A. M. 1955. The relation of Valsa kunzei to cankers on conifers. Phytopathologv. 45:686-692. Wright, E. 1957. Cytospora canker of Rocky Mountain Douglas-fir. Plant Disease Reporter. 41:811-813. / A. B. C. E. PART I THE HOST, PATHOGEN, SYMPTOMS, AND HISTOLOGY Pattern of Symptom Development in the Field Effect of Temperature on Conidia Germination Effect of Exogenous Nutrients on Conidia Germination Effect of Temperature on Mycelial Growth Histological Observation of Cytospora Canker INTRODUCTION In its native range, Colorado blue spruce (£1933 pungens Engelm.) grows in the deep ravines of the central and southern Rocky Mountains. There its aesthetic and watershed values far outweigh its importance for commercial timber. Despite its Rocky Mountain origin, blue spruce has become a commonly planted ornamental conifer in the north temperate zone. Its symmetric full crown, wide adaptability and striking blue color are responsible for its great popularity. Cytospora canker caused by Cytospora kunzei Sacc. is the most destructive disease of blue spruce in Michigan. The perfect stage of this species has been described as Valsa kunzei Fr. (6,9,10,11,12). The taxonomy of this genus has been discussed (12). The most readily recognized symptom associated with Cytospora canker of blue spruce is the accumulation of dead branches on diseased trees. Definite cankers are not formed. However, upon closer examination, infected branches exhibit cankers with heavy resin flow. Removal of resin and outer bark reveals pycnidial and/or perithecial fruiting bodies. There are also brown, dead areas of inner bark and 8 cambium (3). When cankered branches are briefly soaked in water, then wrapped with wet paper towels, yellowish, whitish or orange spore tendrils exude from the fruiting bodies. These tendrils consist of a mass of spores embedded in a matrix (8). If wetness persists for some time, tendrils are not formed and spore masses exude into the sur- face moisture, and the matrix surrounding them is quickly dissolved. In 1936, Gilgut (2) reported that Cytospora canker of blue spruce is a bark and cambium disease which invades and turns cortical and cambial tissue brown. The wood beneath the bark is not discolored by the fungus. However, his studies were basically observational, rather than histological. The work reported here was initiated to determine the nature of symptom development in the field, the optimum environmental requirements for conidia germination, and mycelial growth, and histological observations of infected branches. MATERIALS AND METHODS A. Pattern of Symptom Development in the Field Two Colorado blue spruce trees on the campus of Michigan State University known to be infected with 9; Egg- zgi were observed over a period of 3 years for new infec- tions and development of symptoms. On each tree cankered branches with resin flow and dead branches were tagged at 9 the beginning of each study period. Trees were monitored from spring until first snowfall for new infections, addi- tional branches with resinosis, and browning or dropping needles. B. Effect of Temperature on Conidia Germination Cankered blue spruce twigs were collected and soaked in a 2.62% sodium hvpochlorite solution for 5 to 10 minutes with continuous agitation. The twigs were washed in running deionized water for 5 minutes, soaked submerged in deionized water for 10 to 30 minutes, wrapped in a moistened paper towel and left to stand for 3 to 8 hours in a moist chamber. The twigs were examined under the microscope for conidial spore masses. Spore tendrils or ooze drOplets were removed with a sterile needle and placed in sterile deionized water to yield a spore suspension. The medium used in all conidia germination studies consisted of 10 g maltose, 20 g agar, 2 9 sucrose, 2 9 glucose, 2 g yeast extract, and 3.5 g potassium nitrate (KNO3) per liter of glass distilled water. To enhance coni- dia germination and the formation of strong germ tubes, the medium was made 2X normal concentration, autoclaved and poured into 55 mm diameter plastic petri dishes and allowed to solidify. Plates were incubated overnight at the designated temperature regimes before inoculating. Two droplets of the spore suspension (2.3 X 106 conidia per m1 of water) were applied to the culture medium 10 and spread over the surface. Cultures were incubated at 0, 10, 20, 24, 27 and 33°C. Conidia were considered germinated when a germ tube equal in length to the largest conidial dimension developed. A total of 100 conidia were counted at random every 8 hours. The number germinated was divided by total number of conidia counted and multiplied by 100 to give percent germination. Ten randomly selected conidia were measured with an ocular micrometer to determine the average germ tube length. These experiments were replicated 3 times. In another experiment, 2 m1 of conidia suspension were exposed to -15°C. Each day, 2 test tubes containing conidia were thawed at room temperature for 30 minutes, placed on culture media, and incubated for 32 hrs at 27°C. Percent germination was determined as explained above. These experiments were replicated 2 times. C. Effect of Exogenous Nutrients on Conidia Germination Freshly harvested conidia of g; kunzei were obtained as described previously. A droplet of conidial suspension was placed in either sterile glass distilled water, a sterile 1% solution of glucose, or sterile blue spruce stem decoction. Preparation of the stem decoction consisted of boiling 100 g of blue spruce stem pieces in 200 m1 of glass distilled water for 30 minutes followed by autoclaving for 20 minutes. The spore suspension was mixed with each of the above solutions in sterile glass despression slides. Two filter paper discs 75 mm in diam were placed in a glass ll petri plate. A glass-U—tube was placed on the filter paper and 5 ml of glass distilled sterile water were added to each plate and sterilized. The depression slides containing the spore suspension were held in the sterile closed petri- dishes to prevent excessive evaporation. The plates were incubated at 27°C and checked for the average germination and germ tube elongation at 8-hour intervals. D. Effect of Temperature on Mycelial Growth Seven mm diam discs of actively growing mycelium from monoconidium and single ascospore cultures were placed in the center of 90 mm diam petri dishes containing medium B agar and incubated at 0, 5, 10, 15, 20, 22, 24, 27, 30, 34, and 36°C. Medium B consisted of 40 9 corn meal agar, 20 g bacto-agar, 2 9 glucose, 2 9 sucrose, 1 g yeast extract per liter of glass distilled water. Colony diam were measured to the nearest mm from 5 plates at each temperature at 24 hr intervals. A similar experiment was conducted with iso- late Colo. 275 from Douglas-fir (3) and identified as Cytospora kunzei. These experiments were replicated twice. E. Histological Observation of Cytospora Canker Small naturally-infected Colorado blue spruce branches were taken from trees and cut into small pieces not over 2.54 cm in length. The specimens were fixed in Jackson's FAA (5) until processed for sectioning. Specimens to be sectioned were either dehydrated or sectioned without 12 dehydrating. Materials to be dehydrated were passed through an ethanol series in increasing concentration (1 change each in 50, 60, 70, 80 and 90%, and 3 changes in 100%) at 12-hr intervals. The materials to be sectioned were softened in glacial acetic acid—alcohol mixture (1) for 3 to 7 days. To enhance softening, liquid detergent was added to the mixture. The composition of the softening solution con- sisted of 20 ml glacial acetic acid, and 80 m1 of 60% ethyl alcohol plus several drops of liquid detergent. Dehydrated materials were sectioned after clearing. The specimens were cleared by passing them through a series of ethanol-xylene solutions (75% ethanol: 25% xylene, 50% ethanol: 50% xylene, 25% ethanol: 75% xylene, and 3 changes 100% xylene) at 3-hr intervals in each solution. Undehydrated materials were softened and rinsed for 24 hours in running water and sectioned. The sections were cut on either a Hooker (Lab Line) fresh tissue microtome, (4) or the sliding microtome. Sections (10-20 um thick) were collected in water, trans- ferred into a watch glass and stained with either 0.1% toluidine blue or 0.1% cotton blue in lactophenol. Sections stained with toluidine were rinsed 3 times in water, blotted dry and mounted in glycerine on a glass slide. Sections stained in cotton blue were immediately rinsed with lac— tophenol to remove excess stain and mounted in clear lacto- phenol for microscopic examination. Most of the sectioning 13 was done in tangential section, since the mycelium could be more readily traced through such sections. Transverse and radial sections were also made. RESULTS A. Pattern of Symptom Development in the Field Observations on disease development on naturally— infected blue spruce in the field indicate that symptoms begin to appear in spring and continue into late fall. The number of branches showing symptoms of infection increased mostly during the summer (Table l). The first symptoms appear as a wet clear gummosis which may or may not be followed by needle drop. As the season progresses, the wet clear ooze hardens into the white resinosis so often diagnostic of Cvtospora canker on blue and Norway spruce. Needle drOp begins in late June with the onset of a green wilt-like symptom and continues through late fall. The needles on infected branches turn brown and finally fall. Remnants of greenish-tan needles may remain attached to the twigs. Sometimes, resinosis develops in late June, but needle drop may not occur until the following year. The sapwood beneath the bark of cankered branches may or may not be discolored. Small branches that have recently dropped their needles have a faint yellowish appearance in contrast to the dark brown appearance of twigs from old 14 TABLE 1 Pattern of Symptom Development on Colorado Blue Spruce in . the Field. Date of Total Number of Cankered Branches Observationa Tree No. 1 Tree No. 2 4/18/77 23 8 7/18/77 25 8 8/22/77 26 8 8/29/77 27 8 9/5/77 30 9 9/12/77 31 9 11/14/77* 31 9 3/20/78 31 9 7/3/78 34 9 7/17/78 42 9 8/14/78 43 9 10/9/78 45 9 11/27/78* 45 9 4/3/79 45 9 7/10/79 47 9 7/24/79 48 9 8/6/79 49 11 11/13/79* 51 11 aDates on which additional branches became cankered or the end and beginning of a study period. *First snowfall. 15 infections. On several of the blue spruce trees where pruning of diseased branches had taken place, several small branches grew directly from the main trunk and on the main branches filling the denuded space on the trunks. However, these branches often became infected with Q; kunzei. B. Effect of Temperature on Conidia Germination No germination of Q; kunzei conidia occurred at either 0 or 10°C (Figure 1). Slow germination occurred at 20 and 33°C. The germ tubes were extremely short at these temperatures. The best germination occurred at 24 and 27°C. Maximum germination and germ tube length was reached at the optimum temperature of 27°C in 32 hours. One or 2 germ tubes were usually formed at one end of most spores, or one on each end of the spore. Considerable hyphal branching occurred at 27°C after incubation for 32 hours. There was reduction and delay in germination when conidia were exposed to various low temperatures at various time intervals and then transferred to 27°C (Table 2). Pre-exposure of conidia to -15°C followed by incubation at 27°C decreased ger- mination by 49-69% (Figure 2). C. Effect of Exogenous Nutrients on Conidia Germination When conidia were placed in the liquid suspension of glass distilled water, 1% glucose, or spruce decoction, ger- mination and germ tube development did not occur in 40 hours or 5 days. MEAN PERCENT GERMINATION AFTER 32 HOURS 16 100 0—0 Percent Germination A--A Germ Tube Length 90 80 70 d 14 ’1‘ - 13 60 / .. 12 \ / \ - 11 50 - - 10 1 \ I \ '1 9 4o - / \ - 8 / 11 7 30 - / .1 6 / - 5 A l A 3 10 — / 2 1 o J l l l O 10 20 24 27 33 TEMPERATURE IN °C Figure 1. Germination of Cytospora kunzei conidia on Double Strength Medium B. GERM TUBE LENGTH AFTER 32 HOURS (,lm) 17 .pwusmmwa ou¢3 cowumoHHowu\mouoam CH mo msumcmfl was» Emma oneo .meHu mops» pwumoHHQwu mms ucwEumwuu some «cowumofiammu\pmucsoo mums monomm compass wcon .Uobm o» mwusumuwmewu ummu EOuw pw>oe wumz mwumam “puma cos3 mewem mmmmsc H.mH O.OO O.O «O.Om :1 Om 0.0 m.Hm H.m O.HH nu O.O ON OH 0.0 0.00 O.O O.NH u- 0.0 OH OH wands; O.O O.OO O.H O.O O OH O.O O.mH m.H O.s u: 0.0 ON m 0.0 O.Om H.m O.O 1. 0.0 OH m wanes: O.O O.OO O.H «O.O O m ms.m O.OH u- O.O in O.O ON O 0.0 OO.m 0.0 O.O u- O.O OH O mmsa>s 0.0 O.mO in O.O O O 1511 AOO Hess lav less lav Amusomv up ow u: mm u: «N mmefie wusmoaxm wupumuwaeme MOON up OAEJV :pmcmp mess auto tcm pfiwv cOMDmcwEuww mopsumuwQEwe 3o; 0» wpsmomxm pound mflmwcoo «cucsx muCQmOuhw mo gnocwq ooze Eumw pcm :oHumCHEumo ucmOHmm cam: N mqmfle MEAN PERCENT GERMINATION AT 27° C AFTER 32 HOURS 100 90 80 7O 60 50 40 30 20 1O 18 llllllllllll 5 1 L 34 678910111213141516 DAYS OF EXPOSURE TO -15° C Figure 2. Effect of Low Temperature Exposure on Germination of Cytospora kunzei Conidia. 10 D. Effect of Temperature on Mycelial Growth Michigan blue spruce Cytospora kunzei isolates did not grow at 0, 5, 10 and 36°C. Good mycelial growth occurred at temperatures between 22 and 27°C. Maximum mycelial growth occurred at 27°C (Figure 3). Valsa kunzei isolates did not grow at 0, 5 and 36°C. Maximum mycelial growth occurred at 27°C. The Douglas-fir isolate of g; kunzei (Colo. 275) like the Michigan isolates did not grow at 0, 5, lO and 36°C. Its maximum mycelial growth, however, occurred at 24°C. The growth rates of the isolates were different. Valsa kunzei exhibited the fastest rate of growth with isolate Colo. 275 being intermediate and g; kunzei the slowest. Both conidia and ascospores gave rise to dull white or cream-colored, appressed mycelium, roughly circular or with fans, and irregular margins. Faint concentric zones of lighter and darker shades of mycelium were also formed. The growth and appearance of Colo. 275 was nearly identical to that of the Michigan Cytospora and Valsa kunzei isolate. However, the mycelium was dark brown with prominent fans. When mycelia from monoconidium and single ascospore cultures were inoculated on medium B containing steam- sterilized or propylene oxide—sterilized blue spurce twigs and incubated at room temperature, they were identical in growth and appearance. Thirty days after inoculation, the monoascospore culture developed globose grayish fruiting bodies but bore no spores. Sixty—nine days after 20 .m 833.2 :0 35:30 355.. a._m_io>. .o 5320 3:322 so 2283th .o .023 .m 2:2“. 0. $3.25..sz OO 3 OO 8 a «a ON 3 O. O .35... 8232a I $3 28. .35... 22.8.3 I .35... 33> Olllo 3 ON an ac on cm as ea SUH 89L 831:“! (NW) UBLBWVIO ANO'IOO NVEW 21 inoculation, the globose fruiting bodies turned brown and bore allantoid conidia, 3 X 4-6um. Mycelia from monoconi- dium isolates, on the other hand, produced abundant conidia. Three months after inoculation, pycnidia appeared and bore typical conidia. Mycelia on medium B without sterilized twigs produced tiny fruiting bodies which never matured. No perithecial fruiting bodies were formed on any medium. E. Histological Observation of Cytospora Canker The fungal hyphae found in the host cells were thin, septate, frequently branched, and grew intracellularly. There was considerable variation in hyphae length and branching in the cells. Branching was more frequent and easily seen in the resin ducts (Figure 4). Fungal hyphae were found to invade outer bark, cortex and secondary phloem, phloem rays, xylem rays and phloem resin ducts. The hyphae also invaded the phellogen and the vascular cambium and its most recent derivatives. Hyphae were found to invade and ramify extensively in the phelloderm and secondary phloem (Figure 5). Occasionally hyphae were found in the tracheids. The hyphae appeared to be inside the tracheid cells and to be growing through the bordered pits (Figure 6 and 7). No hyphae were observed in the pith and/or xylem resin canals. No haustoria-like structures were found penetrating or asso- ciated with any of the host cells. Fungal hyphae were not observed in non-infected branches (Figure 8). No abnormal anatomical or morphological features were noted. Figure 4. Figure 5. Tangential Section. Cytospora hyphae (arrows) in the phloem resin canals of infected Colorado blue spruce twigs. R = resin canal. 400x. Tangential Section. Hyphae of Cytospora canker fungus (arrows) in the phelloderm of infected Colorado blue spruce twigs. 400x. 22 Figure 6. Figure 7. Tangential Section. Cytospora hyphae (open-arrows) in the xylem of infected Colorado blue spruce twigs. b = bordered pits. 400x. Tangential Section. Cytospora hyphae (Open-arrows) in the xylem of infected Colorado blue spruce twigs. t = xylem tracheid, c = cir- cular bordered pit. 400x. 23 Figure 7 . Figure 8. Tangential Section. Phloem resin canals of non- infected Colorado blue spruce twigs. R = resin canal. 400x. 24 25 For example, in healthy and cankered branches, the height of the uniseriate xylem rays varied from 1 to 10 cells. There was no evidence of gum secretion or occlusion of cells. Host reaction to the presence of the fungus was not ascertained in this study. DISCUSS ION The data on the development of Cytospora canker symp- toms on naturally-infected blue spruce indicate resinosis and needle drop begin in late spring and continue into late fall. The greatest symptom expression occurs during the summer. These results are in agreement with those reported by Gilgut in 1936 (2) on blue spruce. Laboratory studies with freshly harvested conidia of g; kunzei indicate that when field temperatures are below 20°C, spore germination will not occur. As temperatures increase above 20°C, more rapid spore germination will pro- bably occur up to the optimum temperature of 27°C. Since germination must occur before infection, at least 32 hours at the optimum temperature of 27°C is required for the maxi- mum number of infections. The bark of £1233 is not smooth and continuous. The outermost layers are broken at certain regions (lenticel-like structures). These regions could allow more moisture to escape, and retain high relative humidity for extended periods of time for germination to 26 take place. Under favorable environmental conditions, they could become ideal infection courts. This phenomenon may explain why infection occurs on older branches with thicker periderm. Temperatures above 33°C may limit germination and subsequent infections. Low and very high temperatures also inhibit mycelial growth. There was no mycelial growth at 0, 5, 10 and 36°C for the Michigan isolates of Cytospora and Valsa. Maximum mycelial growth occurred at 27°C. The results of these studies indicate that both the Cytospora (imperfect stage) and Valsa (perfect stage) have similar growth curves and temperature requirements. However, they grow at different rates. They are probably similar in these two conditions to other Cygospora spp. and Valsa spp. on different host plants. Rohrbach and Luepschen in 1968 (7) reported that cardinal temperatures for pycnidiospore germi- nation of g;_leucostoma were 4 to 10, and 27 to 32°C. In their studies, maximum germination occurred in 24 hrs at 27°C. A saturated atmosphere and a carbon source were necessary for germination and germ tube production but not for pycnidiospore swelling. Ascospore germination of 23153 kunzei has been reported by Marsden (6) and Wehmeyer (10). In his study, Marsden (6) reported that ascospores increased in size, became oval, oblong and sometimes dumb-bell shaped. Two or more oil droplets were visible. Germ tubes formed at both ends of most spores, and infrequently, one or three germ tubes formed. The hyphae began to branch 10 to 24 hrs after germ tube formation. 27 The fact that conidia did not germinate or swell in their allantoid shape while in free glass distilled water, 1% glucose solution or spruce decoction suggests that free moisture is inhibitory to conidial germination. This is probably due to the low oxygen concentration in liquid medium since germination did occur when conidia were sprayed on top of solid medium B. High relative humidity and the presence of dew are possibly critical for conidial germination, once the requirement for optimum temperature has been fulfilled. Perithecial fruiting bodies were not formed on steri- lized blue spruce twigs. Only the pycnidial fruiting bodies (imperfect stage) were produced in culture when sterilized twigs were inoculated with either the monoascospore or the monoconidium cultures. Marsden (6) obtained more luxuriant mycelial growth on steam-sterilized twigs of Colorado blue spruce than on Norway spruce. More and slightly larger pycnidia deve10ped on those twigs inoculated with conidia than those inoculated with ascospores. Wehmeyer in 1924 (10) obtained mature perithecial stromata on twigs of Thuia occidentalis L. inoculated with ascospores. At the same time, single ascus and single spore cultures on 6 percent oat agar gave rise to the imperfect stage of the fungus, which was not found on the twig cultures. He concluded that the necessity of 2 sexual strains for the formation of peri- thecia was eliminated since perithecia and ascospores were 28 produced from single spore culture. The results reported in this section prove that both the perithecial and the pycnidial fruiting forms are stages of the same fungus since both the single ascospore and monoconidium cultures always produced the pycnidial stage known as Cytospora kunzei. Data on the histological studies of other Cytospora canker diseases are lacking. However, the results of this preliminary study allow us to make some general observations. The intracellular nature of the hyphae found in the parenchyma cells, phellogen, phelloderm, phloem rays, and xylem tracheids and rays could account for the lack of any visible swelling or shrivelling of cankered branches. On the other hand, phellem cells are compactly arranged without intercellular spaces. It is possible, therefore, that penetration and hyphal invasion of these cells is also intracellular. The observation of extensive intracellular hyphal ramification in the cells of the phelloderm, secondary phloem and phloem parenchyma suggests a nutritional rela- tionship between the fungus and the host. Cells of the phloem are killed, and nutrient translocation stops. The girdling of the branch results in its death and the death of the small branches distal to sites of resin production. Hyphal invasion of the resin canals may stimulate the host to secrete c0pious amounts of resin in response to infection. The observation of fungal hyphae in phloem rays 29 and pit pairs of xylem tracheids suggests important impedance of cross-transfer of materials between adjacent cells. One cannot make a positive conclusion about the role of fungal hyphae found in these cells. The results obtained in this study neither support nor negate the hypothesis of xylem dysfunction as a cause of distal symptoms. Evidence for, or against, this hypothesis can only be derived from xylary fluid movement studies. Although wilting (drooping) of infected branches is not a typical symptom of y; kunzei infection, one should keep in mind that blue spruce is a rigid woody plant and even if wilt occurred, it could not be observed. In addition, there was no evidence of gum secre- tion or occlusion of xylem cells. However, substances, e.g. gum or metabolic products of V; kunzei, could impact visco- sity to xylary fluid and interfere with cross-transfer of materials between adjacent cells. 10. 11. 12. 30 LITERATURE C ITED Gifford, E. M. 1950. Softening refractory plant material embedded in paraffin. Stain Technology. 25:161-162. Gilgut, C. J. 1936. Cytospora canker of spruces. National Shade Tree Conf. Proc. 12:113-119. Hinds, T. E. and J. L. Stewart. 1965. Cytospora canker recurrence on Douglas-fir in Colorado. Plant Disease Reporter. 49:481-482. Hooker, W. J. 1967. A microtome for rapid preparation of fresh sections of plant tissue. PhytOpathology. 57:1126-1129. Jackson, L. W. R. 1947. Methods for differential staining of mycorrhizal roots. Science. 105:291-292. Marsden, D. H. 1948. PhytOpathological notes. A Valsa associated with cvtospora canker of spruces. Phytopathology. 38:307-308. Rohrback, K. G. and N. S. Luepschen. 1968. Environ- mental and nutritional factors affecting pycnidio- spore germination of Cytospora leucostoma. Phytopathology. 58:1134-1138. Strong, F. C. 1953. Spruce branch canker. National Shade Tree Conf. Proc. 29:30-35. Waterman, A. M. 1955. The relation of Valsa kunzei to cankers on conifers. Phytopathology. 45:686-692. Wehmeyer, L. E. 1924. The perfect stages of the Valsaceae in culture and the hypothesis of sexual strains in this group. Michigan Acad. Sci., Arts and Letters. Paper. 4:395-412. Wehmeyer, L. E. 1926. A biologic and phylogenetic study of the stromatic Sphaeriales. Amer. Jour. Bot. 13:575-645. Wehmeyer, L. E. 1975. The pyrenomycetous fungi: Mycologia Memoir, No. 6 250 p. PART II PLANT - WATER RELATIONSHIPS AND SITES OF VALSA INFECTION A. Effect of Drought Stress on Cytospora Canker Development B. Infection Through Wounds C. Infection Through Needles INTRODUCTION Greenhouse inoculation of healthy Colorado blue spruce trees with mycelium from monoconidium cultures consistently failed to produce any cankers, yet Cytospora canker is very common. The common occurrence of this disease is difficult to account for without assuming that the diseased trees had been or are being subjected to some predisposing agent(s). The literature on Cvtospora diseases of many hosts stresses the role of predisposition in disease development (5,6,7,10,11,12,13,14,15,17,18,19,22,23,24). Bier reported that reduced bark moisture in woody plants could result in significant increases in diseases caused by weak pathogens (1,2,3,4). There was a close correlation between the development of bark cankers and the moisture content in living bark. Bloomberg (6) suggested a direct relationship between moisture content of poplars and resistance or susceptibility to Cytospora chrysosperma (Pers.) Fr. Wright (22,23) and Jorgensen and Cafley (13) suggested that drought predisposes trees growing on poor sites and with poorly developed root systems to infection by Q; kunzei. Cytospora species require wounds to gain entry to a 31 32 host. All workers have found wounds to be necessary for the development of Cytospora canker on spruce trees in artifi- cial inoculation studies (8,9,13,19). Gilgut (8) concluded that failure to secure infection without wounding suggests ’that Cypospora kunzei is a wound parasite. He found no visible wounds on naturally infected Norway spruce, yet abrasion wounds are easily noted on infected and healthy Norway and Colorado blue spruce trees. In the case of Colorado blue spruce in Michigan, the major predisposing factors appear to be drought and wounds. The purpose of these studies were to determine: 1) the effect of drought stress, 2) the necessity of host injury as a prerequisite for Cytospora canker development, and 3) to determine sites of infections as a result of artificial inoculations of healthy Colorado blue spruce trees. MATERIALS AND METHODS A. Effect of Drought-Stress on Cytosppra Canker Development Six five-year old potted Colorado blue spruce trees in the greenhouse were drought-stressed by giving them only 1000 ml of water once a week for a period of 10-12 weeks during the summers of 1978 and 1979. Two trees each were inoculated at the beginning of the drought-stress period with discs of mycelium from single ascospore or monoconidium cultures of Valsa kunzei. The remaining 2 trees served as 33 the drought-stressed non-inoculated controls. Six non- drought-stressed trees were also treated as above. Non- drought-stressed trees received 350 m1 of water per day. Four experimental branches and one control branch were inoculated on each tree in 1978; six experimental and one control branches were inoculated on each tree in 1979. A total of 24 trees and 72 branches were inoculated. Inoculation zones were swabbed with 2.62% sodium hypochlorite solution, rinsed with sterile distilled water and wounded through the bark. The inoculum in the form of mycelial blocks was placed onto the wounded zones, wrapped with sterile cheesecloth and covered with parafilm. Control branches were treated in the same manner but received water agar blocks. The wrappings were removed after 2 weeks. The amount of disease was evaluated routinely, and final data on resin production, needle dron and fungus fruitification were recorded after 12 weeks. B. Infection Througp Wounds Wounded and inoculated branches in the non-drought- stressed tree experiment described above served as the wounded and inoculated experimental branches. In 1978, 4 branches on each of 4 non-drought-stressed trees and on 2 trees in 1979 treated in the same manner except for wounding served as non-wounded treatments. A total of 6 trees and 24 non-wounded branches were inoculated. Evaluation of disease was made 10-12 weeks after inoculation for resin production, 34 needle drop and for the formation of fruiting bodies. C. Infection Through Needles Twelve potted Colorado blue spruce seedlings in the greenhouse received a 48-hour pre-inoculation misting period in a mist chamber. Thirty needles on 3 seedlings were individually inoculated with either 0.01 ml of a suspension containing 3.8 x 107 freshly harvested conidia per m1 of water or 2.0 x 106 freshly harvested ascospores per ml of water. An additional 60 needles on 6 seedlings were inocu- lated with sterile glass distilled water. All the needles were left attached to the seedlings. Inoculated seedlings were watered, covered with a plastic bag and left in the mist chamber for another 48-hour period. The seedlings were placed on a bench in the greenhouse and the plastic bags removed after 4 days. The plants were routinely checked for symptoms of infection. These experiments were repeated twice. RESULTS A. Effect of Drought-Stress on Cytospora Canker Infection A11 trees inoculated in 1978 with monoascospore culture inoculum and drought-stressed suffered death and needle drop of the 8 inoculated branches (Table 1). None of the non-inoculated drought-stressed trees suffered death or needle drop of the branches. Five out of 8 inoculated Humwuuev O0.0 u m .Hm>ma mo.o up n EOuu ucmuwuwwp maucmofimficmfim no: m“ a 35 prCCpo3lc0cv HompCpozlcocv m mm\w N m H N wwaoome Eswpwcooocoe mfiamoafi muommoommocoe COHMHDUOCw ICOC mfiaoome Esfipwcooocoe poumapoocwlcoc mHHmowe muommoommocoe pwumasoocHIcoc mHHwomE Eswpficooocos cwumasoocHicoc mHHwo>E muommoomwocoe amp pom nouns HE omm xomz pom nouw3 HE coca monocmun monocmup pwuoomcfi pwumasoocfilcoc NO .oz can popcso3 mo .02 mnma ucoEQOHw>mo uwxcmo wuoam0u>o co mcficcso3 pcm mmouumiunm50ua mo uowmmm cmuMHpoocH monocmun «0 .02 H mamdfi ucwsfiuwmxw Epasoocfi \mmouu mo .02 yo mama ucmEummua 36 branches on non-drought-stressed trees suffered death or needle drop of inoculated branches. Data for 1979 show that all 12 branches on drought-stressed seedlings inoculated with monoascospore culture of y. kunzei suffered death and_ needle drOp within 43 days after inoculation (Table 2). The first symptoms of infection appeared 23 days after inoculation. None of the non-inoculated drought-stressed trees or branches which served as controls suffered death or needle drop. Seven out of 12 branches on non-drought- stressed trees inoculated with monoascospore culture inoculum developed cankers in 63 days. Thus the time from inocula- tion until cankers developed was longer in the non-drought- stressed trees than in the drought-stressed trees. None of the branches on control trees developed cankers. Typical resinosis on infected branches was evident. Re-isolation from artificially inoculated branches yielded cultures iden- tical to the cultures used as original inoculum. Pycnidia production was rare. Drought-stressed and non-drought- stressed seedlings inoculated with monoconidium culture ino- culum were not infected. Three isolates from each of blue spruce and Norway spruce were tested. Growth resumed when the drought stressed seedlings were placed on a regular watering schedule. B. Infection Through Wounds The results given in Tables 1 and 2 indicate that branches which had been wounded prior to inoculation with .1ummuuec OHO.O u a .Hm>mH HO.O an a soup acmummuHO sHucmonHOOHm mH m mHHmomE o Hpmpcsoslcocv v H EsHpHcooocOE mHHoome o Hpmpcsozlcocv v H ouommoommocos o m o H pwumHsoocchoc mHHwONE o N NH N EpHpHcooocoe o m o H pwumeoocchoc amp mHHmoma pom nouns nNH\h N NH N wuommoommocoe HE omm o m o H poumHsoocchoc mHHwome o N NH N EpHpHcooocoe o m o H pquHsoocchoc xwmz mHHmume pom uwuma ImNH\NH N NH N wuommoommocoe HE boOH monocmup monocmun cmDMHsoocH ucmEHuwmxm EsHsoocH acmEumwuB pwuowmcH pquHsoocchoc monocmup no .02 \mwwuu mo .02 mo maze mo .02 paw popcsos mo .02 Nan ucmsmon>mo umxcmo muOQm0u>U co mchcsoz can mmwuumiunm50un mo uommwm N mqmflfi 38 monoascospore culture inoculum were differentially cankered in drought-stressed and non-drought-stressed trees. Inoculation with monoconidium culture inoculum failed to produce infection and wounded branches healed over with much callus formation. Non-wounded, and non-drought-stressed, inoculated branches remained healthy and appeared free of cankers. C. Infection Through Needles Periodic examinations of the inoculated needles indi- cated no infection 3 months after inoculation. There is no evidence to indicate that Valsa infection occurs through the needles. DISCUSSION The results indicate that drought-stress in the host influences Valsa infection and subsequent canker development. These findings are supported by recent work in Canada (l,2,3,4,6) on the relation of bark moisture to the development of canker diseases on various hosts. For example, Bier (4) reported the threshold levels of bark moisture for susceptibility to Hypoxylon canker by Populus tremuloides, g. trichocarpa and Sglig spp. were 75-77 percent. Maintenance of the relative turgidity of the bark above 80 percent arrested canker development. Bloomberg (6) studied the moisture relations of three poplar varieties. He observed greater resistance to canker disease caused by 39 Cytospora chrysosperma in poplar hybrids than in g. trichocarpa. He attributed this resistance to high bark moisture in the hybrids. Although direct water potential or relative bark moisture was not measured in the study reported here, indirect evidence for influence of drought-stress comes from the fact that growth resumed when drought- stressed trees were placed on a regular watering schedule. That Cytospora canker of Colorado blue spruce increases a bark moisture decreases should be investigated. The yglgg stage appears to be the infective stage. This finding is supported by the work of other investigators. Marsden (16) was able to show that cankers and death of Norway spruce branches occurred following ino- culation with mycelium from single spore cultures of yglgg. He did not, however, test the pathogenicity of the species of Cytospora which causes the canker disease of spruce. He was unable to obtain sufficient fertile perithecia to deter- mine the importance of the ascospores in the natural spread of Cytospora canker. Waterman (19) obtained infection on Douglas-fir, and blue, Norway and white spruce using myce- lium and mature pycnidia from isolates of Valsa kunzei var. piceae. She did not report details of conidial inoculations. As reported previously (8,13,16,19) infec- tion occurred only when the host plants were wound-inoculated. The findings of this study, however, conflict with those reported by other investigators regarding infections 40 of spruce with conidia of Cytospora kunzei. Gilgut (8) suc- ceeded in obtaining artificial infection of wounded Norway sprucein the field with freshly harvested spores or myce- lium of g. kunzei. He did not state whether the origin of the culture was pycnidia or perithecia. His spore suspen- sion was made by dissolving several freshly exuded spore tendrils in distilled water. His mycelial inoculum was obtained from a single spore culture from spore suspension. Jorgensen and Cafley (13) were able to induce infec- tion on white and Norway spruce with mycelium, pycnidiospores, or ascospores. The monoascospore cultures used in the inoculation experiments reported here always produced pycnidia on medium containing sterilized blue spruce branch twigs. These cultures were infective. Therefore, knowledge of the original culture and the type of fruiting structure(s) from which the inoculum was obtained is extrememly important. Both types of fruiting bodies exude spores in indistinguishable tendrils on wetting. The possibility of mixed inoculum is conceivable. Since perithecia were rare on naturally-infected Norway spruce, only mycelial inoculum was used in the drought- and non-drought-stressed studies. Therefore, the question of whether ascospores are capable of infecting blue spruce was not answered. The conflicting results regarding infection of spruce by mycelium and conidia of Cytospora kunzei may result from differences in host susceptibility, isolate virulence, host reaction to wounding and host 41 physiology at the time of inoculation. Colorado blue spruce may be less susceptible than white and Norway spruce and may react to wounding in different ways. Although it has not yet been established that the conidial stages found on these hosts are identical, and extensive cross-inoculation studies have not been attempted, there is little evidence to suggest host specificity among the isolates. For example, the ascospore cultures used in this study were obtained from Norway spruce and were pathogenic to blue spruce. Three Cytospora isolates from each of blue and Norway spruce were tested and found to be non-infective. If conidia and myce- lium of g. kunzei are capable of infecting blue spruce, it is possible that this research did not duplicate the host and/or environmental conditions essential for successful inoculation. The question arises as to the role of conidia in the life cycle of Q. kunzei. Although they germinate and pro- duce mycelial colonies, strong corroborating evidence of infection either from conidia or from mycelial colonies derived from them is lacking and should be investigated. The results of this study suggest they probably are not involved in the infection process. Pycnidia arise in the same tissue as the perithecia. However, the two types of fruiting bodies have never been found in the same stroma (21). Possibly conidia serve as gametes in hybridization of compatible perithecial initials. With the exception of 42 Wehmeyer (20), none of the other investigators (8,13,19) has been able to induce perithecial formation in culture. Investigations of Cytospora canker have been concerned with describing canker development. The details of perithecial and pycnidial development in y. kunzei have not been reported. Failure to obtain reproducible results calls for a study to elucidate their developmental morphology. The inoculation methods used in these studies were similar to those used in the studies mentioned above. However, sterilized blue spruce twigs with the test fungus growing on them were used as the inoculum instead of myce- lial blocks. Spruce twigs provided a mass inoculum effect as well as a good and abundant food base for fungus establishment. Therefore, failure of Q. kunzei to infect could not have been due to failure of conidia to germinate but rather due to failure of germinated conidia to become established. A reliable inoculation method is needed to clarify the host range of Q. kunzei. The method reported here could be standardized to make it possible to secure reproducible results. The results of wound and needle inoculations support the hypothesis that wounds are important for Cytospora canker development. However, they do not exert a general prediSpositional influence on the tree. For disease development, the tree must also be weakened by some other factors, e.g., drought-stress. That insects are present and 43 may play a role in infection must be demonstrated in future studies. The atmosphere inside insect wounds would remain suitable for spore germination. Such wound sites may serve as infection courts. In addition, natural products in spruce bark may prove important for germination. 10. 44 LITERATURE C ITED Bier, J. E. 1959. The relation of bark moisture to the development of canker diseases caused by native, facultative parasites. I. Cryptodiaporthe canker on willow. Can. J. Bot. 37:229-238. Bier, J. E. 1959. The relation of bark moisture to the development of canker diseases caused by native, facultative parasites. II. Fusarium canker on black cottonwood. Can. J. Bot. 37:781-788. Bier, J. E. 1959. The relation of bark moisture to the development of canker diseases caused by native, facultative parasites. III. Celphalosporium canker of western hemlock. Can. J. Bot. 37:1140-1142. Bier, J. E. 1961. The relation of bark moisture to the development of canker diseases caused by native, facultative parasites. IV. Pathogenicitv studies of Cryppodiaporthe salicela (Fr.) Petrak, and FusariUm Iateritium Nees., on Populus trichocarpa Torrey and Gray, g. 'rogusta', P. tremuloids Michx., and Salix spp. Can. J. Bot. 39:139- 144. Bloomberg, W. J. 1962. Cytospora canker of poplars: Factors influencing the development of the disease. Can. J. Bot. 40:1271-1280. Bloomberg, W. J. 1962. Cytospora canker of poplars: The moisture relations and anatomy of the host. Can J. Bot. 40:1281—1292. Bloomberg, W. J. and S. H. Farris. 1963.1 Cytospora canker of pOplars: Bark wounding in relation to canker development. Can. J. Bot. 41:303-310. Gilgut, C. J. 1936. CytOSpora canker of spruces. National Shade Tree Conference Proc. 12:113-119. Gilgut, C. J. and O. C. Boyd. 1933. Cytospora canker of Picea spp. Phytopathology. 23:11. Helton, A. W. 1961. Low temperature injury as a con- tributing factor in Cytospora invasion of plum trees. Plant Disease Reporter. 45:591-597 ll. l2. 14. 15. 16. 17. 18. 19. 20. 21. 22. 45 Hinds, T. E. and J. L. Stewart. 1965. Cytospora canker recurrence on Douglas-fir in Colorado. Plant Disease Reporter. 49:481-482. Hubert, E. E. 1920. Observations of Cytospora chry- sosperma in the northwest. Phytopathology. 10: 442-447. Jorgensen, E. and J. D. Cafley. 1961. Branch and stem cankers of white and Norway spruce in Ontario. Forestry Chronicle. 37:394-400. Kable, P. R., P. Fliegel, and K. G. Parker. 1967. Cytospora canker on sweet cherry in New York State: Association with winter injury and patho- genicity to other species. Plant Disease Reporter. 51:155-157. Layallee, A. 1964. A larch canker caused by Leucostoma kunzei (Fr.) Munk ex kern. Can. J. Bot. 42:1495-1502. Marsden, D. H. 1948. Phytopathological notes. A Valsa associated with Cytospora canker of spruces. Phytopathology. 38:307-308. Poyah, A. H. 1921. An attack of poplar canker following fire injury. Phytopathology. 11:157-165. Scharpf, R. F. and H. H. Bynum. 1975. Cytospora canker of true firs. USDA Forest Pest Leaflet. 146:1-5 Waterman, A. M. 1955. The relation of Valsa kunzei to cankers on conifers. Phytooathology. 45: 686-692. Wehmeyer, L. E. 1924. The perfect stages of the Valsaceae in culture and the hypothesis of sexual strains in this group. Michigan Acad. Sci., Arts and Letters. Paper. 4:395—412. Wehmeyer, L. E. 1926. A biologic and phylogenetic study of the astromatic Sphaeriale. Amer. Jour. Bot. 13:575-645. Wright, E. 1957. Cytospora canker of Rocky Mountain Douglas-fir. Plant Disease Reporter. 41:811-813. 23. 24. Wright, E. 1957. Zentmyer, G. A. 46 Cvtospora canker of cottonwood. Plant Disease Reporter. 41:892-893. 1941. Cytospora canker of Italian cypress. PhytOpathology. 31:896-906. A. B. C. PART III DISPERSAL OF ASCOSPORES AND CONIDIA OF VALSA KUNZEI Trapping of Rain Dispersed Conidia and Ascospores Trapping of Air-borne Conidia and Asocospores Environmental Factors Affecting Conidia and Ascospore Numbers Spore Isolation and Spore Viability INTRODUCTION Fungi such as Cytospora spp. that form their conidia in a pycnidium are generally assumed to release their spores in mass in response to wetting. Wind-blown rain and loca- lized Splashing of rain have been suggested as mechanisms of conidial dispersal in Cytospora spp. (5). The Valsa stage of this fungus also occurs on cankered branches of Colorado blue (Picea pungens Engelm.) and Norway spruce (P. abies (L.) Karst) interspersed with pycnidial stromata (6). The centrum development of Valsa spp., Family Diaporthaceae sensu Miller (7), is such that at maturity the asci become detached from the base of the centrum and float in the central cavity of the perithecium (8). Wehmeyer (14) has recently placed Valsa in the Family Valsaceae. This family has allantoid and hyaline ascospores. Ascospores of Valsa ceratosperma have been reported to be discharged from the perithecia by two mechanisms (10). Under continuous wet conditions, the ascospores ooze from the perithecium in mass much the same way that the conidia are released. Forcible discharge of ascospores was also demonstrated to occur, and free water was required to ini- tiate the process. Bertrand and English (1) studied the release and dispersal of conidia and ascospores of Valsa 47 48 leucostoma. Conidia were reported to be water-borne and dispersed by the wind-blown rain. Ascospores were both air- borne and water-borne. Hodgkiss and Harvey (2) have suggested that forcible discharge of ascospores occurs in Diapgrthe spp. Calonectria spp. have been reported to show passive or forcible ascospore discharge depending on the environment (4). Daldinia concentrica is also known to liberate ascospores by forcible discharge or by oozing (3). In the field, spore discharge by Hypgxylon rubininosum, H. fuscum, Nectria ditissima, Lopadostoma turgidum and Diatrypella aspera was largely confined to the rain period (12). Sordaria macrospora has been reported to discharge spores over the temperature range of 5° - 30°C (13). The purposes of this study were to monitor the release and dispersal of ascospores and conidia of Valsa kunzei and to determine the environmental conditions necessary for these processes in the field. MATERIALS AND METHODS Location Ascospores and conidia of Valsa kunzei were trapped from cankered Colorado blue and Norway spruce at Baker's Natural Wood Lot at the Michigan State University (MSU) campus, East Lansing, and at Sharp Nursery, Albion, Michigan, respectively during 1978 and 1979 study periods. Spore trapping was initiated in either mid March or early 49 April and terminated at first snowfall. A. Trapping of Rain Dispersed Conidia and Ascospores Plastic 13.5 cm diameter funnels were positioned beneath active Cytospora cankers. A length of tygon tubing connected the funnel to a one gallon plastic collecting container. Containers were changed on a weekly basis. The water collected from each trap was filtered through six layers of cheesecloth and mixed thoroughly on a Waring commercial blendor. A 25 m1 aliquot from each trap was centrifuged at 12,000 RPM for 15-20 minutes on an IEC clinical centrifuge (Damon/IEC Division, Needham Heights, Massachusetts). The resulting pellet of spores was adjusted to 10 ml by volume with distilled water. This was then mixed on a Voltex mixer for 5-10 seconds and the number of spores in an aliquot counted in a Levy-Hausser Hemacytometer AO (American Optical, Scientific Instrument Division, Buffalo, NY). The Sharp Nursery trapping station also included seven day weather recording instruments e.g. a hygrothermograph (Bendix Corporation, Baltimore, MD), tipping bucket rain gauge (Weathermore Corporation, Sacremento, CA) and DeWitt leaf wetness meter (M. DeWitt Co., Hengelo, The Netherlands). Instruments to record rain- fall, temperature, relative humidity and leaf wetness were not set up at Baker's Natural Wood Lot due to lack of limited access to this site. 50 B. Trapping of Air-borne Conidia and Ascospores A Burkard Volumetric Recording Spore Trap was used to sample the air for the presence of ascospores and conidia of Valsa kunzei from early April or late March until the first snowfall during the 1978 and 1979 study periods, respectively. The trap was placed in the center of four diseased Norway spruce trees bearing both pycnidia and perithecial stages of Valsa kunzei. The trap was set at ground level at 1.67m, 1.12m, 1.07m, and 1.22m from the trees. The spore intake orifice was about 20 inches above the soil surface. The vacuum pump serving the trap drew air through the trap orifice at a rate of 10 liters/minute. The suction fan of the trap was powered by a 12 volt motor with a 12 volt car battery as its power source. The trap allowed a continuous record of spore catches for intervals of up to seven days. A continuous record of rainfall, temperature, relative humidity and leaf wetness was maintained near the spore trap. For spore counting, the tape was cut into lengths of 48mm which is equivalent to 24 hours of spore trapping. Cut sections were mounted on clean microscope slides with two or three drOps of water and stained with lactOphenol and cotton-blue. The tapes were scanned using a Nikon microscope (Nikon, Inc., Instrument Group, Ehrenreich Photo-Optical Industries, Inc., Stewart Ave., Garden City, New York) at 400x magnification. The spore counts were made on an hourly basis. 51 C. Environmental Factors Affecting Conidia and Ascospore Numbers The Pearson's Corelation Coefficient and multiple regression analysis were performed on 1978 and 1979 data. These computations were made using the SPSS - Statistical Package for the Social Sciences (VOgelback Computing Center, Northwestern University, Version 7.0) run on the Model 6500 Computer (Control Data Corporation) at MSU. The environmen- tal parameters considered in correlation coefficients and in the development of the regression equation to account for the variability in spore numbers were: 1) mean daily temperature, 2) hours of 100% relative humidity, 3) hours of rainfall, and 4) hours of leaf wetness. D. Spore Isolation and Spore Viability Cankered Colorado blue and Norway Spruce twigs with c0pious amounts of resin flow were collected biweekly from either March or April until the first snowfall in late November during the study period 1977-1979. Ten randomly selected twigs were soaked in 2.62% sodium hypochlorite solu- tion for 5 to 10 minutes with continuous agitation. The twigs were washed in running deionized water for 5 minutes, soaked submerged in deionized water for 30 minutes, wrapped in moistened paper towel and left to stand overnight in a moist chamber. The twigs were examined under the microscope for perithecia, ascospores, pycnidia, and conidia. From each isolation period, the percentage of twigs with any one 52 of the fruiting bodies and spores was determined. Spore viability was determined by placing spores on a petri dish containing medium B agar and incubating at room temperature for 4-7 days. The resulting colonies were compared with each other and with several isolates obtained previously. RESULTS A. Relative Populations of Water-borne Conidia and Ascospores During 1978, the first conidia were caught at Baker's Natural Wood Lot (MSU) during the week from March 20-27 (Figure 1). Spore counts increased to an average peak of 30 x 105 conidia/ml during the week of May 8-15. Conidia counted remained numerous throughout the spring, and count- able numbers of conidia were trapped for 25 weeks during rain throughout the year. The results of 1979 studies at the same trapping site are presented in Figure 2. A trend similar to that obtained in 1978 was observed. Conidia were trapped for 20 weeks during rain throughout the year. The first conidia were caught during the week of March 27 - April 3. This was also the week with the highest mean number of conidia counted (11.3 x 105 conidia/ml). Conidial count remained high in spring except during weeks with the lowest volume of water collected. Conidia continued to be trapped throughout the summer until first snowfall in November. No apparent second peak of conidia production could be identified for the period from summer through fall. 53 :3 00L x um) ebnr ens Jed xeeM 10d pezoeuoo :ewmugeu go ewmoA noon ('3 wo O ‘9 0 '- v- o: o N c ID V n N - T I l I l l 1 TI I l 1 I ll'lMOW 1811:! "F L fl WlflllOO “I'M 0N E 8 P X ‘1' 8 ==: 9.33.1109 4033M °N DONOIIOO 1.03M 0N P'lOOIIOO ”WM 0N 9.83.1100 ”I'M ON pozoouoo mm on xumpna —-> pus-nos mm on pus-nos mm on ”WIND 1.33M 0N ”33.1190 ”I'M ON 9.30.1100 MUM 0" l Jljl Iowan—o 1111 GONG 10 I 8 R 8 $2 - ( 901 X) 200M nd mm go nu: Jed wanes eipguoo :o Jequmu ueew lZ/lt‘OZ/lt OZ/ll'CLIlt Sl/U'OOIH, 90/”‘00/01 OSIOl’CZ/Ot CZIOPDt/Ot 91/0l'00/Ol 00/01‘30/01 ZO/Ot’SZ/GO SB/GO'Il/GO Ol/OO'lL/BO HIM/00 70/00'08/00 OZ/W‘tZ/OO MIMI/00 ”/8010/00 [.0/80’10/10 WIlO’VZ/lo VZ/lO‘ll/lO lt/tO‘OL/lo 01/10‘80/10 80/1018!” Oil/00'0”” 0U00‘8U00 Zl/OO’SO/OO 90/90'08/90 08/90'33/90 32/90‘9 £190 91/90'00/90 OOIWLO/QO 10/90'92/70 98/701 LIFO ll/VO'Ol/VO 01/9920!” 8017018180 :z/co-oz/so ‘5 3 re kunzei Caught lrom Cankered Colorado 7 . F lgure 1. Number 01 Water-borne Conidle of C 103 Blue Spruce Branches-MSU, Mlchlgen, 54 = oog x (gm) sdnr engg Jed neeM 10d pagoeggoo regemugeu go eumgoA ueew O a: O h c to Q 0 N ", m e n N v- o o a o‘ o o o o c o l l l l T I l l T T l I I 1 ggegmouegugg —-> pomuoo ”I'M ON P'PNIOO ”WM 0N POROIIOO 1.83M 0N 9.1301100 1.83M 0N PCWOIIOO ”WM 0N ”DOIIOO ”WM 0N 9.39.1100 1010M 0N 9.19.1100 1.83M 0N bi . 3 fl . 9.8301100 ”WM 0N ”PMIOO 1.33M 0N 90330II°O ”WM 9N P'lOOIIOO ”I'M oN 9.83.1103 1.83M 0N "”99" —-> W Ts \ 5 901001100 ”I'M 0N E 8 In D p K (9 ;. q ' 4 1 1 1 1 1 1 1 1 1 1 1 I N v- 0 a D N 0 In C 4') N P 0 e- P v- - (901 x) )geeM red JegeM go nu 10d gufinea egpguoa go requmN ueew 03/ll'0l/ H 01/11'00/ Ll 00/”‘03/01 08/0l'03/01 S310l’01/0l 0t/01’00/0l 60101'30/01 30/01’93100 93/00'0l/00 0t/00'll/00 “/00'70160 70/00'03/00 0310013100 131 00'71-100 ”10010100 1010010110 £0110'73110 y 73/101l140 “140'01110 01110120110 001(0'03/00 03100111100 0l100‘3 £100 3l100’90/00 90/00‘63190 63190‘33/90 33190'9l/90 91/90'00/90 0019010190 l0/90'73/70 7317016170 ll/70'0L170 0l170'00/70 0017013100 3 3 Figure 2. Number of Water-borne Conldla 01 C toe Blue Spruce Branches-MSU. Mlchlgan, ra kunzel Caught lrom Cankered Colorado 55 No conidia were trapped during the winter months. No ascospores were caught at the MSU trapping site during the 2-year study period. Ascospores and conidia were trapped in 1978 and in 1979 in spore traps located at Sharp Nursery. In 1978 ascospore catches ran from late April through July (Figure 3). The highest ascospore catch occurred during the week of April 29-May 6. No ascospores were caught after July 1. Conidia were trapped for 24 weeks from April 15 until November 25, 1978. The highest number of conidia trapped were caught between April 15-22, and remained abundant throughout spring until the week of June 24-July 1. Thereafter, numbers of conidia declined but remained present throughout the study period. In 1979 the first and the greatest number of ascospores were caught during the week of April 20-27 (Figure 4). No ascospores were trapped after June 15. Conidia on the other hand, were trapped for 26 weeks from March 30 until November 16. Conidia were first trapped bet- ween March 30-April 6. Conidia trapped reached an average peak of 4.9 x 105 conidia/ml in the week of April 20-27. Again as in 1978, conidia were most abundant in the spring and less abundant during summer and fall. B. The Occurrence and Importance of Air-borne Conidia and Ascospores Ascospores and conidia were first trapped on April 9 in 1978 (Table l). The last ascospores were trapped on 56 1:: 001 x (Iw) anr 111513 19d )geeM red pagoeggoo Iegemugeu go OLUMOA ueew O In a :3 N v- .- 3 G N 0 In an N 1- O I I I I I I I I I I r I I j "314400918113 —9 % fl sz/II-eI/u r g—fi cI/II-II/II fi 11/11'70/ L L peIoeggoa IeIeM 0N 70/ L l-OZ/OI r __1 sum-1:101 ‘ L310£°71101 ; 71/0110/01 L 10/01'00160 ; 08/60'83/60 . fl SUSHI/60 5 fl OHM/60 pegoeuoo IeIeM 0N w/eo-zoxeo 901301100 1013M 0N 30/ 80°93100 r “IMIIBO L 5110011100 31100'90100 ; fl 90/80°62/to fi fi alto-nae “’O b zzuo'smo In; ‘2 POIOMIOO ”I'M ON Silto-OOILO " 0 g. g ‘ fl 00/10'10/10 ‘2 3 fi ' lO/lO’tZ/OO 8 2 I 7319011100 I a pomuoo mm on u/oo-OI/oo 01/90'60/90 ”WMIOO 1010M 0N 00/0013/90 lZ/So-OZISO "g woquna _, oz/so-eI/so g P'lOOIIOO 1089M 0N 2119010190 «.2 so/so-ez/vo 2 901091103 mm on sz/vo-zz/vo ‘. ZZ/tttil/OO peIoeuoo mum on 91/70-00/70 ] l l .1 l l l l, l '3 :1 s s a 2 m ' " " ° ; (901 xi 1190M Jed 1019M go qu red qunea serodsoosv pue egpguoo go requmN ueew .09 .14 .03 .00 .00 2.04 .75 .111.10 .00 .54 .16 .00 .27 .00 .11 Rainlali inches .43 In In .00 .07 .67 .55 .00 .55 .00 .34 .00 .25 .24 .00 .25 .00 kunzei Caught from Valsa Cankered Norway Spruce Branches-Albion, Michigan, 1978. m = missing or unavailable data. Figure 3. Number oi Water-borne Ascospores and Conidia oi 57 a OOIXIIw) sfinr 1115!: led )geaM 19d pagoeggoo .IeIEMugeu go euIngoA ueew 3323332223883333833 IIIrFrIIIIIITTII ggegmoue gang _. 91/11-60/ it peIaeuoo mum on 60/ i t-ZO/ ti « ZO/ll-SZ/Oi 93/01-61/01 EI/or-zI/OI :1/01-90/01 : 1! semi-salsa 90le100 MUM 0N 83/60'13/60 901001100 MvM 0N Iz/eo-n/so E 71/6010/60 IMoOIIoo MOM 0N t0/60'iC/80 It/co-vz/ao vz/ao-u/ao e umo-oueo . oven-80190 comma/Lo 13/10'03/10 ,5; 901001100 MOM on ozuo-cI/zo “a: E eI/zo-ao/zo g g 90/10-62/90 «3 § sz/so-n/oo O 0 ° “ zzmo-smo I B s1/9o-90/90 e coma-10190 —- Iona-salsa E sz/so-aI/so neerqpna 4 91190-11190 901901103 McM 0N XII/so-vo/so r valso-zz/vo £3170‘03170 DEPOHOO J“WM 0N 0317011170 “ €i/70'SOI7O d fl 00/70‘00/00 l l l l I l l I (901 x) IgeeM red IegeM i0 iw Jed Iqbnea seIodsoosv pue egpguoa go quuInN ueew 2b .00 .05 .00 .00 .36 .95 .43 .72 .40 .00 m .73 .62 .00 .44 .41 .00 .57 .02 .34 .07 .55 m .411.12 .04 .00 .07 .601.31 .061.43 .61 Reinieii inches Figure 4. Number oi Water-borne Ascospores and Conidia oi Valsa kunzei caught irom Cankered Norway Spruce Branches-Albion, Michigan, 1979. m = missing or unavailable data. 58 TABLE 1 Number of Air-borne Ascospores and Conidia of Valsa kunzei Caught at Albion, Michigan in a Burkard Spore Trap. 1978. Date Number of Conidia Number of Ascospores April 9a 540 292 April 10 3002 3292 April 11 304 284 April 18 248 669 April 20 5000 2272 April 21 260 56 May 4 264 20 May 5 4272 2236 May 6 274 124 May 8 m m May 9 m m May 10 m m May 11 m m May 12 m m May 13 budbreak m m May 14 m m May 20 m m May 30 36 0 June 6 152 0 June 7 48 4 June 12 8 0 June 17 12 0 June 18 128 0 June 19 20 0 June 20 232 0 June 21 52 0 June 22 248 0 June 24 96 0 June 28 76 0 June 29 208 0 June 30 84 0 July 1 32 0 July 2 32 0 July 4 64 0 July 9 12 0 July 23 88 0 July 26 272 0 July 27 8 0 August 2 725 0 August 3 240 0 August 8 216 0 August 9 88 0 59 TABLE 1 Continued Date Number of Condidia Number of Ascospores August 11 244 0 August 19 304 0 August 27 84 0 August 28 1092 0 September 12 1160 0 September 13 160 0 September 14 684 0 September 15 96 0 September 17 196 0 Spetember 18 80 0 September 21 88 0 September 24 32 0 September 27 116 0 October 3 104 0 October 4 72 0 October 5 132 0 October 6 152 0 October 12 92 0 October 13 64 0 October 15 36 0 October 16 32 0 October 25 232 0 October 26 184 0 November 6 156 0 November 7 12 0 November 13 40 0 November 14 84 0 November 23b 4 o aDate on which trapping began. bDate on which the last spores were trapped. mMissing or unavailable data. 60 June 7. Conidia were caught throughout the season. Numbers of air-borne conidia exceeded those of air-borne ascospores. Peak conidia and ascospore catches occurred in April, several weeks before budbreak. Both spore types exhibited a diurnal-nocturnal periodicity and spore discharge occurred during both daylight and darkness. Peak conidia discharge occurred at 3 am, thereafter discharge declined. Secondary afternoon and evening peaks occurred at 2 pm and 8 pm, respectively (Figure 5). Peak ascospore discharge occurred at 6 pm with other peaks at 5 am, 3 pm, and 8 pm (Figure 6). Air-borne ascospores were first trapped on March 30 in 1979 and the last ascospores were caught on June 10 (Table 2). On the other hand, air-borne conidia were first trapped on March 30 and continued to be trapped until October 29. The number of air-borne conidia exceeded the number of air-borne asc05pores by as much as 329 times. Distinct diurnal and/or nocturnal periodicity was not observed. Conidia discharge reached a peak at 4 am. Several secondary peaks were observed for 8 am, 8 pm, and 11 pm (Figure 7). The ascospore discharge cycle, on the other hand, reached a peak at 8 pm. Small secondary peaks occurred at 1 and 4 am (Figure 8). C. Environmental Factors Affecting Conidia and Ascospore Numbers Table 3 shows Pearson's Correlation Coefficients determined from the 1978 and 1979 Burkard spore trap data. 61 .32 63.5.: .332 52.33%... 2250 .35... 83330 2:27.? .o 33:52 .m 2:9”. >40 “—0 m2: 22 2002 ooéw 8.“? OoéN «I . \sfirdalg \ieurlIHII. [or APl a \d [Id 4 s 1 If; \\ Oil. 4| 4\ e e e I \\’ 4 l . < z a .. 1. . o o o I o o — .l . 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O+OO+OO+OFOOOOOOOOOCC OHOOHOCHOHOOHHCOCO +-+4-+-+4-+-++-+-+c>+-++-+-++ \OLONMMNMQ‘NNHONI‘QMIBQ‘ «wH umoEm>oz N uwnEw>oz ma umnouoo w umo0uoo Hm omnEmummm n uwosmumwm em umsms< ca umsmse em sage ma sage mm mean ma mash H moon ma >mz e um: em adage ma kudd om noun: ucwmwum mwuoamoomw pom ucmmmua mwuoamoomm wanmfl> mflowzufiuwa :ufis mowse wmuoom wanmfi> macwcowa nufi3 mmwsa mwpficoo cam mama cowuomflaoo .mnma .cmmwnofiz .c0woad um museum >m3uoz cwuommcfi muoam0u>u EOuu pwHQEmm mowse oa\mwuomm manmw> Tam mousuoouum newuwoum nuwz wmfise b mqmflP 76 DISCUSSION The results indicate that both conidia and ascospores may or may not be present in infected blue spruce during the same season. When both Spore types were present, conidia were always dominant. Conidia were also the most commonly isolated spore type from cankered branches. Conidia were released throughout the year except in winter, whereas ascospores were released in spring and early summer only. Both conidia and ascospores were air- borne and water-borne. The number of water- and air-borne conidia always exceeded the number of water- and air-borne ascospores. Peak production of ascospores and conidia occurred several weeks before budbreak. This study did not determine the specific mode of ascospores discharge. They may ooze from the perithecium and be splashed up into air currents or they may be forcibly shot from the perithecium into air currents. Although pycnidia are generally assumed to release spores only in response to wetting, wind-blown rain could result in conidia being trapped in the Burkard spore trap. This phenomenon could be tested in future stu- dies by monitoring rainfall and wind velocity and direction on a continuous basis. Both spore types are released during day light and darkness. They exhibit a distinct diurnal-nocturnal periodicity with considerable variation as to when peak release occurs. The one hour difference in the time when 77 peak discharge occurred over the 2-year study period is due to sampling error. The results indicate that conidia and ascospore release is markedly influenced by the environment. However, the environmental parameters considered in the multiple regression equation are highly correlated with each other. For example, rainfall affects temperature, relative humidity and leaf wetness. Temperature, on the other hand, influences relative humidity. These two parameters together or individually influence the leaf wetness period. Therefore, all the variables are not included in the com- putation of the regression equation. Efficient trapping and counting methods contributed to the higher (R2) value obtained. Conidia and asc05pores isolated throughout the year were found to be viable. The number of perithecia with viable ascospores in nature is very small compared to the number of pycnidia and conidia produced. This may suggest that the Valsa (ascospore) stage develOps several years after a branch is infected. In their studies, Bertrand and English (1) reported that only the Cytospora (conidial) stage developed in the two years following the death of a branch from Cytospora canker. The Valsa (ascospore) stage deve10ped three or more years later. Fewer conidia and ascospores were trapped during the 1979 study period than in 1978 at the Albion site. This reduction is explained by the fact that many diseased Norway spruce trees were cut and destroyed during the 1978-79 78 winter. This activity reduced the amount of available inoculum. There are very few reported studies of spore disper- sal in Cytospora spp. Luepschen and Rohrbach (5) collected previously liberated spores of Cytospora leucostoma from infected peach trees by washing cankered areas with water. They reported liberation of viable spores throughout the year, with the highest counts occurring in summer. However, the act of washing the cankered areas may have stimulated liberation of spores from pycnidia. In their study, spore viability was affected by both temperature and moisture. The work with Valsa kunzei reported here is supported by the study of Bertrand and English (1) working with Valsa leucostoma. They reported that conidia were released in all seasons whereas ascospores were released in spring only. In their studies, water-borne conidia were found to be 10 to 4,000 times more common than water-borne ascospores. Ascospores and conidia were water-borne and air-borne. They concluded that conidia were the dominant spore type throughout the year and in the absence of ascospores may serve as primary inoculum. In this study only the ascospores were found to be infective. 10. ll. 79 LITERATURE CITED Bertrand, P. F. and H. English. 1976. Release and dispersal of conidia and ascospores of Valsa leucostoma. Phytopathology. 66:987-991. Hodgkiss, I. J. and R. Harvey. 1969. Spore discharge rhythms in pyrenomycetes. VI. The effects of climatic factors on seasonal and diurnal periodi- cities. Trans. Br. Mycol. Soc. 52:355-363. Ingold, C. T. 1971. Fungal spores, their liberation and dispersal. Clarendon Press, Oxford. 302 p. Linderman, R. G. 1974. Ascospore discharge from perithecia of Calonectria theae, g; crotalariae, and Q; kyotensis. PhytOpathologv. 64:567-569. Luepschen, N. S. and K. G. Rohrback. 1969. Cytospora canker of peach trees: spore availability and wound susceptibility. Plant Dis. Reporter. 53: 869-872. Marsden, D. H. 1948. Phytopathological notes. A Valsa associated with Cytosoora canker of spruces. Phytopathology. 38:307-308. Miller, J. H. 1949. A revision of the classification of the ascomycetes with special emphasis on the pyrenomycetes. Mycologia. 41:99-127. Munk, A. 1953. The system of the pyrenomycetes. Dansk Bot. Arkiv. lS(2):l-163. Ouellette, G. B., J. M. Conway and G. Bard. 1965. Frequence et intensite du chancre cvtosporeen dans les plantations d'epinette du Quebec (in French, English summary). Forestry Chronicle. 41(4):444- 453. Saito, I., O. Tamura, and M. Takakuwa. 1972. Asco- Spore dispersal in Valsa ceratosperma, the causal fungus of Japanese apple canker (in Japanese, English summary). Ann. PhytOpath. Soc. Jap. 38:367-374. Walkey, D. G. A. and R. Harvey. 1966. Spore discharge rhythms in pyrenomycetes. 1. A survey of the periodicity of spore discharge in pyrenomycetes. Trans. Br. Mycol. Soc. 49:583-592. 80 12. Walkey, D. G. A. and R. Harvey. 1968. Spore discharge rhythms in pyrenomycetes. IV. The influence of climatic factors. Trans. Br. Mycol. Soc. 51:779-786. 13. Walkey, D. G. A. and R. Harvey. 1968. Spore discharge rhythms in pyrenomycetes. V. The effect of tem- perature on spore discharge in Sordaria macrospora. Trans. Br. Mycol. Soc. 51:787-789. l4. Wehmeyer, L. E. 1975. The pyrenomycetous fungi: Mycologia Memoir No. 6. 250p. PART IV CONTROL OF CYTOSPORA CANKER Preliminary in vitro Screening of Fungicides Active Against Cytospora kunzei Field Evaluation of Fungicides for Control of Cvtospora Canker INTRODUCTION The removal of cankered Colorado blue spruce branches reduces the amount of inoculum present but does not prevent subsequent infection of the remaining healthy branches (6). The pruning of affected blue spruce branches is, therefore, recommended by extension (7) and tree maintenance agencies for control of the disease. Waterman in 1955 (6) was unsuc- cessful in controlling Cytospora canker by either spraying infected white spruce (Picea glauca Moench) VOss and blue spruce (Picea pungens Engelm.) with a 4-4—50 Bordeaux mix- ture plus casein (spreader) or by cutting out infected bark and cambium of affected areas. Since then there has been no chemical control program tested for control of Cytospora canker on blue spruce (5). Luepschen (4) obtained beneficial results in controlling Cytospora canker when peach trees were sprayed with a combination of benomyl and oil before artificially inoculating the limbs with Cytospora leucostoma (Pers.) Sacc. The wide distribution and severity of Cytospora canker in Michigan makes this disease a major concern for homeowners, nurserymen, and Christmas tree growers. Since no chemical spray program has been tested for control of 81 82 Cytospora canker of blue spruce, experiments were conducted to evaluate fungicides for control of the disease and to determine the degree of control that could be expected from a fungicide spray and pruning program. MATERIALS AND METHODS A. Preliminarytig_g;§£gyscreening for fungicides active against Cytgspoga EHBZSE Eight fungicides were tested using the poison agar technique. The poison agar medium was prepared by dissolving concentrations of fungicides on an active ingre- dient basis (1, 10, 20, 100, 500, and 1,000 ppm) in warm liquified agar and mixing in a commercial blendor. Fungicide amended agar plates were each inoculated with a 7mm agar disc containing actively growing monoconidial cultures of Q. kunzei. Five plates per fungicide con- centration plus 2 unamended controls were employed. Measurements of mycelial growth were made to the nearest millimeter at 24 hour intervals to assess the amount of inhibition. Experiments were repeated twice. The fungi- cides and formulations tested were: Arbotect 20-S(Thiabendazole)- (26.6% 2-(4-thiazolvl) pegsimidazole hypOphosphite), Merck and Company, Benlate (Benomyl) - (50% methyl 1-(butylcarbomyl)-2- benzimidazolecarbamate), DuPont Agrichemicals. Bravo 6F (chlorothalonil) - (40.4% tetrachloro isophthalonitrile), Diamond Shamrock Corporation. 83 Dithane M-4S - (80% zinc-ion-Maneb), Rohm & Haas Company. Kocide 6F - (Cupric Hydroxide), Kennecott COpper Corporation. Lignasan BLP (MBC phosphate) - (0.7% methyl 2-benzimidazolecarbamate phosphate), DuPont Agrichemicals. Mertect Flowable (Thiabendazole) - (42.28% 2-(4- thiazolylbenzimidazole), Merck and Company, Inc. Topsin M 70 WP (Thiophanate methyl) - 70% ThiOphanate methyl (dimethyl 1,2-phenv1ene) bis (iminocarbonothioyl) bis carbamate, Pennwalt Corporation. B. Field Evaluation of Fungicides for Control of Cvtospora Canker Thirty Colorado blue spruce trees naturally-infected with Cvtospora canker were selected at random for the spray trials. Trees were located in 7 planted groves at Ella Sharp Park, Jackson, Michigan. These trees were approxi- mately 49 years of age. The criteria for selection were: 1) the presence of obvious symptoms of recent and natural infections and 2) accessibility with spray equipment. The fungicides tested and rate of application were: Arbotect 20—S soluble liquid (1.125 gal. in 100 gal. of water), Benlate 50% wettable powder (5 lb. in 100 gal. of water), Bravo GP or Bravo 500 (.55 gal. in 100 gal. of water) and Topsin M 70% wettable powder (3.57 lb. in 100 gal. of water). The chemicals were applied at l4-day intervals, and 5 sprays were applied each year. Sprays were applied on 5/3, 5/17 (budbreak), 5/31, 6/14, 6/28/78 and on 5/9 84 (budbreak), 5/23, 6/6, 6/20 and 7/5/79. Fungicides were applied with a power sprayer using a handgun. A11 sprays were applied in mid-morning to avoid excessive drift and contact to afternoon park visitors. Each fungicide tested was used on two groups of trees. In one group, all cankered branches were pruned and destroyed; in the other group, diseased branches were not pruned and served as a source of inoculum. There were three pruned and three non-pruned trees per group per fungicide. In all, 24 trees were sprayed with fungicides. The control group consisted of three pruned and three non-pruned trees which were sprayed with water only. The effectiveness of the spray material was evaluated each year by counting the number of cankered branches showing symptoms of infection. Counts were made at l4-day intervals beginning in July and ending at first snowfall in November during the two-year study period. The severity of Cvtospora canker in the test plots was estimated by the development of a disease index (D.I.). The index was based on cankered branch counts from July 1978 to April 1979 and from April to late November in 1979. The difference in cankered branches rather than the total number of cankered branches per tree was used in calculating the mean number of cankered branches in each treatment. The D.I. was calculated by the following formula (2): 85 '-—- ——g ' (Sum of Cankered Branches‘) d Number of Trees Evaluate D. I. = X 100 --— Tree age - 20 "J The amount of Cytospora canker in susceptible blue spruce tends to increase year after year, due to new infection. Dividing the canker counts by an age factor minimizes the effect of age on the disease index. Dividing by this age factor results in an approximation since it assumes equal yearly increase in the amount of disease, which is probably not the case. Twenty years is subtracted from the age of the tree, because Cytospora canker generally first occurs on mature trees. The effectiveness of each fungicide treatment was determined by the calculation of a percent disease control (P.D.C.) from the disease indexes calculated as shown above. The P.D.C. was calculated by the following formula (1): P.D.C. = (L D.I.ck - D.I.tr x 100 D.I.ck D.I.ck is mean disease incidence in water-sprayed control trees and D.I.tr is mean disease incidence in fungicide-treated trees. The effect of this transformation is to relate the efficacy of a candidate material to that of water. When the P.D.C. equals 100, disease is not present in fungicide treated trees; when P.D.C. equals 0, treated trees have the same amount of disease as the controls; when P.D.C. is a negative number, fungicide treated trees have more disease than the water-sprayed controls. 86 RESULTS A. Preliminary in vitro Screening for Fungicides Active W Results of poison agar screening tests for fungistatic and fungicidal activities against 9; kunzei are given in Figure l. Mycelial growth was inhibited to varying degrees by all the fungicides tested. None of the fungicides tested completely inhibited mycelial growth at all the rates tested. The greatest inhibition of mycelial growth was recorded with Mertect, Bravo 6F, Topsin M, and Benlate. Arbotect 20-S, Bravo 6F, Kocide 6F and Mertect showed complete inhibition with rates of 1000 ppm a.i. B. Field Evaluation of Fungicides for Control of Cytospora Canker The total and mean number of cankered branches on fungicide treated trees from July 19, 1978 through November 16, 1979 are given in Appendix E. Data showing the mean number of cankered branches on fungicide treated Colorado blue spruce are summarized in Table 1. Symptom expression on newly infected branches continued as the season progressed. During 1978, non-pruned trees develOped fewer additional cankered branches (forty-nine) than pruned trees (sixty-three). During 1979, however, non-pruned trees developed more cankered branches (forty-eight) than pruned trees (thirty-eight). Trees treated with the same fungicide were located within the same grove and, although these trees Mean Colony Diameter (mm) Mean Colony Diameter (mm) Diameter (mm) Mean Colony Mean Colony Diameter (mm) 87 25- 20 10 I I I l 0‘ i 1000 500 100 20 10 1 1000 500 100 20 10 Arbotect 20-S ppm a.i. Benlate ppm a.i. 25 20- 10- l I I 1Ic 0 « . 1000 500 100 20 10 1000 500100 20 10 Bravo ppm a.i. Dithane-m-45 ppm a.i. 25r- .- 20- 10- I I l l 0« . 1000 500 100 20 10 1000 500100 20 1o 1 Kocide ppm a.i. Lignasan ppm a.i. 25- 20- '- 10... - o . 1000 500 100 20 1o 1 1000 500100 20 1o. 1 c Mertect ppm a.i. Topsin M ppm a.i. Figure 1. Effect of Various Fungicides on Mycelial Growth of Cytospora kunzei. Measurements shown were made 10 days following Inoculation of Fungicide Amended Medium 8 plates. C = Control. 88 TABLE 1 Mean Number of Cankered Branches Showing Symptoms on Fungicide Treated Colorado Blue Spruce from Julv to November. Rate per Mean Number of 100 gallons DiSeased Branchesé Treatment of water Pruned Non-Pruned Arbotect zo-s 1.125 gal 5.33b 10.33b 11.66C 15.33C Benlate 50% WP 5.0 lb 1.00b 4.33b 4.66c 8.00C Bravo 6F 0.55 gal 2.00b 4.00b 4.33C 6.33C Topsin M 70% WP 3.57 lb o.oob 9.33b 3.66c 12.33C Water 1.00b 6.66b 6.00c 9.00C Total Number of Cankered 63.00 49.00 Branches Arbotect 20-S 1.125 gal 2.66d 2.0d 7.00e 7.66e Benlate 50% WP 5.0 lb 0.00d 0.00d 2.00e 1.33e Bravo 500 0.55 gal 0.006 2.335 5.66e 3.00e Topsin M 70% WP 3.57 lb 0.00d 1.00d 2.00e 2.66e Water 1.00d 0.66d 4.66e 8.00c Total Number of Cankered Branches 38.00 48.00 3Determined by counting all cankered branches on 3 trees per treatment at first snowfall. bFirst day of evaluation. July 19, 1978. CFirst snowfall and last day of evaluation. 1978. dFirst day of evaluation. July 13, 1979. eFirst snowfall and last day of evaluation. 1979. 89 were selected at random, a strong grove effect is evident. Therefore, meaningful statistical tests were not calculated from this data. To overcome the problem of inherent resistence and the lack of randomness due to the grove effect, trees that failed to develop disease during the study periods were not included in the calculations of D.I. and P.D.C. Data on D.I. and P.D.C. are presented in Table 2. The resultant disease index shows a reduction in the amount of disease in 1979 as compared to 1978 in each of the treat- ments except in the Bravo-pruned, Arbotect 20—S non-pruned and in the non-pruned control treatments. Trees sprayed with Arbotect 20-8 in both 1978 and 1979 had more disease than the'water-sprayed control trees. This suggests that Arbotect 20-S is phytotoxic to blue spruce when applied as a foliar spray, hence may increase susceptibility to infection. In 1978, non-pruned trees treated with Benlate and Topsin M developed more cankered branches than the water-sprayed control trees. However, in 1979 the same materials recorded 81.01% and 76.29% control respectively. On the other hand, pruned trees treated with these two fungicides showed improved disease control in 1979 compared to 1978. Their performance was consistent. Disease control by Bravo 6F (Bravo 500) was inconsistent over the two—year study period in both the pruned and non-pruned treatments. In 1978, pruned control trees developed more cankered branches than did non-pruned control trees. In 1979, £98938 9: E confined no: 89. 8089: 288. 8 8:3 umfi moods Shh as .8} GB .23 58588 a\m :0 we... seem} .3} .Hm\m . Qmwunpsnv .5}. .m\m co panama trauma .mHnSuwufi xwmslm as common some Tongan mums museum w>E .5853 m 05m: ugmuam meson m cows omen pom pofimmw 9.83 >mumm mo 931m ma >Hmum5x0umnkm 90 o o 2.: «add w 623598»sz 6580 amen 8.9. NEW. 86 .2 Ea as so» z 582. 3.5. 8.5 «22 med 4mm 86 8m 965 8.8 8.3 mm}. a: A: c.m a: «cm 8328 EST 84:: 3.3 8.: 48 m3; mnom “89.82 c o soda «2: Afismudmnuflms Hoomwamw 8.8- 8.8 3...: «23 .3 EA as was z 588. o 8.8 soda 3:3 .86 $5 .8 88m E; u 8.8 2.2 8.2 a: 9m a: 8m 3255 3:9... smear «TS Nada 48 mafia muom ”.0382 8:551:02 possum Cogumlcoz possum umumz mo mwpwofimwmw QHOHEOU wwmmmwa ucwouwm omwchcH ommwmwa mcoaanw o3 uwm 3mm .22 oza m2: £525 «song so .HEEB mvémmHo BEBE oza mflaozH SE 6deng N Hams. 91 however, pruned control trees developed fewer cankered branches than the non—pruned controls. DISCUSSION Studies on chemical control of Cytospora canker in spruce are lacking. The purpose of the studies reported here is to determine whether control of Cvtospora canker is possible with pruning (cultural practice) and/or fungicides. Laboratorv screening permitted fungicides to be com- pared on the basis of toxicity to g. kunzei. The results showed that g. kunzei is apparently fairly tolerant of the fungicides tested. However, at 1000 ppm a.i. some of the chemicals completely inhibited mycelial growth. Field data have shown that foliar application of Benlate 50% wettable powder and TOpsin M 70% wettable powder reduced the incidence of Cytospora canker. Their improved performance in 1979 over 1978 can be explained in two wavs. First, disease develoPment in 1979 infections was drasti—. cally reduced by 1978 fungicide treatments. Second, the poor results in 1978 mav have been due to heavy disease pressure before the tests were begun. The early application of spravs was attempted with the hOpe that early Spraying would enhance Cytospora inhibition. Data indicate that pruning cankered branches does not reduce infection in the remaining healthy branches. This may be due partially to the fact that pruned trees were 92 surrounded by many infected and non-pruned trees with lots of inoculum. Pruning all diseased branches on the 230 trees in the park would have been impractical. However, the effect of pruning may not be apparent in 2 years. Future studies could focus on the effectiveness and timing of a pruning program. Waterman (6) was unable to control the disease on indi- vidual branches by cutting out all bark and cambium in the cankered areas and scraping off the margins of the cankers. Callus formation was rapid and covered the exposed wood. However, new infections formed girdling cankers between the treated areas and the branch tips. This method of control, however, would be difficult and impractical. Strong (5) achieved reasonable success in controlling Cytospora canker with the continuous surveillance of blue spruce and Norway spruce trees for the appearance of dying branches, prompt removal and burning of cankered branches, and fertilization and watering of trees after removal of cankered branches on trees that had only one or two branches infected when first found. Luepschen (4), using a combination of benomyl and oil, obtained 98 and 80 percent control of Cytospora canker on peach trees artificially inoculated with Cytospora leucostoma in May and June respectively. In another study, Harder and Luepschen (3) obtained significant control of Q. leucostoma when the growth retardant SADH (Succinic acid-Z-Z dimethylhydrazide) was applied with benomyl. Arbotect 20-S, Benlate, Bravo and Topsin M have 93 previouslv not been field-tested for control of Cvtospora canker on spruce. Topsin M and Benlate appear to offer a promising method of disease control. They are nonphytotoxic and are relatively inexpensive. They should, however, be tested further and used in combination with cultural prac- tices as suggested bv Strong (5). This would both reduce the inoculum and improve the aesthetic value of the tree(s). Since the success of a fungicide testing program depends on the amount of natural infection that deve10ps, techniques involving artificial inoculation should be deve- loped to increase the amount of disease that occurs under natural conditions. Five sprays per season are economically impractical. Additional tests will be required to determine the minimum number of sprays, the timing of sprays to coincide with natural infection period(s), and the lowest concentrations of active ingredients for protection against the Cvtospora fungus. Observation indicates that blue spruce has con— siderable genetic variability, and identification of disease resistant cultivars would be a starting point for genetic improvement of this species. A seed collection from seemingly resistant blue spruce in plantations, lawns, parks and native stands should be made with the objective of seeking resistance to Valsa kunzei. 94 LI TERATURE C I TED Beer, S. V. 1978. Techniques for field evaluation of spray materials to control fire blight of apple pear blossoms. In Methods for evaluating plant fungicides, nematicides, and bactericides. The American Phytopathological Society, Publisher. 141p Bertrand, P. F. and H. English. 1978. Evaluation of Cytospora canker severity in French prunes. In Methods for evaluating plant fungicides, nematicides and bactericides. The American Phytopathological Society, Publisher. ldlp Harder, H. H. and N. S. Luepschen. 1975. Synergistic effect of SADH and benomyl against peach Cytospora canker. Hort. Science. 10(1):77-78. ‘ Luepschen, N. S. 1976. Use of benomyl sprays for sup— pressing cytospora canker on artifically inoculated peach trees. Plant Disease Reporter. 60(6):477-479. Strong, F. C. 1953. Spruce branch canker. Natl. Shade Tree Conf. Proc. 29:30-35. Waterman, A. M. 1955. The relation of Valsa kunzei to cankers on conifers. Phytopathology. 45:686—692. Waterscheidt, M. and F. Laemmlen. 1977. Cytospora canker of spruce. Coopertive Extension Service. Michigan State Universtiy. Extension Bulletin E-1078. APPENDICES 9S .5333 H3 “.53quma Hgmanmocmumaou .8 Hgmanmp .mnmm u 353mm... .mba u 53$.“me ”ufifioflmwoo connmmwumwu may you moan.» m Hmugoo .amum sumo um 853.2, mo mwmflmcm 3 ~53pr Swmmmuwmu on» no 83> mp .mmum comm um 85:? mo mammamsm Ham Eopwwum . mo mmoummom cmmm5w3 Ema no 350: prumuwgmu ammo 28: mmrmm .T I .l I. .I .I. unnumcou ooo. cmmzébfi Hmmmmf So. am N cameo. 3.3a. Hammcfimu mo mason 3325: mfiumamm coo. «Somdbma webcmé o mm H mmvwa. mmvma. wooa mo mason wucmowmficmwm om ucmfiofiwmwoo om dmzmfimwu commmmuowu mmcmno mm Swmmwumwhn 3396 coflmmmugmm who mumsvm m or» Oufi >55 mo uwpuo 5 mfimwuflw .33 688803 magnum—2 mo 35sz on”. 0:308: mumuwpaumm may we wank/HMS... 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