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AND THE POST HARVEST DISORDER OF COLORADO BLUE SPRUCE (PICEA PUNGEWS GLAUCA ENGELM.) presented by . Melissa Jane Igoe has been accepted towards fulfillment of the requirements for M. S . degree in Horticulture Mafia. Major professor Date ’l’ig"fil 0—7639 MS U is an Affirmative Action/Equal Opportunity Institution ’ ”Homer 3 I Michigan. State ‘ L University 1 *fi PLACE IN RETURN BOX to remove this checkout from your record. TO AVOID FINES return on or before date due. DATE DUE DATE DUE DATE DUE “l MSU Is An Affirmative Action/Equal Opportunity Institution — . emails-9.. THE RELATIONSHIP BETWEEN PHOMOPSIS OCCULTA TRAV. AND THE POST HARVEST DISORDER OF COLORADO BLUE SPRUCE (PICEA PUNGENS GLAUCA ENGELM.) By Melissa Jane Igoe A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Horticulture 1991 ABSTRACT THE RELATIONSHIP BETWEEN PH OMOPSIS OCCULTA TRAV. AND THE POST HARVEST DISORDER OF COLORADO BLUE SPRUCE (PICEA PUNGENS GLAUCA ENGELM.) By Melissa Jane Igoe Using artificial inoculation techniques, Phomopsis occulta Trav. was shown to be a causal agent of a post-harvest disorder that plagues nursery grown Colorado blue spruce in Michigan. Field-grown trees declined significantly following fall harvest. Initially, post-harvest symptoms were characterized by needle drop from lower branch areas where inconspicuous stem cankers had developed. In spring, newly developed shoots appeared wilted, then browned and died. In 1988, a drought year, August-harvested plants were less symptomatic than later harvest dates. In 1989, a more normal year, August-harvested plants were more symptomatic than later harvested plants. Various fertility regimes had little affect on the symptom severity of harvested plants during the first year of the study. Plants of the highest foliar nitrogen content during the second year of the study were less symptomatic than plants that were denied fertilizer for two years. Benomyl showed potential as a chemical control. ACKNOWLEDGMENTS I need to thank my major professor,,Dr. Curtis Peterson, for his guidance, support and kind regard during the course of my studies. My guidance committee members, Drs. Arthur Cameron, David Roberts, and David Smitley, are sincerely thanked for their help. I also thank Dr. Robert Schutzki and the students who unselfishly gave their assistance: Laura Finck, Beth Engle, Susan Gruber, Tim Wood, Diana Dostal, and Nathan Lange. I also thank my friend, Mark Cook, for his physical assistance and tolerance over the past few years. A sincere thank-you to Zelenka's Evergreen Nursery for their cooperation during this study and for the generous donation of materials used in my research. iii TABLE OF CONTENTS Page LIST OF TABLES ................................................................................ v LIST OF FIGURES ............................................................................... vii INTRODUCTION ..................................................................... ' ............ 1 LITERATURE REVIEW ........................................................................ 3 Summary .................................................................................... 16 Literature Cited ........................................................................... 18 CHARTEEJ PATHOGENICITY, GROWTH, AND CONTROL OF PHOMOPSIS OCCULTA TRAV. ASSOCIATED WITH THE POST-HARVEST DISORDER OF COLORADO BLUE SPRUCE (PICEA PUNGENS GLAUCA ENGELM.) Abstract ............................................................................. 23 Introduction ....................................................................... % Materials and Methods ........................................................ 27 Results and Discussion ........................................................ 34 Literature Cited .................................................................. 55 W INFLUENCE OF HARVEST DATE AND FERTILIZER RATE ON THE SEVERITY OF THE SHOOT BLIGHT DISEASE OF COLORADO BLUE SPRUCE (PICEA PUNGENS GLAUCA, ENGELM.) Abstract ............................................................................. 57 Introduction ....................................................................... 58 Materials and Methods ........................................................ 61 Results and Discussion ........................................................ 64 Literature Cited .................................................................. 76 iv Table 1.1 1.2 1.3 1.4 1.5 1.6 LIST OF TABLES QHAEI‘EEJ Page Pathogenicity Experiment One. The effect of inoculation and reduction of 10 cm root plugs to 4 cm on the development of lesions in one-year-old wound-inoculated Colorado blue spruce seedlings, six months after inoculation ............................................................. 36 Pathogenicity Experiment Two. The effect of inoculation and reduction of 10 cm root plugs to four cm on the lesion length (mm) at two wound locations on one-year-old Colorado blue spruce seedlings after five months incubation .............................................................. 37 Pathogenicity Experiment Two. Symptom development on one-year-old Colorado blue spruce seedlings one month after wound-inoculating and reducing 10 cm root plugs to four cm or leaving roots intact ..................................... Pathogenicity Experiment Three. Efficacy of wounding, inoculating, and sealing wound with Parafilm, in the production of lesions at two locations on one-year old Colorado blue spruce, after four months incubation ....... 39 Pathogenicity Experiment Four. Mean lesion length measured three months after Colorado blue spruce seedlings were inoculated within the center of the terminal leader when roots were left intact or when 10 cm root plugs were trimmed to four cm .................... 40 Pathogenicity Experiment Five. Mean number of shoots blighted per tree on injured one-year-old Colorado blue spruce seedlings sprayed with 1.74 X 104 conidia per milliliter of Phomopsis occulta, four months after inoculation ................................................................ 41 1.7 1.8 2.1 2.2 Pathogenicity Experiment Six. Mean number of blighted shoots on one-year-old Colorado blue spruce seedlings, with tips that were wound-inoculated or surface- inoculated with a concentrated conidial suspension (1.47 X 106 conidia per milliliter), three months after inoculation ................................................................ Mean symptom severity on 15-18 inch Colorado blue spruce, field grown in a commercial nursery and known to be naturally inoculated with P. occulta, when treated with a fungicide in the summer of 1989 and evaluated eight months after hand-digging in the fall ................................................................... WI Average monthly rain levels (inches) at the nursery in Ottawa County, Michigan ........................................ The effect of nitrogen fertilization on the growth, appearance and foliar nitrogen content of field-grown, fall harvested, Colorado blue spruce naturally infected with Phomopsis occulta .............................................. 42 71 74 Figure 1.1 1.2 2.1 2.2 LIST OF FIGURES Page CHARIEBJ Effect of temperature on the radial mycelial growth of one isolate of Phomopsis occulta. Measurements shown were made ten days after PDA plates were inoculated with 10-mm-diameter disks of mycelia. Each treatment was replicated 20 times. The LSD (P=0.05) for comparing means is 1.85 mm ...................................................... Effect of four fungicides on the radial mycelial growth of Phomopsis occulta growing in fungicide amended PDA cultures incubated at room temperature for six days. Each treatment was replicated 20 times. The LSD (P=0.05) for comparing means is Benlate=1.3, Chipco=1.6, Daconil=2.0, and Kocide=2.1 ........................................ 52 W The monthly visual rating of Colorado blue spruce infected with Phomopsis occulta and harvested in the fall of 1988 where 1 = no symptoms of disease, 2 = less than one- third of the plant surface exhibiting symptoms of needle loss or discoloration, 3 = more than one-third, but less than two- thirds of the plant surface showing signs of disease, 4 = more than two-thirds of the surface showing needle loss, needle discoloration, extensive cankering, and death of shoots, and 5 = entire plant dead. Observations were recorded during the first few days of each month. Points represent the monthly rating of trees averaged over all fertilizer treatments ................................................................. 67 The monthly visual rating of Colorado blue spruce infected with Phomopsis occulta and harvested in the fall of 1989 where 1 = no symptoms of disease, 2 = less than one- third of the plant surface exhibiting symptoms of needle loss or discoloration, 3 = more than one-third, but less than two- thirds of the plant surface showing signs of disease, 4 = more than two-thirds of the surface showing needle loss, needle discoloration, extensive cankering, and death of shoots, and 5 = entire plant dead. Observations were recorded during the first few days of each month. Points represent the monthly rating of trees averaged over all fertilizer treatments ........ 68 vii INTRODUCTION Colorado blue spruce (Picea pungens glauca Engelm.) is a popular evergreen tree valued in the Midwest for its bluish-green foliage and perfect symmetry. Several hundred thousand are harvested annually in Michigan and are sold as landscape specimens or as Christmas trees. Colorado blue spruce sales in Michigan account for three percent of the total nursery stock sales in the state. Landscape plants are field grown and harvested in the spring or in the fall. Trees are commonly available in sizes that range from 12-15 inches to 3-4 feet and larger. To ensure early spring shipping from Michigan, smaller sizes are commonly over-wintered in quonset style structures while larger sizes are heeled-in. Since 1986, Michigan nursery growers have been reporting poor post- harvest quality of Colorado blue spruce. The rapid decline of trees after harvest has rendered up to 30% of the plants unsalable. Plants that appear healthy in the field begin to shed needles from the lower third of the plant within a few weeks after harvest. Plants that remain in the field are symptomless except for an occasional flagging branch. Cutting plant roots during the harvest procedure can reduce the vigor of plants. Plants that are weakened by environmental stresses such as nutrient deficiency, drought, flooding, extreme temperatures, or transplanting; suddenly become susceptible to otherwise nonaggressive pathogens (38). Samples of Colorado blue spruce trees exhibiting post-harvest damage were submitted to the Michigan State University Plant Diagnostic Clinic. Stem cankers were revealed by scraping away the bark. Samples of this cankerous tissue consistently yielded cultures of Phomopsis occulta. In 1943, P. occulta was considered only a saprophyte by Hahn (14, 15), although White (45) reported in 1929 two cases of spruce blight caused by P. occulta. White reported that various species of spruce were susceptible to a blight resembling juniper blight caused by Phomopsis juniperovora. In one case spruce were transplanted late and excessively irrigated. In the other case seedlings were grown in overcrowded beds. Sanderson and Worf have provided recent evidence of the susceptibility of spruce to P. occulta, proving the pathogenicity of this fungus (36). Sanderson and Worf artificially inoculated four spruce species and found that Colorado blue spruce was the most susceptible of the 4 spruce species tested. The purpose of this thesis was to determine if there is a causal relationship between Phomopsis occulta and the post-harvest disorder among Colorado blue spruce grown in Michigan, and to study the effects of certain cultural practices upon the severity of the disorder. LITERATURE REVIEW Phomopsis Sacc. was originally included as a subdivision of a much larger genus--Phoma, by Saccardo in 1884 (14). Saccardo later (1905) described Phomopsis as its own genus (14). The separation of the two taxa was based on the recognition of two distinct spore types produced by Phomopsis (14). Phoma produced only one type (14). The filiform spores produced by Phomopsis had previously been disregarded by Saccardo, who in 1905 was still uncertain of the origin and function of these structures (14). In 1911, Diedicke confirmed that the filamentous spores were true spores and designated them as beta spores (14). The beta spores are long, slender and booked at the apex; guttules (oil droplets) are absent. Alpha spores are cylindrical, with a rounded taper at each end. Alpha spores usually contain two oil droplets (guttules), one at each end. Hahn regarded the beta spores as non-functional bodies because they resisted germination when conditions conducive to germination were provided (14). Over 400 taxa of Phomopsis have been described (41). Many are pathogens of agricultural and horticultural crops. Wehmeyer (43, 44) collected considerable cultural evidence that linked Phomopsis with Diaporthe, which is now accepted as the perfect stage of Phomopsis. Wehmeyer recognized many species of Diaporthe to be weak parasites at best, while Phomopsis stages caused serious diseases on citrus fruits, 4 legumes, and potatoes (44). Phomopsis causes blights of azalea, blueberry, and juniper; stem cankers of gardenia, cottonwood, European black alder, and Russian olive; cane, leaf and berry spot of grape; and stem end rot of citrus fruits. Phomopsis is also commonly reported living as an endophyte or saprophyte on many conifers (4, 13, 14, 15, 35, 40). Phomopsis Fruit Rots Phomopsis vaccinii Shear causes a fruit rot of blueberry (Vaccinium corymbosum L.) (20). Milholland (20) observed that infections were obtained with artificial inoculations from the time the fi'uit was in the small green stage until it was ready for harvest; but rot symptoms were not seen until the fruit had matured. In reviewing New York extension newsletters, Pscheidt and Pearson (32) found that in years with high levels of rainfall (>100 mm) during bloom, P. viticola (Sacc.) Sacc. was able to incite a fruit rot disease of grape (Vitis sp.). Pscheidt and Pearson (32) inoculated grape clusters at all stages of development and found that berries were most susceptible just after bloom. Inoculation of clusters at later stages did not show significantly more fruit rot than in the controls. However, as in Phomopsis fruit rot of blueberry, this disease does not manifest itself until the fruit has matured (32). Phomopsis citri Fawcett is found in stem end rots of oranges grown in Florida (2). A similar Phomopsis is found in lemons grown in California (9). The California Phomopsis is much less virulent than the Florida Phomopsis. In California, Phomopsis was only found on 1) a few twigs 5 after careful searching in the orchard and 2) on old mature fruits in storage (9). P. citri infected Florida oranges only after the abscission layer formed, thereby creating a natural entrance for the fungus (2). Blights, Cankers, and Diebacks Caused by Phomopsis Although Phomopsis has been implicated in several fruit rots, most diseases caused by Phomopsis can be characterized as blights, cankers and diebacks. Literature concerning some of these diseases will be reviewed, followed by a review of Phomopses specifically on conifers. Phomopsis vaccinii causes a serious canker and dieback disease that affects 20% of the highbush blueberries in Michigan (25). In North Carolina, Phomopsis twig blight reduces blueberry yield by 2-3 pints per bush (19). Conidia are released from pycnidia in lesions of infected blueberry bushes during the spring from flower budbreak through petal fall (19, 25). The blueberry in North Carolina is primarily infected where conidia fall on the flower buds. After infection the fungus enters the vascular system and can proceed 50-150 mm down the stem (19). Wilcox (46) found that when young plants were sprayed with spores, P. vaccinii entered at the tip of succulent new growth, rapidly grew downward, and finally girdled the second-year wood. The rapid downward progression averaged 5.5 cm. in 2 months. While succulent tissue was killed within five weeks after inoculation, woody tissue inoculated with spores or mycelium developed only small lesions. Wilcox (46) found that P. vaccinii was virulent on succulent shoots regardless of wounding. Parker (25) reported 6 that wounding was necessary to obtain an infection with mycelia or conidia in two year-old stems inoculated from spring to fall. Phomopsis viticola causes a severe cane and leaf spot disease of grapevine during wet springs in California (7). Lake Erie region grape growers in New York saw greater disease development during a 1986 spring of heavy rains than in 1987 when pre-bloom conditions were dry (31). Until 1978, it was thought that P. viticola caused the "dead-arm" disease in grapes. Then it was proven that Eutypa armeniacae Hansf. and Carter caused the "dead-arm" disease and not P. viticola (21). Gardenia cankers are caused by Phomopsis gardeniae Hansen and Barrett (30). Phomopsis gardeniae enters the gardenia through wounds inflicted during cultivation or insect feeding (30). For propagation ' purposes, commercial growers remove leaves on cuttings before sticking the cutting into the rooting medium (30). Cankers formed on rooted cuttings at wound sites created by leaf removal (30). Phomopsis macrospora T. Kobayashi and Chiba causes a stern canker on poplar in Minnesota (11) and on cottonwood (Populus deltoides) in Mississippi (10). French and Bergdahl (11) reported a natural occurence of Phomopsis canker on 84% of nursery grown 'Robusta' poplar. Most of the diseased trees had basal cankers not aerial cankers. Although cankered trees were rarely ln'lled, diseased trees could not be sold. Occasionally, infected plants were sold when lethal cankers were not evident. Trees that developed cankers during bare-root storage were a complete loss (11). 7 P. macrospora was one of three pathogenic fungi responsible for lethal cankers on young cottonwoods in Mississippi (10). Wound inoculations performed in September, 1963, on meted cuttings resulted in girdling of 75% of the plants held in a greenhouse and 100% of those held in a lathhouse. Plants that were wound inoculated and held in a growth chamber did not die. This pathogen may not be serious unless the plants are stressed, but Filer (10) found no relationship between the level of bark moisture and the incidence of disease when bark turgidity was measured on 2 samples every 2 months. Filer (10) suggests that light, temperature and humidity strongly influence rate of disease development. In Michigan, Phomopsis elaegni Arnold and Carter is responsible for most cankers found on Russian-olive (18). Maffei and Morton (18) usually found cankers at the base of epicormic branch clusters or at branch tips. Infections were always obtained when spores or mycelium were used to artificially wound inoculate Russian-olive seedlings. However, they found pathogenicity variable among different isolates used as inoculum. In 1983, Oak and Dorset (22) first reported Phomopsis alnea (Sacc.) Hoehn. causing a canker on European black alder (Alnus glutinosa) in a field planted for seed production. The field began to decline within several months after planting. Basal cankers were found on all sides of the main stem. Preliminary inoculations with Phomopsis conidia on unwounded succulent stems did not result in infection. When water-stressed plants were wound inoculated, small localized cankers developed. The primary symptom was an inhibition of callus formation at wounds. Epidemic years 8 were attributed to poor planting sites, temperatures of 20-30 C and frequent rainfall in the fall prior to the epidemic. Bedwell reported Phomopsis among other fungi occurring on Asiatic chestnuts planted at 112 sites in 22 states (1). Bedwell tested the pathogenicity of Phomopsis by wound inoculating twigs of two-year-old plants. The typical dieback and canker resulted only when plants were wounded and inoculated. Cankers were often found at the intersection of the twig and limb. Plants that were dormant or just brealn'ng bud were most susceptible to wound inoculations. Phomopsis Occurring on Conifers For purposes of differentiation, Hahn extensively studied, in nature and in culture, eight species of Phomopsis occurring on conifers (13, 14, 15). Within species of Phomopsis, Hahn found pycnidial stromatic structure variable in culture, but found growth characteristics stable in culture (14). Size and shape of spores were consistent in nature and in culture. Characterization of spores can be relied on for species identification (14). However, there was some variability in the relative numbers of alpha and beta spores produced (14). Hahn (13, 14) accounted for the presence of Phomopsis on many coniferous hosts: Abies, Chamaecyparis, Cupressus, Juniperus, Larix, Picea, Pinus, Pseudotsuga, Sequoia, Taxodium, and Taxus. The most well studied of the Phomopses occurring on conifers is Phomopsis juniperovora Hahn, a widespread virulent parasite which devastates Eastern red cedar, Juniperus virginiana L. Hahn (13) found that P. juniperovora could also parasitize other coniferous hosts as vigorously as it infected J. virginiana. Artificial wound inoculations were performed with P. juniperovora and resulted in canker formation and tip blight of the various host plants. Infections showed signs of expansion within five to tens days of inoculation of young stems; and quickly girdled small stems less than one-half inch in diameter. Growth was primarily in a longitudinal direction within the inner bark, killing the cambium, and staining the wood. Entire plants were killed when the fungus entered the main stem through a lateral. Older trees are rarely killed since only the smaller stems are girdled (28). Peterson and Hodges (28) explained that P. juniperovora infects new foliage and then spreads to the stem tissue. As the needles age, they become less susceptible to infection (26, 28). Pycnidia can form within 3 to 4 weeks and appear partially embedded in needles and stems (26, 28). Viable spores can be produced for up to two years after infected tissue has died (28). High humidity and high temperatures enhance infection and disease development on junipers (26, 28). Growth of P. juniperovora in culture is optimum at 24-26 C (26). Schoeneweiss (37) reported severe disease development occurring in the cool, wet springs of 1966-1968 in Illinois. Epidemics have occurred with the use of overhead irrigation in fields with a few infected plants (13, 28). When Hahn began his study of coniferous Phomopses (13), he observed that P. juniperovora and Phomopsis occulta Trav. were 10 morphologically very similar. Both were found on hosts belonging to Cupressaceae, although only the former was considered parasitic (13, 15). The alpha spores of the two species are very similar, but a significant difference was found in the spore length. The alpha spores of P. occulta are shorter (14). The straighter beta spore of P. juniperovora is distinguished from the hooked beta spore of P. occulta (14). The most obvious difference was illustrated when the two fungi were grown in culture. Bright orange crystals are formed in cultures of P. juniperovora, while cultures of P. occulta on the same medium are dull colored (15). Hahn found P. occulta widely distributed in Europe and North America; and living on fourteen genera of conifers (14). In rare instances, Hahn associated the perfect stage of P. occulta with Diaporthe conorum. Hahn further demonstrated that cultures of monoascospore isolates of D. conorum gave rise to P. occulta (14). In turn, monopycnidiospore cultures gave rise to the ascogenous form (14). D. conorum is now accepted as the perfect stage of P. occulta. It is commonly found in Europe, but its presence in North America has not been substantiated (15). After considerable study, Hahn (15) concluded that P. occulta is a saprophyte, or secondary organism, infecting host plants after injuries caused by frost, transplanting, drought, or after infection by other parasitic fungi. P. occulta has been found living saprophytically on all plant parts: cones, leaves, stems, and trunks (14). Sieber (40) isolated P. occulta fiom twigs of healthy and diseased Norway spruce (Picea abies (L.) Karst.) and white fir (Abies alba Mill.) at three sites in Switzerland. P. occulta was 11 found more frequently on diseased Norway spruce than on healthy Norway spruce at one site which was warmer and dryer. Sieber did not consider this coincidence as evidence for the pathogenicity of P. occulta. However, there was a report in 1927 of P. occulta causing a blight on Colorado blue spruce (Picea pungens Engelm.) that was similar to the disease of juniper caused by P. juniperovora (45). Even after this report, the organism was not considered a pathogen. In 1986, Sanderson and Worf (36) reported the pathogenicity of P. occulta, which caused a shoot blight of Colorado blue spruce in Wisconsin. Symptoms were observed on nursery plants and included downward curling of expanding shoots, browning of the needles and tips, and subsequent death of the tip or entire shoot. Sanderson and Worf (36) found small cankers and sap exudate on diseased branches. They ‘ also found pycnidia on dead shoots and dead needles. Sanderson and Worf (36) sprayed a P. occulta spore suspension to inoculate healthy, one-year-old Colorado blue spruce seedlings. Symptoms appeared within 13 days after inoculation. Thirty percent of the plants became symptomatic. Greatest symptom development was achieved at a higher temperature (25 C) and higher humidities (7 5-90% RH). These researchers suggested that plants may be susceptible to infection for only a short time in the spring, when conditions are cool and relatively dry. Infection may occur without further symptom development, until the weather becomes warmer and more humid. This correlates well with the more commonly observed symptoms of disease during the warmer months of June and July (36). 12 Artificial inoculation of several coniferous hosts has proven the potential pathogenicity of P. occulta (36). Sanderson and Worf (36) obtained symptom development when Picea abies, P. glauca, and P. obovata as well as P. pungens were inoculated with spores of P. occulta. P. pungens showed much greater symptom severity than any of the other susceptible spruce investigated. Abies balsamea and Abies concolor were not susceptible. Predisposing Factors and Latent Infections In reviewing the literature on Phomopsis diseases, many studies have shown that the wounding or weakening of the host plant encourages disease development. Hahn (13) reported that transplanting, cultivating and pruning wounds predisposed plants to attack by P. juniperovora. In 1961, Raniere (34) attributed the sudden occurrence of extensive cane blighting and death of blueberry plants by Phomopsis canker and Botryosphaeria canker to low temperature injury. Damage predisposed "plants to invasion by various weak pathogens normally unable to cause any measurable injury to vigorous plants (34)." Brown (3) found that Phomopsis was a weak pathogen on oak and hickory trees and required wounds to enter the host plant. Wounding or injury as a consequence of drought, low temperatures, mechanical injuries, animal damage, or poor planting site was a prerequisite for the development of a parasitic relationship between Phomopsis and Asiatic chestnuts (1). In such instances, the fungus may have penetrated the host without 13 the development of symptoms until the presence of predisposing elements. This condition is termed a latent infection (42). Fungi enter susceptible and resistant hosts with equal frequency (40, 39). After entry, disease development depends on the environmentally influenced, genetic response of the host to the presence of the organism (40). Predisposition refers to the environmental factors that affect the susceptibility of the host (40, 38). Verhoeff (42) described latent infections of fungi in fruits that appeared during senescence. Phomopsis has produced latent infections in fruit of blueberry (20), grape (32), cranberry (6), and citrus fruits (2, 9). Symptoms of fruit rot did not appear until the fruit was wounded or began to senesce. P. batatae, Harter and Field, P. phaseoli (Desm.) Grove and P. sojae Lehm. were shown to infect seedlings of sweet potato (Ipomoea batatas (L.) Lam.) and 16 legumes when artificially inoculated (17). However symptoms did not appear until the onset of senescence. Cerkauskas and Sinclair (5) used paraquat to detect latent infections in soybeans. Formation of pycnidia was observed two weeks before pycnidia were seen on untreated controls. Pscheidt and Pearson were also able to utilize paraquat to aid in detection of P. viticola infections in grape vines and fruits (31, 32). Shoots 2.5 cm. long were inoculated but did not develop symptoms. An application of paraquat resulted in 78% of the shoots showing disease symptoms after 1 week (31). Schoeneweiss (38) observed that canker and dieback diseases were more prevalent on plants subjected to environmental stress before symptom development. The organisms attacking environmentally stressed plants 14 were often nonaggressive pathogens or facultative parasites (38). The most common environmental stresses predisposing woody plants to disease are drought, freezing, and in nursery or landscape plants-- transplanting (38). Botryosphaeria dothidea is considered a weak pathogen that invades peach trees through wounds or lenticels followed by a latent infection that can last from two weeks to many months (33). Pusey (33) drought stressed one-year-old potted peach trees. Plants that were inoculated and watered daily showed no sign of disease until 19 weeks after inoculation. Plants that were denied daily irrigation for six days out of an eight day cycle began to exude gum 5 weeks after inoculation. Plants showing different levels of water stress at the time of inoculation, showed no difference in disease development. When water stress was imposed 2-6 months after inoculation disease severity was significantly increased. Colorado blue spruce is predisposed to Cytospora canker by drought (16, 39). Kamiri and Laemmlen saw the development of greater numbers of cankers, produced in a shorter time, in drought stressed trees than in the well watered controls (16). Schoeneweiss (39) reported that bark cankers appeared on wound-inoculated stems of 5-year-old spruce when plant water potentials fell to between -20 and -30 bars. In the same study, spruce were not predisposed to Cytospora canker by freezing temperatures between -20 and -30 C. Control of Phomopsis When English covered freshly pruned branch stubs of Kadota fig trees with plastic caps, Phomopsis canker was controlled by 60% (8). When 1% phenyl mercuric acetate or a Bordeau paste was painted onto pruning wounds, in addition to plastic caps, 75% control was attained (8). When pruning was performed later in the dormant season (April) cankering was reduced (8). Cucuzza and Sell found sodium arsenite applied as a dormant treatment controlled Phomopsis cane and leaf spot on grapevines. Satisfactory control was also achieved with a dormant treatment of dinoseb and treatment with captan at 100% budbreak (7). Pscheidt and Pearson found that two applications of mancozeb during bloom, or two treatments with captan during shoot growth, significantly reduced fruit rot and rachis lesions on grapes (32). In another study, Pscheidt and Pearson found that hand pruning grape vines reduced the amount of disease compared to those vines that are hedged. Hedging creates more nodes and more dead branches, increasing the level of inoculum (31). Phomopsis blight of juniper could easily be controlled by planting resistant plants once they are identified. Schoeneweiss found notable variation in resistance to P. juniperovora among species and among some cultivars within species of host plants (37). The progeny of 86 J. virginiana were evaluated for resistance to P. juniperovora. Progeny from twenty of the selected trees were found to have some degree of resistance (27). Peterson and Hodges suggest avoiding 1) planting juniper seed next 16 Peterson and Hodges suggest avoiding 1) planting juniper seed next to beds containing juniper stock, 2) planting on poorly drained areas, 3) overhead irrigation that will not allow plants to dry before nightfall, 4) use of shade frames that can prolong wet periods 5) using junipers or other hosts as windbreaks (28). Chemical control of P. juniperovora has been achieved in several trials using benomyl to control (12, 23, 24). Otta and coworkers obtained in vitro control of P. juniperovora with benomyl which restricted growth and pycnidia formation. Chemical control in the field resulted in a significant decrease in disease severity on individual infected trees, the percent infected trees bearing pycnidia, the amount of diseased tissue bearing pycnidia, the percent pycnidia with spores, and the spread of infection from inoculum sources. Although benomyl did decrease the production of pycnidia and production of spores within the pycnidia, it did not inhibit germination of spores. Foliar applied benomyl was not redistributed systemically but roots translocated low levels of benomyl through out the plant. Successful recommendations were to "spray weekly with benomyl during the entire growing season and to rogue all plants with any dead foliage every 7-10 days (24)." Benomyl must be applied often enough to assure protection of new growth (29). Summary From a review of the literature, it is clear that some species of Phomopsis merit pathogen status since they cause many different symptoms on a wide range of host plants. Phomopsis species range in 17 Phomopsis seem to infect new succulent growth while woody tissue is less susceptible. Often disease is related to stress or wounding and latent infections are sustained for variable periods. P. juniperovora is considered a virulent pathogen causing serious losses, but control measures have been recommended. Little is known about the pathogenicity of P. occulta, which until recently was considered only a saprophyte. Little is known about the control of P. occulta. 9. LITERATURE CITED Bedwell, J .H. 1937. Twig blight of Asiatic chestnut, especially that caused by Phomopsis. Phytopathology 27:1143-1151. Brown, GE. and W.C. Wilson. 1967. Entry of stem-end rot fungi in Florida oranges. Phytopathology 57:805 (Abstr.). Brown, NA. 1938. The tumor disease of oak and hickory trees. Phytopathology 28:401-411. Carroll, G.C. and FE. Carroll. 1978. Studies on the incidence of coniferous needle endophytes in the Pacific Northwest. Can. J. Bot. 56:3034-3043. Cerkauskas, RF, and J .B. Sinclair. 1980. Use of paraquat to aid detection of fungi in soybean tissues. Phytopathology 70:1036-1038. Crowley, DJ. 1923. Preliminary report of rots of the cranberry in Pacific County, Washington. Phytopathology 13:509-510. Cucuzza, J .D., and Sall, MA. 1982. PhomOpsis cane and leaf spot disease of grapevine: Effects of chemical treatments on inoculum level, disease severity, and yield. Plant Disease 66:794-797. English, H. 1958. Physical and chemical methods of reducing Phomopsis canker infection in Kadota fig trees. Phytopathology 48:392 (Abstr.). Fawcett, HS 1922. A Phomopsis of citrus in California. Phytopathology 12:107 (Abstr.). 10. Filer, T.H., Jr. 1967. Pathogenicity of Cytospora, Phomopsis, and Hypomyces on Populus deltoides. Phytopathology 57:978-980. 11. French, D.W., and Bergdahl, DR. 1983. Phomopsis canker of 'Robusta' poplar. J. of Arboriculture 9(6):151-152. 12. Gill, D.L. 1974. Control of Phomopsis blight of junipers. Plant Dis. Reptr. 58:1012- 1014. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 19 Hahn, G.G. 1926. Phomopsis juniperovora and closely related strains on conifers. Phytopathology 16:899-914. Hahn, G.G. 1930. Life-history studies of the species of Phomopsis occuring on conifers. Part 1. Trans. Brit. Mycol. Soc. 15:32-93. Hahn, G.G. 1943. Taxonomy, distribution, and pathology of Phomopsis occulta and Phomopsis juniperovora. Mycologia 35:112-129. Kamiri, L.K., and FF. Laemmlen. 1981. Effects of drought-stress and wounding on Cytospora canker development on Colorado blue spruce. J. of Arboriculture 7(5):113-116. Kulik, M.M. 1984. Symptomless infection, persistence, and production of pycnidia in host and non-host plants by Phomopsis batatae, Phomopsis phaseoli, and Phomopsis sojae, and the taxonomic implications. Mycologia 76(2):274-291. Maffei, H.M., and Morton, H.L. 1983. Phomopsis canker of Russian- olive in southeastern Michigan. Plant Disease 67:964-965. Milholland, RD. 1982. Blueberry twig blight caused by Phomopsis vaccinii. Plant Disease 66:1034-1036. Milholland, RD, and Daykin, ME. 1983. Blueberry fi'uit rot caused by Phomopsis vaccinii. Plant Disease 67:325-326. Moller, W.J., and AN. Kasimatis. 1978. Dieback of grapevines caused by Eutypa armeniacae. Plant Disease 62:254-258. Oak, S.W., and Dorset, RD. 1983. Phomopsis canker of European black alder found in Kentucky seed-production areas. Plant Disease 67:691-693. Otta, J .D. 1974. Benomyl and thiophanate methyl control Phomopsis blight of eastern redcedar in a nursery. Plant Dis. Reptr. 58:476-477. Otta, J .D., D.J. Fiedler, and V.H. Lengkeek. 1980. Effect of benomyl on Phomopsis juniperovora infection of Juniperus virginiana. Phytopathology 70:46-50. Parker, RE. 1976. The epidemiology, etiology and chemical control of Fusicoccum and Phomopsis cankers of highbush blueberry. PhD dissertation, Mich. State Univ., E. Lansing, Mich. 73 pp. Peterson, G.W. 1973. Infection of Juniperus virginiana and J. scopulorum by Phomopsis juniperovora. Phytopathology 63:246-251. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 39. Peterson, G.W. 1986. Resistance to Phomopsis juniperovora in geographic seed sources of Juniperus virginiana. In: Recent research on conifer needle diseases. Gulfport, Mississippi. October, 1984. USDA Forest Service General Technical Report GTR WO-50, 65- 69. Peterson, G.W., and GS. Hodges, Jr. 1982. Phomopsis blight of junipers. USDA Forest Service Leaflet 154. Peterson, G.W., and J .D. Otta. 1979. Controlling Phomopsis blight of junipers. Am. Nurseryman 149(5):15, 75, 78, 80-82. Pirone, RP. 1938. Where are gardenia cankers initiated? Phytopathology 28:597-598. Pscheidt, J .W., and Pearson, R.C. 1989. Effect of grapevine training systems and pruning practices on occurrence of Phomopsis cane and leaf spot. Plant Disease 73:825-828. Pscheidt, J .W., and Pearson, R.C. 1989. Time of infection and control of Phomopsis fruit rot of grape. Plant Disease. 73:829-833. Pusey, PL. 1989. Influence of water stress on susceptibility of nonwounded peach bark to Botryosphaeria dothidea. Plant Disease 73:1000-1003. Raniere, LC. 1961. Observations on new or unusual diseases of highbush blueberry. Plant Dis. Reptr. 45:844. Rowan, SJ. 1982. Tip dieback in southern pine nurseries. Plant Disease 66:258-259. Sanderson, P. G, and G. L. Worf. 1986. Phomopsis shoot blight of Colorado blue spruce. J. Environ. Hort. 4(4): 134-138. Schoeneweiss, DR 1969. Susceptibility of evergreen hosts to the juniper blight fungus, Phomopsis juniperovora, under epidemic conditions. J. Amer. Soc. Hort. Sci. 94(6):609-611. Schoeneweiss, DP. 1981. The role of environmental stress in diseases of woody plants. Plant Disease 65(4):308-314. Schoeneweiss, DP. 1983. Drought predisposition to Cytospora canker in blue spruce. Plant Disease 67:383-385. 40. 41. 42. 43. 44. 45. 46. 21 Sieber, TN. 1989. Endophytic fungi in twigs of healthy and diseased Norway spruce and white fir. Mycol. Res. 92(3):322-326. Sutton, BC. 1980. The Coelomycetes. Commonwealth Mycological Institute. Kew, Surrey, England. 696 pp. Verhoeff, K. 1974. Latent infections by fungi. Annual Rev. Phytopathol. 12:99-110. Wehmeyer, LE. 1927. Cultural life histories of Diaporthe II. Mycologia 19:165-183. Wehmeyer, LE. 1933. The genus Diaporthe Nitschke and its segregates. Univ. Mich. Stud. 349 p. White, RP. 1929. Juniper blight. Ann. Rept., N.J. Agri. Expt. Sta. 270- 272. Wilcox, MS. 1939. Phomopsis twig blight of blueberry. Phytopathology 29:136-142. CHAPTERI PATHOGENICITY, GROWTH, AND CONTROL OF PHOMOPSIS OCCULTA ASSOCIATED WITH THE POST-HARVEST DISORDER OF COLORADO BLUE SPRUCE (PICEA PUNGENS GLAUCA ENGELM.) ABSTRACT Phomopsis occulta Trav. was shown to be a causal agent of a post- harvest disorder that affects nursery grown Colorado blue spruce (Picea pungens glauca Engelm.). Symptoms of the post-harvest decline appear several weeks after fall harvest. Symptoms are characterized by needle loss primarily from the lower third of the plant and inconspicuous stem cankers occurring in all areas of the plant. This has been a serious concern for Michigan nursery growers who rouge thousands of symptomatic spruce each season. All samples collected from symptomatic harvested plants yielded cultures of Phomopsis occulta. Koch's postulates were satisfied by means of artificial inoculation of healthy Colorado blue spruce seedlings. Both mycelia and conidia of P. occulta were shown to cause symptoms similar to those symptoms on field-harvested spruce. Wounding and simulated harvest were significant factors in the development of lesions on plants inoculated with a mycelial slurry. Wounding was not required for the penetration of stem tissue by conidia. Cankers formed most rapidly on younger tissue regardless of the inoculum type. Optimal growth of P. occulta in potato dextrose agar (PDA) cultures occurred at 25°C. When mycelial plugs were grown on fungicide amended PDA, benomyl stopped growth at all concentrations tested. Iprodione, chlorothalonil, and cupric hydroxide reduced growth of P. occulta only at 24 higher concentrations. Cupric hydroxide stimulated growth at lower concentrations. When fungicides were sprayed on naturally inoculated field plants, adequate control was not achieved with any of the fungicides. While these fungicides may be effective’when used to protect healthy plants, they were not effective in controlling the development of cankers in infected harvested plants. INTRODUCTION In 1986, Michigan nursery growers noticed a post-harvest decline of Colorado blue spruce (Picea pungens glauca, Engelm.) trees of sizes ranging from 30-40 cm to 2-3 m in height. Plants that remained in the field appeared symptomless except for an occasional flagging branch. Affected plants displayed symptoms several weeks after harvest. Symptoms were characterized by needle loss concentrated on the lower part of the plant. Extreme cases resulted in plant death. Since 1986, Michigan nursery growers have reported severe annual losses of harvested Colorado blue spruce. In one report, unmarketable plants resulted in a loss of 30% of the harvested crop. Additional losses occur because many affected trees require cosmetic pruning before sale, trees must be sold at a lower cost because of poor form, dead plants must be rogued out, warranteed plants must be replaced and the growers reputation is questioned. Symptoms of this disorder appeared similar to those caused by a common canker-causing pathogen of mature Colorado blue spruce, Cytospora kunzei Sacc. var. picea Waterman, which is considered the most destructive disease of Colorado blue spruce in Michigan (4). Samples of I spruce afi'ected by the post-harvest disorder were submitted to the Michigan State University Plant Diagnostic Clinic. Samples yielded cultures of Phomopsis only. % The only previous study suggesting that Phomopsis is a pathogen of spruce was done in 1986 by Sanderson and Worf (7). By means of artificial spore inoculation, Sanderson and Worf found that Phomopsis occulta caused disease symptoms on four spruce species: Picea abies, P. obovata, P. glauca cv. 'Densata', and P. pungens glauca. Colorado blue spruce was the most susceptible of the four spruce in their research. This study was initiated to determine whether Phomopsis is a pathogen of young spruce, determine the role of Phomopsis in the post- harvest disorder of Colorado blue spruce in Michigan, find the optimal temperature for growth of the suspect organism, and measure the effects of chemical controls on the growth of Phomopsis. MATERIALS AND METHODS Koch's Postulates were used to verify a causal relationship between P. occulta and the post-harvest disorder of Colorado blue spruce. Requirements include isolation of suspect organism from symptomatic plants, inoculation of healthy plants resulting in the reproduction of symptoms found on initially symptomatic plants, and resiolation of suspect organism from artificially inoculated plants. Isolations. Samples were collected in 1988 and 1989 from nursery- grown Colorado blue spruce, harvested from a large Michigan nursery in the fall of each respective year. Fifty samples exhibiting die-back symptoms were collected each year from ten plants that displayed symptoms of post- harvest decline. Samples were prepared by rinsing under running tap water for several minutes, surface sterilizing for ten to fifteen minutes in 20% bleach (5.25% NaOCl), and then rinsing twice with sterile water. The outer bark was removed and 4 small sections of each shoot were taken from the margin of the canker and were plated directly on potato dextrose agar (PDA) in 90-mm-diameter Petri plates. PDA cultures were incubated at room temperature for seven days before evaluation. Inoculum. Several isolates of Phomopsis occulta were obtained from the margins of expanding cankers of symptomatic Colorado blue spruce. Cultures were grown on PDA and incubated at room temperature for fourteen days. Two fourteen day old cultures of the same isolate were 28 randomly chosen and then macerated for two minutes in a commercial blender with 30 milliliters of sterile water to produce a mycelial inoculum. Sterile PDA inoculum was prepared in the same manner, macerating two Petri plates of sterile PDA. Several cultures of each of five isolates were incubated under continuous fluorescent lighting to encourage pycnidia formation. Sixteen- day-old cultures were flooded with sterile water to obtain a conidial suspension. The water was allowed to sit for thirty minutes; the surface of the culture was scraped using a sterile teasing needle; and the suspension was filtered through four layers of cheesecloth. The concentration of alpha conidia was determined using a hemacytometer. Pathogenicity. Six inoculatiOn experiments were conducted in 1990 and were performed as completely randomized designs. Mycelial inoculum was used in the first four experiments and a conidial suspension was used as inoculum in the last two trials. Fresh inoculum was prepared for each experiment as previously described. Half of the plants in each treatment were selected for reisolation when the experiments were terminated. Samples were prepared for isolation and were cultured on PDA as described previously. Reisolation from symptomatic plants and comparison to the original culture was the last step in verifying Koch's Postulates. One-year-old Colorado blue spruce plugs were potted in a peat-based, soilless commercial mix (Baccto, Michigan Peat Company) three months prior to inoculation. Seedlings were placed in a greenhouse with sixteen 29 hour supplemental fluorescent lighting and computer controlled day/night temperatures set at 240 C / 200 C. Seedlings were well established at the time of inoculation. In the first trial, (started Jan. 8, 1990) 5 trees were wounded 10 cm above the soil line by pushing a sterile pin, 1 mm in diameter, through the stem. Needles were clipped off of the main stem within 1 cm surrounding the wound. A sterile hypodermic needle was used to inject inoculum into the wound until a small amount of inoculum was seen exuding from each side of the wound. Five control trees were wounded in the same manner, but received an injection of sterile PDA. All wounds were wrapped with a small strip of Parafilm. To simulate a harvest procedure, the root mass of ‘ half of the inoculated trees and half of the control trees was reduced by cutting away the lower six cm from a 10 cm long root plug. Treatments were arranged as a completely randomized design in a 2 X 2 factorial experiment. Trees were observed periodically and at the end of six months, lesion length was measured by scraping away the outer bark and measuring the length of the necrotic region. A second trial was conducted (started Feb. 5, 1990) using the aforementioned treatments but two wounds were inflicted on each plant instead of one to investigate the susceptibility of tissues of different ages. The first wound was located 10 cm above the soil line and the second wound was located on the terminal leader, in the center of the most recent flush of growth. Both wounds on one plant were treated alike. Ten plants were inoculated with Phomopsis, and 10 were inoculated with sterile PDA. The 30 lesion length at each wound location was measured five months later. In order to develop a successful inoculation technique, the third inoculation experiment (started Feb. 5, 1990) was conducted to determine which factors would be most effective in producing disease symptoms. Wounding, sealing the wound with Parafilm, and inoculating, at two locations on the plant were evaluated. These factors composed 16 treatments in a 4-way factorial experiment with ten replications in a completely randomized design. Each plant was wounded in a lower and an upper location as described previously. Lesion length was measured after a four month incubation time. The final mycelial inoculation experiment (started Feb. 15, 1990) investigated three factors: inoculation, wounding, and simulated harvest. Plants were wounded only within the most recent flush of growth of the terminal leader. Five plants served as replicates in each of eight treatments in a 2 X 2 X 2 factorial experiment. Lesion length was measured three months after inoculation. Two techniques of wounding were used to determine if wounds were required for conidial penetration (started Feb. 15, 1990). Ten plants were wounded by cutting needles and 10 plants were wounded by pruning branches. Ten control plants remained unwounded. Five branches on each of ten plants were wounded by pruning three cm from the tips of branches. Another set of ten trees was injured by cutting needles in half on five branches per tree. A conidial suspension of 1.74 X 104 conidia per milliliter, was sprayed on half of the trees with an atomizer until small 31 droplets formed and fell from branches. Control plants were sprayed with sterile water to the point of runoff. Each plant was covered with a clear polyethylene bag for 72 hours. Plants were periodically observed for the development of symptoms. The number of cankered branches was recorded four months after inoculation. Data were analyzed as a completely randomized 2-way AOV with 10 plants per treatment. One month later (March 13, 1990) another inoculation trial was initiated, using a spore concentration of approximately 1.47 X 106 conidia per milliliter. Spores were brushed directly on three branches per tree. Control trees were brushed with sterile water. In a third treatment, the spore suspension was injected through syringe needle wounds into three different stems per tree, four cm from the branch tips. Control trees were injected with sterile water. There were twenty plants in each treatment, and three branches treated per plant. All plants were covered with clear polyethylene bags for 72 hours. Plants were evaluated when the experiment was terminated three months later. Efi'ect of temperature on growth. The radial growth of one isolate of Phomopsis occulta was measured at the following temperatures: 0, 5, 10, 15, 20, 25, and 30 0C. Mycelial plugs, 10 mm in diameter, taken from the margin of 10-day-old cultures were placed in the center of 90-mm-diameter Petri plates containing PDA. Plates had been incubated over-night at the assigned temperature prior to inoculation. Twenty replicate plates were incubated in the dark at each temperature. Radial growth was measured every 24 hours. 32 In-vitro fungicide screening. The poison agar technique was used to evaluate four firngicides: benomyl (Benlate: 50% methyl 1-(butylcarbamoyl)- 2-benzimidazolecarbamate, Dupont Agrichemicals), iprodione (Chipco 26019: 23.3% 3-(3,5-dichlorophenyl)-N- (1-methylethyl)-2,4-dioxo-1- imidazolidine-carboxamide, Rhone-Poulenc Inc.), chlorothalonil (Daconil 2787: 40.4% tetracholorisophthalonitrile, Fermenta, Plant Protection), and cupric hydroxide (Kocide 606: Kennecott Copper Corporation). Fungicides were added to warm PDA for final concentrations of 1, 10, 100, and 1000 ppm active ingredient. Fungicide amended PDA, in 90-mm-diameter Petri plates were inoculated with 10-mm mycelial plugs cut from the margin of 10-day-old cultures. Twenty PDA plates were prepared for each concentration of each fungicide, and 20 unamended plates were used as controls. Radial growth was measured every 24 hours. Field evaluation of fungicides. The same fungicides used in the in vitro study were sprayed on a field of 38-45 cm Colorado blue spruce trees located in a large commercial nursery. A nursery worker separately mixed, 454 g of Benlate, 454 g of Chipco, 1 liter Daconil, and 2 liters Kocide with 379 liters of water each to cover 46.45 m2. Sprays were applied to one field divided into beds. Each bed contained three rows of trees spaced 50 cm apart with trees in a row spaced 45 cm on center. Treated beds were separated by two unsprayed beds in order to act as a buffer in case of fungicide drift. One bed of control plants did not receive a fungicide application. Three fungicide sprays were applied by a trained nursery applicator in 1989 on June 24, July 24, and August 30. 33 Ten plants from each treatment were randomly selected by the researcher, hand dug and placed in pots by nursery workers in November, 1989. Plants were immediately transported to Michigan State University, Horticulture Teaching and Research Center in East Lansing, Michigan. Plants were placed in unheated quonset style houses covered with 4-mil white polyethylene and stored for the winter. Plants were periodically monitored until final evaluation July 1, 1990. RESULTS AND DISCUSSION Isolations. All samples collected in 1989 and 1990 fiom symptomatic Colorado blue spruce yielded cultures of P. occulta and rarely any other fungus. Pycnidia were formed in PDA cultures within 10 days, when isolates were incubated at room temperature under continuous fluorescent lighting. Within fourteen days, pycnidia were exuding tendrils of alpha and beta conidia typical of Phomopsis species. The identification of Phomopsis occulta was confirmed by Dr. David L. Roberts and Dr. Alvin Rogers of the Department of Botany and Plant Pathology, Michigan State University. Identification was based on conidia size and morphology of conidia. 4 Until recently, P. occulta was only considered a common saprophyte found growing on many conifers (2, 3, 11). Nearly all stress-related diseases are caused by organisms that usually grow as saprophytes on the host plant (8). Suspicious of the pathogenicity of P. occulta arose in 1929, when White published a New Jersey report of stressed P. pungens with blighted tips from which P. occulta was isolated (12). In 1986, firm evidence of the potential of P. occulta to cause disease was reported by Sanderson and Worf who artificially inoculated four species of spruce and two species of fir with conidia of P. occulta (7). The inoculum was isolated from Colorado blue spruce that were found growing in nurseries and landscapes in Wisconsin. Suspect plants were showing necrosis and death of new 35 growth. This was the first report of P. occulta causing disease on Colorado blue spruce in Wisconsin. There are no previous reports of P. occulta causing disease on spruce grown in Michigan, hence this is the first report of Phomopsis shoot blight on Colorado blue spruce in Michigan. Pathogenicity. Inoculated plants in all pathogenicity trials were significantly more symptomatic than control plants (Tables 1.1-1.7). Plants that were inoculated with mycelia 10 cm above the soil line on the main stem in the first pathogenicity experiment were slow to show any symptoms. After six months, the mean lesion length of inoculated plants was significantly longer than the lesion length in control plants (Table 1.1). Wounding caused by piercing the main stem with a 1 mm pin caused some splitting of the stem. Wounded stems inoculated with sterile agar slowly closed while seedlings inoculated with P. occulta showed an inhibited callus formation in wounded zones when compared with control plants. The wound of inoculated plants was characterized by an open lesion with inconspicuous necrotic tissue extending beyond the open lesion. The extended canker was measured by carefully scraping away the outer bark with a razor blade and then measuring the length of the necrotic zone. In experiment one, removing approximately 60% of the root mass of plants had a significant effect on the mean lesion length when plants were also inoculated (Table 1.1). Lesion length in plants that were inoculated with sterile agar was unaffected by root reduction. After six months the greatest lesion length achieved was 14.4 mm, but none of the plants showed Table 1.1. Pathogenicity Experiment One. The effect of inoculation and reduction of 10 cm root plugs to 4 cm on the development of lesions in one- year-old wound-inoculated Colorado blue spruce seedlings, six months after inoculation. _MeanLesiQnLensth_(mm)__ Inoculum Roots Intact Roots Trimmed .SterilePDA 3.7 6.0 P. occulta 8 .2 14 .4 Source of variation Inoculation (I) ** Root treatment (Rt) ** I X Rt * fl" Significant at P=0.05, 0.01 respectively, according to F test. Table 1.2. Pathogenicity Experiment Two. The effect of inoculation and reduction of 10 cm root plugs to four cm on the lesion length (mm) at two wound locations on one-year-old Colorado blue spruce seedlings after five months incubation. WW— fleundLothw Winn Roots Roots Roots Roots Inoculum Intact Trimmed Intact Trimmed SterilePDA 4 3 4 2 2.8 4 4 Phomopsis occulta 8 . O 32 .2 53 . 3 64 .3 Source of variation Inoculum (I) ** Root treatment (Rt) NS I X Rt NS Location (L) ** I X L ** Rt X L NS I X Rt X L NS 1! Each tree was wounded 10 cm above the soil line, identified as wound location one, and in the center of the most recent flush of terminal leader growth, identified as wound location two. NS,“ Nonsignificant or significant at P = 0.01, respectively, according to the F test. Table 1.3. Pathogenicity Experiment Two. Symptom development on one- year-old Colorado blue spruce seedlings one month after wound- inoculating and reducing 10 cm root plugs to 4 cm or leaving roots intact. No. Trees With Blighted Inoculum Terminal Leadersx Sterile PDA Roots intact 0 Roots trimmed 0 Phomopsis occulta Roots intact 5" Roots trimmed 9‘ X A total of ten plants per treatment were each inoculated on the main stem 10 cm above the soil line and in the center of the terminal leader. ‘Values marked with an asterisk are significantly different from each other and from the control treatments at P = 0.05 according to the Chi- square analysis. 39 Table 1.4. Pathogenicity Experiment Three. Efficacy of wounding, inoculating, and sealing wound with Parafilm, in the production of lesions at two locations on one-year-old Colorado blue spruce, after four months incubation. i n mm) W Inoculum Parafilm Exposed Parafilm Exposed Sterile agar Wound location onex 3 .3 3 4 0 0 Wound location two 3 8 4 4 0 0 Phomopsis occulta Wound location one 9 .0 13 .7 o 0 Wound location two 4 2 . 9 6 6 . 2 0 0 Source of variation Inoculum (I) ** Wounding (W) , ** IXW ** Parafilm (Pf) NS IXPf NS WXPf * IXWXPf * Location (L) ** IXL ** WXL ** IXWXL ** PfXL NS IXPfXL NS WXPfXL NS IXWXPfXL ** X Each of ten plants per treatment was wounded in two locations. Each wound per plant was treated in the same manner. Wound location one is defined as the wound inflicted on the main stem 10 cm above the soil line. Wound location two is the wound located in the center of the terminal leader. NS, *, ** Nonsignificant or significant at P = 0.05, 0.01, respectively, according to F test. Table 1.5. Pathogenicity Experiment Four. Mean lesion length measured three months after Colorado blue spruce seedlings were inoculated within the center of the terminal leader when roots were left intact or when 10 cm root plugs were trimmed to four cm. Wound Surface Inoculum , Inoculated Inoculated Sterile PDA Roots intact 2 . 4 0 .0 Roots trimmed 2 . 8 0.0 Phomopsis occulta Roots intact 2 1 . 8 7 . 6 Roots trimmed 3 2 . 6 0 . O Source of variation Inoculum (I) ** Wounding (W) ** I X W ** Root treatment (Rt) NS I X Rt NS W X Rt NS I X W X Rt NS NS,“ Nonsignificant or significant at P = 0.01, respectively, according to F test. 41 Table 1.6. Pathogenicity Experiment Five. Mean number of shoots blighted per tree on injured one-year-old Colorado blue spruce seedlings sprayed with 1.74 X 104 conidia per milliliter of Phomopsis occulta, four months after inoculation. _Msan_N.umheLo£Blighted.Shth Stem Needle No Inoculum Injury Injury Injury Sterile water 0.0 1.8 0.0 ‘ Phomopsis occulta 4.7 7.1 8.8 Source of variation Inoculum (I) ** Wounding (W) NS I X W NS NS,“ Nonsignificant and significant at P = 0.01 respectively, according to F test. Table 1.7. Pathogenicity Experiment Six. Mean number of blighted shoots on one-year-old Colorado blue spruce seedlings, with tips that were wound-inoculated or surface-inoculated with a concentrated conidial suspension (1.47 X 106 conidia per milliliter), three months after inoculation. Wound Surface Inoculum Inoculated Inoculated Sterile water 0.4 0.0 Phomopsis occulta 2.9 4.2 Source of variation Inoculum (I) ** Wounding (W) NS I X W * NS,*, ** N onsignificant or significant at P = 0.05, or 0.01 respectively, according to F test. 43 symptoms of needle loss or tip death, in fact all the plants produced new growth. When plants were wounded in two locations, lesions that developed on younger tissue were significantly longer than lesions that developed on older stems (Table 1.2). Location of wound was not a significant factor in the development of lesions when plants were inoculated with sterile agar. This indicates that younger tissue may be more susceptible to colonization by P. occulta than older stems. The effect of root reduction on lesion length in this experiment was only significant when comparing the lesions that developed at the older region of wound-inoculated plants (Table 1.2). Lesions in plants with reduced root systems were much longer than lesions in plants without root disturbance. Although the effect of root reduction on lesion length in inoculated terminal leaders was not significant, 90% of the plants that were inoculated and had roots trimmed developed girdling cankers that killed terminal leaders within one month after inoculating (Table 1.3). Only 50% of plants that were wound-inoculated without root treatment developed lethal girdling cankers. This difference was significant according to the Chi-square analysis at the 5% confidence level. None of the control plants developed cankers. Growth of cankers was primarily longitudinal, but when roots were trimmed, small, young stems were quickly girdled. Pathogens that cause stem cankers are mostly nonaggressive pathogens that only attack weakened or wounded hosts (8). Host vigor is reduced by stresses such as drought, flooding, freezing, defoliation, and 44 transplanting. Cutting plant roots reduces stored food reserves, and reduces the absorptive surface area for taking up water and nutrients (9). Transplanting will induce stress in most plants until an adequate root system can be reestablished (8). The experimental simulation of harvest stress in Colorado blue spruce in the previously discussed experiments has shown that stressed plants were more symptomatic than nonstressed plants. Drought stressed Colorado blue spruce have also been shown to be more susceptible to CytOSpora canker, caused by Cytospora kunzei Sacc. var. picea Waterman, when artificially wound-inoculated (4, 10). When all permutations of inoculating, wounding, and sealing with Parafilm, were evaluated as techniques for creating disease symptoms at two wound sites, all factors were found to be dependent on one another (Table 1.4). Parafilm was found to have a significant effect only when plants were also wound-inoculated. When wounds were wrapped with Parafilm, the mean lesion length was shorter than lesion length at wound zones without Parafilm. The moist wound environment maintained by a Parafilm wrap could have either inhibited growth of the pathogen, encouraged growth of competitor organisms, or enhanced compartmentalization efforts by the plant. The greatest mean lesion length, 66.2 mm, was achieved by leaving the wound exposed (not wrapped with Parafilm) and by wound-inoculating the younger region of the main stem. As demonstrated in an earlier experiment, (experiment two), the newer tissue was more susceptible to colonization than older tissue. Half of the plants that were wound 45 inoculated developed girdling cankers on the terminals. In the absence of wounding, no lesions developed and the mycelial inoculum was unable to penetrate even the newest most succulent tissue. The surface inoculum was still viable after four months of inoculating as indicated by reisolation from the surface. The role of wounding was further demonstrated when only one wound per plant was inflicted on the terminal leader (Table 1.5). Plants that were wounded and inoculated developed the longest lesions. When plants were unwounded no lesion developed except when a mean lesion length of 7.6 mm was obtained when plants were surface inoculated while roots remained intact. This large value can be explained by accidental wounds that were inflicted on several plants when needles were clipped from the terminal leader at the inoculation point. This was done to facilitate the wrapping of the wound with Parafilm. The new growth was very tender and easily injured by scissors that slightly pulled needles from the stem. No terminal leader was girdled in any of the treatments in this fourth pathogenicity experiment. Of the four experiments discussed previously, the only treatments that caused girdled terminal leaders were those that involved two wounds on the main stem. When two wounds were inflicted and the roots remained intact, 15 out of 30 plants developed girdling cankers (experiment two and three). When roots were trimmed, 9 out of 10 plants developed girdling cankers (experiment two). Although comparisons between experiments are of limited value because 46 experiments were performed at different times with different isolates, plants that were wounded in two locations developed girdling cankers on the terminal leaders while those plants wounded only in an upper or lower location did not, regardless of root pruning. The lower wound on the main stem may have been stressing the terminal leader tissue and predisposing it to colonization. Root pruning would further aggravate this condition. P. occulta was successfully reisolated from all samples of discolored lesion tissue that had been originally wound-inoculated with the same fungus. Cankers extended primarily longitudinally, although some staining of the xylem tissue was evident within the first 1 mm of stem tissue. P. occulta was isolated from discolored xylem tissue. In the previously discussed experiments, P. occulta mycelia was shown to be infectious, causing small cankers that developed slowly. The conidial suspension was shown to be even more infectious (Table 1.6, 1.7). When plants were sprayed with a 1.74 X 104 conidia per milliliter suspension, new shoots had very small purplish cankers on the tips within 10 days of inoculation. After three weeks, cankers were barely visible as purple-bronze streaks extending backwards from the tips. Ten uninjured plants sprayed with the conidial inoculum developed an average of 8.8 shoots blighted per tree after four months. An average tree contained only 15 tips per tree (n: 140). Wounding did not produce significantly more shoots blighted per tree as might be expected if wounding aided penetration. Plants were successfully inoculated when concentrated amounts of conidia were brushed directly on the surface of the most susceptible tissue 47 or injected into syringe wounds (Table 1.7). The main effect of wounding was not a significant factor in symptom development. However, the interaction between inoculating and wounding was significant. When unwounded plants were inoculated with conidia, 4.2 blighted shoots developed per tree while those that were injected with conidia developed only 2.9 blighted shoots per tree. Since only three branches were treated per tree some branches on trees that were surface inoculated were either contaminated by unnoticed dripping when applying spores with a paintbrush or developed natural infections. Injection of conidia with a syringe was a cleaner technique that left a smaller volume of the conidial suspension on each plant. Two plants that received injections of sterile water were also contaminated by P. occulta. P. occulta was recovered from the few dead shoots on these control plants and from all shoots that were symptomatic. Wounding is necessary for the infection of shoots by mycelia but not for the infection by spores. In nurseries, open wounds are commonly created by pruning practices. Open wounds were experimentally shown to be more susceptible to canker development than those wounds covered by Parafilm. If Colorado blue spruce are pruned when highly susceptible new growth is present, the plants are at risk of infection by P. occulta mycelia growing saprophytically on the stem. Also, it may be possible for pruned shoots to become inoculated by contaminated pruning tools. Furthermore, pruning plants would increase the quantity of new growth, thereby greatly increasing the number of potential infection sites for conidia. If pruning is 48 to be done, waiting until growth has matured and hardened would provide fewer chances for infection. Effect of temperature on growth. Optimal growth of P. occulta occurred in cultures incubated at 25° C (Figure 1.1). Moderate growth was observed at 15° C, 20° C and 30° C. No growth occurred in cultures incubated at 0° C and 5° C after 30 days. These low temperatures were not lethal. When cultures were brought to room temperature, growth resumed. If growth of P. occulta within host plants is also optimal at 25° C, symptom development would be expected to be greatest during warmer months. Sanderson and Worf observed greater symptom development on artificially inoculated Colorado blue spruce when plants were incubated at around 25° C and at a relatively high humidity (7). They suggest that plants may be susceptible to infection for a short time in the spring but that further symptom development does not occur until the weather becomes warmer and more humid. In-vitro fungicide screening. Benlate was the only fimgicide that showed complete control of the fungus at all concentrations (Figure 1.2). The mycelial plug was killed by Benlate concentrations of 10, 100, and 1000 ppm active ingredient. Chipco and Daconil provided good control at 10 and 100 ppm a.i., and complete control at 1000 ppm a.i. Kocide controlled growth at 1000 ppm a.i. but gave no control at 1, 10, and 100 ppm a.i. Kocide slightly encouraged growth of P. occulta at lower concentrations when compared to control cultures. Figure 1.1. Effect of temperature on the radial mycelial growth of one isolate of Phomopsis occulta. Measurements were made ten days after PDA plates were inoculated with 10-mm-diameter disks of mycelia. Each treatment was replicated 20 times. The LSD (P=0.05) for comparing means is 1.85 mm. Qo mash/EMQEMH on mm cm mm or m. _ _ — _ _ _ Rm om; Toe row too 10x. tom tom OO— sxvo 0L 33m (ww) aaiawwo ANO‘IOO NVBW 51 Figure 1. 2. Effect of four fungicides on the radial mycelial growth of Phomopsis occulta growing in fungicide amended PDA cultures incubated at room temperature for eight days. *Significantly different from the control at P = 0.05 according to Tukey's HSD. Each treatment was replicated 20 times. The LSD (P=0.05) for comparing means is Benlate: 1.3, Chipco: 1.6, Daconil: 2.0, Kocide=2.1. ...o E8 .__zoommmmO mmm _ 4.2.... Hmm>moz Bo Sm F r P \ s f P ,. ..N In ..t Siam e822: «$552 I. ..4 Siam Gui? $880 I oeumm 59%: Emzmamm I «taste e322: 5:02 To u b p P b n n ONLLVEI Figure 2.2. The monthly visual rating of Colorado blue spruce infected with Phomopsis occulta and harvested in the fall of 1989 where 1 = no symptoms of disease, 2 = less than one-third of the plant surface exhibiting symptoms of needle loss or discoloration, 3 = more than one- third, but less than two-thirds of the plant surface showing signs of disease, 4 = more than two-thirds of the plant surface showing needle loss, needle discoloration, extensive cankering, and death of shoots, and 5 = entire plant dead. Observations were recorded during the first few days of each month. Points represent the monthly rating of trees averaged over all fertilizer treatments. _ m:.<0 ZO_._.<>mmmmO 43.. 22. >32 and. «<2 >02 FOO Qmm A mnumm #82:: E553? I Siam 5.13%: Emobo is onumm e822: mumzmimm I Noam”. Box/«<1 5:02 I mmm _ Dada Hmm>m