“WWWNWllHlmlMWWW“WIHIMM 110 038 __THS Wests mes MICHIGAN STATE ”3” \ll l\\\\l\ll\lllflll\ \ l l\\\\ \\\l 3\\1293 00882 4074 l This is to certify that the thesis entitled CULTURAL AND CHEMICAL CONTROL OF PHOMOPSIS SHOOT BLIGHT OF COLORADO BLUE SPRUCE (PICEA PUNGENS GLAUCA) presented by Susan Marie Gruber has been accepted towards fulfillment of the requirements for MS. degree in .HOflicultuLe. Major professor Date 7/ “7/4'6i V 0-7639 MS U is an Affirmative Action/Equal Opportunity Institution LiBRARY Michigan State University PLACE IN RETURN BOX to remove thie checkout from your record. TO AVOID FINES retum on or before due due. DATE DUE DATE DUE DATE DUE __J[:—L '-[_‘ T— MSU Ie An Affinnetive ActioNEquel Opportunity inuitution cWMG-M CULTURAL AND CHEMICAL CONTROL OF PHOMOPSIS SHOOT BLIGHT OF COLORADO BLUE SPRUCE (PICEA PUNGENS GLA UCA). By Susan Marie Gruber A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Horticulture 1992 ABSTRACT CULTURAL AND CHEMICAL CONTROL OF PHOMOPSIS SHOOT BUGHT OF COLORADO BLUE SPRUCE (PICEA PUNGENS GLAUCA). By Susan Marie Gruber Phomopsir occulta Trav., causal agent of shoot blight of Colorado blue spruce (Picea pungens glauca), was isolated from six spruce species in Michigan nurseries. Oiltural and chemical methods for control of Phomopsis on Colorado" blue spruce were tested. Fall root pruning prior to spring digging increased postharvest symptom severity, while spring root pruning before fall harvest was beneficial. Improving postharvest root regeneration did not alleviate symptom severity on naturally infected field grown plants. Overwintering spruce inside a polyhouse reduced disease severity compared to those held outdoors. In vitm and greenhouse screening of fungicides showed several compounds have potential as chemical controls. Spruce shoots inoculated two weeks after budbreak were more susceptible to tip blight than those at earlier or later stages of development. Postharvest applications of benomyl were effective in reducing disease severity. for Anne ACKNOWLEDGMENTS - My major professor, Dr. Om Peterson, has been supportive and encouraging throughout this 131'ij and I thank him for all his time and tolerance. The members of my guidance committee - Drs. Muralee Nair, Dave Roberts, and Bob Schutzki, have each given generously of their time, advice, and resources. Their interest in my research and professional goals has been very helpful, and is greatly appreciated. Zelenka’s Evergreen Nursery generously supported this project, and provided plant materials, supplies, and the time and help of stafi' members. Thanks to all of you; this research is also the fruit of your labors. I can’t list all of my fellow grad students who have shared their knowledge and good humor with me over the last two years, but thank-you especially - Bill, Jim, Sue, Diana, Shawn, Ann and Mark. Vicki Buell and Tom Fink spent many hours on this research, and I thank them for their hard work. Thanks also to the staff of the HTRC, John, Lou, Gary, and Bill. You guys are the best! MyhusbandKarlhasworkedandwaitedforthe lasttwoyears, andlthank him for his love and friendship, and for reminding me how good it would feel when this is finished. Finally, I thank our parents for their love and all they have done over many years to make this possible. iv TABLE OF CONTENTS page LIST OF TAB! FS vi LIST OF FIGURES viii INTRODUCTION 1 Literature Cited 4 CHAPTER I THE EFFECT OF CULTURAL PRACTICES ON THE SEVERITY OF PHOMOPSIS OCCULTA (TRAV.) CANKER DEVELOPMENT IN COLORADO BLUE SPRUCE (PICEA PUNGENS GLA UCA ENGLM.). Abstract 7 Introduction 8 Materials and Methods 1 1 Results and Discussion 16 Literature Cited 33 CHAPTER II INCIDENCE, HOST RANGE, AND CHEMICAL MANAGEMENT OF PHOMOPSIS CANKER OF COLORADO BLUE SPRUCE (PICEA PUNGENS GLAUCA) IN MICHIGAN. Abstract 37 Introduction 38 Materials and Methods 41 Results and Discussion 47 Literature Cited 5 7 Table 1.1 1.2 1.3 1.4 1.5 1.6 LIST OF TABLES CHAPTER I Page Properties of media used in the root regeneration study. ............. 13 Root regeneration: Phomopnlr symptom severity on naturally infected, field-grown Colorado blue spruce, when treated with 1000 ppm IBA and potted in wood chip medium or field soil. Trees potted October 27, and evaluated December 17, 1990. ..... 23 Root regaterntion: Shoot moisture content of naturally infected, field-grown Colorado blue spruce, when treated with 1000 ppm [BA and potted in wood chip medium or field soil. Trees potted October 27, and evaluated December 17, 1990. ..... 24 Root regeneration: Root and shoot moisture content of naturally infected, field-grown Colorado blue spruce, when treated with 1000 ppm [BA and potted in wood chip medium or field soil. Trees potted October 27, 1990 and evaluated May 22, 1991. 27 Root regatemtt'on: Phomopsit Symptom severity on naturally infected, field-grown Colorado blue spruce, when treated with 1000 ppm [BA and potted in wood chip medium or field soil. Trees potted October 27, and evaluated May 22, 1991. ................ 29 Root regeneration: Symptom severity on naturally infected, field-grown Colorado blue spruce, when treated with 1000 ppm [BA and potted in wood chip medium or field soil. Trees potted October 27, and evaluated August 15, 1991. 30 Table 1.7 Table 2.1 usr or TABLES (cont) CHAPTER I Page The :1?ch of Waring and benomyl applications: Phomopsis' symptom severity on naturally infected, field-grown Colorado blue spruce when treated with benomyl once in June, or monthly in June, July, and August 1990, harvested October 11, 1990, and overwintered outdoors or inside a poly covered quonset house. Plants evaluated August 15, 1990. ......... 3 1 CHAPTER II Page In vitro fungicide screening: Properties of the tested fungicides. 45 Field test of benomyl: Phomopstk symptom severity on naturally infected, field-grown Colorado blue spruce when treated with benomyl in the field and after harvest. Trees harvested October 18, 1990, and evaluated August 15, 1991. 56 1.1 1.2 1.3 1.4 1.5 LIST OF FIGURES CHAPTERI Root pruning 1: June, July, and August evaluations of Phomopsit symptom severity on naturally infected, field- grown Colorado blue spruce root pruned October 26, 1990 and harvested April 3, 1991. Page 17 Root priming II: Phomopsis symptom severity on naturally infected, field-grown Colorado blue spruce root pruned ' ' April 24, 1991 and harvested October 24, 1991. Trees were " placed in a heated greenhouse November 7, 1991, and evaluated 5, 8, and 11 weeks after harvest. Root regateration: Number of white roots > one millimeter in length on field-grown Colorado blue spruce, when treated with 1000 ppm [BA and potted in wood chip medium or field soil. Trees potted October 27, and evaluated December 17, 1990 . Root regeneration: Fresh weight of new roots (as a percentage of root system fresh weight) on field-grown Colorado blue spruce, when treated with 1000 ppm [BA and potted in wood chip media or field soil. Trees potted October 27, and evaluated May 22 1991. 18 -22 Ovmvintering: Phomopsir symptom severity evaluations for June, July, and August on naturally infected, field-grown Colorado blue spruce harvested October 1 1, 1990, and overwintered outdoors or inside a poly covered quonset house. Plants evaluated August 15, 1990. (Data averaged over 32 fungicide treatments). Figure 2.1 2.2 2.3 1131' or FIGURES (cont) CHAPTER II Page Severity of Phomopsis shoot blight symptoms on seedlings of Norway, Colorado, white, and Spartan spruce, evaluated six weeks after inoculation. (RP) = root system reduced by approximately 60% before inoculation. 49 Plant gmwth and pathogen susceptibility: Percent of shoot tips blighted on Colorado blue spruce seedlings at six stages of shoot development 4 weeks after inoculation with conidia of Phomopsis occulta. Stages of shoot growth: DORM a dormant, BRK = budbreak, 2, 4, 6 and 8 weeks of shoot growth after budbreak. 5 1 In vitro fimgicide screening: Radial growth of Phomopsis colonies on fungicide amended agar after 10 days of incubation at 25 ' C. PRO= prOpiconazole, TRD stridiamefon, BENabenomyl, BOR-bordeaux, TRF-trifiorine, MANa-mancozeb, MYC-myckwbutanil, FEN- fenarimol. 53 INTRODUCTION Members of the genus Picea (spruce) are widely used in landscapes and as Christmas trees. In recent years the production of spruce, particularly Colorado blue spruce (Picea pwrgensglauca) in Michigan has been affected by shoot blights and stem cankers caused by the imperfect fungus Phornopsis occulta ('Trav.) (12). P. occulta is not a newly described, or newly introduced organism. reports of this fungus and it’s association with conifers were first described by Funkel who isolated it from cone scales of spruce (Picea encelrea) in Germany in 1875 (6). Other reports of Phantom occulta infestations were published in the late 1920’s in conjunction with research on P. junipemvora. The pathogenicity of P. occulta on spruce was suggested, but was not investigated (21,22). G.G.Hahn reported the isolation of P. occulta from 14 coniferous genera including Picea, and found it widely distributed in the United States and Europe (8). Hahn considered this fungus to be only slightly parasitic, although he held the position that shoot blight agents Phomopsis titujae and Phomopsis criptomeriae were actually strains of P. occulta. He also stated that other physiologic strains of the fungus may indeed be plant pathogens. P. occulta has been categorized in references as a saprophyte or secondary pathogen, on the basis of Hahn’s later 2 work which showed the organism failed to parasitize red cedar (9,11,18). There are many references to Phomopsir occulta and other Phomopsis sp. as endopyhtes of conifer needles and non pathenogenic members of the microflora found on cones, twigs, and bark (5,7,10,17). Phomopsis species have also been reported to cause cankers and tip blights of Douglas-Fir (10). In the late seventies and early eighties, growers in the state of Wisconsin experienced losses in the production of young spruce due to a shoot blight which caused ‘severe damage’ to Colorado blue spruce liners, costing one grower an estimated 525,000 per year (1). Researchers from The University of Wisconsin isolated P. occulta from infected trees in these growing areas, and demonstrated that it was a causal agent of tip blight on blue spruce seedlings (16). Michigan growers also experienced losses of field grown blue spruce during the early eighties. Lower branches of the trees became defoliated following harvest, or in the field during periods of stress, such as the 1987 and 1988 growing seasons, when the state experienced a flood followed by a severe drought. Severe shoot blight symptoms were also found on larger sizes of blue and white spruce in Christmas tree production. Isolation of fungi from samples submitted to the Michigan State University Plant and Pest Diagnostic Lab consistently yielded cultures of Phomopsis occulta. Research done by Igoe confirmed that this fungus was a causal agent of the postharvest disorder of Colorado blue spruce (12). Inoculation trials have also provided information about the susceptibility of spruce species to Phomopsis. Sanderson and Worf inoculated Siberian 3 (P. obovata), Black Hills (P. glauca ‘Densata’), Norway (Babies), and Colorado blue spruce, and found that all four species were infected, but Colorado blue spruce was the most susceptible (16). A pattern of symptomless infection and disease development following the incidence of stress is common among members of the genus Phomopnls A (13,14,15,20). The correlation between root injury and severity of P. occulta infection on Colorado blue spruce was demonstrated in studies by Igoe, who provided a review of the genus, and the role of stress in disease development (12). Management strategies for the suppression of P. occulta in spruce production were sought by the nursery industry to reduce losses which have exceeded the economic tolerance of many Michigan growers. The fungus has been successfully controlled by one Wisconsin nursery by applying benomyl fungicide (23), but this single management tool is not a reliable long term method of disease control. Considering the great potential for fungicide resistance to develop under such conditions (2,3,4,19), a combination of cultural and well designed chemical controls will provide the most successful control of P. occulta. This research was undertaken to develop cultural and chemical strategies for long term disease management of P. occulta on Colorado blue spruce. Cultural practices designed to reduce harvest and postharvest stress were examined for their effect on the development of latent infections. Fungicidal compounds were tested, and experiments were carried out to help determine the most effective timing of fungicide applications. 10. LTTERATURECITED Amos, R. 1990. HRI solves a costly problem for the industry. New Horizons in Horticulture, Horticultural Research Institute, Washington DC. 1:1. Dekker, J. 1976. Acquired resistance to fungicides. Ann. Rev. Phytopath. 14:405-428 Delp, CJ. 1980. Coping with resistance to plant disease control agents. Plant Dis. 64:652-657 Delp, CJ. 1988. Fungicide resistance problems in perspective. In: Fungicide Resistance in North America. APS Press, St. Paul, Minnesota. p.4-5. Fokkema, NJ. and van den Heuvel, J. Eds. 1986. Microbiology of the phillosphere. Cambridge University Press, London. Funkel, L 1875. Diaponhe occulta (Fckl). Nke. Symb. Mycol. Nachtr. 11123-34. Gregory, S. C. 1984. Micro-organisms isolated from wounded stems of Picea sabbath. Trans. Br. Mycol. Soc. 83: 683-725. Hahn, 6.6. 1930. Life history studies of the species of Phomopsis occurring on conifers. Trans. Br. Mycol. Soc. 15 :32-92. Hahn, 6.6. 1943. Taxonomy, distribution, and pathology of Phomopsis occulta and P. junipemvora. Mycologica 35:1 12- 129. Hansen, EM. and Hamm, PB. 1988. Canker diseases of Douglas-fir seedlings in Oregon and Washington bareroot nurseries. Can. J. For. Res. 18: 1053-1058. 11. 12. 14. 16. 17. 18. 19. 21. 5 Heptig, GK. 1971. Diseases of Forest and Shade Trees of the United States. USDA Forest Service Agriculture Handbook 386. US. Government Printing Office, WashingtOnDC. Igoe, MJ. 1991. The relationship between Phomopsis occulta (Trav.) and the post harvest disorder of Colorado blue spruce (Picea pungens glauca Engelm.). MS Thesis, Michigan State University, East Lansing. Kulick, MM. 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. Mycologica 76:274-291. Parker, P. E. and Ramsdell, DC, 1977. Epidemiology and Chemical Control of Phomopsis Canker of Highth Blueberry. Phytopathology 67: 1481-1484. Pscheidt, J.W. 1989. Time of Infection and Control of Phomopsis F nrit Rot of Grape. Plant Disease 73:829-833. Sanderson, PC. and Worf, 6.1... 1986. Phomopsis shoot blight of Colorado blue spruce. J. Environmental Hort. 4:134-138. Sieber, TN. 1989. Endophytic fungi in twigs of healthy and diseased Norway spruce and white fir. Mycol. Res. 92:322-326. Sinclair, W.A., Lyon, I-LH. and Johnson, W.T. 1987. Diseases of Trees and Shrubs. Cornell University Press, Ithaca, NY. p.144. Staub, T. and Sozzi, D. 1984. Fungicide resistance: A continuing challenge. Plant Dis. 68:1026-1031. Verhoeff, K. 1974. Latent infectiom by fungi. Ann. Rev. Phytopathology. 12:99-110. White, RP. 1929. Juniper blight. Ann. Rept., NJ. Agri Expt. Sta. 2mm Wilson, M. 1925. The Phomopsis disease of conifers. Grt. Brit. For. Comm. Bul. #6. HM. Stationary Office, London. Worf, 6.1., and Patton, RF. 1987. Research Summary. Department of Plant Pathology, The University of Wisconsin - Madison, p.3. CHAPTER] THE EFFECT OF CULTURAL PRACTICES ON THE SEVERITY OF PHOMOPSIS occur. TA ('I'RAV.) CANKER DEVELOPMENT IN COLORADO BLUE SPRUCE (PICEA PUNGENS GLA UCA ENGLM.). ABSTRACT Cultural management techniques were examined for their effectiveness in reducing the severity of postharvest Phomopsis disease symptoms on Colorado blue spruce (Picea pungens glwca). Root pruning ten year old (30-36") blue spruce by cutting roots with a landscape spade in October prior to April harvest resulted in more severe symptom expression following harvest. In contrast, root pruning in April prior to October harvest significantly reduced symptom severity. Postharvest root regeneration of field grown blue spruce was significantly improved by treating root systems with 1000 ppm IBA (indole-3-butyric acid), or by potting plants in a wood-chip container medium instead of native field soil. Improved root regeneration had no effect on the severity of subsequent canker development. Overwintering fall field-potted blue spruce inside a polyhouse reduced both mean symptom severity and number of dead plants compared to those kept outdoors. INTRODUCTION Long term solutions to disease problems are most successful if integrated pest management (IPM) techniques are employed. Although IPM is more widely applied to insect pests, a combination of chemical and cultural tools should be applied to manage plant pathogens in order to minimize the use of chemical agents. This is important for environmental and economic reasons, but also in helping to delay or prevent the occurrence of resistance to chemical controls (5). Cultural control of plant vigor can be effective in reducing damage caused by plant pathogens, because stress plays an important role in plant susceptibility to infection and disease development. (23,24). A relationship between stress and the development of shoot blights and stem cankers has been reported for many species of Phomopsis and their woody hosts (14). The severity of P. occulta shoot blight of Colorado spruce as related to plant stress was first observed by White in 1925 who found the fungus causing tip blight on trees in overcrowded and flooded beds (29). Hahn, who studied in detail the pathogenicity of P. occulta observed that it acted as a pathogen only on wounded or stressed conifers (11). A survey of the disease in Michigan nurseries have correlated well with these findings. Igoe demonstrated this effect by root 9 wounding artificially inoculated Picea pungens seedlings. Root pruned and intact plants had the same number of infections, but the amount of canker development and number of dead terminal shoots was significantly greater in the seedlings with injured root systems. Igoe’s research also showed that time of harvest (spring vs. fall), and levels of nitrogen fertilization programs had little effect on the severity of P. occulta symptoms on naturally infected, field grown plants (14). While water stress from drought or flooding has been observed by growers to cause outbreaks of P. occulta in the field, the stress of harvest is usually most severe, and is also potentially more manageable by growers. Harvest stress in spruce is mainly the result of two factors: 1) large reduction of the total root mass at the time of digging, and 2) slow regeneration of the root system after harvest. Root mass reduction at harvest is generally controlled in nurseries by root pruning trees one or more seasons prior to digging. Many studies have demonstrated increased survival of spruce seedlings due to root pruning and root wrenching prior to harvest (12,17,22). Few reports have been published on the effect of root pruning larger conifers. In one study, Watson (28) improved the harvested root mass of landscape sized Colorado spruce by pruning the root system 4 years prior to harvest. Improving root regeneration following potting, balling, or transplanting has also been examined by many researchers. Much of this work has focused on environmental conditions including light, temperature, and media fertility and physical properties. Media aeration and bulk density influence the amount of root 10 regeneration in conifers (19). Spruce seedling have been shown to perform poorly in compacted, low oxygen media, and in soils with high bulk density (8). Applications of IBA (indole-3-butyric acid, a synthetic auxin) to roots of harvested trees is another potential method of improving the postharvest plant status. Carter and Tripepi observed a significant increase in root number, weight, and length when 1000 ppm IBA was applied to fall harvested (3 + 2) Colorado blue spruce seedling. While they concluded such treatment was not economically justified, the economic value of reduced symptom severity on plants with P. occulta could justify the expense of IBA treatment if losses from the disease were significantly reduced (4). Another source of difficulty in maintaining plant vigor is the handling and storage of potted plants during the winter. Harvesting spruce in fall for early spring sales is a common practice in Michigan nurseries. Winter storage of these plants outdoors can cause stress fiom root injury, desiccation and freezing. Indoor storage in quonset style polyhouses may help to alleviate these problems, but may simultaneously create an environment more favorable for fungal growth and canker development. Higher temperatures and relative humidity occur inside the structures, particularly during periods of warm weather in late fall and early spring. Such conditions have been shown to favor the development of Phomopsis blight of Colorado spruce (20). MATERIALS AND METHODS Root Pruning. Plants used in this experiment were 24-36", naturally infected Colorado blue spruce randomly selected fi'om a uniform field at a large commercial nursery in Ottawa County, Michigan. Previously these trees had been root pruned between rows only, with a tractor and U-blade late in September or early October 1988, 1989, and 1990. A landscape spade was used to cut roots starting just under the edge of the first whorl of branches (approximately 30 cm from the trunk), and cutting at an angle of about 60 ' . Roots were cut around the whole circumference of the trees to the depth of the spade (35cm). Twenty trees were treated on October 26, 1990, and harvested on April 3, 1991, along with 20 untreated controls from the same farm. Disease symptom severity was visually M rated on the 15‘“ of June, July, and August using a scale of 1 to 5, where 1 = symptomless, 2 = less than-one third of the plant showing needle loss, 3 = one- third to two-thirds of the branches affected, 4 = more than two-thirds of the plant showing needle loss, cankering, and death of shoots, and 5 = dead, as described by Igoe (14). The root pruning procedure was repeated on April 24, 1991. Ten root pruned trees and controls were harvested on October 24, 1991. The trees were moved to a heated greenhouse (24 ’ C/75 ' F day, 18 ' C/ 65 ' F night) on November 7, 1991 to accelerate symptom development. Symptoms were visually rated on December 1 and 20, 1991 and January 13, 1992, (5, 8, and 11 weeks after harvest). 11 12 Root Regeneration. Two hundred 15-18" Colorado blue spruce (Picea pungent glauca) seedling were harvested on October 26, 1990 from a uniform field in a large commercial nursery in Ottawa County, Michigan. None of the harvested plants showed any symptom of Phomopsis infection, but were growing in an area known to contain high levels of Roccrdta inoculum. The trees were bare rooted and placed in groups of 8 - 10 in 30 gallon plastic bag with wet burlap wrapped around the roots to prevent desiccation during transport to East Lansing. Immediately upon arrival the trees were heeled into moist peat moss in a 1.5 ' C (35 ' F) cooler and held overnight. The following day plants were removed from the peat beds, and the root systems were washed with running water. All root systems were trimmed to a uniform size for planting in a Classic 1600TM pot. Lateral roots were cut to 12 cm from the central axis, and the vertical length of the root mass was standardized at 20 cm. A small number of white roots were found on 12 trees, and were removed during this process. The experiment was set up as a 3x2 factorial, with 30 replications. The roots were treated with a ten second dip in one of the following solutions: 1000 ppm IBA (indole-3 butyric acid, Sigma Chemical Corporation) in a 10% EtOH solution in water, 10% EtOI-I, Or water. Immediately following treatment, the spruce were potted in either soil from the growing area, or in a container medium. Properties of these two potting media are summarized in Table 1.1. 13 Table 1.1 Properties of media used in the root regeneration study. I 0 Le 11.. ONTAINER MIX . 40% hardwood chips Granby fine sandy 40% rice hulls MEDIA PROPERTY “ 10““ 1362,1332? pH 6.5 7.0 soluble salts (mmhos) .75 .58 nitrate (ppm) 32 3 'phosphonrs (ppm) 0.5 5.8 l potassium (ppm) 25 93 mldmgpm) 86 57 magnesium (ppm) 20 22 sodium (ppm) 10 7 [ chloride (ppm) 9 11 zinc (ppm) 18 24 manganese (ppm) 1 1 31 copper (ppm) 1 1 iron (ppm) 83 95 I organic matter % 4.6 (digestion) 50.8 (ignition) % solids 76.94 37.03 % air space 5.41 29.87 % water space 17.65 33.10 ’ Classification from: Soil Survey of Ottawa County, U.S. Soil Conservation Service, USDA, Washington DC. + Media chemical properties determined by the Soil Testing Laboratory, Michigan State University. ‘ Physical properties of media calculated using containers identical to those used in the experiment. 14 The potted plants were placed in randomized complete blocks in an Open quonset style polyhouse at the Horticulture Teaching and Research Center at Michigan State University. Poly cover (white 4ml, 18% shade) was placed on the house on November 8, 1990, and removed on April 2, 1991. Root and shoot fresh and dry weights were measured from 20 trees at the initiation of the experiment. Shoot weights for this experiment were taken from 3 branches collected from the top whorl of the tree. Measurements of root regeneration were made On December 17, 1990 and May 22, 1991. Ten plants per treatment were measured at each sampling. In December, the number of white roots over 1 mm were counted, and fresh and dry weights of the shoots were measured. In May, white roots were separated from the rest of the root system for analysis. Data on white (new) and brown (old) root fresh and dry weights, and shoot fresh and dry weights were collected. Symptom severity was measured on the plants when root data was collected. A complete set of treatments was held for evaluation of disease severity in June, July, and August 1991. Symptoms were visually rated during the summer on a scale of 1 (no disease symptoms) to 5 (dead), as previously described. Over-wintering. Eighty 12-25" Colorado blue spruce were harvested from a commercial nursery in Ottawa County, MI on October 1 1, 1990. None of these trees had P. occulta disease symptoms. Forty plants had been treated in the field with benomyl applied by nursery personnel as Benlate 50wp 22kg/3781 of water 15 (11b/100 gal) once a month in June, July, and August of 1990. Forty trees received a single application in June. Twenty plants from each group were overwintered inside the polyhouse. The remaining 20 were overwintered just outside the house, on a gavel surface identical to the inside. The plants were arranged in a randomized complete block design with four blocks of five replications in each location. All containers were moved as close together as possible for overwintering when the house was covered with poly. The trees were spaced out in spring when the plastic was removed. Disease symptoms were visually rated during the summer on a scale of 1 (no disease symptoms) to 5 (dead), as previously described. I "‘ RESULTS AND DISCUSSION Root Pruning. Trees root prunediin October prior to field potting in April were significantly worse than control plants. In the field prior to harvest, and for three weeks following potting, there was no evidence of canker development or needle cast on any of the plants. Following bud break and shoot expansion, difl'erences between treatments became quite obvious. As illustrated in Figure 1.1, the average root pruned plant was not suitable for sale by June, and the quality continued to decline throughout the summer. The untreated controls developed much milder symptoms early in the season, and never became as severely diseased as the root pruned plants. Trees which were root pruned in April prior to October field potting were also symptomless at harvest. Needle loss on the lower limbs of trees in both treatments began after one week in the geenhouse. Eight weeks after harvest new gowth was beginning, and defoliation had become severe on many trees. By eleven weeks, the difference between treatments was pronounced. In contrast to the fall experiment, symptoms were significantly less severe on the root pruned plants in this goup (Figure 1.2). There is a need for more research into the preharvest conditioning of landscape sized spruce. There is limited information available in the liturature on the periodicity of root gowth in Colorado blue spruce, or the effects of root pruning and other cultural practices on root development. 16 17 t3 \ \ \ \ \ L5" / / 1 ./ / ./ JUNE JULY AUGUST - Roorr PRUNBD §\\\ CONTROL Figure 1.1. Root pruning I: June, July, and August evaluations of Phomopsis symptom severity on naturally infected, field-gown Colorado blue spruce root pruned October 26, 1990 and harvested April 3, 1991. 18 SYMPTOM SEVERITY / / r .f ./ fi/ WEEKS WEEK8 WEEKII - Roor PRUNBD CONTROL Figure 1.2 Root pnming II: Phomopsis symptom severity on naturally infected, field-gown Colorado blue spruce root pruned April 24, 1991 and harvested October 24, 1991. Trees were placed in a heated geenhouse November 7, 1991, and evaluated 5, 8, and 11 weeks after harvest. 19 Root gowth for white (P. glauca) and Sitka spruce (P. stichensit) generally occurs in two peaks during the gowing season. The first peak occurs from early spring until shoot expansion begins, the second begins after shoot gowth in midsummer (7,9,15). Root gowth capacity of Colorado blue spruce has been shown to increase from August to October (21). Root regeneration in waiters depends mainly on current photosynthate, and not on starch reserves (18,27). Trees root pruned in October and harvested the following April did not have the benefit of the gowing season to regenerate roots. The improved quality of spring treated plants may be due to increased density of roots in the harvested root system compared to the fall root pruned plants. Root pruning in early September would also allow more time for the regeneration of roots than late October treatment. Many gowers root prune in the fall because spring root pruning can reduce subsequent top gowth (3,13), and because the spring season is very busy. However, shoot gowth on the root pruned plants in these experiments was comparable to controls. July root pruning has been shown to increase long term survival of spruce seeding (6,17). Also, spring transplanting of young Colorado blue spruce results in no loss of quality (26). If further research shows that disease severity were reduced by spring root pruning, this practice may deserve further consideration where P. occulta is causing postharvest losses. Reduction in top gowth one or two seasons prior to harvest would be helpful if top pruning, and the resulting number of wounds were reduced. Needle and twig injuries 20 provide an entry point for P. occulta mycelia to colonize tissue and cause cankers (14). If the number of fibrous roots inside the root ball is increased, the trees should experience less stress, and be more resistant to canker development after , harvest (2,23,25). Large increases in the amount of roots inside the root ball have been reported for landscape sized Colorado blue spruce which were root primed only once, several years before harvest (28). Multiple root pruning before harvest may actually be detrimental (10). More testing of the frequency and timing of root pruning landscape sized spruce is needed, since spring root pruning shows some potential to improve the performance of spruce infected with P. occulta. Root Regeneration. The first evaluation of postharvest root regeneration was made six weeks following potting on December 17, 1990. At that time, there were very small white tips of newly formed roots visible on most plants. The white roots were formed primarily at the tips of uncut fibrous roots. The cut ends of the large primary roots of several plants had small amounts of callus formation, but the secondary roots were the site of most new root development. Very few roots had white tips over 5m when the data were collected. The size and quantity of root regowth during this period may have been geater than indicated by this data. A drop in the media temperatures occurred during the two weeks previous to the evaluation, causing rapid suberization of the new roots, and making them impossible to distinguish from the existing root system. 21 Difi'erences between the treatment means are large, but are not statistically significant due to high variation within treatments (Figure 1.3). The variation between blocks was not significant for any variable in this experiment. There was very little symptom development before the first sampling, and all treatments were similarly afiected (Table 1.2). Shoot moisture content was similar for all treatments, and comparable to the samples measured at the beginning of the experiment (Table 1.3).. Evaluation of treatments was repeated on May 22 1991. Most of the spruce had begun the spring gowth flush, indicating the end of the first peak period of root gowth. New roots were numerous on most trees, and originated primarily from fibrous secondary roots as indicated by the December observations. The cut ends of large primary roots occasionally gave rise to new roots, but these were usually short and contributed little to the mass of new roots. Trees harvested for this study were seedling, and therefore varied in size and mass of the root system. In order to reduce the impact of their variation on the results, data presented in Figure 1.4 are fresh weight of new roots as a percentage of the entire root system. IBA treatment and media type significantly affected the amount of root regeneration. There was no significant interaction between factors. A significant improvement in root system regeneration was achieved through the use of a root dip in a solution containing 1000 ppm IBA, confirming results obtained by Carter and Tripepi on younger blue spruce (4). NEW ROOTS > 1mm LENGTH Figure 1.3 Root regeneration: Number of white roots > one millimeter in length on field-gown Colorado blue spruce, when treated with 1000 ppm IBA and potted in wood chip medium or field soil. Trees potted October 27, and evaluated December 17, 1990. Table 1.2. Root regeneration: Phomopsis symptom severity on naturally infected, field-gown Colorado blue spruce, when treated with 1000 ppm IBA and potted in wood chip medium or field soil. Trees potted October 27, and evaluated December 17, 1990. Symptoms of Sampled Plants" Auxin Treatment Container Mix Field Soil 1000 ppm IBA in 10% 1.5 1.8 EtOH 10% EtOH 1.2 1.2 water 1.7 1.1 Source of Variation Media NS Auxin NS Media x Auxin NS ‘ Symptom severity rated on a scale of 1 (no symptoms) to 5 (dead); trees rating higher than 2 would be considered unsalable. NS = Not significant at P - 0.05, according to F test. Table 1.3. Root regeneration: Shoot moisture content of naturally infected, field- gown Colorado blue spruce, when treated with 1000 ppm IBA and potted in wood chip medium or field soil. Trees potted October 27, and evaluated December 17, 1990. Shoot Moisture Content (% of fresh weight) Auxin Treatment Container Mix Field Soil 1000 ppm IBA in 10% 58.04 56.63 EtOH 10% EtOH 56.19 57.90 water 55.37 57.16 Pre-test samples" 54.84 Source of Variation Media NS Auxin NS Media x Auxin NS " Measurements from twenty plants taken at the beginning of the experiment. NS =- Not significant at P = 0.05, according to F test. NEW ROOT WEIGHT (% of total) Figure 1.4 Root regeneration: Fresh weight of new roots (as a percentage of root system fresh weight) on field-gown Colorado blue spruce, when treated with 1000 ppm IBA and potted in wood chip medium or field soil. Trees potted October 27, and evaluated May 22 1991. 26 In their work, 1000 ppm IBA in 100% ethanol solution reduced root regeneration by over 50% compared to IBA in 20% ethanol. Reduction in new root fresh weight was also observed in this study, but ethanol and water controls were not statistically different. Root regowth and regeneration was also improved by potting the trees in container medium rather than field soil. This result is not surprising considering the physical properties of the two media. Several researchers have demonstrated the harmful effects of poor aeration and high bulk density media on the gowth and development of conifer seedling (8,16,19). Root and shoot moisture data from the May evaluation are shown in Table 1.4. Shoot moisture levels were similar for all treatments, and were approximately 15% higher than the November controls and plants sampled in December. This difierence between fall and spring sampled plants is consistent with the Stage of gowth during the evaluations and with normal seasonal variations in plant water content. It is doubtful that this difference occurred as a consequence of the new root gowth. Treatment differences would be expected if there were any correlation with the amount of new root gowth. There were also no significant differences between treatments for root moisture content. The goal of this study was to determine if the severity of Phomopsis symptom development could by reduced by improving the postharvest root regeneration of blue spruce. Unfortunately, the expression of Phomopsis shoot blight symptoms was not significantly affected by the amount of root regeneration. Table 1.4. Root ' n: Root and shoot moisture content of naturally infected, field-gown Colorado blue spruce, when treated with 1000 ppm IBA and potted in wood chip medium or field soil. Trees potted October 27, 1990 and evaluated May 22, 1991. Moisture Content (% of fresh weight) New Roots Shoots Auxin Treatment Mix \Soil Mix Soil 1000 ppm IBA in 10% 92.24 91.57 71.80 61.50 EtOH 10% EtOH 91.15 89.98 63.63 70.26 water 92.10 91.43 65.17 71.04 Source of Variation Roots Shoots Media NS NS Auxin NS NS Media X Auxin NS NS NS = Not significant at P = 0.05, according to F test. 28 The evaluation of plants sampled in May, and of a third complete set of treatments observed through the summer showed no meaningful reduction in symptom severity in any of the treatments (Table 1.5 and Table 1.6). The visible shoot blight symptoms did not appear serious on these plants until late May. By this time the new shoots were expanded, temperatures were higher, and the trees were transpiring rapidly. Phomopsis cankers can expand under the bark of woody hosts without causing blight symptoms until the host is under stress. The lesions did not develop enough during the cool months of early spring to interfere with root regeneration, but became injurious following bud ‘ break. Cultural control of this disease would be more successful if efforts address reducing stress during harvest, and preventing the gowth of incipient infections. Over-wintering Study. The winter storage conditions of these spruce proved to be an important factor in their performance during the following gowing season. The difference in treatments is due mainly to the increased number of severely or fatally afiected trees. Table 1.7 shows the number of trees in each goup which were unsalable (living), and the number which were dead by August. Spruce which were overwintered outdoors developed symptoms earlier than the other goup, and continued to decline through the season (Figure 1.5). Wind desiccation and freezing injury to the root systems of these plants may have resulted in water stress, which is known to make conditions more favorable for canker development (25). 29 Table 1.5. Root regarerntion: Phomopsis Symptom severity on naturally infected, field-gown Colorado blue spruce, when treated with 1000 ppm [BA and potted in wood chip medium or field soil. Trees potted October 27, and evaluated May 22, 1991. Symptoms of Sampled Plantsx Auxin Treatment Container Mix Field Soil 1000 ppm IBA in 10% 1.8 1.7 EtOH 10% EtOH 1.9 1.6 water 1.4 1.7 Source of Variation Media NS Auxin NS Media x Auxin NS ‘ Symptom severity rated on a scale of 1 (no mptoms) to 5 (dead); trees rating higher than 2 would be considered unsalable. NS - Not significant at P =- 0.05, according to F test. 30 Table 1.6. Root regeneration: Symptom severity on naturally infected, field-gown Colorado blue spruce, when treated with 1000 ppm IBA and potted in wood chip medium or field soil. Trees potted October 27, and evaluated August 15, 1991. Symptom Severityx Auxin Treatment Container Mix Field Soil 1000 ppm IBA in 10% 1.9 2.0 BtOH 10% EtOH 1.8 2.3 water 1.3 1.5 Source of Variation Media NS Auxin NS Media x Auxin NS ‘ Symptom severity rated on a scale of 1 (no symptoms) to 5 (dead); trees rating higher than 2 would be considered unsalable. NS = Not significant at P =- 0.05, according to F test. 31 Table 1.7. The efi'ects of overwintering and benomyl applications: Phomopsis symptom severity on naturally infected, field-gown Colorado blue spruce when treated with benomyl once in June, or monthly in June, July, and August 1990, harvested October 11, 1990, and overwintered outdoors or inside a poly covered quonset house. Plants evaluated August 15, 1990. Symptom Unsalable Dead Treatment Severityx (living) Inside / One Spray 1.6 1 1 Inside / Three Sprays 1.7 l 0 Outside / One Spray 2.65 1 6 Outside / Three 2.05 2 2 Sprays Source of Variation (Tmt Means) Overwintering ” Method Benomyl Applications NS Overwintering x NS Benomyl ‘ Symptom severity rated on a scale of 1 (no symptoms) to 5 (dead); trees rating higher than 2 would be considered unsalable. NS = Not significant at P = 0.05, according to F test. ” - Significant at P- 0.01, according to F test. 32 1.8“ 1.6: "P \X\\\\ SYMPTOM SEVERITY 1.4- / / I 1.2 ./ ./ ./ JUNE JULY AUGUST - UNPRO'I'ECTED \\\\\\\ POLYHOUSE Figure 1.5. Overwintering: Phomopsis symptom severity evaluations for June, July, and August on naturally infected, field-gown Colorado blue spruce harvested October 1 1, 1990, and overwintered outdoors or inside a poly- covered quonset house. Plants evaluated August 15, 1990. (Data averaged over fungicide treatments). 10. LITERATURE CITED Atkinson, D. 1980. The distributiOn and effectiveness of the roots of tree crops. In: Horticultural Reviews, Volume 2. Jules J anick, Ed. AVI Publishing, Westport, Conn. p. 424-590. Bier, J .E. 1964. The relation of some bark factors to canker susceptibility. Phytopathology. 54:125-248. Buse, LJ. and Day, RJ. 1988. Morphological effects of root pruning and wrenching white spruce transplants. N. J. App. For. 5:245-247. Carter, J .E., and Tripepi, RR. 1989. Lifting date influences the ability of auxins to promote root regeneration of Colorado spruce. J. Environ. Hort 7: 147-150. Delp, CJ. 1988. Fungicide resistance problems in perspective. In: Fungicide Resistance in North America. APS Press, St. Paul, Mn. p.4-5. Eis, S. and Long, LR. 1971. Lateral root pruning of Sitka spruce and hemlock seedling. Can. J. For. Res. 2:223-227 Ford, E.D., and Deans, J .D. 1977. Growth of a Sitka spruce plantation: Spatial distribution and seasonal fluctuations of lengths, weights and carbohydrate concentrations of fine roots. Plant and Soil. 47:463-485. Fuchigami, LH. and Moeller, F.W. 1978. Root regeneration of evergeen plants. Combined Proceeding of the International Plant Propagator’s Society-283949. Gilman, E.F. 1990. Tree root gowth and development. L Form, spread, depth and periodicity. J. Environ. Hort. 8:215-220. Gilman, ER 1990. Tree root gowth and development. 11. Response to culture, management, and planting. J. Environ. Hort. 8:220-227. 33 11. 12. 13. 14. 15. 16. 17. 18. 19. 21. 34 Hahn, G.G. 1943. Taxonomy, distribution, and pathology of Phomopsis occulta and P. junipemvora. Mycologica. 35 : 112-129 Harris, R.W., et al. 1971. The influence of transplanting time in nursery production. J. Amer. Hort. Sci. 109:827-831. Hobbes, S.D, Stafi'ord, S.G., and Slagle, R1. 1987. Undercutting conifer seedling: efi'ect on morphology and field performance on draughty sites. Can. J. For. Res. 17:40-46. Igoe, MJ. 1990. The relationship between Phomopsis occulta (Trav.) and the postharvest disease of Colorado blue spruce (Picea pungent glauca Engelm.). MS Thesis, Michigan State University, East Lansing, MI. Johnson-Flanagan, AM., and Owens, IN. 1985. Root gowth and root gowth capacity of white spruce (Picea glauca (Moench) Voss) seedlings. Can. J. For. Res. 15:625-630. Leyton, and Rousseau. 1958. Root gowth of seedling in relation to aeration. In: The Physiology of Forest Trees. K. Thimann, Ed. pp.467- 475. Mullin, RE 1966. Root pruning of nursery stock. For. Chron. 42:256-264. Philipson, J .J . 1988. Root gowth in Sitka spruce and Douglas-fir transplants: dependance on the shoot and stored carbohydrates. Tree Phys. 4:101-108. Provost, M. and Bolgghari, HA. 1990. Growth and rooting ability of black spruce of two origins as a function of apparent soil density and soil water properties. Can . J. For. Res. 20:185-192 Sanderson, P.G., and Worf, 6.1... 1986. Phomopsis shoot blight of Colorado blue spruce. J. Env. Hort. 4: 134- 138. Schutzki, R. 1989. Efi'ects of water stress on Picea seedling. PhD Dissertation, Department of Forestry, Michigan State University, East Lansing, MI. Singh, 0., Sharma, H.P.,and Sharma, SK. 1984. Effect of root clipping on the gowth of transplanted spruce seedling. J. Tree Sci. 3:149-152. 24. 35 Shoenweiss, D.F. 1978. The influence of stress on diseases of nursery and landscape plants. J. Arb. 4:217-225. Shoenweiss, D.F. 1981. The role of environmental stress in disease of woody plants. Plant Dis. 65 :308-3 14 Shoenweiss, D.F. 1981. Infectious diseases of trees associated with water and freezing stress. I. Arb. 7:13-18. Tripepi, R.R., and Carter, J .E. 1989. Growth and quality of Colorado spruce transplanted during the spring gowth flush. J. Env. Hort. 4:151-154. van den Driesshe, R. 1987. Importance of current photosynthate to new root gowth in planted conifer seedling. Can. J. For. Res. 17:776-782. Watson, G.W. 1987. The effect of root pruning on the root system of nursery trees. J. Arb. 13:126-130 White, RP. 1929. Juniper blight. Ann. Rept, NJ.Agi. ExpLSta. p.270-272. CHAPTER II INCIDENCE, HOST RANGE, AND CHEMICAL MANAGEMENT OF PHOMOPSIS CANKER OF COLORADO BLUE SPRUCE (PICEA PUNGENS GLA UCA) IN MICHIGAN. 36 ABSTRACT Phomopsis occulta was isolated from spruce tissue collected in several counties across southern lower Michigan. Five species of spruce were found to be hosts, including Colorado blue (Picea pungent glauca), white (P.glauca), Black Hills (P.g. ‘Densata), Norway (P. abies), and Engelmann spruce (P. engelmanii). Inoculation showed that Spartan spruce (P. pungens X P. glauca) is also susceptible. Colorado blue spruce seedling at six stages of shoot development were inoculated with conidia of P. occulta. Shoots which expanded for two weeks following budbreak were more susceptible to shoot blight than dormant plants, as well as those inoculated after 4, 6, or 8 weeks of shoot expansion. In vitro screening of eight fungicides in amended potato dextrose agar (PDA) shoWed that P. occulta was completely inhibited by 1 ppm benomyl, 10 ppm myclobutanil and propiconazole, 100 ppm triadimefon, trifiorine, mancozeb, and 1000 ppm bordeaux. F enarimol supressed gowth, but provided incomplete control at 1000 ppm. Benomyl was applied to field gown plants before and after fall field potting. Sprays in July and August prior to digging had no significant effect on disease development. Sprays applied on the first day after harvest, or three times during shoot expansion in the spring following harvest reduced symptom severity. 37 INTRODUCTION Shoot blights and stem cankers caused by Phomopsis occulta result in serious production and postharvest losses of spruce (Picea sp.) gown in Michigan landscape and Christmas tree nurseries. Colorado blue spruce (P. pungens glauca) has been the most severely affected species in recent years. Postharvest canker development results in substantial losses and increased production costs which have caused some gowers to reduce the number of blue spruce entering the production cycle. Long term suppression of P. occulta will be achieved by implementing a progam which combines several appropriate fungicides and proper timing to use the least amount of pesticide for adequate control. This strategy, combined with effective cultural control measures, should be adopted in order to avoid fungicide resistance (5, 19,22). Chemical control of Phomopsis occulta was examined by Igoe (10) who reported the in vitro activity of four fungicides against Phomopsis occulta. Her research showed that ipriodine and chlorothalonil were very inhibitory to mycelial gowth and benomyl arrested gowth of the fungus at lppm. However, the fungicides failed to reduce symptoms on naturally infected, field gown plants which were sprayed once a month in June, July, and August prior to fall digging. 38 39 The failure of those treatments was probobly due to the presence of advanced cankers in the trees before treatment. The effectiveness of benomyl in the field has been reported by a gower in Wisconsin, who has successfully managed Phomopsis on their spruce crop by applying 2.2kg of benomyl/3581 of water (11b/100gal) three times during budbreak and shoot expansion at approximate 3 week intervals to protect the plants from infection (1). However, an effective long-term progam to control P. occulta should not rely on a single frmgicide. Many fungicides have been tested against other species of Phomopsis which cause blights and cankers on woody hosts which may have potential for controlling P. occulta. Benomyl is the primary compound used in the control of Phomopsis diseases of juniper, blueberry, arizona cypress and russian olive (2,1 1,13,18,20). Piperazine controls stem cankers on gape and blueberry, but failed to reduce the severity of juniper tip blight (14). Phomopsis fruit rot of gape can be controlled by two applications of mancozeb (15). Fungicide applications should be timed to protect the crop during periods of maximum susceptibility. These conditions occur when inoculum level is high and the host plant is at a stage of gowth which is vulnerable to infection. For most species of Phomopsis, the optimum conditions for infection and colonization include warm, humid weather and a host with tender new gowth (4,8,9,12). The mycelia of P. occulta gows most rapidly at around 25 ' C (77 ' F) (10), which is close to the peak temperature for gowth and conidia germination in many Phomopsis species (8,16,15). Sanderson and Worf found the highest rates of 4o infection of Colorado blue spruce when seedling were incubated at warm temperatures (28' C/ 82' F) and high relative humidity (17), though Incipient infections may also form during less ideal conditions (10,17). The experiments described in this chapter were designed to investigate an effective fungicide progam for the suppression of Phomopsis occulta in Colorado blue spruce production by determining the most susceptible stage of shoot gowth for infection. An attempt was also made to understand the relative activity of several fungicides, and the effectiveness of pre and postharvest benomyl spray applications. MATERIALS AND METHODS Host Range and Distribution in Michigan. During May and June of 1990, samples of symptomatic spruce tissue were collected from nurseries in eastern, central, and western lower Michigan. Samples were washed with running water, soaked in 10% bleach for 5 minutes, then rinsed repeatedly in sterile water. The outer bark was removed and small sections were taken from canker margins and placed on potato dextrose agar (PDA) plates. The cultures were incubated for 5- 7 days before evaluation. Susceptibility of Four Spruce Species to P. occulta. Seedling trees of Norway spruce (Picea abies), Colorado blue spruce (Picea pungens glauca), white spruce (Picea glauca), and Spartan spruce (Picea pungent x Picea glauca) were purchased as 6-8" A-G (accelerated gowth) plug from Van’s Pines, West Olive, MI. All the trees were gown in a peat based media (Baccto, Michigan Peat Company) under accelerated optimal gowth conditions (3). The trees were at approximately the same stage of shoot expansion when inoculated (three weeks after budbreak). Sixteen plants of each species were inoculated. Eight trees of each species were root pruned by removing the pot and cutting the root ball in half, severing approximately 60% of the roots. Inoculum was prepared from sporulating cultures of P. occulta by placing 3 ml of sterile deionized water onto the plates and scraping the surface gently with a sterile glass rod. The resulting suspension was filtered through a sterile triple 41 42 layered cheesecloth and the concentration of a conidia determined with the aid of a hemacytometer. The suspension was further diluted with sterile water to a final concentration of approximately 121x107 a conidia/ ml. The inoculum was sprayed onto the seedling with a spray bottle until droplets formed on the stem and leaves (about 30 ml per plant). The control trees were sprayed similarly with sterile deionized water. All plants were placed on a geenhouse mist bench and covered with a 4ml poly tent. Mist was kept on for 30 second intervals every 8 minutes for 72 hours. The mist heads were shielded to prevent water splash from inoculated plants to controls. Temperature in the chamber was 30' :t 5'C (86' t 8'F), with 88% relative humidity. Disease symptoms were visually rated 6 weeks after inoculation. The rating are based on ascaleof1t05,where 1 a nosymptoms, and5= dead. Plant Growth and Pathogen Susceptibility. Colorado blue spruce seedling used in this experiment were purchased as 6-8” A-G (accelerated gowth) plug from Van’s Pines Nursery in West Olive, MI. They were potted Classic 600 nursery containers using a peat based media (Baccto, Michigan Peat Company). The seedling were gown in a geenhouse under accelerated optimal gowth conditions (3) for 4 months. The photoperiod was then reduced to 8 hours to allow budset. On March 10 1991, 90 plants were moved to a 4.4'C (40'F) vernalization cooler with an 8 hour photoperiod. Fifteen trees were moved from the cooler to the geenhouse every 2 weeks beginning on July 15 1991. After two weeks in the geenhouse the spruce were just beginning to break bud. 43 Bud development within each goup was quite uniform, and each successive set responded in the same way. When the last set was brought to the geenhouse on September 23 1991, 10 trees from each goup were sprayed with a conidial suspension of P. occulta. Six goups of seedling were treated. The stages of shoot development were: dormant, breaking bud, and 2, 4, 6, and 8 weeks of shoot gowth after budbreak. By eight weeks of expansion, lateral buds appeared to be fully formed with bud scales present. A suspension containing 7.1X10‘5 a (alpha) conidia/ ml was prepared from sporulating cultures of P. occulta gown on PDA. The conidia were harvested by placing 3 ml of sterile deionized water onto the plates and scraping the surface with a glass rod. The suspension was filtered through several layers of sterile cheesecloth, and the concentration of spores was determined with the aid of a hemacytometer. The solution was sprayed onto the seedling with a spray bottle to the point of saturation. Control plants were sprayed similarly with sterile deionized water. After inoculation, all plants were placed on a geenhouse mist bench and covered with a 4ml poly tent. Mist was applied automatically for 30 second intervals every 2.5 minutes. The mist heads were shielded to prevent water splash from inoculated plants to controls. Temperature in the chamber was 30' z 5 'C (86' :t: 8'F). Relative humidity was maintained at approximately 92%. After 72 hours the mist was turned off, and poly cover removed from the bench. Disease severity was determined four weeks later by counting the number of infected branches per tree. 44 In Vitro Fungicide Screening. Fungicide amended agar was used to test the relative effectiveness of several fungicides in vitro. This ‘poison agar’ technique was used to evaluate the eight fungicides listed in Table 2. 1. Potato dextrose agar (PDA) was combined with each compound to produce standard 100x15 mm petri plates containing 20 ml each of agar amended with 1, 10, 100, or 1000 ppm of fimgicide. Ten plates were prepared for each treatment, including 10 unamended PDA controls. Plug of P. occulta were removed from 20 day old cultures using a 10 mm cork borer. The top half of each disk was transferred to the agar, where it was placed inverted in the center of the plate. Plates were placed in plastic bag in randomized complete blocks, and incubated in the dark at 25 - C Radial growth of each colony was measured after 10 days of incubation. The experiment was repeated once using 5 replicate plates per treatment. Field Test of Benomyl. An experiment was conducted to test the effectiveness of benomyl applications on 15-18" Colorado blue spruce at various intervals before and after harvest. Benomyl was applied as Benlate 50 WP at 2.2kg/ 3581 of water (11b/100 gal) for all treatments. Preharvest sprays were applied by trained nursery personnel using a tractor sprayer. Postharvest treatments were applied with a hand sprayer equipped with a flat fan nozzle. The benomyl was sprayed to the point that droplets formed on the foliage. The experiment was set up as a 2x2x2 factorial. Plants were arranged in the holding area in randomized complete blocks. 45 Om NA .0552: cam—GEES- cameos -méEofloLCEoévéA—Eosqo~o39~Ye 385:3 Boo antigen—+2853 use: a. B 9. tag- canonaocoaoieaeaene fiancee? :23. 962 mum ouggggnoeoifio -8053 canoe—BE Be.— .6 8a 2352 . Aoegoaaeéoeoeieo om e a .ocoEoE.~.~.~Yae.E353834;7:5 8:an Oz..— Senna has 8863 cacao Ea seas 3&8 58.8 see .828 Eon—«£8 “zone—egos as on Absences BEE aces. 3882. see .6 228m oeonsBeAEaeaEifi a? an .5I-_E.oa_?m.m-$oeonaecozver escapee see: cease . soaecegeiaaeoe crosses. om 3 .2Essa—incense28.46%.— o_o§_8_aoa $8.36 scene m2 F = .0016). The most severe symptoms appeared on plants which had undergone 2 weeks of shoot gowth prior to inoculation (Figure 2.2). These seedling were significantly more susceptible to colonization and canker development than plants in the other 5 51 %BLIGHTEDTIPSITREE \ \ \ i\ \ \ \ 4L e ./ ./ ./ ./ ./ . DORM BRK TWO FOUR SIX EIGHT WEEKS OF SHOOT EXPANSION Figure 2.2 Plant growth and pathogen susceptibility: Percent of shoot tips blighted on Colorado blue spruce seedling at six stages of shoot development. Plants evaluated 4 weeks after inoculation with conidia of Phomopsis occulta. Stages of shoot gowth: DORM = dormant, BRK = budbreak, 2, 4, 6 and 8 weeks of shoot gowth after budbreak. 52 goups. This does not prove that spruce are immune to infection by P. occulta during dormancy or after the Shoots have hardened off. The Phomopsis fungi are known to form incipient infections without producing symptoms until conditions are favorable. However, the tissue which is the most vulnerable to canker development would likely .provide favorable conditions for an increased percentage of the incident conidia to successfully penetrate the host tissue under field conditions. Fungicide sprays should be timed to protect the new gowth from conidia penetration, and to suppress the development of existing infection sites when the plants are most susceptible. A series of applications of protectant fungicides should commence immediately at the first indication of bud break, and continue until the new shoots are fully developed, 8-10 weeks after budbreak. Though P. occulta conidia may be present later in the season, the number and severity of resulting infections should not result in unacceptable aesthetic or physiological damage. If the levels of incipient infections are reduced, and the trees are under no severe stress, they will perform well following harvest. In Vitro Fungicide Screening. Data from both replications of this study were pooled for analysis and presentation in the following discussion. All of the compounds tested in this experiment were effective in inhibiting P. occulta mycelial gowth at high concentrations (Figure 2.3). The colonies were inhibited completely at 1000 ppm a.i. of all the compounds except fenarimol, where colonies gew only about 7mm in radius in 10 days. ._oEm._a=o_uZm—m .==2=ao_u>8u0>2 £3858u2<2 6389:"th innovuoouMOm .Saoaoauzmm douofiamvmcuun—E .o_ona=8_eounuO~—m .8283an 2 30852 San— sone—8 Eocene 5 Sea 08E 2 autumn—Eb mam 3:095. 88 3 .0. mm 3 5:33.: Lo 99% S San Home vacuum—a 0239.3 no mow—28 humofioam Lo 532w 36am "meanest“. samurai can; S .WN 2:3"— . 6 .n ..m m .2 N N D D 3 w m ”A m a m m .3 sz—QMEOZH m>ED< 2mm 83 E—Qmmozm m>ED< 2.: 8— 53 ”i: \‘ S M? my anmwMMLNMm (W) mono mm (m) mono mavu EHQNMDZ— m>E< SEQ S Euflmdczm m>E< 2A: fl _ 54 Propiconazole, benomyl, and myclobutanil were clearly the most effective at very low levels (1 ppm). Data obtained by Igoe for ipriodine and chlorothalonil are in agreement with the results found in this study for piperazine (10). Field Test of Benomyl. The trees in this experiment showed few symptoms of infection in May, but following budbreak new shoots began to wilt, and needles were dropping by mid June. The symptom development for most treatments progessed steadily throughout the season. Cankered branches were completely defoliated by the August evaluation. The August evaluation data are presented in Table 2.2. The fact that July and August applications of benomyl in the field did not provide any additional control was also observed in another study (Chapter 1). The June spray may have been beneficial since shoots are more susceptible to infection when succulent foliage is present. Benomyl applications on the first day after harvest and three times during shoot expansion after harvest were statistically significant factors in reducing disease severity. One fungicide treatment after harvest reduced average symptom severity from as well as trees which received three benomyl sprays the spring after harvest. It is important to note that these data are averaged over all other treatments. It is doubtful that postharvest applications alone will provide satisfactory control of P. occulta. The notable result of this study is the gadient in disease severity which decreases with additional applications of fungicide. Continuous spraying with fungicide helped to prevent the advance of the fungus. Trees in the treatment which received all 55 seven sprays remained symptomless until the July evaluation, then showed only the slightest disease expression during the following months. However, it is unlikely such applications would be cost effective or practical. in nursery production and subsequent handling. Retarding the gowth of existing cankers is an inefficient strategy. Chemical control should be applied to protect the trees from infection. This approach will provide better control with less pesticide input and labor. The long term suppression of P. occulta with chemical control agents will be most successful if anti-resistance strategies are adopted at the initiation of the . control progam. Minimizing the total use of fungicides and particularly the use of any one agent reduces the selection pressure on the pathogen, delaying or preventing the buildup of resistant strains (6,22). Methods for management of resistance to benzamidole fungicides include cultural practices, resistant genotypes, and the use of a variety of fungicides (7). Lacking effective cultural control methods or resistant blue spruce, the primary management tool available in the case of Phomopsis is the use of several difierent fungicides. Companion fungicides can be used in alternation, mixtures, or combinations of mixtures and alternations. The fungicides should be chosen from two or more classes of chemicals with different modes of action, in order to avoid cross-resistance (21) 56 Table 2.2. Field test of benomyl: Phomopsis symptom severity on naturally infected, field-gown Colorado blue spruce when treated with benomyl in the field and after harvest. Trees harvested October 18, 1990, and evaluated August 15, 1991. Symptom Severityx Sprayed 1‘t Day Unsprayed 1*"t Day 1W6 Postharvest Postharvest 3 Applications During Shoot Expansion/ Field Applications YES NO YES N 0 June Only 1.2 1.3 1.4 2.1 June, July, and August 1.0 1.2 1.2 1.8 Source of Variation Field Ns 1St Day Postharvest ‘ ‘ (PH) Field x PH NS Shoot Expansion“, (SE) '- Field x SE NS PH x SE Ns Field x PH x SE _ NS ‘ Symptom severity rated on a scale of 1 (no symptoms) to 5 (dead); trees rating higher than two would be considered unsalable. Benomyl applied at budbreak, then three weeks, and seven weeks later. NS, " = nonsignificant at P= 0. 05, and significant at P= 0.01 respectively, according to the F test. 10. LITERATURE CITED Amos, R. 1990. Production Manager, Evergeen Nursery, Sturgeon Bay, WI. Personal communication. Anonymous. 1991. Fruit spraying calendar for commercial gowers. Extension Bulletin E 0145, Michigan State University, East Lansing, MI. p.61 Bongarten, BC, and Hanover, J.W. 1985. Accelerating seedling gowth through photoperiod extension for genetic testing: a case study With blue spruce (Picea pungens). For. Sci. 31:631-643. Braun, AJ. 1972. Correlation of gapevine shoot gowth with maturity od Phomopsis viticola pycnidia. Phytopathology. 62:521 (Abstract). Dekker, J. 1976. Acquired resistance to fungicides. Ann. Rev. Phytopathology. 14:405-428 Delp, CJ. 1980. Coping with resistance to plant disease control agents. Plant Dis. 64:652-657 Delp, CJ. 1988. Resistance management strategies for benzamidoles. In: Fungicide Resistance in North America. APS Press, StPaul, Minnesota. p.41-43 Hahn, G.G. 1943. Taxonomy, distribution, and pathology of Phomopsis occulta and P. juniperovom. Mycologica 35:112-129. Hansen, EM. and Hamm, RB. 1988. Canker diseases of Douglas-fir seedling in Oregon and Washington bareroot nurseries. Can. J. For. Res. 18: 1053-1058. Igoe, MJ. 1990. The relationship between Phomopsis occulta (T rav.) and the postharvest disease of Colorado blue Spruce (Picea pungens glauca Engelm.). MS Thesis, Michigan State University, East Lansing, MI. 57 11. 14. 15. 16. 17. 18. 19. 20. 21. 58 Marsh, R.W. Ed. 1977 . Systemic Fungicides. Longnaanc. NY, New York. Morin, 1.. Watson, AK, and Reeleder, RD. 1990. Effect of dew, inoculum density, and spray additives on infection of field bindweed by Phomopsis convulvus. Can. J. Plant Path. 12:48-52. Parker, RE. and Ramsdell, D.C., 1977. Epidemiology and Chemical Control of Phomopsis Canker of Highbush Blueberry. Phytopathology 67:1481-1484. Peterson, G.W., and Hodges, CS. 1975. Phomopsis blight of junipers. Forest Pest Leaflet 154. USDA Forest Service, Washington, DC. Pscheidt, J .W. 1989. Time of Infection and Control of Phomopsis Fruit Rot of Grape. Plant Disease 73:829-833. Rupe, J .C. 1990. Effect of temperature on the rate of infection of soybean seedling Phomopsis Iongicolla. Can. J. Plant Path. 12:43-47. Sanderson, P.G. and Worf, G1. 1986. Phomopsis shoot blight of Colorado blue spruce. J. Environmental Hort. 4: 134-138. Smyly, WB. and Filer, TH. 1973. Benomyl Controls Phomopsis Blight on Arizona Cypress in a Nursery. Plant Disease Reporter 57:59-61. Staub, T. and Sozzi, D. 1984. Fungicide resistance: A continuing challenge. Plant Dis. 68:1026-1031. Stewart, J. and Worf, G. Fungicidal control of Phomopsis canker of Russian Olive. Fung. Nem. Test Res. 38:185 Urecht, PA. 1988. Companion products for use in fungicide resistance strategies. In: Fungicide resistance in North America, C.J. Delp, Ed. p 95-97. Wade, M. 1988. Strategies for preventing or delaying the onset of resistance to fungicides and for managing resistance occurrences. In: Fungicide resistance in North America, C.J. Delp, Ed. p 14-15. "Illillllllllillllilli