llWlHlllWW1ININIHIWIHIHIWIWWHHHHI llllllljlzll\\\\\\\\\\\\\\\\\l\\\lllllllllWill 3104239 mzszs - A,” . 5-“ it LIBRAR Y ms" BA lib)? L93 9!; r3 3;;- Um?) I! at; if}? *1 '5': §‘~.' “v1 (.31 p}. This is to certify that the thesis entitled EUTYPA DIEBACK OF GRAPE (VITIS LABRUSCA L.) CAUSED BY EUTYPA ARMENIACAE I. INFECTION AND CONTROL STUDIES II. FLUORESCENT ANTIBODY STUDIES presented by Elie Hy Gendloff has been accepted towards fulfillment of the requirements for M.S. figmein Botany and Plant Pathology fl‘mrz/ Cl /€4wc¢/t’:‘ 1-7/7 Major professor Date 9L. ZS; I781 0-7639 OVERDUE FINES: 25¢ per day per item RETURNING LIBRARY MATERIALS: Place in book return to remove charge from c1 rculctton records EUTYPA DIEBACK 0F GRAPE (VITIS LABRUSCA L.) CAUSED BY EUTYPA ARMENIACAE I. INFECTION AND CONTROL STUDIES II. FLUORESCENT ANTIBODY STUDIES By Elie Hy Gendloff A THESIS submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Botany and Plant Pathology 1982 ABSTRACT EUTYPA DIEBACK 0F GRAPE (VITIS LABRUSCA L.) CAUSED BY EUTYPA ARNENIACAE_-__' I. INFECTION AND CONTROL STUDIES II. FLUORESCENT ANTIBODY STUDIES By Elie Hy Gendloff Fungicide field trials were conducted for two years in a com- mercial vineyard at Lawton, MI. BenlateR 50%NP sprays of l.2 to 9.6 g/L gave significant control of Eutypa armeniacae ascospore infection of pruning wounds made on two-year-old wood. Other treatments gave little or no control. Artificially induced frost injury and simulated mechanical harvester injury sites were not susceptible to g, armeniacae infection. Differences in infection were found among treatments varying in temperature regimes after inoculation. Antisera were made to whole cell and cell wall preparations of g, armeniacae. Specificity of these antisera was fairly poor when various fungi were stained on glass slides with rhodamine isothiocyanate-conjugated antisera, but was improved by cross- adsorption with Phomopsis viticola. Cross sections of grape wood, inhabited by various fungi, were stained directly and indirectly with these antisera. Hyphae in the indirectly stained wood sections fluoresced brighter than hyphae in directly stained wood sections. To my parents ii ACKNOWLEDGEMENTS I would like to express my sincere appreciation to Dr. Donald Ramsdell for his guidance, support, encouragement and friendship during this work. I would also like to thank Dr. Clyde Burton for his sup- port and suggestions. I would also like to acknowledge the aid of Dr. Burton, Dr. Alfred Saettler and Dr. Wayne Neidlich for serving on my guidance committee. To Dr. Leo Mericle and especially Dr. James Asher, I extend my appreciation for the use of their equipment. Also, thanks go to Dr. Karen Baker for aid in fungal culture identifica- tion. I would also like to acknowledge the Michigan Grape Growers Association for their interest and support. Finally, I would like to thank all my friends and col- leagues for making my Master's research a valuable and enjoyable experience. TABLE OF CONTENTS LIST OF TABLES LIST OF FIGURES . PART I INFECTION AND CONTROL STUDIES INTRODUCTION . LITERATURE REVIEW MATERIALS AND METHODS . Fungicidal Control Trials, Year l Fungicidal Control Trials, Year 2 . Spring Frost Damage as a Possible E. armeniacae Infection Site . . . Simulated Mechanical Harvester Induced Injury as a Possible §_. armeniacae Infection Site . . Effect of Various Environmental Conditions on Infection of Potted Vines with g, armeniacae Isolations from the Vicinity of a Canker on Infected Vines . . . . . . . . . . . RESULTS Fungicidal Control Trials, Year 1 Fungicidal Control Trials, Year 2 . Spring Frost Damage as a Possible E. armeniacae Infection Site . . Simulated Mechanical Harvester Induced Injury as a Possible §_. armeniacae Infection Site . Effect of VariousTEnvironmental Conditions on Infection of Potted Vines with E} armeniacae Isolations from the Vicinity of a Cafiker onTInfected Vines . . . . . . . . . . DISCUSSION BIBLIOGRAPHY . iv Page vi viii 12 12 15 15 16 17 18 20 20 27 27 30 30 32 36 PART II FLUORESCENT ANTIBODY STUDIES INTRODUCTION LITERATURE REVIEW . MATERIALS AND METHODS Antigen Production . . . Immunization and Bleeding Schedule . Determination of Titer . Preparation of Fluorescent Antisera. Fluorescent Antibody Staining of Fungi on Glass Slides . . . . . . Staining of Fungi in .wood Sections . Observation of Material RESULTS Antiserum Titer Levels . Specificity of Fluorescent Antibody to Fungi on Glass. Slides . . Staining of Hyphae in Wood DISCUSSION BIBLIOGRAPHY Page 40 42 54 54 55 56 56 58 61 62 62 52 64 78 84 Table LIST OF TABLES PART I Cumulative Totals, Fungicide Trials, Year 1. Eut armeniacae Pruning Wound Infection (Lawton, MI Four’Blocks Combined. Cumulative Totals, Fungicide Trials, Year 1. Eut armeniacae Pruning Infection (Lawton, MI). BlocE 5 Excluded. . Analysis of Variance, Fungicide Trials, Year 1. Eut a armeniacae Pruning Hound Infection (Lawton, MI). Four Blocks, Excluding Fusarium lateritium Treatment Duncan' 5 Multiple Range Test, Fungicide Trials, Year Eut a armeniacae Pruning Wound Infection (Lawton, MI). Four Blocks, Excluding Fusarium lateritium Treatment. Difference Between Treatments at Both Inoculation Times . . . . Analysis of Variance, Fungicide Trials, Year L Eutypa armeniacae Pruning Wound Infection (Lawton, MI). Block 2 and FUsarium lateritium Treatment Excluded. Duncan' 5 Multiple Range Test, Fungicide Trials, Year 1. Eutypa armeniacae Pruning Wound Infection (Lawton, MI). Block 2 and Fusarium lateritium Treatment Exlcuded. Difference Between Treatments at Both Inoculation Times . Cumulative Totals, Fungicide Trials, Year 2. Eut a armeniacae Pruning Wound Infection (Lawton, MI Analysis of Variance, Fungicide Trials, Year 2. Eutypa armeniacae Pruning Hound Infection (Lawton, MI). Duncan' 5 Multiple Range Test, Fungicide Trials, Year 2. Eutypa armeniacae Pruning Wound Infection (Lawton, MI). Differences Between Treatments vi Page 21 22 25 25 26 28 29 29 Table Page 10. Eutypa armeniacae Infection of Inoculated Pruning Mounds Subjected to Various Environmental Conditions in Growth Chambers . . . . . . . . . . . . 21 PART II 1. Fluorescence of Fungi on Slides . . . . . . . . 63 2. Fluorescence of Fungal Hyphae in Grape Hood-- Indirectly Stained . . . . . . . . . . . . 77 vii Figure LIST OF FIGURES PART II Transmitted light view of Eutypa armeniacae mycelium stained with RITC-conjugated whole cell antiserum . . . . . . . . . . . Epifluorescence under fluorescence filters of Eutypa armeniacae mycelium stained with RITC- conjugated whoTe cell antiserum Transmitted light view of Fusarium lateritium mycelium stained with RITC-conjugated whole cell antiserum . . . . . . . . . . Epifluorescence under fluorescence filters of Fusarium lateritium mycelium stained with RITC- conjugated Whole céll antiserum Epifluorescence under fluorescence filters of Alternaria sp. mycelium stained with RITC- conjugated whole cell antiserum Epifluorescence under fluorescence filters of Alternaria sp. mycelium stained with RITC-conjugated cell wall antiserum Epifluorescence under fluorescence filters of Phomopsis viticola mycelium stained with RITC- conjugated celleall antiserum . Epifluorescence under fluorescence filters of Phomopsis viticola mycelium stained with RITC- conjugated ceTT'wall antiserum that has been cross-adsorbed with Phomopsis viticola Autofluorescence of cross section of grape wood under RITC fluorescence filters (as described in Materials and Methods) . . . . viii Page 65' 65 65 65 67 67 67 67 70 Figure 1D. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. Autofluorescence of cross section of grape wood under FITC fluorescence filters (blue interference 455- 490 nm excitation filter set, FT 510 chromatic beam splitter and LP 520 barrier filter) Transmitted light view of cross section of grape wood not stained. (Arrow shows hypha ) Autofluorescence under fluorescence filters of same unstained cross section of grape wood as in Figure 1] O O O O O O O O O O O Transmitted light view of cross section of grape wood indirectly stained with rabbit normal serum. (Arrow shows hypha. ) . . . . . . Autofluorescence under fluorescence filters of same normal serum-stained cross section of grape wood as in Figure 13 . . . . . Transmitted light view of cross section of grape wood directly stained with RITC-conjugated cell wall antiserum. (Arrow shows hyphae. ) . Autofluorescence under fluorescence filters of same directly stained cross section of grape wood as in Figure 15 . . . . . . . . Transmitted light view of cross section of grape wood indirectly stained with cell wall antiserum. (Arrow shows hyphae.) . . . . . . . Autofluorescence under fluorescence filters of same indirectly stained cross section of grape wood as in Figure 17. (Arrow shows hyphae.) . . Transmitted light view of cross section of grape wood indirectly stained with cell wall antiserum. (Arrow shows hypha behind pith cell wall.) . . . . Autofluorescence under fluorescence filters of same indirectly stained cross section of grape wood as in Figure l9. (Arrow shows hypha behind pith cell wall. . . . . . . . . . . . . . . ii Page 70 70 70 72 72 72 72 74 74 74 74 PART 1 INFECTION AND CONTROL STUDIES INTRODUCTION Eutypa dieback, also referred previously as "dying arm" (31) or "dead arm" (37,41), is an important disease of grapevine world- wide, causing cankering and necrosis of woody tissue. It has been reported in New York (2), Japan (17), Michigan (46), New Zealand (13), Australia (in 13), Ontario (48), California (26), Greece (19), Mexico (44) and France (in 44). Eutypa armeniacae Hansf. and Carter has only recently been proven to be the causal agent (27,30), due to early studies which had associated the leaf and cane spotting organism Phomopsis viticola with that role (12,41). In Michigan, Eutypa dieback has been estimated to occur in about 10% of the mature grapevines (45) and in California, disease levels of 81% have been reported (31). The purpose of this study is to follow up on the work of Trese (45) by conducting fungicidal control trials with compounds that were found in that work to be inhibitory to E, armeniacae jg_vitro, and by repeating experiments conducted in that report that assessed frost damage and mechanical harvester injury as sites of E, armeniacae infection. These studies also incorporate assess- ments of E, armeniacae infection and control factors determined to be important in other situations. Included here is an assessment of the biotic agent Fusarium lateritium Nees (shown effective on apricots in Australia) and determinations of wound susceptibility variation that may be influenced by variations in time of inocula- tion, age of wound and temperature after inoculation. LITERATURE REVIEW The ascomycete Eugypg_armeniacae Hansf. and Carter is described in detail by Carter (3). Briefly, the perfect stage is found on dead grape or apricot wood in the form of many perithecia imbedded in a carbonaceous black stroma. Numerous asci develop through a centrum of disintegrating pseudoparenchymatous tissue. Paraphyses are numerous but obscured in the mature perathecia by the very numerous asci. Eight allantoid, nonseptate ascospores with a pale yellowish brown color, 7 to 11 by 1.5 to 2pm, are produced in each ascus. After a wetting period, these spores are forceably discharged up to 1 mm as an octad, all eight held together by the mucilaginous contents of the ascus (3). Muller and von Arx (34) place EgEypg_in the Diatrypaceae of the Sphaeriales. The imperfect stage of E, armeniacae is Qytosporina sp. of the family Sphaeropsidaceae in the order Sphaeropsidales. The pycnidia, which are produced in culture as well as on wood, are black and subglobose with a very short ostiole or apical pore. Conidia (scolescospores) are very numerous, hyaline, bent to arcu- ate, filiform, and 18-25 by 1 pm (3). Eutypa armeniacae has been found infecting many woody plant species. Beside being an important pathogen of grape and apricot, E, armeniacae has been found to cause a dieback of Ceanothus sp. (22,32), western choke cherry (l4), prunes (3), lemon (18), and 3 manzanita (36). Experimental mycelial inoculations have caused disease in peach, nectarine and plum by Carter (3), and almond and peach by English and Davis (20). Ascospore inoculations have caused disease in peach and almond (8). Tamarix sp., almond and apple have been found bearing the perithecial stage (4). The grapevine disease referred to as "dead-arm," "dying arm," or "dieback" is most noticeable during early spring growth, when the affected leaves take on a dwarfed, cupped, and chlorotic appearance. In subsequent years, the arms on which these symptoms occur die. Cutting through the wood of infected branches reveals a brown canker with a distinct margin between cankered and healthy tissue. These symptoms were first observed by Reddick who, in 1914 (41) concluded that this disease was caused by the fungus Cryptosporella viticola. He included as symptoms caused by this fungus "small reddish brown or black spots on the green shoots, petioles, peduncles, and leaf veins" which may become very deep and extensive on the canes. Coleman (12) found a constant association of the dieback symptoms with pruning wound lesions but his inocula- tion studies were inconclusive. Goidanich (in Pine [37]) renamed the pathogen Phomogsis viticola based on the presence of a and B spores, which are typical of the genus Phomopsis. Pine (37) agreed and further elucidated the cultural characteristics of E, viticola. The first association of E, armeniacae with the grape die- back was made by Carter (4), who could isolate E, armeniacae from margins of cankers on infected vines. Nillison, et al. (48) noted that E, viticola could easily and rapidly infect green tissue but pruning wound infections with E, viticola occurred in only 25% of the inoculations, and these infections did not become large lesions. Further indications of a separation of the leaf and cane symptoms with the dieback symptoms were reported by Moller, et a1. (31) in 1974, and Dye and Carter (13) and Braun, et a1. (2) in 1976. These three papers reported consistent isolation of E, armeniacae from margins of cankers of infected vines. Finally in 1978 (27) and again in 1981 (30) Moller and Kasimatis reported completion of Koch's postulates, proving that E, armeniacae causes the dying arm symptoms. In both cases, an E. armeniacae culture isolated from a dieback infected grapevine was used to make mycelial inoculations to fresh pruning wounds of Vitis vinifera cv. Grenache. Typical spring dieback symptoms as well as cankers were induced after at least two and a half years by this treatment. Isolation of’E, armeniacae from the cankers fol- lowed. Contrary to the relatively recent discovery that E, armeniacae (Cytospgrina) causes a dieback of grape, Cytosporina has been recognized as the causal agent of a disease of similar etiology on apricot since 1938 when Adam (1) reported on extensive research done on this "gummosis" or "dieback" disorder. This research included the demonstration that pruning wounds were neces- sary for infection. Subsequently, Carter (3) showed that the perfect stage was E; armeniacae. That same paper also demonstrated that the scolecospores were non-infectious and that asc05pore dis- charge only occurs after a stomatal wetting. Since that time there has been extensive research in Australia and California on infection and control of E, armeniacae on apricot. English and Davis (15,16) conducted histopathological studies of apricot infection by E, armeniacae. They found that the fungus first invades the young xylem, then moves out through the cambium and bark, at which point a canker develops. They also demonstrated that cambial inoculations were generally ineffective, due to an apparent inactivation of the fungus in the cambial tis- sues. Phloem inoculations were ineffective. Ascospore trapping studies in Australia (23) and California (40) showed a seasonal periodicity in spore release, with a large reduction occurring in late fall and early winter (May to July in Australia). Related to these studies, Carter (3) and Ramos, et a1. (40) noted that in dry apricot growing areas (less than about 30 cm annual rainfall) perithecia are absent. Ramos, et a1. were able to trap spores in these areas, however, and eliminated the possibility that alternate hosts of the pathogen (i.e., grape and Ceanothus sp.) were the source of these spores. They concluded that the ascospores were dispersed over long distances via prevailing winds from areas where perithecia were present to the drier areas where they induced a great deal of disease. Studies to determine the amount of inoculum needed to cause infection on apricot were also conducted in Australia (8) and California (29). In Australia it was found that 10 ascospores per wound could infect 43% of pruning wounds inoculated whereas 100 ascospores could infect 83%. This is in contrast to the California study which reported that both 10 and 100 asc05pores could infect 98% to 100% of wounds inoculated. The same California workers (39) also found that the California isolate was significantly more viru- lent than the Australian isolate, with single ascospore inoculations capable of causing more infection and more extensive discoloration. Reduction in susceptibility of wounds over time was studied initially in Australia (7) and then California (39). In Australia, wounds were found to be susceptible for less than two weeks in early winter if not protected from rain but more than one month if covered with a rainproof shelter. It was hypothesized that the reduction in susceptibility was due to natural microflora colonizing the wound and taking up the sites that the pathogen would otherwise infect. Price (38) followed up on this hypothesis and determined that there was a peak in microbial colonies on the apricot wound surface at 12 days on unsheltered trees and 15 days on sheltered trees, although the final amount of microbial colonies was the same. The California study (39) revealed a seasonal variation in length of susceptibility of pruning wounds, with susceptibility in fall longest (at least 42 days after pruning) and susceptibility in spring shortest (less than 14 days). Length of susceptibility was also found to vary by temperature (resistance to infection is acquired much quicker at 20°C than at 3°C) and humidity (wounds subjected to higher humidity became resistant faster). These workers postulated physiological factors relating to wound healing as the reason for decrease in susceptibility over time. Control studies of E, armeniacae infection on apricot were mainly done in Australia. They have taken a number of approaches, including escape of inoculum, preventive fungicide treatment, and biological control agents. As discussed before, there is a lull in ascospore release in late fall. Presumably for this reason, it was found (6,21) that natural infection was greatly reduced over other times of the year when pruning was done in early winter (June in Australia). This rec- commendation was made (22), although infection was still possible, especially with large wounds (6). Therefore, studies aimed at pre- venting infection through fungicide treatment of fresh pruning wounds were initiated. By this time, capper containing chemicals had already been found to be ineffective in preventing infection (4). Therefore, evaluations were made on the efficacy of 12 different fungicides in preventing infection (24). Only benomyl gave significant control when compared to the unsprayed check treatment. Benomyl again demon- strated control in a follow-up trial (25) when applied to pruning wounds by hand held sprayer. However, separate studies in Australia (11) and in California (33) revealed that benomyl applied by high volume orchard sprayers was ineffective in preventing infection. The only feasible control method found in the California study was hand application of benomyl at high concentration (2.4% w/v) using a paint brush or hand atomizer. Biological control studies were also conducted in Australia as a follow-up of the previously postulated idea that microbial colonization prevents establishment of E, armeniacae on pruning wounds (7). Fusarium lateritium was isolated from a pruning wound in which E} armeniacae failed to become established after inocula- tion with 100 ascospores (5). Other studies in that paper demon- strated some prevention of establishment of E, armeniacae on fresh pruning wounds by E, lateritium. Subsequent studies (9) established that E, lateritium produced a non-volatile, diffusable inhibitor of E, armeniacae germination and growth in agar plates; was especially effective against E, armeniacae establishment on pruning wounds when introduction of the pathogen was delayed; and was about ten times as tolerant to benzimidazole fungicides as E, armeniacae. Finally (10), workers found that when benzimidazole fungicides and E, lateritium were used together, the former would prevent establish- ment of inoculum arriving to the infection site early, whereas the latter would prevent establishment of late arriving (six days or later after pruning) ascospores. Another agent that may hold promise for E, armeniacae control is Gliocladium roseum, which has been reported to be mycoparasitic on E, armeniacae (42). Compared to the extensive infection and control work done with E; armeniacae on apricot, there has been little work done deal- ing with infection and control of this pathogen on grapevine. This is because it has only recently been established that E, armeniacae is the cause of the grape dieback disease (27,30). 10 Spore release studies in Michigan (46) and in New York (35) strongly established that a lull in ascospore release occurs during the summer months in those areas, in contrast to the late fall lull observed on apricot in California (40) and Australia (23). This would preclude adjustment of the pruning schedule in Michigan and New York (as was partially successful with apricots in Australia [22]) to a time of low inoculum presence due to the need for dormant season pruning. Pruning schedule adjustment was suggested as a con- trol measure in California by Petzoldt, et a1. (36) who found that E, vinifera pruning wounds were more susceptible and susceptible for a longer period of time in December than in March. Trese (45), however, found greater susceptibility in February than in December in Michigan. Susceptibility of pruning wounds on different age wood was investigated by Moller and Kasimatis (28). They found that one-year- old wounds were significantly less susceptible to infection than older wounds. In another California study (36) and in a Michigan study (45), however, this difference was not apparent. One investigation (29) demonstrated protection of grape pruning wounds using 0.2 lb/gal. (active ingredient) benomyl paint. The report stated that a lower concentration was ineffective for this purpose. In another report, Trese (45) found that the compound BenlateR 50%NP strongly inhibited mycelial growth of a Michigan isolate oqu, armeniacae at the concentration of 0.1 ug/ml in poison R, BravoR, CaptanR agar tests, and the compounds Difolatan , and 11 PhaltanR strongly inhibited ascospore germination at the same con- centration. Clearly, a number of studies are needed to elucidate more completely parameters of E, armeniacae infection and possible control measures on grapevine. One cannot assume that the etiology of this pathogen is the same on grape as on apricot, especially in such areas as Michigan and New York where there are no commercial apri- cots to serve as a source of inoculum and where climatic conditions are very different than in apricot growing areas. As stated before, differences in spore release patterns have already been found between the two areas, supporting this assertion. MATERIALS AND METHODS Fungicidal Control Trials, Year 1 To examine the possibility of using fungicides on fresh pruning wounds of grapevine, Vitis labrusca L. 'Concord,‘ to pre- vent the establishment of Eutypa armeniacae ascospores, fungicide field trials were conducted in December, 1979 and repeated in January, March and April of 1980. A 10-year-old commercial vineyard located about three miles south of Lawton, Michigan was used for these trials. No evidence of previous infection with Eutypa dieback could be found in this study or by Trese (45) in this vineyard. The vines were pruned since establishment into a bilateral cordon system, which facilitated mechanical harvesting. Twenty pruning wounds per vine were made just above a node or branch on two-year-old wood. Any vines so small that twenty pruning wounds on two-year-old wood could not be obtained were eliminated from the trials. After pruning, each vine was sprayed with 0.5L of a suspen- sion of either BenlateR 50%NP (benomyl) at the rate of 1.2 or 4.8 g/L (l or 4 lb/100 gal.) water, DifolatanR4F (captafol) at the rate of 10 or 20 m1/L (4 or 8 qt./100 gal.) water, or a water control (equivalent to 79.26 gal./acre or 741.3 L/ha.). In the March and April trials, a sixth treatment of 500 macroconidia per pruning 12 13 wound of Fusarium lateritium suspended in 5 ul distilled water was applied as a possible biological control agent. The E, lateritium culture was provided by M.V. Carter (Department of Plant Pathology, Waite Agricultural Research Institute, University of Adelaide, Glen Osmond, South Australia). This inoculum was scraped off cultures growing in potato dextrose agar (Difco Laboratories, Detroit, MI 48201) and suspended in distilled water. Concentrations were deter— mined using a hemacytometer. There were five vines per treatment randomly chosen along the rows. R Benlate was selected for these trials because tests con- ducted by Trese (45) demonstrated a strong inhibition of E, armeniacae mycelial growth on agar incorporated with BenlateR. R Benlate also has shown some control of E, armeniacae in previous studies on apricot (24,25,33) and grape (29). DifolatanR was selected because Trese (45) also demonstrated a strong inhibition of E5 armeniacae ascospore germination on agar incorporated with DifolatanR. Fusarium lateritium was selected because studies by Carter and Price (9,10) demonstrated some control of E, armeniacae on apricot using E, lateritium in field trials in Australia. After treatment, 10 of the 20 pruning wounds per vine were inoculated with 500 ascospores of E, armeniacae suspended in 5 ul distilled water. The other 10 pruning wounds were inoculated 14 days later when possible. In the January trial, however, it was necessary to make the second inoculation 35 days after pruning instead of 14 days due to the absence of above freezing temperatures during that period. An inoculum of 500 ascospores was chosen 14 because Trese, et al. (46) were only able to obtain an overall infection of 14.3% on two-year-old wood when inoculated at various times of the season with 250 asc05pores. Ascospores were obtained for these inoculations by soaking mature stroma of E, armeniacae in distilled water for 10 minutes then, one day later, making freehand sections of the stroma with a razor blade, cutting through numerous perithecia, and suspending these freehand sections in a few ml of distilled water. After a few seconds of shaking followed by a one hour wait, the suspension was again shaken and the ascospore concentration was determined with a hemacytometer. In no cases were spores other than those of E, armeniacae seen during microscopic examination of the spore sus— pension. Germination of these ascospores was always betwen 90 and 97%. In the December, 1979 and January and March, 1980 trials the temperature was always between 0 and 5°C during the pruning, spray- ing and inoculating and in the April, 1980 trial the temperature was about 10°C during these events. Isolations and identifications of E, armeniacae taken from the pruning sites were made from 9 to 13 months after inoculation after the method of Trese (45). Each cane was cut off 6 to 10 cm below the original pruning wound. Using aseptic techniques, the canes were then split lengthwise and ten small wood chips were removed from 1 to 5 cm below the pruning would and placed on 2% potato dextrose agar amended with 100 ug/ml streptomycin sulfate. After 3 to 6 days, any fungal colonies resembling E, armeniacae 15 were transferred to fresh potato glucose agar (an extract of 200 9 potatoes, 8 9 glucose, and 20 g agar in 100 ml distilled water). These plates were placed under cool white fluorescent light (GE F15T8CN) and soft black light (GE F30T8SB) with a 14 hour daylength. After about one month, the presence of E, armeniacae cultures were confirmed by the microscopic presence of scolecospores. Fungicidal Control Trials, Year 2 Based on preliminary results from the first year trials, the year 2 trials were started on February 17, 1981. The same vine- yard was used as in the year 1 trials. The trial was conducted as a 4 by 4 factorial experiment. BenlateR 50%NP (benomyl) at the rate of 1.2, 4.8, or 9.6 g/L (1, 4, or 8 1b/100 gal.) water and a water control was applied after pruning as before. There were only 15 pruning wounds per vine in this trial, however. The pruning wounds were then inoculated as before on day l, 14, 28, or 56 after pruning and spraying. The temperature was between -2 and 5°C for the first three inoculation periods and 22°C for the fourth inoculation period. There were three randomly selected vines per treatment. Isolation and identification of E, armeniacae was made as before, six to seven months after pruning and spraying. Spring Frost Damage as a Possible E. armeniacae Infection Site Sixty potted four-year-old vines of E, labrusca L. 'Concord' were placed in a growth chamber (Sherer-Gillett 00., Marshall, MI 49068) held at a constant 16°C on May 18, 1980, when new spring growth on these vines was from 5 to 20 cm long. Over the next 16 seven days the temperature was slowly brought down to near 0°C. The vines were then subjected to a temperature of -5°C for 2 hours, followed by a rise to 18°C over a 2 hour period. Ten control plants were also subjected to the same temperature regime except for the temperatures below 0°C. These control plants and ten plants that were subjected to freezing temperatures were then inoculated with a spore suspension containing 1000 E, armeniacae ascospores per m1 distilled water (prepared as previously described). Inoculations were made by misting the vines with a DeVilbis No. 15 hand atomizer, applying 3 ml of the spore suspension uniformly to each vine. After inoculation, a plastic bag containing a wet paper towel was put over the plants for 24 hours to maintain free moisture and was then removed. Inoculations as just described were then done on ten plants each on 1, 3, 7 and 14 days after freezing. Areas of the plant were tagged where the new spring growth had died as a result of the freezing. Isolations were made after about 12 months by examining ten sites per vine, selected to obtain samples from all ages of wood. All sites where new spring growth had died as a result of the freezing were included here. Five wood chips from each site were removed using aseptic technique and assessed for E, armeniacae as previously described. Simulated Mechanical Harvester Induced Injury as a Possible E. armeniacae Infection Site The easternmost (most downwind) row of a l7-year-old, 0.2ha. vineyard of E, labrusca L. 'Concord' in which about 5% of the vines 17 bore mature stromata of E, armeniacae was used for this experiment. The vines were beaten by hand with a 1m long metal bar on October 9, 1980, which was at the same time that mechanical harvesting was being done in Michigan. Two hundred wounds from this treatment, which resembled wounds made by mechanical harvesters, were tagged. One hundred of these wounds were inoculated with 500 ascospores of, E, armeniacae as described previously. The other one hundred wounds were uninoculated. Rain sufficient to release ascospores of E, armeniacae (45) occurred 2 and 5 days after wounding. Isolations to determine the presence of E, armeniacae near the site of inoculations were made about seven months later. They were done as described for the fungicide trials. Effect of Various Environmental Conditions ' on Infection of Potted Vines with E. armeniacae Forty-two potted vines as described previously were pruned at three sites just above a node or branch on two-year-old wood in early 1981. Treatments 1 and 2 were pruned on January 18, Treatment 3 on January 20, Treatment 4 on January 29, and Treatment 5 on February 28. Just after pruning, the pruning wounds were inoculated with 500 ascospores of E, armeniacae as described before. After inoculation, a plastic bag containing wet paper towel was put over the vines for 24 hours to maintain free moisture and then removed. The vines were then subjected to the following environmental condi- tions: 18 Number of Treatment Vines l. Held at 3-8°C until April 17, 1981 then put outside 8 2. Held at l-3°C until April 17, 1981 then put 10 outside 3. Held at -5°C seven days,then l-3°C until April 17, 8 1981 then put outside 4. Held at -5°C 30 days,then l-3°C until April 17, 8 1981 then put outside 5. Held at 10°C seven days,then 1-3°C until April 17, 8 1981 then put outside Seven to eight months after inoculation, isolation and identification of E. armeniacae from near the pruning wounds were made as described for the fungicide trials. Isolations from the Vicinity of a Canker on Infected Vines This experiment was conducted to determine if E, armeniacae is present in healthy tissue adjacent to cankered wood. This know- ledge would help the grower determine where to cut off infected vines to remove all traces of E, armeniacae. Four mature vines, which in the previous spring had exhibited typical symptoms of Eutypa dieback, were cut off near ground level on October 10, 1981. They were then sawed into numerous pieces, exposing the discolored canker on the mature wood. After surface sterilization with 0.5% sodium hypochlorite, lO wood chips were aseptically transferred as described previously from the margins of the canker and each 0.5 cm away from the canker to the edge of the 19 surface. The presence of E, armeniacae from each of these areas was determined as previously described. RESULTS Fungicidal Control Trials, Year 1 Table 1 shows cumulative totals of infection by E, armeniace over all four blocks (times of replication of the experiment). Included here is percent infection, percent reduction of infection compared to the control, and a X2 value indicating the significance of the difference in infection of the various treatments vs. the control. Part A shows these parameters for both inoculation times summed together. Part B shows the data for the inoculation on the day of pruning and spraying. The data for the second inoculation are not included here because, as stated previously, the second inoculation was conducted at a different time after pruning and spraying (35 days instead of 14 days when the experiment was con- ducted in January) than when the experiment was conducted the other three times. Because of this, Table 2 is included, showing the same parameters as Table l with the January trial excluded. Part A again shows the data for both inoculation times and part B shows the totals for the second inoculation time (14 days after pruning and spraying). Table 3 shows an analysis of variance of the data for all feur blocks, analyzed as a randomized block, split plot design (43), with the time of inoculation after pruning and spraying as the split. This analysis was possible because Bartlett's test (43) showed no 20 221 TABLE l.--Cumulative Totals, Fungicide Trials, Year 1°. Eutypg armeniacae Pruning Hound Infection (Lawton, MI). All Four Blocks Combined. Infected 1 Reduction of Infection 2 Treatment Total IEBEETEIEU x Infected Compared to Control x vs Control A. Inoculation on day of pruning and spraying or day 14 or 35 after pruning and spraying,,inclusive. Control 657353 24.06 - - BenlateR 505w? 53/366 14.46 39.67 10.06b 1.2 g/L Benlate“ 50m 26/347 7.49 66.90 31.61 4.8 g/L DifolatanR 45 67/362 18.51 23.13 2.91 10 m1/L DifolatanR 45 57/364 15.66 34.97 7.99 20 I1/L g, lateritium 29/162 15.93 33.85 4.27 B. Inoculatiggfon day of prggjng and spraying. Control 64/173 36.99 - - BenlateR 5059? 33/166 17.74 52.04 15.89 1.2 g/L Benlatea sozup 177179 9.5 74.32 35.93 4.8 g/L 01mm".R 45 43/165 23.24 37.17 7.42 10 ml/L 011613236R 45 39/167 20.86 43.61 10.66 20 ml/L E, lateritium 22/93 23.66 36.04 4.33 .This experiment was conducted in a healthy lO-year—old commercial vineyard of V. labrusca L. 'Concord.‘ Twenty pruning wounds were made per vine on two-year-old wood. There were flve vines per treat- ment. Each vine except for the F. lateritium treated vines was sprayed after pruning with 0.5 L of its respective treatment. Each wouna on the E, lateritium treated vines was inoculated with 500 macroconidia of F. lateritium. Ten of the 20 pruning wounds per vine were inoculated with 500 ascospores of E, armeniacae Th 5 pl disfilled water on the day of pruning and spraying. The other 10 pruning wounds per vine were inoculated as described above on day 14 or day 35 (in the case of Block 2) after pruning and spraying. The four dates of pruning and spraying were December 5, 1979 (Block 1), January 15, 1980 (Block 2), March 13, 1980 (Block 3) and April 17, 1980 (Block 4). Positive infections were determined by tissue isolations onto PDA 9-13 months after treatment. bCritical x2 values: g_- 0.10 - 2.71; g_- 0.05 - 3.64; g_- 0.01 - 6.63; g,- 0.005 - 7.66. 22 TABLE 2.--Cumulative Totals, Fungicide Trials. Year 1. Eutypg armeniacae Pruning Hound Infection (Lawton, MI). Block 2 Excluded. Infected 1 Reduction of Infection 2 Treatment Total IEBEUIEEEB % Infection Compared to Control X '5 Control A. Inoculation on day of prgpingiggg spraying or day 14 after pruning and sprgying,ginclusive. Control 60/162 22.90 - - Benlate“ 50¢HP 33/271 12.16 46.61 9.14a 1.2 g/L eeniateR soxwp 19/266 7.09 69.04 24.69 4.8 g/L Difolatan“ 45 54/271 19.93 16.97 <1.00 lO mllL DifolatanR 45 44/277 15.66 30.66 3.62 20 ml/L g, lateritium 29/182 15.93 30.44 2.63 B. Inoculation 14 days aftergpruning and spraying. 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