main@111mywmmmmmm * 31.,_W__77411875 Lm‘ I University t This is to certify that the dissertation entitled Mycological and Pathological Examinations of Cytospora Canker of Spruce presented by Tyre John Proffer has been accepted towards fulfillment of the requirements for _Eh..D_.___degree in Wlant Pathology Major professor Date 1 November, 1985 ucll.‘.--‘or .: A - r1 .n . , . , 042771 MSU LIBRARIES “ RETURNING MATERIALS: Place in book drop to remove this checkout from your record. FINES will be charged if book is returned after the date stamped below. MYCOLOGICAL AND PATHOLOGICAL EXAMINATIONS OF CYTOSPORA CANKER OF SPRUCE By Tyre John Proffer A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Botany and Plant Pathology 1985 ABSTRACT MYCOLOGICAL AND PATHOLOGICAL EXAMINATIONS OF CTYOSPORA CANKER OF SPRUCE By Tyre John Proffer Colorado blue spruce [21233 pungens] is a valuable landscape orna- mental. Mature trees are often progressively disfigured by Cytospora canker. Cytospora canker is caused by Leucostoma 32252; (syn. gals; 52255;]. The anamorphic form, geucocytosporg‘gugggi (syn. Cztospora 52952;] is most frequently observed. Several lines of research were undertaken to examine this fungal pathogen and disease. A collection of 487 isolates of L. 52252; were isolated from 196 cankers on 121 trees growing at several different sites. Leucocxtospora 53952; was isolated from blue spruce as well as from five other coniferous host species. Vegetative compatibility is an important biological feature of fungi. In E. 52252;. a4 vegetative compatibility groups (vc-groups), including eight multi-merge groups were identified. Vegetative com- patibility groups did not segregate during conidiogenesis. The distri- bution patterns of vc-groups seemed to indicate a role for conidia in the dissemination of this pathogen. Isolates of L. 52352; from widely separated sites within Michigan, as well as between Michigan and Colorado isolates, were found which were in a common vc-group. Some of the in vitro growth responses of ;. kunzei were also Tyre John Proffer examined. The optimal water potential for mycelial growth was from -14 to -15 bars. The water potential of test media was adjusted by the addition of sucrose, mannitol, NaCl, KCl. or polyethylene glycol. NaCl and KCl proved to be unsuitable osmotica for use with L. £23523. Leucocytospora kunzei grew well in 215:2 over a pH range of 5.0 - 7.0, optimal growth occurred at a pH of 6.2. Leucocytospora 52253; is well adapted to the internal environmental conditions of its spruce host. In inoculation trials, using sixteen Cytospora g. lat. isolates. those isolates of 5. 522521 from spruce and one from eastern white pine caused canker formation on wound inoculated branches of blue spruce. Cytospora g. lat. isolates from deciduous tree hosts did not incite canker formation on inoculated branches. Both excised and intact branches of mature trees were utilized. Excised branch inoculations reliably and accurately reflected responses in inoculated intact branches. Isolates of g. kunzei started from conidia were infective. DEDICATION I dedicate this dissertation to my wife and best friend, Linda Kay Leach. Her love, patience, understanding, and support have made this effort possible. I also want to dedicate it to our children, Anna Eru and Nathan Grey Leach-Proffer, who have added so much to our lives. ii ACKNOWLEDGMENTS I would like to thank the members of my graduate committee for their advice and assistance during the course of this research. Their encouragement and support is greatly appreciated. I would like to particularly thank Dr. John Hart for his help and physical support, and Dr. Donald Ramsdell for his assistance in preparing this thesis. Dr. Henry Imshaug and Dr. Karen Klomparens (Baker) both provided me with invaluable insite and encouragement and served as excellent resource people for me. I would also like to thank Mrs. Phyllis Robertson for her help and friendship. Dr. Gene Safir and Ms. Karol Elias are acknowedged for their advice and use of equipment for the water potential work. This research was supported by a grant from Dow Gardens in Midland, Michigan. I would like to thank the Dow Foundation and Mr. Douglas Chapman for that support and also for the opportunity he afforded me to expand my background with disease diagnosis. Mr. Chapman has played a key role in my professional development. I would also like to thank the staff of Dow Gardens for their input and friendship. My experience at Dow Gardens was very rewarding, both professionally and personally. I would like to also acknowledge Mr. Sturdevant for his interest and support. iii TABLE OF CONTENTS LIST OF TABLES . LIST OF FIGURES. SECTION 1. SECTION 2. SECTION 3. GENERAL INTROUUCTION . LIST OF REFERENCES. VEGETATIVE COMPATIBILITY GROUPS IN LEUCOCYTOSPORA KUNZEI. . INTRODUCTION. MATERIALS AND METHODS. . Isolate collection . . . . . . . Isolation and culturing of L. kunzei . Coding system. . . . . . VC-groups: Isolate conditioning. VC-groups: Isolate pairing VC-groups: Evaluation . VC-groups: Conidiogenesis. RESULTS . . . Isolate collection . Cultural morphology. . . Vegetative compatibility groups. VC-groups: conidiogenesis. . Vegetative compatibility groups: distribution patterns DISCUSSION LIST OF REFERENCES. WOUND INOCULATIONS OF EXCISED AND INTACT BRANCHES or BLUE SPRUCE WITH LEUCOCYTOSPORA KUNZEI AND OTHER CYTOSPORA g. Egg. ISOLATES INTRODUCTION. iv Page vii ix 10 12 12 12 13 13 13 16 16 16 16 31 31 31 38 41 48 A9 SECTION 4. SECTION 5. MATERIALS AND METHODS. Isolates . . . . . Excised branch inoculations . Intact branch inoculations Evaluation. RESULTS DISCUSSION . LIST OF REFERENCES. WOUND INOCULATIONS OF EXCISII AND INTACT BRANCHES OF BLUE SPRUCE WITH CONIDIAL ISOLATES OF LEUCOCYTOSPORA KUNZEI. . . INTRODUCTION. MATERIALS AND METHODS. Isolates . Inoculations . . . Experiment No. 1 Experiment No. 2. Evaluation. RESULTS DISCUSSION LIST OF REFERENCES. THE EFFECT OF WATER POTENTIAL ON THE IN VITRO GROWTH OF LEUCOCYTOSPORA KUNZEI . INTRODUCTION. MATERIALS AND METHODS. The test isolates l’test plates. Water potential determination Water potential and growth Evaluation. . . . RESULTS DISCUSSION LIST OF REFERENCES. Page 51 51 51 54 54 SS 58 65 67 68 68 68 70 q I 70 71 71 71 77 79 80 80 80 80 81 81 81 83 86 Page SECTION 6. IN VIVO WATER POTENTIALS IN BLUE SPRUCE TREES WITH AND WITHOUT A PRIOR HISTORY OF CYTOSPORA CANKER. . . . . . . . . . . . . . 88 INTRODUCTION. . . . . . . . . . . . 89 MATERIALS AND METHODS. . . . . . . . . 90 Study sites . . . . . . . . . . 90 Sampling . . . . . . . . . . . 90 Measurements of water potential. . . . 91 RESULTS . . . . . ‘. . . . . . . . 91 DISCUSSION . . . . . . . . . . . . 95 LIST OF REFERENCES. . . . . . . . . . 97 SECTION 7. THE EFFECT OF PH ON THE IN VITRO GROWTH OF LEUCOCYTOSPORA KUNZEI. . . . . . . . . 98 INTRODUCTION. . . . . . . . . . . . 99 MATERIALS AND METHODS. . . . . . . . . 99 Isolates . . . . . . . . . . . 99 pH test media. . . . . . . . . . 100 Evaluation. . . . . . . . . . . 100 RESULTS . . . . . . . . . . . . . 100 DISCUSSION . . . . . . . . . . . . 102 LIST OF REFERENCES. . . . . . . . . . 105 APPENDICES . . . . . . . . . . . . . . . . 106 Appendix A. Culturing Leucocytospora kunzei: Notes and observations. . . . . . 107 Appendix B. Cytospora canker of spruce: Notes and observations. . . . . . 108 Appendix C. Other fungi frequently isolated from branches of blue spruce . . . . . 109 Appendix D. Scanning Electron Microscopy. . . . 110 vi LIST OF TABLES Table Page SECTION 2. VEGETATIVE COMPATIBILITY GROUPS IN LEUCOCYTOSPORA KUNZEI 1. Collection sites from which isolates of Leucocytospgrg kunzei were obtained . . . . . 15 h.) List of isolates of Leucocytospora kunzei: original hosts and source . . . . . 18 3. Vegetative compatibility groups in Leucocytosporg kunzei. . . . . . . . . . . . . . . 33 a. Multi-merge vegetative compatibility groups in Leucocytospora kunzei. . . . . . . . . . 40 5. Vegetative compatibility groups isolated from more than one host species . . . . . SECTION 3. WOUND INOCULATIONS OF EXCISED AND INTACT BRANCHES OF BLUE SPRUCE wITH LEUCOCYTOSPORA KUNZEI AND OTHER CYTOSPORA é- LAI- ISOLATES 1. Origin and Identification of the 16 Cytospora spp. Isolates used to Inoculate Blue Spruce . . 52 Is.) The Z canker initiation on inoculated branches of blue spruce caused by isolates of Cytospora 5. lat. . . . . . . . . . . . . . . 57 3. Comparison of canker lengths on excised and intact branches of blue spruce inoculated with four isolates of Leucocytospora kunzei . . . . S9 4. Host effects on excised branch inoculations with four Leucocytosporg kunzei isolates . . . . . S9 5. Percent canker initiation on excised branches of blue spruce inoculated with Leucocytosporg kunzei isolates: branches collected during different seasons . . . . . . . . . . . . . . 60 vii Table Page SECTION 4. WOUND INOCULATIONS OF EXCISED AND INTACT BRANCHES OF BLUE SPRUCE WITH CONIDIAL ISOLATES OF LINCOCYTOSPORA KUNZEI 1. Inoculations of excised and intact branches of blue spruce with isolates of Leucocygospora kunzei started from infected tissues or conidia. Experiment No. 1 . . . . . . . . . . . 72 2. Inoculations of excised and intact branches of blue spruce with isolates of Leucocygospora kunzei started from infected tissues or conidia. Experiment No. 2 . . . . . . . . . . . 73 3. Mean canker lengths on excised branches of blue spruce inoculated with tissue and conidia] iso- lates of Leucocytospora kunzei. Experiment No. 2 . . . . . . . . . . . 74 SECTION 6. IN VIVO WATER POTENTIALS IN BLUE SPRUCE TREES WITH AND WITHOUT A PRIOR HISTORY OF CYTOSPORA CANKER 1. Water potential in blue spruce branches taken from trees with and without cankers caused by Leucostoma kunzei. Site No. 1, East Lansing, Michigan . . . . . . . . . . . . . . 92 Ix.) 0 Water potential in blue spruce branches taken from trees with and without cankers caused by Leucostoma kunzei. Site No. 2, East Lansing. Michigan . . . . . . . . . . . . . . 93 3. Horizontal gradient of water potential along the branches of blue spruce with and without cankers caused by Leucostoma kunzei. Site No. 2, East Lansing, Michigan. . . . . . . . . . 94 viii LIST OF FIGURES Figure Page SECTION 2. VEGETATIVE COMPATIBILITY GROUPS IN LEUCOCYTOSPORA KUNZEI l. The coding system used to identify isolates of Leucocytospora kunzei. . . . . . . . . . 14 2. Vegetative compatibility pa1r1ng plates . . . 17 3. Determination of vegetative compatibility groups. 32 A. The multi-merge response. . . . . . . . . 37 5. Vegetative compatibility groups found at more than one site . . . . . . . . . . . . 39 SECTION 3. WOUND INOCULATIONS OF EXCISED AND INTACT BRANCHES OF BLUE SPRUCE WITH LEUCOCYTOSPORA KUNZEI AND OTHER CYTOSPORA g. 551. ISOLATES 1. Canker initiation on an excised branch of blue spruce inoculated withC Cytospora 3. lat. isolates . . . . . . . . . . . . 56 SECTION 4. WOUND INOCULATIONS 0F EXCISED AND INTACT BRANCHES OF BLUE SPRUCE WITH CONIDIAL ISOLATES OF LEUCOCYTOSPORA KUNZEI 1. Induced sporulation of Leucocytospora kunzei on spruce. . . . . . . . . . . . . . 69 SECTION 5. THE EFFECT OF WATER POTENTIAL ON THE IN VITRO GROWTH OF LEUCOCYTOSPORA KUNZEI 1. The effect of water potential on the i2 vitro growth of Leucocytospora kunzei . . . . . . 82 ix Figure Page SECTION 7. THE EFFECT OF PH ON THE IN VITRO GROWTH OF LEUCOCYTOSPORA KUNZEI The effect of pH on the IQ vitro growth of Leucocytospora kunzei. . . . . . . . . . 101 The response of four isolates of Leucocytosporg kunzei to pH: Growth rates. . . . . . . . 103 SECTION 1 General Introduction GENERAL INTRODUCTION Colorado blue spruce (IIggg pungens Engelm.) is a popular and widely planted ornamental conifer, prized for its color and dense pyramidal growth form (2). Although native to the Rocky Mountains in the western United States, blue spruce have been planted in the mid- western and northeastern part of the country as landscape trees (2). Blue spruce is unimportant as a timber species and is generally used for landscape specimens or as 'shelterbelts' or 'hedgerows’ along pro- perty or lot lines. In the Midwest and Northeast, mature trees are often affected by Cytospora canker (7.14.19). It is of interest to note that reports of Cytospora canker on blue spruce in its native range are lacking. The diffuse branch cankers associated with this disease cause the death of affected limbs (17). The needles on the affected limbs are first slightly off-colored and then progressively redden and even- tually fall. Sometimes on blue spruce, the needles take on a purplish tint. Generally the lower branches are affected initially, often a single bottom limb will first be affected, which is often overlooked by the property owner. The disease, however, progressively affects limbs higher in the tree, spreading upward and laterally (3,5,7,17). Trunk cankers and tree mortality are rare in blue spruce (5,19). The aesthetic damage however, caused by the death of the branches. has a major impact on the value of this generally large and prominent landscape tree. Cytospora canker of blue spruce is caused by Leucostoma NggggI (Fr.) Munk ex Kern (13) [syn. ygIgg NggggI] and is most frequently encountered in its anamorphic form. Leucocytospora NggggI (Sacc.) Urban [syn. Cygospora NggggI] (1,3,18,20). There are conflicting references in the literature dealing with this fungus. First, there are nomen- clatural problems. For example, the combination Leucocytosporg 33933; is not listed in the CMI Index of Fungi, although Urban (18) has been cited as making the combination. Author citations for Leucostoma kunzei are also not in total agreement (1,3,13,20). Secondly, Leucostoma and Leucocytospora have been considered to be subgroups within EgIgg and Cygospora respectively, by some authors (19). The darkened con- ceptacle of compact tissues delimiting the perithecial and pycnidial stromata, however, puts this pathogen of spruce into the genus Leucostoma with its anamorphic form Leucocytospora (1,3,13,20). Leucostoma IggggI was used by Barr (1) to illustrate the genus. Pathologists should utilize this taxonomic classification. In the United States. I. NggggI also affects other species of spruce (3,6,7,9,15,19) as well as some other species of conifers such as: eastern hemlock [Igggg canadensis (L.) Carr.]; European larch [Larix decidua Mill.]; Douglas-fir [Pseudotsuga menziesii (Mirb.) Franco]; eastern white pine [Pinus strobus L.]; and balsam fir [Abies balsamea (L.) Mill.] (3,8,13,19,21). Branch cankers are common in these other hosts, as in blue spruce. In addition, tree mortality has been reported on Douglas-fir (8,21) and Engelmann spruce [gIggg pengelmannii Parry] (6). Cytospora canker of blue spruce was first reported in 1933 by Gilgut and Boyd (5). They described the symptoms of this desease as well as isolated the pathogen, Cytospora Nggggi (identified by Wehmeyer). Gilgut in 1936 (4) fulfilled Koch's Postulates with the pathogen on Norway spruce [Picea abies (L.) Karst.] utilizing wound inoculations with spore suspensions (presumably conidial). The telemorph, ygIgg EggggI, was associated with cankers on blue and Norway spruce by Marsden (15) in 1948 (again the fungus was identified by Wehmeyer). The work of Alma Waterman published in 1955 (19) remains as one of the foundation works on this disease and fungus. She compared fruiting structures and isolates in culture,of EgIgg NggggI and the anamorphic Cytospora NggggI from various coniferous hosts. Although slight differences in perithecial stromata indicated three varieties within the species, pycnidial stromata were variable and not sufficiently different to indcate that more than one species was involved. She noted that the stromata development and marginal conceptacle were characteristic of Leucostoma and Leucocytospora as described by Von HUhnel, but still used ygIgg and Cvtospora for her classification scheme. In 1981, Kamiri and Laemmlen (12) reported on some of the epidemiological features of this fungus. They found conidia to be common and to be produced and viable essentially throughout the growing season, while ascospores were rare and seemed to be released only for a short time in the spring. They also studied some of the IE_!I££2 responses of the fungus in respect to temperature optima for mycelial growth and conidia germination. Several workers have noted an association between drought stress and the severity of this disease on spruce and other hosts (6,9,19.21) in the field. Inoculation studies by Kamiri and Laemmlen in 1981 (11) and by Schoeneweiss in 1983 (16) verified these observations. Water stress did increase the susceptibility of inoculated 5 year-old trees to canker formation by I. NggggI. Inoculation studies have also lead to some disagreement as to whether conidia are capable of causing canker initiation. While some authors found inoculations utilizing mycelium of isolates started from either conidia of ascospores to be infective (9,16,19). Kamiri and Laemmlen (11) indicated that only mycelium started from ascospores was capable of inciting canker formation on inoculated trees. Attempts to control Cytospora canker of spruce using fungicides have been ineffective or inconclusive (10,19). The current control strategies are those recommended by Strong (17) in 1953 and include: removing infected branches and burning them, continuous monitoring for new sympotmatic branches, and fertilization and watering. Leucocytosporg kunggI has been isolated from non-symptomatic tissues (14). For that reason, the term 'affected tree' is used in preference to 'infected tree', to describe trees in which there is active canker expansion taking place. It appears that trees which show no symptoms of Cytospora canker are in fact already colonized to some extent by the fungus. Leucostoma $2353; may be part of the normal bark mycoflora, but can cause canker formation under certain predisposing stresses. The purpose of this research was to broaden the available infor- mation dealing with the fungus Leucostoma NggggI and the disease Cytospora canker of blue spruce. Many of the areas investigated were done to supplement or answer questions posed by previous findings. Areas of investigation ranged from basic research in the IN gIggg growth re- sponses of I. Iggggi to more applied pathogenicity studies. In addition, a new avenue of investigation dealing with vegetative compatibility in I. kunzei was undertaken which has provided both basic information, and has applications and implications for plant pathology. By increasing the amount of information available, this research should contribute to our efforts to manage this serious disease of an important landscape ornamental. 10. 11. 13. LIST OF REFERENCES Barr, M. E. 1978. The Diaporthales in North America. Mycologia Memoir No. 7. J. Cramer Pub. Lehre, Germany 232 p. Dirr, M. A. 1975. Manual of Woody Landscape Plants. Stipes Pub. Co. Campaign, Illinois 826 p. Funk, A. 1981. Parasitic Microfungi of Western Trees. Canadian Forestry Service. Victoria, B.C., Canada 190 p. Gilgut, C. J. 1936. Cytospora canker of spruces. National Shade Tree Conf. Proc. 12:113-119 Gilgut, C. J. and O. C. Boyd. 1933. Cytospora canker Of Picea spp. Phytopathology 23:11 Hawksworth, F. G. and T. E. Hinds. 1960. Cytospora canker of Engelmann spruce in Colorado. Plant Disease Reporter 44:72 Hepting, G. H. 1971. Diseases of Forest and Shade Trees of the United States. Agricultural Handbook 386. USDA Forest Service 658 p. Hinds, T. E. and J. L. Stewart. 1965. Cytospora canker recurence on Douglas-fir in Colorado. Plant Disease Reporter 49:481 Jorgenson, E. and J. D. Calfey. 1961. Branch and Stem Cankers of white and Norway Spruce in Ontario. For. Chron. 37:394-400 Kamiri, L. K. 1980. Cytospora canker of Colorado blue spruce: etiology, symptomology. epidemiology and control. Ph. D. Thesis. Michigan State University, East Lansing, Michigan 103 p. Kamiri, L. K. and F. F. Laemmlen. 1981. Effects of Drought- Stress and Wounding on Cytospora Canker Development on Colorado blue spruce. J. Arboric. 7:113-116 Kamiri, L. K. and F. F. Laemmlen. 1981. Epidemiology of Cytospora canker caused in Colorado blue spruce by Valsa kunzei. Phytopathology 71:941-947 Kern, H. 1955. Taxonomic studies in the genus Leucostoma. Papers of the Michigan Acad. of Sci., Arts, and Letters Vol. XL 14. 15. 16. 17. 18. 19. 21. Manion, P. D. 1981. Tree Disease Concepts. Prentice-Hall Inc. Englewood Cliffs, New Jersey 399 p. Marsden, D. H. 1948. A Valsa associated with Cytospora Canker of Spruces. Phytopathology 38:307-308 Schoeneweiss, D. F. 1983. Drought Predisposition to Cytospora Canker in Blue Spruce. Plant Disease 67:383-385 Strong. F. C. 1953. Spruce branch canker. National Shade Tree Conf. Proc. 29:30-35 Urban. Z. 1958. Revise ceskoslovenskych zastupcu rodu Valsa, Leucostoma, a Valsella. Rozpr. Ceskosl. Akad. Ved 68,12, 101 S. Waterman, A. M. 1955. The Relation of Valsa kunzei to Cankers on Conifers. Phytopathology 45:686-692 Wehmeyer, L. 1975. Pyrenomycetous Fungi. Mycologia Memoir No. 6. J. Cramer Pub. Lehre, Germany 250 p. Wright, E. 1957. Cytospora canker of Rocky Mountain Douglas- fir. Plant Disease Reporter. 41:811-813 SECTION 2 Vegetative compatibility groups in Ieucocytosporg kunzei INTRODUCTION Colorado blue spruce (£1222 pungens Engelm.) is a popular and widely planted landscape ornamental in the northeastern and midwestern United States. Cytospora canker of blue spruce is an important and common disease of mature blue spruces in these areas (13.22). The diffuse branch cankers characteristic of this disease are caused by Leucostoma IggggI (Fr.) Munk ex Kern [syn. EgIgg 52952;] which is most frequently seen in its anamorphic form, Leucocysospora NggggI (Sacc.) Urban [syn. Cytospora IggggI] (5.9.23). LeucocyEospora EggggI also occurs on other species of spruce and various conifers (13.22). Although rarely fatal. the loss of branches due to this pathogen seriously de- tracts from the aesthetic value of this important landscape species. Current control recommendations call for the removal of infected limbs as they occur. Being prized for their conical crown, which extends symmetrically down to ground level. the removal of branches also detracts from the overall appearance of these specimen trees. Attempts to use fungicides to control this pathogen have not been successful or were inconsistent in their action (18.22). Promising results have been achieved in controlling the diffuse cankers caused by Cryphonectria parasitica (Murr.) Barr [syn. Endothia parasitica] on American chestnut using a biological control. 'Hypo- virulent'isolates of Q. oarasitica exist which are less virulent than the normal isolates and trees can often survive and compartmentalize 10 11 infection. The 'hypovirulence' trait can be transmitted from a hypo- virulent isolate of the fungus to a healthy virulent isolate II 31353 or Ig,nyg (3,4,14,15,19). One potential benefit of developing a biological control for I. IggggI on blue spruce similar to the 'hypovirulence' system on American chestnut is that it may be able to prevent the aesthetic damage associated with both the disease and the current control strategy. If hypovirulent-like isolates of I. 522521 could be established on blue spruces then they may be able to convert any virulent isolates of I. IggggI present or introduced later. thus preventing limb losses. Because of their high value and use as ornamental trees, labor and monetary considerations would probably be tolerable. Transmission of hypovirulence in Q. parasitica requires an anastomosis between donor and recipient isolates and the transfer of a double stranded RNA 'virus-like particle'(3,4,14,15,19). Vegetative compatibility refers to the ability of two hyphae to make contact, fuse. and exchange nuclear and/or cytoplasmic materials (19). In 9. parasitica there are many vegetative compatibility groups (vc-groups) (1.19.21). Vegetative compatibility. or rather incompatibility, has been shown to limit the effectiveness of using hypovirulence as a biological control, due to its regulation of the anastomosis process. Vegetative compatibility groups (vc-groups) have been documented in several fungi (1.6.8.10,11. 12.16.17.20) and may present a complicating factor in developing a biological control program. The main purpose of this investigation was to examine vegetative compatibility in I. IggggI. A large collection of I. Iggggi isolates was needed to accomplish this. Additionally during the culturing of 12 the isolates. visual screening for irregular isolates of unusual cul- tural morphology could be conducted. Hypovirulent isolates of I. parasitica have often had an altered cultural morphology (19). MATERIALS AND METHODS Isolate colIection- Cankered branches of blue spruce and other conifers were removed from the trees. The trees were located in various Michigan cities and from one site near Colorado Springs. Colorado. Most of the trees were located in residential areas. while some were from cemetary plantings. Records were maintained of the species of tree. the location, and the proximity of other cankered trees. The collections were made from 1983 through 1985. Isolatiog_§pd culturigggof L. IggggI- Cankered branches were brought into the laboratory and isolations were made either from infected host tissue or from conidial cirrhi. For tissue isolations, the cankered branch was surface disinfested by wiping with 957. ethanol and flaming. The bark was then aseptically removed. Pieces of the underlying dis- colored cambial/cortical tissues were removed and placed in petri plates containing Potato Dextrose Agar (PDA). When possible, isolations were made from tissues at the interface of healthy and diseased areas. For conidial isolations, cankered branch segments were soaked in a 5% sodium hypochlorite solution for 10 minutes. rinsed with distilled water. and placed in a moist chamber. Cirrhi of conidia could soon be seen on the surface of the branch and could be collected. Isolates were started from masses of conidia and/or from a single conidium. Single conidium isolates were obtained by a combination of a dilution series 13 of a spore suspension and streak plating the conidial suspension onto PDA. Isolate records were maintained which indicated the host of the isolate. the particular canker on the tree. and in some cases. the location along the cankered branch where the isolate was taken. Cultures were transfered to a spruce decoction agar maintenance medium (SBA). SBA is prepared by soaking approximately 100 g of spruce bark shavings and twigs in 1000 ml of distilled water at 95°C for 45 minutes. The solution was filtered through four layers of cheesecloth and 20 g of Difco Bacto-Agar was added per liter of decoction fluid. This was then sterilized in an autoclave and later poured into 100x15 mm petri plates. The identity of the resulting cultures was verified through microscopic observation of the developing fruiting bodies and by cultural morphology on PDA. SBA. and 22 Malt Extract Agar (MEA). Colonies with unusual cultural morphology were looked for. Coding svstem- A coding system was devised to keep track of the isolates. The code has four main parts. The key to the code is shown in Figure l. The site codes are listed in TEELE_L- YQLEIOUPS‘ isolate conditioning; Pilot experiments showed that for optimal results, the I. IggggI isolates to be paired needed to be preconditioned. Each isolate was transfered to, and grown on 2% Water Agar (WA) prior to the pairing step. Isolates were allowed to grow in the dark at 26“C. Isolates of I. 522321 grow slowly on WA but remain viable and conditioned as long as the WA medium is not allowed to dry out. VC-groups: isolate pairing- The isolates were paired to determine vc-groups. adapting the systems used with other fungi (1). Plugs of each isolate to be paired were cut from the WA conditioning plates 14 DG-iACl) L--- The number in the parentheses represents a particular isolate from a given canker. This letter represents a particular canker on a given tree. The number after the hyphen represents a particular tree from a given site. The first part of the code identifies the site from which the isolates were collected. FIGURE 1. The coding system used to identify isolates of Leucocytosporg kunzei. 15 TABLE 1 Collection sites from which isolates of Leucocytospora kunzei were obtained. Site Code Location BF Northwest corner of Beaumont and Forest Roads, Michigan State University. East Lansing. MI COL Colorado Springs. Colorado1 DG Dow Gardens, Midland, MI EL East Lansing, MI F Frankenmuth. MI FK Franklin, MI H Haslett, MI HL Holt. MI M Midland. MI MAS Mason, MI MSU Michigan State University, East Lansing, MI OK Okemos. MI Richville, MI V Vassar, MI BC Bay City, MI 1 These isolates of I. kunzei came from a forest stand of Engelmann spruce growing at Kenosa Pass, Pike National Forest. a natural planting of spruce. These isolates are the only ones isolated from 16 (the plugs were 5 mm in diameter) and transfered to 100x15 mm petri plates containing 40-45 ml of PDA. Plugs were placed 1 cm from each other. Twenty one plugs could be placed on each pairing plate. A standardized grid pattern was used (Figure 2) to facilitate: placement and record keeping. All isolates were paired to themselves (as controls) and to each of the other isolates. There were two replicates of each pairing plate. The pairing plates were sealed with Parafilm H®. kept in low light at 26°C. and examined daily. y9;groups: evaluation- VC-groups were determined as previously described by Anagnostakis (1). Colonies of isolates within the same vc-group merge together. A black. barrage-like, reaction zone develops along the line of contact between two isolates contained in different vc-groups. !§;groups: conidiogenesis- Vegetative compatibility among conidial isolates taken from cankers and from tissue isolates from the same canker were examined. In a separate experiment, 80 single conidium isolates obtained from a mass of conidia produced by a single pycnidium were cultured and paired for vc-grouping. The pycnidia were being produced on an excised branch inoculated with an isolate of I. IIQIEI [EL-16B(5)l and sporulation was induced as previously described. RESULTS IsgIage collection- A total of 487 isolates of I. 522221 were isolated and cultured (Table 2). The isolates were collected from a total of 196 cankers from 121 trees. Most of the isolates were from blue spruce. Isolates of I. IggggI were also isolated from Engelmann, white. and Norway spruce; eastern white pine: eastern hemlock; and 17 FIGURE 2. Vegetative compatibility pairing plates. A standardized grid was used to maintain consistent distances between the isolate plugs. A total of 21 plugs could be used on one pairing plate. 18 TABLE 2 List of isolates of Leucocytospora kunzei: original host and.source. Isolate Host Species Sourcea BC-1A(1) Picea pungens 'lB(1) " -3A(1) " -3B(2) " Flt-3H"! BF-6A(1) " -6A(2) " -6A(3) " ~6B(1) " -6B(2) " -6B(3) " -OB(4) " -6C(1) " -6E(1) " BF-7A(1) “ ~7A(2) " ~7B(1) " -7C(1) " -7C(2) " -7C(S) " -7C(6) " -7C(7) " -7D(1) " -7D(2) " -7D(3) " -7D(5) " MC -7D(10) " SC -7D(11) " SC -7E(1) " T -7E(2) " MC -7E(10) " SC -7F(1) " T -7F(2) " T -7F(3) " MC -7G(1) " T -7G(5) " T -7G(6) " T HHHZHHHHHEHHHHHHHZHH Souces of isolate: T-infected tissue. MC-mass of conidia. SC-single condidium. 19 TABLE 2 (cont'd.) Isolate Host Species a Source BF-7H(1) -7H(2) -7H(3) -7H(4) -7I(1) -7I(2) BF- WP COL-1A(1) -1A(2) -1A(3) -1A(4) -1A(5) COL-3A(1) -3A(2) -3A(3) -3A(4) ~3A(S) COL-4A(1) -4A(2) ~4A(3) -4A(4) COL-SAll) -5A(2) -SA(3) COL-6A(1) -6A(2) -6A(3) -6A(4) -6A(6) -6A(7) DG-1A(1) -1A(2) -1A(3) -IR(1) -lB(2) DG-2A(1) -2A(2) -2A(3) Picea pungens T Pinus strobus Picea engelmannii H r-i HHHHHHHHHHHHHHHHHHHHHHH r-lv-ir-Ji-lr-Jr-IHD-J a Sources of isolate: T-infected tissue. conidium. MC-mass of conidia, SC-single 20 TABLE 2 (cont'd.) Isolate Host Species Source:a DG-3A(1) Picea pungens -3A(2) " ~3A(3) " '3A(4) " DG-4A(1) Picea glauca -4A(2) " -4B(2) " -4D(1) " -4D(2) " EL-ZB(3) Picea pungens -ZB(4) " EL-3A(1) " -3A(2) " -3A(3) " -3A(4) " EL-8A(1) Picea abies -8A(2) " -8A(3) " EL-9A(l) " -9A(2) " -9B(1) " -9B(2) " -9B(3) " -9C(1) " EL-10A(1) Picea pungens -1OB(1) " -1OB(2) " EL-12A(1) " -IZB(1) " -lZB(2) " EL-13B(1) Pseudotsuga menziesii -13B(2) " EL-14A(1) Picea pungens -14A(2) " '14A(3) " '14A(4) " EL-15A(3) " ~15A(4) " EL-16B(1) " -16B(2) " Hear-3P3v-ir-ir-ir-Jt-ir-JD-JHHHHHHHHHHHHHHHHHHH r-lt-ir-ir-Jr-Jr-Jt-lv-ir-i .a Sources of isolate: T-infected tissue, MC-mass of conidia. SC-single conidium. 21 Table 2 (cont'd.) Isolate Host Species Sourcea EL-16B(3) Picea pungens T -16B(4) " T EL-17A(1) " T -17A(2) " T ‘17A(3) " T -17B(1) " T '17B(3) " T '17B(4) " T '17B(5) " T -17B(6) " T EL-21A(3) Picea glauca T -ZlB(l) " T -ZIB(2) " T -21B(3) " T EL-22A(2) " T ‘22A(3) " T EL-23A(1) Picea pungggs T '23B(1) " T '23B(2) " T EL-24A(Z) " T EL-25A(1) " T -25A(2) " T -25A(3) " T EL-26B(l) Picea glauca T '26B(2) " T EL-27A(1) " T -27A(2) " T -27A(3) " T '27B(1) " T '27B(2) " T -27C(1) " T -27C(2) " T -27D(l) " T °27D(2) " T EL-28A(l) " T '28A(2) " T EL-29A(1) " T -29A(2) " T '29B(1) " T ‘29B(2) " T a Sources of isolate: T-infected tissue. MC-mass of conidia, SC-single conidium. 22 TABLE 2 (cont'd.) Isolate Host Species Sourcea EL-29C(1) Picea glauca T -29C(2) " T EL-3OA(1) " T -30A(2) " T -30A(3) " T -30B(1) " T -3OB(2) " T -30B(3) " T -3OB(4) " T -30C(1) ” T -30C(2) " T EL-31A(1) Picea pungens T -3lB(1) " T ~318(2) " T -31C(1) " T -31C(2) " T F-1A(l) " T -1A(2) " T F-2C(1) " T -2C(2) " T F-3A(3) " T F-4A(1) " T '4A(2) " T -4B(1) " T -4B(2) " T F-SB(1) " T -SB(2) " T F-6A(1) ” T ~6A(2) " T F-7A(1) " T -7A(3) " T -7A(4) " T F-8B(1) " T -88(2) " T -88(3) " T F-9A(l) " T -9A(2) " T -9A(3) " T F-10A(1) Picea abies T a Sources of isolate: T-infected tissue. MC-mass of conidia. SC-single conidium. 23 TABLE 2 (cont'd.) Isolate Host Species Source F-10A(2) Picea abies F-11A(1) " F-12A(1) " -12A(2) " -12A(3) " -12A(4) " HHHI—it-ir-J FK-1A(1) Picea pungens -1A(2) " '1A(3) " -1A(4) " '1A(5) " -1B(1) " -1B(2) " —IB(3) " -lB(4) " -1C(2) " -1D(1) " -1D(2) " -1D(3) " FK-2B(1) " -2B(2) " HHHHHHHHHHHHHHH H-IB(1) " H-2B(1) Picea glauca H-3A(3) " H-4A(2) PiCea pungens H'5A(2) " '5B(2) " -5C(l) " ‘5C(2) " -SC(5) " -5D(1) " -5D(2) " -5D(3) " H-6A(1) " -6A(2) " H'7D(1) " _7D(2) H H-8A(1) Picea glauca -8B(1) " HHHHHHHHHHHHHHHHHH a Sources of isolate: T-infected tissue. MC-mass of conidia. SC-single conidium. 24 TABLE 2 (cont'd.) Isolate Host Species Source; H-8B(2) Picea glauca -8C(1) " -8C(2) " H-9A(1) Picea pungens -9A(2) " -9B(1) " -9B(2) " H-10A(l) " -10A(2) " -IOB(1) " -1OB(2) " -10C(1) " H-11A(l) " -11A(2) " -llB(1) " -11C(1) " -11C(2) " H-12A(l) Picea glauca HL-1B(1) Picea pungens -lB(2) " -lB(3) " ‘8‘“) H HL-2C(1) " '2C(2) " ‘2C(3) " -2C(4) " HL-3A(1) Picea abies '3A(2) " '3A(3) " -3A(4) " -3A(6) " '3A(7) " HL-4A(2) Picea pungens -4B(1) " -4B(2) " -4B(3) " -4B(4) " '4C(1) " ‘4C(3) " HHHHHHHHHHHHHHHHHHHHH HHHHHHHHHHHHHHHHHH Sources of isolate: T-infected tissue. MC-mass of conidia, SC-single conidium. 25 TABLE 2 (cont'd.) a Isolate Host Species Source- HL-4D(1) Picea pungens -4D(2) " -4D(3) " HL-SC(1) " -SC(2) " -5C(3) " -5D(1) " -SD(2) " -SD(3) " HL-6A(1) " -6A(2) " -6A(3) " -6A(4) " HL-7A(1) " -7A(2) " -7A(3) " -7A(4) " -7B(1) " -7B(2) " -7C(1) " -7C(2) - " -YC(3) " -7C(4) " HL-8A(1) Tsuga canadensis -BB(1) " -88(2) " -8B(3) " -8B(4) " -8B(S) " -8B(6) " v-lr-ii-ir-lr-ir-iI-lt-it-ir-lt-lr-lt-lHHHHHHHHHHHHHHHHH M-1A(1) Picea pungens -1A(Z) " -1A(3) " M-2A(1) " -2A(2) " M-3A(1) Picea abies -3A(2) " M-4B(1) Picea pungens -4B(2) " HHHHHHv-Jr-JD-J 3 Sources of isolate: T-infected tissue, MC-mass of conidia. SC-single conidium. 26 TABLE 2 (cont'd.) Isolate Host Species Source.a M-5A(1) Picea pungens M-6B(2) " M-7A(3) " -7A(4) " -7B(1) " -7B(2) " -7B(3) " l-il-Jr-JO-it-ir-ir-i MAS-1B(3) " MAS-2A(1) Picea abies MAS-3A(5) " -3A(6) " -3A(7) " -3A(8) " MAS-48(1) Picea pungens -4B(2) " -4B(3) " '4B(4) " -4B(5) " -4B(6) " -4D(1) " -4D(2) " -4D(3) " -4D(4) " -4D(5) " MAS-5A(1) " -5A(2) " -SB(1) " -5B(2) " -5B(3) " -SB(4) " -SB(5) " ~5B(6) " -SC(1) " -SC(2) " -5C(3) ” -SC(4) " Habit-38*]HHHHHHHHHHHHHHHHHHHHHHH MSU-1A(1) " MSU-2A(1) " 0-36 .a Sources of isolate: T-infected tissue. MC-mass of conidia. SC-single conidium. 27 TABLE 2 (cont'd.) Isolate Host Species Source-a MSU-2A(2) Picea pungens -2A(3) " -2B(1) " -ZB(2) " -2B(3) " MSU-3A(1) " MSU-4A(l) " MSU-SB(1) Picea abies ’SB(2) " MC -5B(3) ” MC -SB(4) " SC -SB(S) " SC ‘53(6) " SC -SB(7) " SC -SB(8) " SC -SB(9) " SC -SB(10) " SC -SB(11) " SC -SB(12) " SC Fir-lD-Jv-li-Jr-Jt-ir-i OK-1A(1) Picea pungens -1A(2) " ‘1B(1) " '1B(2) " -1B(3) " OK-2A(1) " _2A(2) H -2C(1) " -2C(2) " OK-3C(1) " -3C(2) " -3C(3) " OK-4A(l) " -4A(2) " -4B(1) " -4B(2) " '4B(3) " -4B(4) " OK-SA(2) " OK-6C(1) Picea gIauca HHHHHHHHHHHHHHHHHHHH a Sources of isolate: T-infected tissue, MC-mass of conidia, SC-single conidium. 28 TABLE 2 (cont'd.) Isolate Host Species Sourcea OK-7A(1) Picea glauca T -7A(2) " T OK-8C(l) Picea pungens T -8C(2) " T OK-9A(l) Picea glauca T -9A(2) " T ~9A(3) " T '9B(1) " T ~9B(2) " T °9C(1) " T -9C(2) " T -9C(3) " T OK-10A(1) " T -10A(2) " T -1OB(1) " T -IOB(2) " T 'IOB(3) " T '10C(1) " T '10C(2) " T OK-11A(1) Picea pungens T ‘11A(2) " T '113(1) " T '1lB(2) " T OK-12A(1) " T -12A(2) " T -1ZB(1) " T '123(2) " T -1ZB(3) " T R-1A(1) " T -1A(2) " T °1B(1) " T -lB(2) " T '1B(3) " T -1B(4) " T R‘2A(1) " T -2A(2) " T '2A(3) " T ‘2A(4) " T °ZB(1) " T 3 Sources of isolate: T-infected tissue. MC-mass of conidia. SC-single conidium. 29 TABLE 2 (cont'd.) Isolate Host Species Sourcea R-2B(2) Picea pungens -ZB(3) " -2C(1) " -2C(2) " -2C(3) " V-1A(1) " -1A(2) " -1A(3) " -1D(1) " -1D(2) " V-ZB(1) " -ZB(2) " -2B(3) " -2C(1) " -2C(2) " '2E(1) " -2E(2) " -2E(3) " V-3A(1) " V-4D(1) " -4E(1) " -4E(2) " V-SB(1) " -SB(2) " -SB(3) " -5C(1) " -5C(2) " -SC(3) " -SC(4) " V-GB(2) " V-7A(l) Picea glauca -7A(2) " -7A(3) " V-8A(1) Picea pungens -8A(2) " -8A(3) " -8C(1) " -8C(2) " -8C(3) " Pit-3t-Jt-Jt-it-JO-lHHHHHHHHHHHHHHHHabit-366688886 60-31-366 a‘Sources of isolate: T-infected tissue. MC-mass of conidia. SC-single conidium. 30 TABLE 2 (cont'd.) Isolate Host Species Sourcea V-9A(1) Picea pungens -9A(2) " -9D(1) " -9D(2) " V-10D(1) " V-11A(2) Picea abies -11A(3) " V-12A(1) Picea glauca V-13A(1) " -13A(2) " -13A(3) " V-14A(1) " -14A(2) " -14A(3) " V-15A(1) Picea pungens -15A(2) " V-168(l) " V-17A(1) " HHHHHHHHHHHHHHHHHI—i a Sources of isolate: T-infected tissue. MC-mass of conidia. SC-single conidium. 31 Douglas-fir. Cultural morphology- The appearance of I. 322222 isolates in culture is affected by the type of medium and by lighting. On all of the media.the colonies were generally appressed, without any 'fluffy' aerial hyphae. Colonies generally started out buff colored and in the light progressively turned amber. and then darkened to a grey/brown. On any given medium the isolates all looked similar. However,there were a few exceptions. Several isolates remained white even under the lights. Isolates which remained white were: EL-10B(1). F-2C(1)(2), V-4D(1). V-10D(1)(2). and HL-7B(1)(2). 8 isolates in all. Isolate EL-21A(3) which was isolated from a white spruce grew slowly in culture. Vegetative compgtibilityggroups- The colonies of the I. 52955; isolates grew out from the WA plugs which were placed on the PDA pairing plates and made contact with the neighboring colonies (Figure 3.). After 7 to 10 days the type of response could be scored. Using the collection of 487 isolates at least 44 vc-groups were identified within I. NEEEEI. Of these 44 vc-groups. 36 single discrete groups were found (Table 3.) and 8 multi-merge groups were noted (Table 4.). Multi- merge groups showed compatible reactions to isolates contained in different vc-groups. which were themselves incompatible (Figure 4.). yg;groups: conidiogenesis- In those cases where isolates of I. IggggI were started from tissue and from conidia produced on the same canker, the isolates were always in the same vc-group. Conidial isolates from a given pycnidium were also in the same vc-group upon testing. In the supporting experiment, 80 monoconidium isolates obtained from the cirrhi of a single pycnidium were paired and all were contained in the same vc-group as the original isolate producing the pycnidium. 32 FIGURE 3. Determination of vegetative compatibility groups. After the pairings are started the colonies expand and make contact. Compatible colonies merge along the line of contact (A). In the incompatible reaction. a black 'barrage-like' reaction line develops along the line of contact of the two incompatible colonies (B). 33 TABLE 3 Vegetative compatibility groups in Leucocytospora kunzei. VC-GROUP Isolates in the vc-group 1. R-1A(1,2) 2. R-1B(1,2.3,4) 3. R-2A(1,2,3.4). R-ZB(1.2.3). R-2C(1.2.3) 4. EL-lOB(1). F-2C(1,2). HL-7B(1,2) 5. F-1A(l,2). F-4B(1,2), M‘3A(l,2). H-9A(1,2). H-11A(1,2) 6. F-3A(3). F-4A(1,2) 7. F-SB(1.2). F-7A(1,3.4). FK-1A(1.2,3.4,5). FK-1B(1,2.3.4). HL-2C(1.2.3,4). HL-3A(l.2,3,4,6,7) 8. F-6A(1.2). F-8B(l,2.3). H-lB(1.2). H-ZB(1). H-3A(3) 9. F-9A(1.2.3). F-12A(l,2.3.4). M-4B(1,2). M-SA(1). M-7A(3.4). M-6B(2). F-10A(l.2). F-11A(1) 10. DG-lA(1,2,3). DG-1B(1,2). DG-2A(1,2.3) 11. DG-3A(1,2,3,4). V-4E(1.2). V-7A(1,2.3). V-8A(1.2.3). EL-17B(1,3.4.5) 12. DG-4A(1.2). DG-4B(2), V-11A(2,3). H-4A(2). H-12A(1). EL-25A(1,2.3) 13. DG-4D(1,2). M-7B(1,2.3). MSU-SB(1,2,3,4,5,6.7,8,9. 10.11.12) 14. M-1A(1.2,3). V-15(1,2). V-16B(1). V-17A(1) 15. M-2A(1.2) 16. MSU-2A(1.2.3) 17. MSU-2B(1,2,3). FK-2B(1,2). EL-16B(1,2,3.4). EL-27C(1,2). EL-28A(1.2). EL-30A(1,2.3). EL-30B(1.2.3.4). EL-30C(1.2). 34 TABLE 3 (cont'd.) VC-GROUP Isolates in the vc-group 18. BC-3A(1). BC-3B(2) 19. EL-3A(1.2.3.4), EL-6A(1.2,3) 20. BF-6A(1,2.3), BF-6B(1,2,3.4), BF-6C(1). BF-6E(1). BF-7A(1.2), BF-7B(1) 21. BF-7C(1.2,5.6.7), BF-7D(1,2.3,4,S,10,11), BF-7E(1,2.10). BF-7F(1.2.3). BF-7G(1,5,6). BF-7H(1.2.3.4), BF-7I(1,2). EL‘29A(1.2). EL-29B(1.2). EL-29C(1.2). EL-31A(1). EL-318(1,2). EL-31C(1.2). EL-26B(1.2), EL-27A(1,2,3), EL-27B(1.2). EL-27D(1,2). EL-21A(3), EL-ZlB(1.2,3), EL-22A(2.3), OK-1A(1.2), OK-lB(1,2,3). OK-4A(1,2). OK-4B(1,2,3,4). OK-5A(2). OK-6C(1), OK-7A(1,2), OK-8C(1,2). OK-9A(1,2.3), OK-9B(1.2), OK-9C(1,2.3). OK-10A(1.2). OK-lOB(1,2,3), OK-10C(1,2), OK-11A(1,2). OK-llB(1,2), OK-12A(1.2), OK-128(1.2.3), BF- WP 22. FK'1C(2)9 FK'1D(19293) 23. V-SB(1,2,3). V-5C(1.2.3.4), V-6B(2). V-13A(1.2.3), V-2C(1,2). V-2E(1.2,3). HL-SC(1.2.3). HL-5D(1,2,3). HL-6A(1.2.3.4), HL-7A(1,2,3,4), HL-7C(1,2.3.4), COL-1A(1,2,3,4,5) 24. V-9A(1,2), V-9D(1,2). V-8C(1,2,3) 25. V-4D(1), V-10D(1,2) 26. V-3A(1), V-12A(1). V-14A(1.2.3) 27. HL-8A(1). HL-BB(1,2,3,4,5.6) 28. MAS-18(3). MAS-2A(l), MAS-4B(1.2,3,4,5,6). MAS-4D (1.2.3.4.5), COL-4A(1,2,3,4) 29. MAS-3A(5.6.7,8) 30. H-5A(2). H-5B(2). H-5C(1,2,5), H-5D(1,2,3). H-6A(1.2), H-8C(1.2). H-10A(1,2). H-lOB(1,2), H-10C(1), H-11C(2) 31. H-9B(1,2), H-llB(1). H-11C(1) 32. EL-138(1.2) 33. EL-23A(1). EL-23B(1.2) 35 TABLE 3 (cont'd) VC-GROUP Iaolates in the vc-group 34. EL-24A(1.2) 35. COL-3A(1.2,3.4,5) 36. COL-5A(1.2,3), COL-6A(1,2.3,4.6.7) 36 TABLE 4 Multi-merge vegetative compatibility groups in Leucocytospora kunzei Multi-merge Isolates in Compatible Late group the group vc-groups incompatibility MMl EL-ZB(3.4), EL-9A(1,2), EL-9B(l,2.3). EL-9C(1). EL-12A(1). HL-1B(1.2,3,4). EL'IZB(1,2), MSU‘1A(1)9 20 33 HL-4A(2). HL-4D(1.2,3), ' HL'4C(193)9 MSU'3A(1)D HL-4B(1,2,3.4), MSU-4A(1) MM2 BC-1A(1). BC-lB(1). V-lA(1,2,3), V-1D(1.2) 1,19 V'ZB(19293) MM3 MAS-5A(1,2), MAS-5B(1,2,3, 4.5.6). MAS-5C(l,2,3,4). 2.10 EL'14A(1929394) MM4 OK-2A(l.2), OK-2C(1,2) 30,MM6.MM7, 31,MMS MM8 MMS OK-3C(1,2.3) 30.MM6.MM7, 31,MM4 MM8 MM6 EL-8A(1,2,3), EL-10A(1). 30.MM4,MM5, 31 EL-lOB(1,2) MM7,MM8 MM7 EL-17A(1,2.3), EL-17B(6) 30,31.MMS MM4 MM6.MM8 MM8 H-8A(1). H-BB(1,2) 30,31.MM4, MM5,MM6,MM7 a Late incompatible reactions occurred between some of the multi-merge groups. much later than normal. In this case the black reaction line did not appear until Most reactions were very visible at 7 days. late incompatibility showed up at 14-20 days. 37 FIGURE 4. The multi-merge response. The isolate in vc-group MM-8 is compatible with isolates in vc-groups 30 and 31. VC-groups 30 and 31 however, are incompatible. 38 Vegetative compatibility groups: distribution patterns Within a canker- Within a single canker.generally all of the individual isolates were contained in a single vc-group. Upon exam- ining 196 individual cankers. in only two cases were isolates obtained from a single canker which were in different vc-groups. Within a tree- Generally. within a single tree when more than one canker was collected, the isolates obtained were in the same vc-group. It was not unusual however. for isolates to be contained in two vc-groups. One tree. BF-7. was sampled extensively. Of the nine cankers examined. seven yielded isolates in a single vc-group while the other two were in a vc-group which was the same as a neighboring tree. Within neighboring trees- Several study sites included more than one tree in the planting in close proximity, Generally trees located near each other yielded isolates in the same vc-group of groups. For example. six trees sampled in a yard in East Lansing [EL-26 through EL-31] yielded isolates in only two vc-groups (vc-groups l7 and 21). Within distant sites- Some vc-groups contained isolates which were collected from different.and in some cases widely separated sites within Michigan, and even included some Colorado isolates (Figure 5). Amonggdifferent tree species- Some of the vc-groups contained isolates which originally came from different tree species (Table 5). The I. Iggggi isolates from eastern hemlock and Douglas-fir however, were not in the same vc-groups as any of the spruce isolates. Iggggythe white isolates- The white isolates formed two discrete vc-groups (vc-groups 4 and 25) and were not compatible with any of the normally pigmented isolates. 39 ]/ SAGINAW Dow Gardens [:;:>(11.12.13)fl F"\C\ Midland gay City (MM-2) (5.9.13.14) Ségjipw ,p 'Richville ' Vassar-(11.12.14.23 'Frankenmuth MM-2) (4.5.7.8.9) COLORADO Michigan . (23.28) Flint East Lansing-(4,11,12.17,21,MM-1,MM-3) l MSU-(13 17 MM-l) : BF-(Zl) ’ I-Haslett-(5.8.12) Pontiac Lansin . Okemos-(Zl) ~Holt-(4, .23 MM- 1) . Mason-(28,MM-3) (7.17)'Franklin Warren ‘ FIGURE 5. Vegetative compatibility groups found at more than one site. VC-groups which contained isolates from different sites are listed by the sites from which they were isolated. 40 TABLE 5 Vegetative compatibility groups isolated from more than one host species. vc-group Hosts isolated from 5. Pic—ea pungens, 3. 5915s 7. P. pungens, g. abies 8. g. pungens, g. gLLca 9. P. pungens, g. abies 11. _P. pungens, g. M 12. g. pungens, g. M, g. gl_at£§ 13. 3. un ens, g. abies, g. glauca 17. g. gaggens, g. glgggg 21. g. pungens, g. glauca, £3225 strobus 23. g. pungens, P. glauca. g. engelmann'i 26. P. pungens, g. glaugg 28. g. pungens, g. abies, g. engelmannii 30. g. pungens. g. glauJ MMl P. pungens, P. M MG _13. pungens, g. alga; DISCUSSION Vegetative compatibility in fungi,as stated by Kulman, "refers to the characteristic enabling two fungi to make contact, fuse, and exchange cytoplasm or nuclear material." (19). Vegetative compatibility therefore is an important biological feature of fungi in that it governs heteroplasmon and heterokaryon formation respectively (7,8,19). Isolates which are vegetatively compatible make up a vc-group. Based on this survey of 483 isolates there appears to be at least 44 vc-groups in Leucocytospora_§ggggi. Large numbers of vc-groups have also been demonstrated in other ascomycetes: Cryphonectria parasitica (1.19.21), Podospora anserina (20), Neurospora grass; (10,12), and several Aspergillus species (6,8,11,16,17). Vegetative compatibility has been found to be homogenic in all examined cases (1,2). That is. in order for two isolates to be veg- etatively compatible they must have common alleles at one or more loci. Based on the number of vc-groups obtained for p. 53222;, there appears to be at least six loci involved in regulating vegetative compatibility in this fungus. This was determined according to the method of Anagnostakis (2), where the number of vc-groups is used to indicate the number of loci involved (where N equals the number of loci, the number of vc-groups = 2N). This leaves the potential for at least 64 vc-groups to occur in L. 52353;. Evidence supports the idea that homologous alleles are not required at all of the compatibility loci for a compatible 41 42 reaction to occur. Alleles at some loci appear to have a quantitative effect while alleles at other loci produce a qualitative effect (3,8,). In L. kggggi the multi-merge groups demonstrate this feature in that they are compatible with more than one vc-group. This has been taken as an indication of close genetic relationships between the vc-groups, with homogenic alleles at many but not all alleles (2). This would indicate that isolates in vc-group 20, vc-group 33. and vc-group HH-l are closely related to each other (Table 6.). Vegetative compatibility governs hyphal anastomosis and the exchange of cytoplasmic or nuclear materials. The main biological impact of this is that it functions as a cellular defense mechanism against genetic infection (7,8). Genetic infection would include such agents as viruses, 'virus-like' particles, and/or defective mutant derivatives of essential cytoplamic elements (7). It would also include other nuclei. Hartl (12) suggests that vegetative compatibility, rather, vegetative incom- patibility, may function to protect an established mycelium adapted to its environment from exploitive nuclei less well adapted to the environmental conditions. Unfortunately vegetative compatibility works against using biological control agents such as viruses or virus-like agents.such as the dsRNA associated with hypovirulence in Q. parasitica. Information gained here on L. kunzei with regards to vegetative compatibility will be useful in approaching future efforts in the biological control of this fungal pathogen. Fortunately multi-merge groups do occur which may serve as future 'delivery agents' for virus or virus-like biological control agents. Encouragingly, it has been noted in Q. parasitica that there is sometimes conversion between some hypovirulent and virulent 43 isolates which are not vegetatively compatible (3.15). This occurs presumably due to the fact that the several loci involved in determination of vegetative compatibility effect different steps in the anastomosis and exchange process. In some cases enough steps may occur to allow the transfer ofauiinfective virus-like agent, such as the ds-RNA particle in Q. parasitica (19). The set of multi-merge isolates which showed some late incompatibility responses may be explained along these lines (Table 4.). Examining the distribution of vc-groups in E. kggggi is not only important in a biological sense but has implications for pathological studies. Several factors seem to indicate that conidia do serve a role in the dissemination of this pathogen. It was shown that conidal isolates of L. kggggi were always in the same vc-group as their original isolate. Vegetative compatibility groups do not appear to segregate during conidiogenesis. This has also been noted with Q, parasitica. No perithecia of E. kggggi_were found during the 3 years of this research, and are rare on blue spruce. It has not been shown if vc-groups segegrate during ascosporogenesis in L. 32253; as they do in Q.parasitica. There was a tendency, at several levels, for a single or few vc- groups to predominate. Cankered branches within a single tree or in neighboring trees often yielded isolates within the same vc-group or groups. Generally within a single cankered branch,the isolates found were in the same vc-group. Trees located further apart showed more variety with regard to vc-groups. However, vc-group 21 didoccur widely in those sites centered around East Lansing (Table 3., Figure 5.). Short range spread via conidia could explain the predominance within cankers, tree, and neighboring trees of a single or few vc-groups. Long distance 44 Spread may be due to ascospores which could account for some of the variety in vc-groups encountered. The tendency for a single or few vc-groups to occur on a tree is similar to the situation with chestnut blight in Europe. On chestnuts in Europe, a single or a few vc-group(s) occur in a tree or in neighboring trees. In the United States there are commony many vc-groups of Q. parasitica within in a tree or single canker. In the United States ascospores have been found to disseminate this fungus but in Europe ascospores are rare and conidia serve as the primary means of dissemination (19). Also of pathological interest, several vc-groups were represented on more than one species of spruce. This in combination with the results of inoculation trials indicate that that most if not all species of spruce may be serving as reser- voirs of inoculum capable of infecting other spruces. Here again vegetative compatibility has applications for pathological studies. Another use of vegetative compatibility is as a marker for inoc- ulation studies. By surveying the vc-groups present before inoculation trials it is then possible to use isolates known to be in different vc-groups for inoculation studies. Using vc-group analysis one can accurately verify the identity of the isolates recovered from any in- oculations done on the tree. In this way vegetative compatibility can serve as a non-mutational marker for epidemiological studies. Using vc-groups as markers would also presumably avoid and unintentional selective pressures which might occur when using mutational markers. No apparent 'hypovirulent' isolates of L. kggggi were found in this survey. The slow growing isolate, EL-21A(3), was used in inocu- lation experiments (isolate WS) and proved to be virulent. The patho- genicity of the white isolates has not been fully determined but it 45 appears that they are also capable of canker formation, at least in a few inoculated excised branches of blue spruce. 10. 11. 12. 13. LIST OF REFERENCES Anagnostakis, S. L. 1977. Vegetative incompatibility in Endothia parasitica. Exp. Mycology 1:306-316 Anagnostakis, S. L. 1980. Notes on the genetics of Endothia parasitica. Neurospora Newsletter 27:36 Anagnostakis, S. L. and P. R. Day. 1979. Hypovirulence conversion in Endothia parasitica. Phytopathology 69:1226-1229 Anagnostakis, S. L. and P. E. Waggoner. 1981. Hypovirulence, vegetative incompatibility and growth of cankers of chestnut blight. Phytopathology 81:1198-1202 Barr, M. E. 1978. The Diaporthales in North America. Mycologia Memoir No. 7. J. Cramer Pub. Lehre, Germany 232 p. Canten, C. E. 1971. Heterokaryon incompatibility in imperfect species of Aspergillus. Heredity 26: 299-312 Canten, C. E. 1972. Vegetative compatibility and cytoplasmic infection in fungi. J. Gen. Microbiol. 72:221-229 Canten, C. E. 1973. Genetic control of heterokaryon incompati- bility and its effect on crossing and cytoplasmic exchange in Aspergillus amstelodami. Genetics 74:40 Funk, A. 1981. Parasitic Microfungi of Western Trees. Canadian Forestry Service. Victoria, B.C. Canada 190 P- Garnjobst, L. and F. Wilson. 1956. Heterokaryosis and proto- plasmic incompatibility in Neurospora crassa. Proc. Natl. Acad. Sci. US 42-613-618 Grindle, M. 1963. Heterokaryon compatibility of unrelated strains in the Aspergillus nidulans group. Heredity 18:191-204 Hartl, D. L., E. dempster, and S. Brown. 1975. Adaptive sig- nificance of vegetative incompatibility in Neurospora crassa. Genetics 81:553-569 Hepting, G. H. 1971. Diseases of Forest and Shade Trees of the United States. Agricultural Handbook 386. USDA Forest Service 658 p. 46 15. 16. 17. 18. 19. 23. 47 Jaynes. R. A. and J. E. Elliston. 1978. Control of Endothia parasitica canker on American chestnut sprouts with hypo- virulent strains. symposium: l978-Morgantown, 122 p. Proceedings of the American chestnut West Virginia University Books Jaynes, R. A. and J. E. Elliston. 1980. Pathogenicity and canker control by mixtures of hypovirulent strains of Endothia parasitica in American chestnut. Jinks, J. L. et a1. 1974. iation in wild populations of Aspergillus nidulans. 21:227-239 Jinks, J. L. and M. Grindle. Phytopathology 70:453-456 Heterokaryon incompatibility and var- Heredity 1963. The genetical basis of hetero- karyon incompatibility in Aspergillus nidulans. Heredit 18:407-411 Ramiri, L. K. (Picea pungens): control. Lansing, Michigan Kulman, E. ‘ b. conversion in Endothia parasitica: Y 1980. Cytospora canker of Colorado blue spruce Ph. D. Thesis, Michigan State University. East 103 p. Etiology. symptomology, epidemiology and 1982. Vegetative incompatibility and hypovirulence State of the art. (i Proc. USDA For. Serv. Am. Chestnut Coop. Meeting. H.C. and W.L. MacDonld, eds. Morgantown. 229 p. Labarere, J. et a1. 1974. of a nonallelic system. MacDonald, W. L. West Virginia University Books, n) Smith Incompatibility in Podospora anserina. Comparative properties of the antagonistic cytoplasmic factors J. of Bacteriology 120:854-860 and M. Double. 1978. Frequency of vegetative compatibility types of Endothia parasitica in two areas of West Virgiia. (in) Symposium: Proceedings of the American Chestnut 1978-Morgantown, West Virginia University Books 122p. Waterman, A. M. 1955. The Relation of Valsa kunzei to Cankers on Conifers. Wehmeyer, L. No. 6. 1975. Pyrenomycetous Fungi. J. Cramer Pub. Lehre, Germany Phytopathology 45:686-692 Mycologia Memoir 250 p. SECTION 3 Wound inoculations of excised and intact branches of blue spruce with Leucocytospora kunzei and other Cytospora s.lat.isolates INTRODUCTION Blue spruce (Picea pungens Engelm.) is widely planted in the mid- western and northeastern United States as a landscape ornamental. The unique coloration and dense pyramidal crown of this tree make it a popular specimen tree (2). Blue spruce however, is often progressively disfigured by Cytospora canker (5,9,15,16). Cytospora canker of blue spruce is characterized by the occurrence of diffuse cankers on the limbs of mature trees (5,7,13,15,16). Cankered branches are commonly covered with resin exudations. Trunk cankers and tree mortality are not common in blue spruce (9.16) but the loss of branches associated with this disease destroys the symmetry and aesthetic value of these large and often strategically located landscape trees. Cytospora canker of spruce is caused by Leucostoma 52253; (Fr.) Munk ex Kern, which is seen almost exclusively in its anamorphic form, Leucocytospora kunzei (Sacc.) Urban (1,3,17). This pathogen is still commonly refered to as Cytospora kunzei in the pathology literature (telemorph- Kalggpkggggl) (5.7.8.9,11,12,15,16). The genera Valsa and Leucostoma and their associated anamorphic forms, Cytospora and Leucocytospora, are closely related. Leucostoma and Leucocytosporg are considered by some to be subgroups within Valsa and Cytospora (1,14). For the purpose of clarity I will use Cytospora in the broader sense, Cytospora g. l§£., which would include fungi in the genus Leucocytospora including L. kunzei. Virtually every mature blue spruce in Michigan is affected by this 49 50 pathogen at some point in maturity. Blue spruce however, exists as a non-continuous population in the region, restricted primarily to widely separated sites as ornamental plantings. The spores of L. ku_n_z_e_i_ and Cytospora g. 133. are disseminated primarily via rainsplash (6,9,10). This combination of factors would seemingly make it unlikely that so many trees would be affected” ‘This suggests the possibility that other tree species may be serving as sources of inoculum capable of infecting blue spruce. Essentially every woody plant species examined has been found to be colonized by some species within Cytospora g. 125. (4,5). The various Cytospora spp. fungi have generally been treated as distinct species in the past, with 400 species 'having been described (14), many on the basis of their host association. There have been few pathogen- icity or host range studies done with these fungi. The variability in Cytospora g. lat. conidiomata is high even on a single host species (14). The possibility exists that we may be dealing with a single or a few Cytospora g. 125. species which have a wide host range but have been treated as distinct species on the basis of their host assoc- iation. Leucocytosporg kunzei may owe its characteristic conidiomata type only to the particular nature of the bark of its spruce hosts, and Cytospora g. lat. isolates from other hosts, upon infecting spruce, may look similar. The purpose of this research was primarily to get some indication as to whether other hosts of Cytospora 3. lat. are serving as reser- voirs of inoculum capable of infecting blue spruce. That is. are Cytospora g. 123. isolates from non-spruce hosts capable of infecting blue spruce. 51 The observed tendency of Cytospora canker of blue spruce to occur predominantly on mature trees (16) poses methodological problems when studying this disease. In Michigan, and other areas in the Midwest and Northeast, most mature blue spruces are located in highly visible and aesthetically important sites. This factor tends to restrict the amount of available material for experimentation. Past inoculation studies have utilized young nursery sized trees for experimentation (8.12.16). yet young trees are rarely affected by this disease. For that reason the applicability of using nursery sized trees for inoc- ulation studies has been questioned (16). Because of the aforementioned criticism, these inoculations were done on mature blue spruces. In an effort to increase the amount of host material available for exper- imentation, a secondary goal of this research was to examine the efficacy of inoculating excised branch segments from mature trees to study the pathogenicity of isolates of Cytospora 3. lat. on blue spruce. MATERIALS AND METHODS Isolates- Sixteen Cytospora 5. lat. isolates were used in these inoculation experiments. Four of the isolates were from spruce hosts and twelve came from other tree species (Table 1). All of the isolates were originally isolated from infected cambial/cortical tissues except the isolate from Norway Spruce which was started from a mass of conidia. The isolates were grown on 22 Malt Extract Agar (22 MEA I 20 g Difco Malt Extract and 20 g Difco Bacto-Agar per liter of distilled water) at 26°C prior to inoculation. Excised branch inoculations- Live healthy branches from mature 52 mcofinam .m wagon msouoso xmo nmwamcm mo manommomxuno .w .eam maHsmom .cuua%z. .coam< mm manoamom>uno .m muoucoouocmuu usasmom . nuoou man .coem< .o mofium>HNm msmmm comma uoaooo vow m maoumooaoa muoamouMoooaoA mofimuom museum nomom m mumH::cm muonmoumw mmofiocmumam woo< magma >m39oz : :ofiumowmwucopH umo: ascwmauo opoo oumHomH ..e.uaoov a manna 54 blue spruce trees were collected and brought into the laboratory. Branches were collected in both the summer and winter months. Branches at least 2 cm in diameter were cut into sections 30-40 cm long and all of the smaller branchlets and needles were removed. The cut ends and branch stubs were sealed with molten paraffin wax to prevent drying. The site on the branch to be inoculated was surface disinfested by dabbing with 952 ethanol and flaming. Flap wounds 7 X 7 mm, were cut in the bark and small slabs of mycelium with the subtending agar medium were placed under the flaps. Sterile MEA was placed into the wounds for the control treatment. The flaps were closed and wrapped with Para- film M®. The inoculated twigs were placed into loosely closed plastic bags to incubate for 7-10 days. A total of 403 inoculations were made on excised branches. Intact branch inoculatiggg- Live. intact branches of mature blue spruces were inoculated in the summer of 1984 and 1985. The wound-flap inoculations were done as described previously. but the surface disin- festing step was omitted. Inoculations were made along the branches between the points were the secondary or tertiary branchlets were growing. The small branchlets were not removed. The inoculations were wrapped with brightly colored tape to facilitate locating the inoculation sites later. The inoculations were examined after 3 weeks or more. A total of 289 inoculations were done on intact branches in the trees. Evaluation- The wound inoculations were evaluated by stripping the bark off the branches and measuring canker development as indicated by a brown discoloration of the cambial/cortical tissues. Cankers were measured along the axis of the branches and at the widest point longitudinally. RESULTS The response of blue spruce to wound inoculations with Cytospora g. 119 was dependent on the particular isolate tested. On excised branches some isolates caused canker initiation. as indicated by the occurrence of expanding areas of discoloration in the cambial/ cortical tissues. Other isolates and the sterile medium control did not cause canker initiation. However, the actual wound flaps were commonly discolored to some extent (Figure 1). Resin was not generally exuded at the inoc- ulation site. Similar results occured on inoculated intact branches, with some isolates causing canker formation. Heavy resin exudations were noted at the inoculation sites where canker initiation was successful. Control inoculations or isolates incapable of initiating canker form- ation did not elicit the heavy resin exudation. Based on their ability to incite canker formation on inoculated branches, the 16 Cytospora g. 135. isolates varied in their pathogenicity to blue spruce. Canker initiation and expansion was seen on both excised and intact branches when inoculated with the isolates taken from spruce or eastern white pine (Table 2). For each of the isolates tested, the percent canker initiation (Number of developing cankers/number of inoc- ulations) was similar on both excised and intact branches. Differences in the virulence of the four L. kunzei isolates from spruce were detected. Inoculations of excised and intact branches from an individual tree by these isolates resulted in cankers of various sizes. Measurements of the developing cankers indicated that the isolate 55 56 FIGURE 1. Canker initiation on an excised branch of blue spruce inoculated with Cytospora g. lat. isolates. Isolate WS which was originally isolated from a spruce caused canker initiation on this inoculated excised branch segment. The other isolates failed to incite canker formation. This branch was evaluated 10 days post inoculation. S7 o.o o.c Houucoo c.o o.o omesm cuonmmum o.o c.o comma woeaoc pom o.o c.o~ nomom o.o c.o magma zmzuoz o.o 9.: xmo nmfidmcm o.o o.o .owunms. .coam< c.o c.o nuoou was .cmam< o.o o.o m~qe< o.c o.o ump~< moon? msosofiooo N.wm o.ma scan mugs: cuoummo c.o c.c xoo~aos cuoummm o6 c5 “8-33:8 mwomacoo nonuo ~.oo n.0a mugs: m.mm o.om xmzuoz n.0m c.mm oomuoHou m.~m o.om moan oomuoaoo moauam monocmum uomucH monocmum vmmfioxm oumHomH one mo cOMumfiuwcH umxcmo N coflumeuacH uoxcmo N umom Hmcwmwuo .mm& .m ouommouxo mo moumaomw an oomsmo oosuom mean no monocmun voumasooca co acqumauaca poxcmo ucoouoe one N mqmdifferences in virulence were indicated among the four L. 52252; isolates from spruce (Table 4). Host effects on canker initiation and expansion were indicated in these experiments. Inoculations of excised branches from two differ- ent blue spruce trees growing at the same site resulted in differences in both the percent canker initiation and in canker expansion (Table 4). Canker initiation and expansion was greater on a tree which was predisposed to Cytospora canker, as indicated by the presence of naturally occurring cankers, than on a previously unaffected tree. The percent canker initiation on excised branches inoculated with the four L. 52552; isolates from spruce was similar regardless of whether the branches were collected in the summer or the winter (Table 5). The increased efficiency seen across Table 5 is probably due to the refinement of the experimental technique. DISCUSSION Wound inoculations of both excised and intact branches of blue spruce indicate that most non-spruce hosts of Cytospora g. léE- are not serving as reservoirs of inoculum capable of infecting blue spruce. None of the Cytospora g. Lag. isolates from deciduous trees were capable of inciting canker formation in inoculated blue spruce trees. Leucocytospora Lungs; isolates from other conifers varied in their pathogenicity to blue spruce. Isolates of L. 52253; from Douglas-fir and eastern hemlock were not capable of inciting canker formation on blue spruce in this study. 59 TABLE 3 Comparison of canker lengths on excised and intact branches of blue spruce inoculated with four isolates of Leucocytospora kunzei. Origin Excised Branches1 Intact Branches1 of 10 days 22 days Isolate (in mm) (in mm) Colorado blue spruce 35.8 t 20.9 26.4 t 12.6 Colorado spruce 27.5 t 10.1 56.0 t 32.9 Norway spruce 62.3 t 10.5 56.8 t 24.4 White spruce 74.9 $21.2 101.0 f 22.7 1The excised branches came from the same tree upon which the intact branches were found, tree BF-8. TABLE 4 Host effects on excised branch inoculations with four Leucocytospora kunzei isolates. Affected trees1 Unaffected trees2 2 Canker Z Canker Isolate Initiation Size (mm) Initiation Size (mm) CBS 88.9 60.4 t 20.0 66.7 30.7 t 11.8 CS 77.8 58.6 t 15.5 33.3 39.0 t 10.1 NS 90.0 55.6 t 20.9 44.4 24.3 t 13.4 WS 88.9 56.6 t 17.0 33.3 26.7 t 18.6 1This tree did have naturally occuring cankers at the onset of the study. 2This tree did not have naturally occuring cankers at the onset of the study. 60 TABLE 5 Percent canker initiation on excised branches of blue spruce inoculated with Leucocytospora kunzei isolates: Branches collected during different seasons. L. kunzei Season during which branches were collected Isolate Summer 1984 Winter 1985 Summer 1985 CBS 77.8 77.8 100.0 CS 75.0 88.9 100.0 NS 62.5 90.0 100.0 WS 100.0 88.9 100.0 61 This agrees with the earlier findings of Waterman (16). However, in contrast to her findings, in this study the L. 52222; isolate from eastern white pine did prove to be pathogenic to blue spruce. This different response could be due to particular isolate differences or could be due to methodology. Waterman (16) utilized nursery trees for her inoculations and she also raised the question of the applica- bility of using nursery trees for inoculation. Using mature blue spruces already predisposed to Cytospora canker. as indicated by the natural occurrence of the disease, is important for studying this disease. Since mature trees are most commonly affected by this disease (7) it seems reasonable that they should be utilized when possible for assessing the pathogenicity or virulence of C as ora g. L35. isolates on blue spruce. No host specificity was detected among the isolates of L. 52352; from the various spruce hosts. Isolates of L. 52552; from white and Norway spruce were capable of inciting cankers of blue spruce upon inoculation. Further evidence in support of this was noted in vegeta- tive compatibility studies with L. 52252;. Isolates of L. 52352; taken from neighboring white and blue spruces were found to be contained in the same vegetative compatibility group at one site. These results have implications for developing a control strategy for Cytospora canker of blue spruce. On the one hand it simplifies the situation in that we probably do not have to consider deciduous trees as potential inoculum sources capable of infecting blue spruce. On the other hand, spruces of various species and potentially other conifers. as seen with eastern white pine, must be considered when developing control strategies for this disease. Even including 62 eastern white pine and all of the spruces as potential hosts and inoculum sources of L. kunzei. the ubiquitous occurrence of this disease through- out Michigan is surprising. Two possible explanations come to mind. In one case. perhaps there is an alternate method of long range dissem- ination. Windborne ascospores have not been reliably documented. Birds could easily be contaminated with the spores of L. 52552;. English sparrows are often seen perching in these trees. The pycnidia exude masses of conidia. which when wet. form slimy. spreading droplets along the surface of cankered limbs. Alternatively, the actual colonization of blue spruce may occur at an earlier time in the tree’s history. Leucocytospora kunzei has been isolated from 'healthy' uncankered tissues. Perhaps infection occurs while the trees are young but cankers do not occur until some predisposing change occurs in the mature trees. If this is the situation, than control strategies will need to protect young trees in spite of the fact that cankers do not generally form on them. Using excised branch inoculations appears to be a reliable and suitable method for catagorizing the pathogenicity and/or virulence of Cytospora g. L35. isolates on blue spruce. The response of both excised and intact branches of blue spruce to the wound inoculations with the various Cytospora g. léE- isolates was similar. No isolate which caused cankers in inoculated trees failed to cause canker initiation on excised branches. Inoculated excised branches also accurately reflected host effects noted in the field. In this examination. branches taken from a blue spruce predisposed to Cytospora canker. as indicated by the occurrence of the disease naturally in the tree, proved to be more susceptible to canker initiation and expansion than excised branches from a tree at the same site which did not have Cytospora canker prior 63 to inoculation. If any differences in the response of inoculated excised and intact branches can be seen.it is that excised branches are more susceptible to canker formation than branches in the trees. This fact is reflected in two areas. In the first. canker expansion is faster in excised branches inoculated with L. @333 than branches left intact on the tree. Secondly, in the only inconsistent response noted in these exper- iments, the L. leucostoma isolate from peach did incite canker formation on excised branches yet failed to incite cankers on intact branches (Table 2). This increased sensitivity in excised branches makes them even more suitable for screening Cytospora g. léE- isolates for being potentially pathogenic to blue spruce. Using inoculated excised branches as a method to study Cytospora canker of spruce offers many benefits. It is a fast (7-10 days). sensitive, and reliable indicator of in yiyg responses of blue spruce to inocul- ation. It utilizes mature tissues which are normally affected by the disease. and one can select branches from trees already predisposed to canker formation. Using these normally affected tissues avoids some of the complicating factors encountered by earlier workers who utilized nursery stock and found that they had to severely drought stress nursery trees in order for any canker formation to occur. This method also increases the amount of material at ones disposal for experimentation. A branch or branches may be collected from a tree and brought into the laboratory.avoiding the aesthetic damage field inoculations cause on these often key landscape trees. Convenience is also a factor in being able to do these tests in the laboratory. One potential application for this technique could be to test for 64 genetic resistance in blue spruce to L. kunzei. Branches could be tested from mature. seed producing trees without introducing or increasing the incidence of the disease in your seed producing stock. ‘4 e 10. 11. 12. 13. LIST OF REFERENCES Barr. M. E. 1978. The Diaporthales in North America. Mycologia Memoir No. 7. J. Cramer Pub. Lehre, Germany 232 p. Dirr. M. A. 1975. Manual of Woody Landscape Plants. Stipes Pub. Co. Campaign, Illinois 826 p. Funk. A. 1981. Parasitic Microfungi of Western Trees. Canadian Forestry Service. Victoria, B.C., Canada 190 p. Grove. W. B. 1935. British stem-and leaf Fungi. Cambridge Univ. Press. Cambridge. England 488p. Hepting. G. H. 1971. Diseases of Forest and Shade Trees of the United States. Agricultural Handbook 386. USDA Forest Service 658 p. Ingold, C. T. 1971. Fungal Spores- Their liberation and dispersal. Clarendon Press. Oxford, England 302 p. Jorgenson, E. and J. D. Calfey. 1961. Branch and Stem Cankers of white and Norway Spruce in Ontario. For. Chron. 37:394-400 Kamiri, L. K. and F. F. Laemmlen. 1981. Effects of Drought- Stress and Wounding on Cytospora Canker Development on Colorado blue spruce. J. Arboric. 7:113-116 Kamiri, L. K. and F. F. Laemmlen. 1981. Epidemiology of Cytospora canker caused in Colorado blue spruce by Valsa kunzei. Phytopathology 71:941-947 Luepschen. N. S. and K. G. Rohrbach. 1965. Cytospora canker of peach trees: spore availability and wound susceptibility. Plant Disease Reporter 53:869-872 Marsden, D. H. 1948. A Valsa associated with Cytospora Canker of Spruces. Phytopathology 38:307-308 Schoeneweiss. D. F. 1983. Drought Predisposition to Cytospora Canker in Blue Spruce. Plant Disease 67:383-385 Strong, F. C. 1953. Spruce branch canker. National Shade Tree Conf. Proc. 29:30-35 65 14. 15. 16. 66 Sutton, B. C. 1980. The Coelomycetes. ,Commonwealth Mycol. Inst. Kew. Surrey, England 696 p. Waterman. A. M. 1937. Cytospora canker of spruce. Plant Disease Reporter 21:55 Waterman, A. M. 1955. The Relation of Valsa kunzei to Cankers on Conifers. Phytopathology 45:686-692 Wehmeyer, L. 1975. Pyrenomycetous Fungi. Mycologia Memoir No. 6. J. Cramer Pub. Lehre, Germany 250 p. SECTION 4 Wound inoculations of excised and intact branches of blue spruce with conidial isolates of Leucocytospora kunzei INTRODUCTION Cytospora Canker of blue spruce (Picea punggns Engelm.) is caused by Leucostoma k_u_n_z;i_ (Fr.) Munk ex Kern [syn. V_a_l£g M]. which is most frequently seen in its anamorphic form, Leucocytospora M (Sacc.) Urban [syn. Cytospora ngggi] (1,2,7). Some workers have indicated that mycelial inoculum from isolates of L. 32952; originating from conidia failed to cause cankers in wound inoculated trees while ascosporic isolates were infective (4). Other workers found conidial isolates to be capable of canker initiation in inoculated trees (3.6.7) . The purpose of these experiments was bore-examine the pathogenicity of conidial isolates of L. 52252; on inoculated intact and excised branches of mature blue spruce trees. MATERIALS AND METHODS Isolates- The isolates of L. kunzei used in these inoculation trials were isolated from either the infected tissues of a cankered spruce branch or from conidia produced on the same branch. Conidia were collected by first soaking the infected branch for 10 minutes in a 52 sodium hypochlorite solution and then rinsing them with distilled water. The cleaned branch was then placed into a moist chamber for 2 hours. Masses of conidia could be soon seen on the surface of the branches (Figure 1.) Conidial isolates were started from either masses of conidia or a single conidium. Isolations were made on Potato 68 69 FIGURE 1. Induced sporulation of Leucocytospora kunzei on spruce. Masses of conidia produced by Leucocytospora kunzei on cankered branches of spruce placed in a moist chamber. 70 Dextrose Agar (PDA). After collecting the spores.the fruiting structures were hand sectioned and examined under the compound microscope to verify that the spores were indeed conidia. Single conidium isolates were obtained by placing a mass of conidia in sterile distilled water. shaking vigorously. followed by serial dilutions umtil discrete single conidia could be observed in a drop of the conidial suspension. The conidial suspension was then streak-plated onto PDA. Prior to inoculations all isolates were transfered and grown on 22 Malt Extract Agar (20 g Difco Malt Extract and 20 .g Difco Bacto-Agar per liter of distilled H20) for 5-7 days at 26°C. Inoculations- The wound inoculations on excised and intact branches from mature blue spruces were done as in previous experiments. Slabs of mycelium from the various isolates, with the subtending agar medium were used as inoculum with sterile medium inoculations for controls. Experiment No. 1 - In experiment No. l, the isolates of L. 52952; were isolated from a cankered branch of a mature blue spruce tree (BF-7) located at the Beaumont and Forest Road site described in previous sections. Inoculations were made on excised branches taken from the same tree at a later time. Four inoculation treatments were used (Table 1). Experiment No. 2 - In experiment No. 2, the isolates of L. kunzei were isolated from the cankered branch of a Norway spruce (L. agigg (L.) Karst.). These isolates came from the same cankered branch as the Norway spruce isolate used in the previous inoculation studies (NS). Inoculations were made on excised branches of blue spruce (from BF-6 which had been naturally infected prior to the onset of this experiment) predisposed to Cytospora canker. Inoculations on intact branches however. due to the limited availability of hosts. were made on two blue spruce trees 71 at the same site which had no prior history of Cytospora canker (BF-2 and BF-3). Several isolates were utilized (Table 2). Evaluation- The wound inoculations were evaluated as previously described. Some of the excised branches in experiment No. 1 were ex- amined at 6 days after inoculation and others at 10 days. The results were combined. The excised branches in experiment No. 2 were examined 10 days after inoculation. The intact branches were inoculated on 13 September. 1984 and were examined on 7 November. 1984. RESULTS The results of these experiments indicate that mycelial inoculum of L. ngggi isolates originating from infected tissues. masses of conidia. or from a single conidium are capable of inciting canker formation on inoculated blue spruce branches (Table 1,2). Additionally,in experiment No. 2,differences in the virulence among. the conidial isolates were indicated (Table 3). Although canker initiation was seen on inoculated intact branches in experiment No. 2, the cankers were very small. In most cases the expansion was limited to only a few millimeters beyond the inoculation flap. Control inoculations failed to cause any expanding discoloration. Due to the limited expansion,no virulence differences were detected among the isolates on intact branch inoculations. DISCUSSION In this study, canker initiation occurred on both intact and excised branches of blue spruce wound-inoculated with mycelial fragments of 72 TABLE 1 Inoculations of excised and intact branches of blue spruce with isolates of Leucocytospora kunzei started from infected tissues or conidia. Experiment No. l Treatment Origin Number 2 Canker initiation1 or of of at ten days post Isolate Culture Inoculations inoculation BF-7D(1) infected tissue 11 72.7 BF-7D(m) mass of conidia 11 100.0 BF-7D(11) single conidium 11 81.8 Control - 7 0.0 1 Z Canker Initiation ' N2. of inoculations with expanding cankers/Ng. of total inoculations. 73 TABLE 2 lnoculations of excised and intact branches of blue spruce with isolates of Leucocytospora kunzei started from infected tissues or conidia. Experiment No. 2 Treatment Origin Excised branches Intact branches or of Z Canker l 2 Z Canker 1 3 Isolate Culture Initiation ’ Initiation ° MSU-58(1) infected tissue 80. 50.0 MSU-5B(4) mass of conidia 80. 50.0 MSU-5B(6) single conidium 100. 83.3 MSU-5B(8) " 100. 50.0 MSU-53(9) " 100. 50.0 MSU-58(10) ” 80. 66.7 MSU-58(12) " 80. 66.7 Control - 0. 0.0 1 Z Canker Initiation - Ng.of inoculations with expanding cankers/ fig. of total inoculations Z A total of five inoculations were made with each isolate. 3 A total of six inoculations were made with each isolate. 74 TABLE 3 Mean canker lengths on excised branches of blue spruce inoculated with tissue and conidial isolates of Leucocytospora kunzei. Experiment No. 2 Treatment Origin Mean Canker or of Length Isolate Culture . MSU-5B(1) infected tissue 37.0 t 18.1 ab1 MSU-5B(4) mass of conidia 42.0 t 7.8 ab MSU-53(6) single conidium 55.8 t 11.4 a MSU-58(8) " 49.2 t 19.5 ab MSU-5B(9) " 32.2 t 11.1 b MSU-5B(10) " 45.5 t 12.2 ab MSU-58(12) ” 48.8 t 28.1 ab 1 Those means followed by the same letter do not differ significantly LSD, g I 0.05 . 75 L. 5% isolates derived from various sources. Cultures originating from infected host tissue. masses of conidia. or from a single conidium were infective. This agrees with the findings of Jorgensen and Cafley (3). Waterman (7). Schoeneweiss (6). and with other inoculation trials presented in this thesis. In earlier inoculation studies done at Michigan State University by Kamiri and Laemmlen (4), monoconidial cultures failed to produce cankers in inoculated 5 year-old trees while mono- ascospore cultures were infective. Whether this was due to specific isolate characteristics or due to experimental technique is debatable. In this dissertation, inoculations with L. 52253; isolates from eastern hemlock and Douglas-fir failed to incite canker formation, so there is precidence for avirulent isolates. Waterman (7) obtained similar results. Additionally, 5 year-old blue spruce trees are not normally affected by Cytospora canker (3,7) and their use in inoculation ex- pteriments has been questioned (7). Inoculating excised branches of blue spruce with L. ngggi isolates accurately approximates the response of inoculations on intact branches in the tree (from the previous section). In experiment No. 2. the single conidium isolate. MSU-5B(9), appeared to be less agressive than the other isolates. Variability in the virulence of other L. ngggi isolates on blue spruce was detected in the previous section. Variability has also been documented among isolates of Leucocytospora leucostoma in- oculated onto their peach host (8). The inoculations of intact branches in experiment No. 2, produced small yet discrete cankers. The trees which were inoculated (BF-2 and BF-3) were at the same site utilized for the other inoculation experiments reported on in this thesis. Neither of these trees had 76 naturally occurring Cytospora cankers at the onset of this study. As indicated by the previous inoculation study.branches from non-predisposed trees showed less canker initiation and expansion upon inoculation. This inoculation experiment also supports that finding. The nature of the response however has not been accounted for and host genetic factors or seasonal effects may be a complicating factor here. These inoculations were done in the fall and it has been reported that L. 52252; grows maximally near 27°C (5), a much warmer temperature than the autumn temperatures in Michigan. Perithecia of L. 52253; were never found on blue spruce during the 3 years of this doctoral research. Other workers as well. have reported that perithecia are rare on blue spruce (5). Pycnidia on the other hand.are common and numerous (2,5,7). Conidia do appear to be infective.based on wound inoculations with conidial suspensions (3). Additional support for considering conidia to be important in the dissemination of this fungus can be found in the vegetative compat- ibility work done with L. 52352; on blue spruce presented elsewhere in this thesis. Conidia appear capable of playing an important role in the spread of this fungus, particularly within a single tree and for short distances (less than 50 meters). \l 0 LIST OF REFERENCES Barr, M. E. 1978. The Diaporthales in North America. Mycologia Memoir No. 7. J. Cramer Pub. Lehre, Germany 232 p. Funk, A. 1981. Parasitic Microfungi of Western Trees. Canadian Forestry Service. Victoria, B.C., Canada 190 p. Jorgenson, E. and J. D. Calfey. 1961. Branch and Stem Cankers of white and Norway Spruce in Ontario. For. Chron. 37:394-400 Kamiri, L. K. and F. F. Laemmlen. 1981. Effects of Drought- Stress and Wounding on Cytospora Canker Development on Colorado blue spruce. J. Arboric. 7:113-116 Kamiri. L. K. and F. F. Laemmlen. 1981. Epidemiology of Cytospora canker caused in Colorado blue spruce by Valsa kunzei. Phytopathology 71:941-947 Schoeneweiss. D. F. 1983. Drought Predisposition to Cytospora Canker in Blue Spruce. Plant Disease 67:383-385 Waterman. A. M. 1955. The Relation of Valsa kunzei to Cankers on Conifers. Phytopathology 45:686-692 Wysong. O. S. and L. E. Dickens. 1962. Variation in Virulence of Valsa leucostoma. Plant Disease Reporter 46:274-276 77 SECTION 5 The effect of water potential on the in vitro growth of Leucocytospora kunzei INTRODUCTION Cytospora canker of blue spruce is caused by Leucostoma Lungs; (Fr.) 'Munk ex Kern [syn. Vngg kunzei]. The anamorphic form of the fungus, Leucocygogpora 53222; (Sacc.) Urban [syn. Cytospora 53255;] is most commonly encountered in the field (1). In the United States and Canada this pathogen has been found to cause branch and occasionally stem cankers on a variety of coniferous hosts (24). Colorado blue spruce (£1925 pungens Engelm.) is an important and valuable ornamental tree species. Mature trees, however, are often seriously disfigured due to the branch mortality caused by L. kunzei. In blue spruce. and with other hosts of L. 52252;, drought stress has been associated with the severity of this disease (11.13.25). In experimental inoculations of Colorado blue spruces, canker formation and fungal colonization was greater in water stressed trees (14.20). The purpose of these experiments was twofold. The first objective was to compliment and expand on the available basic information on the factors affecting the in yiggg growth of L. 52953; (15). The second objective was to provide evidence as to whether the increased severity and canker expansion seen in association with drought stress and low tree water potentials was due to some predisposing effect on the host or whether it could be directly accounted for by the growth response of L. kunzei to water potential (Y ). 79 MATERIALS AND METHODS The test isolate- .A selected isolate of Leucocytospora,kugggi [EL-2B(3)] from a cankered branch of a blue spruce growing in East Lansing. Michigan. was grown in petri plates containing 22 Malt Extract Agar (22 MEA I 20 g of Difco Malt Extract and 20 g of Difco Bacto- Agar per liter of distilled H20). After 5-7 days. 6 mm plugs were transfered from near the margins of the colony to the water potential test plates. W’test plates- The water potential of the‘f test plates was adjusted to a range of water potentials by the addition of various osmotica or a matricum to the base 22 MEA medium. Leucocytospora 52952; grows very uniformly on this medium and forms colonies which are uniform from the center of the colony to the margins. For these experiments the‘* test media were prepared by adjusting the water potential of the distilled water fraction by the addition of either sucrose. mannitol. NaCl, or KCl as osmotica or a matricum, polyethylene glycol 8000 [Mol. wt. 6000-8000] (21). With the osmotically or metrically adjusted solutions, a 22 Malt Extract medium was made. A solid 22 MEA medium was prepared with all osmotica. The matricum, polyethylene glycol (PEG), prevents the solidification of an agar medium (18). so a liquid 22 Malt Extract broth culture was utilized. Water potentiaLgdetermination- The water potentials of the test media were determined prior to inoculation using a dew point hygrometer. Wescor model HR-33(T) with C-52 sample chambers (Wescor Inc.. Logan, 80 81 Utah, 84321). Measurements with the dew point hygrometer were made at room temperature using the 'dew point mode' as described in the HR-33(T) instruction manual. There were four replicates at each water potential treatment in all experiments. The total range of the water potentials of the 4'test media was from -0.7 bars to -110.0 bars. The water potential of the base 22 MEA was -0.7 bars. Water potential and growth- After transfer of the inoculum plugs to 100x15 mm petri plates containing 25 ml of w’test medium. the plates were sealed with Parafilm®and grown at 26°C in the dark. The liquid broth cultures consisted of 150 ml of medium in a 300 ml Erlenmeyer flask. These were inoculated. fitted with foam stoppers, and placed on a shaker at room temperature (23°C t 2°C) under ambient laboratory lighting. Evaluation- The growth of L. ngggL was evaluated by measuring increases in colony diameter daily on solid media and by colony weight in liquid media. Increases in colony diameter.oyer a 48 hour period when all the isolates were actively growing, were used for the com- putations. The total weight of the colonies in the liquid medium was recorded after 1 week. The results of each experiment were statis- tically analyzed using analysis of varience. RESULTS The in vitro growth of L. kunzei was affected by the water potential of the culture medium and was dependent on the type of osmoticum or matricum utilized. The growth curves (Figure 1) for L. kunzei in response to water potential were similar. thought not identical when the medium 82 .eoussx muoqmoumooosoq mo cusouw ouu«> mw one so Hmeucouoe “mom: mo someom one .s mxauHs «mmoo c/«\\)/a oumvcmum coma )/ .oo~ 3830mm ZDZHx -15 bars) the growth of L. 522! L was inhibited more in the liquid media (Figure 1). It is of interest to note that Hellkvist et al (10), while measuring the water potentials in Sitka spruce, noted solute potentials in spruce shoots ranging from -13 to -24 bars. This range of water potentials is near the growth optimum of L. 52952;. It appears that L. Lungs; is well adapted to its conifer hosts. While NaCl and KCl have been used as osmotica with other fungi (4.22.23) and have not been noted as being particlarly toxic to fungi (6), they do not appear to be suitable osmotica for use with L. 53253;. Fungal growth was inhibited at water potentials well above those noted with the other osmotica and matricum tested here. Growth was inhibited at water potential values higher than reported for many fungi (4,6,7. 22.23). Several authors have noted the increased occurrence and severity of Cytospora canker on spruce and other hosts in relation to drought stress (11.13.14.25). Schoeneweiss tested that observation by inoculating Colorado blue spruces which were drought stressed and held at known water potential values (20). The role of the environment in predisposing plants to particular pathogens is well documented and accepted in plant pathology (5,19). Drought and the resulting water deficits it causes in plants has been shown to predispose a variety of woody and herbaceous 85 plants to disease. Several examples of drought stress and "bark moisture deficits" predisposing woody plants to canker-causing fungi can be found (2.3,8.14.16,20). A complicating factor however, which needs to be examined. is the possibility that the pathogens may have growth optima at lower water potentials which may account for the increased colonization and canker sizes on water stressed trees directly (20). Examples have been reported of plant pathogens which grow better at lower water potentials (7). Ln giggg growth studies address this issue. With Cytospora canker of blue spruce,the evidence indicates that drought and low tree water potentials predispose spruce to canker formation and expansion by L. M. In his experiments. Schoeneweiss found significantly greater canker expansion and fungal colonization on inoc- ulated trees at -30 compared to -20 bars (20). In contrast, in these experiments the Lg_y$££g growth of L. ngggL was significantly lower at -30 versus -20 bars (g - .01). The increased growth of L. 52953; noted by Schoeneweiss cannot be accounted for simply by the growth response of L. Lungs; to water potential changes. Whether the nature of the predisposition to canker formation in drought stressed trees is related to changes in sugar-carbohydrate levels in the inner bark as noted by Hodges (12) in drought stressed loblolly pines, or is due to some other factors.has yet to be addressed. 10. 11. LIST OF REFERENCES Barr, M. E. 1978. The Diaporthales in North America. Mycologia Memoir No. 7. J. Cramer Publ. Lehre, Germany 232 P- Bagga, D. K. and E. B. Smalley. 1974. The development of Hypoxylon canker of Populus tremuloides: Role of interacting environ- mental factors. Phytopathology 64:658-662 Bier, J. E. 1964. The relation of some bark factors to canker susceptibility. Phytopathology 54:250-253 Brownell, K. H. and R. W. Schneider. 1985. Roles of metric and osmotic components of water potential and their inter- action with temperature in the growth of Fusarium oxvsporum in synthetic media and soil. Phytopathology 75:53-57 Colhoun, J. 1973. Effects of environmental factors on plant disease. Ann. Rev. Phytopath. 11:343-364 Cook. R. J. 1973. Influence of low plant and soil water potentials on diseases caused by soilborne fungi. Phytopathology 63:451-458 Cook, R. J. and R. I. Papendick. 1972. Influence of water potential of soils and plants on root disease. Ann. Rev. Phytopath. 10:349-374 Crist, C. R. and D. F. Schoeneweiss. 1975. The influence of controlled stresses on susceptibility of European white birch stems to attack by Botryosphaeria dothidea. Phytopathology 65:369-373 Griffen, D. M. 1977. Water potential and wood decay fungi. Ann. Rev. Phytopath. 15:319-329 Hellkvist. J., G. P. Richards, and P. G. Jarvis. 1974, Vertical gradients of water potential and tissue water relations in sitka spruce trees measured with the pressure chamber. J. Appl. Ecol. 11:637-667 Hinds. T. E. and J. L. Stewart. 1965. Cytospora canker recurence on Douglas-fir in Colorado. Plant Disease Reporter 49:481 86 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 87 Hodges. K. D. and P. L. Lorio Jr. 1969. Carbohydrate and nitrogen fractions in the inner bark of loblolly pines under moisture stress. .Can. J. Bot. 47:1651-1657 Jorgenson, E. and J. D. Calfey. 1961. Branch and Stem Cankers of white and Norway Spruce in Ontario. For. Chron. 37:394-400 Kamiri. L. K. and F. F. Laemmlen. 1981. Effects of Drought- Stress and Wounding on Cytospora Canker Development on Colorado blue spruce. J. Arboric. 7:113-116 Kamiri, L. K. and F. F. Laemmlen. 1981. Epidemiology of Cytospora Canker Caused in Colorado Blue Spruce by Valsa kunzei. Phytopathology 71:941-947 Landis, W. R. and J. H. Hart. Cankers of ornamental crabapples associated with thsalospora obtusa and other microorganisms. Plant Disease Reporter 51:230-234 Marsden. D. H. 1948. A Valsa associated with Cytospora canker of Spruces. Phytopathology 38:307-308 Mexal, J. and C.P.P. Reid. 1973. The growth of selected mycorrhizal fungi in response to induced water stress. Can. J. Bot. 51: 1579-1588 Schoeneweiss. D. F. 1975. Predisposition. stress. and plant disease. Ann. Rev. Phytopath. 11:193-211 Schoeneweiss. D. F. 1983. Drought Predisposition to Cytospora Canker in Blue Spruce. Plant Disease 67:383-385 Steuter, A. A., A. Mozafar, and J. R. Goodin. 1981. Water potential of aqueus polyethylene glycol. Plant Physiology 67:64-67 Sung, J. -M. and R. J. Cook. 1981. Effect of water potential on reproduction and spore germination by Fusarium roseum 'Graminearum', 'Culmorum'. and 'Avenaceum'. Phytopathology 71:499-504 Tresner, H. D. and J. A. Hayes. 1971. Sodium chloride tolerance of terrestial fungi. Appl. Microbio. 22:210-213 Waterman. A. M. 1955. The Relation of Valsa kunzei to Cankers on Conifers. Phytopathology 45:686-692 Wright, E. 1957. Cytospora canker of Rocky Mountain Douglas- fir. Plant Disease Reporter 41:811-813 SECTION 6 L3 vivo water potentials in blue spruce trees with and without a prior history of Cytospora canker INTRODUCTION Colorado blue spruce (LL52; pungens Engelm.) is a popular and widely planted landscape ornamental in the midwestern and northeastern United States. The aesthetic value of these trees however, is often markedly reduced due to Cytospora canker. Cytospora canker of blue spruce is caused by Leucostoma ML (Fr.) Munk ex Kern [syn. M kunzei] which is most frequently seen in its anamorphic form. Leucocygospora 52252; (Sacc.) Urban [syn. Cygospora 52252;] (1). The diffuse branch cankers caused by this pathogen characteristically kill the lower branches of mature trees first and then 'proceed to affect branches. higher in the tree. Trunk cankers in blue spruce are uncommon (9). In Michigan and other areas, canker development is rare in seedlings and juvenile tress less than 20 years old (6). At a given time, mature trees at the same site are generally not affected to the same degree. Several workers have indicated a relationship between drought stress and the occurrence and severity of Cytospora canker on spruce and other coniferous hosts (3,4,9). In other experiments the water potential of blue spruce trees inoculated with L. kunzei has been shown to affect canker development (5.7). The purpose of this experiment was to investigate whether there is any simple relationship between the water status of blue spruces in the field and the differential occurrence of Cytospora canker on those trees. 89 MATERIALS AND METHODS Study sites- Two study sites were used in this experiment. Both sites were on the properties of Michigan State University in East Lansing, Michigan. These sites were selected because of the presence of mature blue spruces in which Cytospora canker was affecting some but not all of the trees. Site No. 1 was located north of Baker Woodlot and Site No. 2 was located at the northwest corner of Forest and Beaumont Roads. In addition, each site contained younger unaffected trees as well. Sggpling- At each site, three mature trees with and without Cytospora canker were sampled. (Note- Site No. 2 was also used for inoculation experiments and L. Lungs; was introduced into one of the three unaffected trees on a few branches. This tree was free of Cytospora canker before inoculation and was placed into the unaffected catagory for this experiment.) A small secondary or tertiary branchlet was removed from two separate. limbs found at approximately breast height on each tree. The branchlets were 20-30 cm in length and were taken from midway along the main branches, near the point where the first needle-bearing branchlets could be found along the limb. The branchlets were shaded at this point by the more distal portions of other surrounding branches which form the dense perimeter of blue spruce trees. All samples were taken from the south- west side of the trees for the sake of uniformity. Branchlets were collected similarly from juvenile trees at site No. 1. but from limbs 90 91 knee to waist high. At site No. 2. the immature trees consisted of naturally seeded. suppressed seedlings less than 33 cm in height. The upper 20-30 cm of these seedlings were collected. In addition at Site No. 2. branch terminals 20-30 cm in length were collected from all of the mature trees for comparison to the branchlets taken within the perimeter of the tree. Immediately upon removal from the trees, the branchlets were enclosed in tightly rolled plastic bags (2) and placed into a cardboard box which was kept in the shade. The branchlets from both sites were collected from 12 noon to 1:30 pm on 3 September. 1985. The weather was sunny with a stiff breeze and the temperature was 28°C. Measurements of Water PotentiaL- The collected branchlets were brought into the laboratory immediately after their collection. Terminal portions. 15 cm in length. were recut immediately before being placed into a pressure chamber. Using a pressure bomb, the water potential of the branches was measured as described previously (2.7.8). RESULTS The average measured water potential of the branchlets from within the crowns of mature trees affected with Cytospora canker and from the mature and immature trees without Cytospora canker was similar within each of the sites (Table 1. 2). The average water potential of all the branchlets from Site No. 1 was -15.1 bars. The average water potential of all the branchlets from Site No. 2 was -13.3 bars. The water potential of branch terminals at Site No. 2 was lower in comparison to the branchlets closer to the trunk (Table 3). No 92 TABLE 1 Water potential in blue spruce branches taken from trees with and without cankers caused by Leucostoma kunzei. Site No. 1, East Lansing, Michigan. Mature Treesa’b Juvenile Treesa’ Branchlet No Canker Previous Canker No Canker 1 14.7 16.8 15.4 2 14.9 15.2 14.6 3 14.8 14.7 14.8 4 15.1 14.6 14.7 S 14.9 15.2 15.4 6 16.0 15.0 15.0 Mean 15.1 2 15.3 z 15.0 z a Water potential figures are given in (- Bars) and were measured using the pressure bomb technique. b Means followed by the same letter do not differ significantly (ANOVA. F test. LSD. Pr.05). ”—- 93 TABLE 2 Water potential in blue spruce branches taken from trees with and without cankers caused by Leucostoma kunzei. Site No. 2, East Lansing. Michigan. Mature Treesa’b’ Juvenile Treesa’b Branchlet No Canker Previous Canker No Canker 1 12.0 13.0 12.2 2 12.2 12.7 12.4 3 13.8 15.6 15.0 4 14.1 14.9 14.9 5 13.5 11.0 13.5 6 13.3 11.1 13.8 Mean 13.2 2 13.1 2 13.6 2 a Water potential figures are given in (- bars) and were measured using the pressure bomb technique. b Means followed by the same letter do not differ significantly (ANOVA, F test. LSD. Pr.05). 94 TABLE 3 Horizontal gradient of water potential along the branches of blue spruce with and without cankers caused by Leucostoma kunzei. Site No. 2. East Lansing. Michigan. Branchletab Branchleta from from Canker midway along terminal a Status limb portion Change none 12.1 13.0 1.1 none 13.9 14.8 0.9 none 13.4 14.4 1.0 present 12.9 14.0 1.1 present 15.3 16.1 0.8 present 11.1 13.0 1.9 a Water potential figures are given in (- bars) and were measured using the pressure bomb technique. b The water potentials in this column are the average of two measured limbs. _m ,_ y__ __ .1 1.13.. E7 n-PPI: 95 significant differences in the measured water potentials was detected between the three categories of trees tested. DISCUSSION The water potentials obtained in this experiment are comparable to those measured in Sitka spruce IL. sitchensis (Bung.) Carr.] in mid-August by Hellkvist et al (2). The water potential gradient observed between branchlets collected along the middle portion of a major limb and from the terminal end of the limb was also noted in that paper. The lower average water potential of the trees at Site No. 1 as com- pared to those at Site No. 2, reflects the more exposed location of that site. The trees at Site No. 1. were more open to the effects of wind and sunshine while the trees at Site No. 2 were more sheltered from the wind and were shaded somewhat by neighboring deciduous trees. In examining the relationship of tree water status to the occurrence of Cytospora canker. an effort was made to examine those tissues initially affected by this disease. Based on observations of over 100 spruces affected by Cytospora canker in the field. L. Lgnggi seems to invade the main branches of blue spruce midway back towards the trunk rather than moving in from the terminal branch tips. For that reason, water potential measurements were taken from branchlets located within the crown of the trees. Lower branches were tested also because of the observed tendency of L. LgnggL to affect the lower branches of blue spruce first (6.9). The type and time of day at which samples were taken were chosen to maximize the water potential stresses experienced 96 by the trees as indicated by Hellkvist et al (2). Drought stress has been implicated as being a factor in the occur- rence and severity of Cytospora canker of spruce by several authors (3.4.5.7.9). Based on these results,however. the water status of a tree alone does not appear to account for the observed apparent diff- erences in susceptibility to Cytospora canker among individual mature trees at a given site. Likewise, the 'field resistance' of young trees to canker formation by L. ku_n_z_ei_ cannot be explained simply by diff- erences in tree water status. Young trees in this study were under the same water stresses as neighboring mature trees with Cytospora canker. At this point, it appears the role that tree water potential plays in the occurrence of Cytospora canker of blue spruce is complex. Tree water status does not appear to singly account for observed differences in the susceptibility of mature trees or immature trees to cankers caused by L. 52952;. Genetic and other physiological factors are probably involved in regulating the time of occurrence and the severity of Cytospora canker of blue spruce. It is possible that under conditions of severe drought stress. differences in tree water potentials could become evident which were not detected in this study. This possibility could be examined in future research by artificially inducing more severe drought stresses on these trees. LIST OF REFERENCES Barr, M. E. 1978. The Diaporthales in North America. Mycologia Memoir No. 7. J. Cramer Pub. Lehre, Germany 232 p. Hellkvist, J., G. P. Richards. and P. G. Jarvis. 1974. Vertical gradients of water potential and tissue water relations in sitka spruce trees measured with the pressure chamber. J. Appl. Ecol. 11:637-667 Hinds, T. E. and J. L. Stewart. 1965. Cytospora canker recurrence on Douglas-fir in Colorado. Plant Disease Reporter 49:481 Jorgenson, E. and J. D. Calfey. 1961. Branch and Stem Cankers of white and Norway Spruce in Ontario. For. Chron. 37:394-400 Kamiri, L. K. and F. F. Laemmlen. 1981. Effects of Drought- Stress and Wounding on Cytospora Canker Development on Colorado blue spruce. J. Arboric. 7:113-116 Manion, P. D. 1981. Tree Disease Concepts. Prentice-Hall Inc. Englewood Cliffs, New Jersey 399 p. Schoeneweiss, D. F. 1983. Drought Predisposition to Cytospora Canker in Blue Spruce. Plant Disease 67:383-385 Scholander. P. F.. et a1. 1965. Sap pressure in vascular plants. n Science 148:339-346 Waterman, A. M. 1955. The Relation of Valsa kunzei to Cankers on Conifers. Phytopathology 45:686-692 '9'“ 97 SECTION 7 The effect of pH on the L3 vitro growth of Leucocytospora kunzei INTRODUCTION Cytospora canker of blue spruce is caused by the fungal pathogen Leucostoma £95531 (Fr.) Munk ex Kern [syn. 3.31.33 M]. This fungus is most frequently seen in its anamorphic form. Leucocygospora £2253; (Sacc.) Urban [syn. Cygospora 52352;] (1). Like all microorganisms, L. 53952; has certain optimal and minimal requirements for growth. In order to understand plant disease it is often of importance to gather basic biological information about the pathogen itself. The purpose of this experiment was to examine the effect of pH on the L2 33532 growth of L. kunzei. MATERIALS AND METHODS Isolates- Four isolates of L. kgnggi were used in this experiment. F? Isolates V-5C(3) and OK-4B(4) were originally isolated from Colorado blue spruce [Picea pungens Engelm.) while isolates EL-30A(1) and EL-8A(2) were originally isolated from white spruce [2. glaugg (Moench) V053] and Norway spruce [2. abies (L.) Karst.], respectively. All of the isolates were from cankered trees growing in Michigan. The isolates were grown in 100x15 mm petri plates containing 22 Malt Extract Agar ( 22 MEA I 20 g of Difco Malt Extract and 20 g of Difco Bacto- Agar per liter of distilled H20). After 5 days. 6 mm plugs were trans- fered from near the margins of the fungal colonies to the pH test media. Leucocytospora kunzei grows very well on this medium (22 MEA) and forms 99 100 colonies which are uniform. The pH of standard 22 MEA is 5.5. pH test media- For this experiment the pH test media were pre- pared by adding sterile solutions of lactic acid or KOH to autoclaved but still unsolidified 22 MEA. The pH value for each test medium was measured with a Beckman pH meter. The pH values for the 14 test media were: 3.0, 3.3, 3.6, 4.1, 4.5. 5.0, 5.5, 6.2. 6.5, 7.0, 7.8. 8.5. and 10.4. Mycelial plugs of the L. 53252; isolates were placed in 100x15 mm petri plates containing 25 ml of the solidified pH test media, parafilmed closed, and grown in the dark at 26°C. There were four replicates at each pH for each of the four isolates of L. 52252; tested. Evaluation- The growth of L. 53953; in response to pH was evaluated by measuring daily increases in colony diameter. The time from the inoculation of the pH test plates to the final measurements was 6 days. The results were analyzed statistically by analysis of variance. RESULTS The pH of the culture medium affects the in vitro growth rate of L. kunzei. The optimal pH for the growth of L. kunzei under these conditions was 6.2 with all of the isolates except OK-4B(4). which had a growth optimum at pH 6.7. Good growth occured over a range of pH values from approximately 5.0-7.0 (Figure l). The in vitro growth 5: of L. kunzei was inhibited during the time course of the experiment on media adjusted to a pH value of 3.0 [3.3 for OK-4B(4)J and at pH values greater than or equal to 8.5. Weeks later however, viable though irregular colonies did develop on those media of extreme pH. The 101 8.. o f + do n. 4F-0 .. . eager a mo «35.. .5: * ”we ”a .. 0!- so .u e. .o .. . a u e. . mHocmm H. are mmmmnn om o: o: are MD apv mr-m>A~v <-mvov ox-smesv #. C. )- 102 The average maximum increase in colony diameter per day for all of the isolates combined was 15.0 mm/day. There were significant differ- ences in growth rates among the four isolates of L. kunzei tested under these environmental conditions (Figure 2). DISCUSSION Under given conditions a fungus will grow optimally over a range of pH values of the culture medium. and will fail to grow at high and low extremes (2). This experiment indicates that L. 52225; grows well on 22 MEA over a range of pH values from 5.0-7.0. Plant pathogenic fungi as a group tend to grow optimally on media adjusted to initial pH values of 5.0-6.5 and seem to form a 'biological type' among the fungi which prefer slightly acidic habitats (2). Two other fungi in the genus Leucostoma. L. 232235 (Fr.) Von thnel and L. persoonii (Nit.) Togashi, which cause cankers on various stone fruits. prefer slightly more acidic conditions with reported optimal Lg yiggg growth at pH values from 4.2-5.2 (3.4). Standard culture media such as Potato Dextrose Agar (PDA) and 22 MEA (pH 5.6 and 5.5 respectively) are suitable for isolating and culturing L. Lgnggi without making pH adjustments. These results cannot be regarded as absolute. The effects of pH on fungal growth are complex in nature and the mode of action leading to growth inhibition may not be the same at high and low hydrogen ion concentrations (2). Indeed, other environmental factors. such as temperature or the specific composition of the medium. can also affect the shape of the pH-growth response curve. For these reasons the results of this experiment cannot be taken as being the absolute effect of mFFFFEFFFFF 5 r4. ._.FFFFFFFFF s 2... ..F.FFFFFFF c \\\\\\\\\\\\\\\ c \\\\\\\\\\\\\ C\\\\\\\\\\\ 'll'l'nlll'nl d\\\\\\\\\\\ c \\\\\\\\. Evan-0.0.0.00 2......FFFF. 33 A _= U“ VX w _ _ . w m m n w nw mm M“ MW mu mw :. m m m H w a. s - b.‘\.\WW\.\W\.\.\W\. . a .. UFFEFEFFF 7. ...\.\.\m\.\.\m\.\.\m\.\.\m .. .. .mFFFFFFFFF. 7. bc\\\\\\\\\\\\\\\\ 16 .WFFFFFFEFFfié e. b cV\\\\\\\\\\\\\\\. .werfififififfié .... c A\\\\\\\\\\\\\\\\ III'II'IIIIIIII'A .WFFFFEE: ..FF .. 3.0 15- 10- 5 Increase _ o 5 VI colony diameter mm/tzlayal 15- pH of culture medium Growth rates. The response of four isolates of Leucoc tos ora kunzei to pH: FIGURE 2. P=.05). t O n e r a S r e t t e 1 m S e h t V. .D d e D. p O t S e u l a V e S O h t L e V e 1 H D. h C a e n .1 h t .1 W significantly different (ANOVA, f-test, LSD. a 103 104 pH on the growth of L. 52923;. These results can however, be useful in suggesting a choice of a culture medium and for indicating possible ecological preferences. Along that line. in a supporting observation. the pH of cold water extracts of three pooled samples of bark/cortical tissues of blue spruce was 6.8. The actual value of the pH of live tissues would be lower. since the pH of the distilled water used was measured to be 7.4. This is within the observed range of the optimal growth of L. kggggl Ln yiggg. The two stone fruit pathogens. L. 53235; and L. persoonii. had lower pH optimums and the pH of peach bark/ cortical tissues was 4.4-5.6 (5). Based on the in 31553 growth response of L. 523531 to pH and this observation, L. 53352; is well adapted to the hydrogen ion environment of the tissues in its spruce host. FEM U“- LIST OF REFERENCES Barr. M. E. 1978. The Diaporthales in North America. Mycologia Memoir No. 7. J. Cramer Pub. Lehre, Germany 232 p. Cochrane. V. W. 1958. Physiology of Fungi. pages 19-23. John Wiley & Sons. New York, New York.. 524 p. Konicek, D. E. and A. W. Helton. 1962. An Optimum environment for culturing of Cytospora isolates from stone fruits. IV. Hydrogen-ion concentration. Mycopathol. Mycol. Appl. 16:243-248 Rohrbach, K. G. and N. S. Luepschen. 1968. Environmental and nutritional factors affecting pycniospore germination of Cytospora leucostoma. Phytopathology 58:1134-1138 Togashi, K. 1928. On the development of two races of Valsa in relation to the hydrogen-ion concentration of peach trees. Japan. Agr. Hort. 3:893-902 W Al‘s-I .l 4 105 APPENDICES \l O 107 APPENDIX A Culturing Leucocytospora kunzei: Notes and observations. The appearance of L. kunzei in culture is dependent on the type of growth medium and on lighting conditions. Lighting tends increase the amount of pigmentation of the colonies. Colonies growing on Potato Dextrose Agar (PDA) under lights often 'staled-out' and ceased to grow before they filled the petri plate. The medium (PDA) was darkened. The pH of darkened PDA was higher than the pH of fresh PDA (7.1 versus 5.6 in one examination). Based on cultural test, L. kunzei grows best on defined media utilizing maltose as a carbohydrate source. Isolates of L. kunzei grown in axenic culture for two years were still pathogenic on inoculated blue spruce branches. Twig or branch cultures are useful if pycnidia production is desired. Leucocytospora kunzei was rarely recovered if the medium on which it was growing had dried down. "Potato chip cultures" are not viable. Leucocytospora kunzei was best isolated from the discolored “ We. AZ". Ll fl‘n AW" 6 LIA-3211‘; TEX-WM} ' A cambial/cortical tissues on branches which were not completely dead. If the discolored tissues were resin soaked. L. kunzei could still be recovered but it required more time. Avoid branches which are totally dead and lack needles. 108 APPENDIX B Cytospora canker of spruce: Notes and observations. Based on observations at homeowner properties. in many cases the occurrence and severity of Cytospora canker was greater on trees with root obstructions or soil related problems. Often trees near sidewalks and driveways would be first to be affected in a given yard or site. Resinosis was not as pronounced on cankered branches of Norway and white spruce as compared to blue spruce. Inoculations with L. kunzei isolates indicated that canker expansion was greatest in inoculated branches 1.5 to 3.0 cm in diameter. Inoculations on larger branches nearer to the trunk caused cankers which expanded but at a slower rate. Inoculations were best done on tissues at least two years old. Inoculations on new shoots generally failed to incite canker formation. Cankered branches were placed on the ground in a brush pile. After one year. L. kunzei could be isolated from those branches which were not in contact with the soil. Few pycnidia however. were observed on the piled branches. 111mm“; LII. 51!. 109 APPENDIX C Other fungi frequently isolated from branches of blue spruce. Aureobasidium spp. Coniothyrium spp. Phomopsis spp. Epicoccum spp. 110 APPENDIX D SCANNING ELECTRON MICROSCOPY 'Leucocytospora kunzei Free-hand sections of a pycnidium. Preliminary photographs. 1.3. through pycnidium. Conidiophores lining the chambers. Conidiophores and ' Conidia. 15000 X