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TO AVOID FINES return on or before date due. __________________—7 DATE DUE _. DATE DUE DATE DUE 1 "9““ x £11,152 v; r s 9 . 1710 fl ‘ ' a MSU Is An Affirmmive Action/Equal Opportunity Institution ABSTRACT SCREENING ASPEN FOR RESISTANCE T0 HYPOXYLON CANKER By John R. French TWO methods of screening aspen (Populus tremuloides and E, grandidentata) for resistance to Hypoxylon canker were tested. These included inoculation of naturally existing clones of aspen with patho- genic isolates of Hypoxylon mammatum, and assay of excised leaves from various host genotypes with host-selective metabolites produced in vitro by the fungus. The inoculation method was evaluated by comparing length of cankers resulting from inoculations with the amount of natural infection in each clone. 1. Pathogenic single spore isolates of 5, mammatum were identified by inoculating branches of trees in 4 clones of f, tremuloides with 12 isolates. The two most virulent isolates and one isolate with low virulence were used to inoculate lOO naturally occurring clones of trembling aspen (Populus tremuloides) and 13 clones of bigtooth aspen (E, grandidentata) in 12 geographic areas of Michigan during late April and early May. The resulting cankers were measured 70 days after inoculation, and canker length among clones located within the 12 areas was subjected to analysis of variance. The two highly virulent isolates caused cankers on 95% and 96% of inoculated branches, and one isolate Produced significantly longer cankers than the other (mean length 59 vs. 45 mm over 100 clones). Cankers ranged from 12 to 14 mm in length. .3. tremuloides clones in northern areas produced significantly shorter \— cankers than those in some southern areas. Significant differences in John R. French length of cankers among trembling aspen clones in 10 areas were observed. Amount of natural infection of Hypopylon canker in each clone was deter- mined at the same time that cankers were measured; infection level per clone ranged from 0% to 58%. Length of cankers resulting from artificial inoculation was not correlated with amount of natural infection among clones in ll of the areas. Lack of correlation may be ascribed to ab- sence of suitable levels of natural inoculum within some of the areas, so that genetic susceptibility was not expressed. Branches of E, grandidentata inoculated with 3, mammatum became infected, and developed cankers similar in size to those on E, tremu: lgjggs, Large amounts of callus were observed on inoculated branches in some clones. Size of cankers following indculation also varied among clones of this species, and mean length ranged from 29 to 69 mm after infection with the most pathogenic isolate. II. 5, mammatum produced toxic metabolites in culture which were host-selective. Toxic preparations caused spreading necrotic lesions when applied to puncture wounds on leaves of E, tremuloides. The preparations also affected leaves of E, grandidentata and E, maxi: mowiczii. Leaves of 10 other woody species were not affected. Toxic metabolites from one isolate of E, mammatum were partially purified, and at least two host-selective components were evident. Lesion diameter was plotted against toxin concentration, using leaves of a sensitive clone of E, tremuloides. The response was linear over a 2,000-fold toxin concentration gradient. Toxic compounds were probably less than 1000 d in molecular weight, and did not lose activity after heating to 120 C. Toxic metabolites were produced under all tested conditions which allowed growth of the fungus. John R. French Isolates of fit mammatum differed in ability to produce host- selective toxic metabolites in culture. Non-pathogenic isolates and those with low pathogenicity in inoculation tests produced the lowest amounts of toxin in culture. Attempts were made to correlate clonal sensitivity to E, mammatum toxin with susceptibility to infection by the fungus. Leaves from 29 clones of E, tremuloides were assayed with toxin preparations, and stems of young trees in the same clones were inoculated with E, mammatum. In general, sensitivity to toxin in leaf assays was not correlated with length of cankers developing from inoculations with the fungus. The lack of correlation in these tests may be ascribed to the juvenile nature of tissues which were inoculated. Additional attempts to correlate sensitivity to toxin with genetic susceptibility to 5, mammatum infection are suggested, because it is not clear whether inoculation with the fungus or tests with its host-selective toxin are good indicators of susceptibility to the disease. SCREENING ASPEN FOR RESISTANCE TO HYPOXYLON CANKER By John Robert French 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 1976 ACKNOWLEDGMENTS Sincere gratitude is expressed to Dr. John H. Hart for his enthu- siastic support of this work. His encouragement of applied research is most timely, and indicative of far-sighted and well-placed priorities. I also acknowledge with appreciation the helpfulness of Drs. J. w. Hanover, R. P. Scheffer, and J. L. Lockwood in providing equipment and guidance for the completion of these studies. Their critical re- view of the manuscript is gratefully remembered. Thanks are also expressed to Dr. P. D. Manion for establishing basic principles and attitudes early in my career. The outgoing assistance and encouragement of Dr. A. L. Schipper is also appreciated. I express deep gratitude to my parents, Robert and Geraldine French, for their aid during the many years of my education, and for their unfailing support of high goals. I also express thanks to my wife, Diane, for her support, en- couragement, and actual assistance in the field during my years of graduate study. My children, Kristin and Stephen also provided immeasurable impetus in this regard. Lastly, Msrs. R. Carr and R. Wilson of the Michigan State University, Office of Research Consultation, are acknowledged for their help in statistical application. ii TABLE OF CONTENTS Page LIST OF TABLES AND FIGURES ...................................... v GENERAL INTRODUCTION ............................................ 1 LITERATURE CITED -- GENERAL INTRODUCTION ........................ 4 PART I INOCULATION 0F ASPEN WITH HYPOXYLON MAMMATUM AS A POSSIBLE METHOD OF IDENTIFYING RESISTANT GENOTYPES INTRODUCTION .................................................... . 7 MATERIALS AND METHODS ........................................... 8 Preparation of Inoculum .................................... 8 Inoculations in 1974, ....................................... 8 Inoculations in 1975 ......................... g .............. 9 Survey of Natural Infection ....................... , ......... ll RESULTS ....................... . ................................. 12 Inoculations in 1974 ....................................... 12 Inoculations in l975 ....................................... 14 DISCUSSION ...................................................... l9 LITERATURE CITED ................................................ 22 PART II VARIATION OF ASPEN IN SENSITIVITY TO A HOST SELECTIVE TOXIN PRODUCED BY HYPOXYLON MAMMATUM INTRODUCTION .................................................... 25 MATERIALS AND METHODS ............................ ............... 26 Host Material .............................................. 26 Pathogen Isolates .......................................... 26 Production of Toxin ........................................ 27 Purification and Extraction of Toxin ....................... 28 Bioassay .. .................................... ............ 28 RESULTS ......................................................... Host Selectivity of Toxic Metabolites ....... ............... Physical Properties and Purification of Toxic Metabolites .. Production of Toxic Metabolites Under Various Conditions ... Comparative Toxin Production by Fungal Isolates ............ Variability of Host Response to Toxin ...................... DISCUSSION .................................. . ................... LITERATURE CITED ................................................ SUMMARY ......................................................... APPENDIX A ...................................................... APPENDIX B ...................................................... iv 50 54 56 58 63 PART I Table 1. Analysis of variance of canker length caused by inoculation of 4 P, tremuloides clones with 6 isolates of H, mammatum during June and July, 1974 .... ..... . ....................... 2. Analysis of variance of canker length caused by inoculation of 100 P. tremuloides clones occurring in 12 geographic areas 0? Michigan ................ ..... ..................... 3. Analysis of variance of canker length caused by inoculation of 13 E, grandidentata clones with 2 isolates of H, mammatum . ............................. .. ................... Figure 1. Locations of 12 geographic areas of Michigan wherein aspen clones were studied during 1975 ............. ...... . ........ PART II Table 1. Sensitivity of various woody species to deproteinized fil- trates of Hypoxylon mammatum cultures ...................... 2. Compounds visible after thin layer chromatography of 1yo- philized toxin from Hypoxylon mammatum spotted on silica gel thin layer plates and eTDted with butanol-acetic acid- water (3:1:1, v:v:v) .............. .. ..... . ................. 3. Response of 5 genotypes of Populus tremuloides to inocula- tions with Hypoxylon mammatum isolate RL5¥2, and to its host-selective toxin .. ...................... . ..... . ....... 4. Response of 24 genotypes of Populus tremuloides to inocula- LIST OF TABLES AND FIGURES tions with Hypoxylon mammatum isolate RL5-2, and to its host-selective tox1n ................................. . ..... Page 13 16 18 IO 33 46 47 Page Figure l. Humid chambers in which leaves were assayed with prepara- tions of Hypoxylon mammatum toxin .... ..... . ................ 29 2. Host-selective toxicity of culture filtrates from Hypg§ylon mammatum isolate RL5-2 ..... . ..... . ........... . ............. 32 3. Migration of toxic components through Sephadex G-lO dextran gel ..... . ..... . .................... ...... .................. 35 4. Sensitivity of leaves of Populus tremuloides clone 5 and of H, deltoides to varying concentration of lyophilized Hypoxylon mammatum toxin ........... . ..... . ................. 39 5. Mycelial dry weight, toxin production, and pH of H, mammatum in cultures incubated up to 42 days .............. . ......... 39 6. Toxin production (upper graph) and mycelial growth (lower graph) by H, mammatum ....................................... 41 7. TDxin production (upper graph) and mycelial growth (lower graph) by H, mammatum in media containing 2.5 or 5.0 9 glucose per liter ...................... . ................... 41 8. Toxin production by 5 isolates of Hypoxylon mammatum ....... 43 9. Toxin production vs. canker length caused by 5 isolates of H, mammatum ........ .............. . ...... . .................. 43 10. Sensitivity of 5 Populus tremuloides clones to lyophilized H, mammatum toxin ................... . ......... . ............ 45 vi GENERAL INTRODUCTION The importance of aspen (collectively, Populus tremuloides Michx. and E, grandidentata Michx.) to pulpwood industries in the United States and Canada has been well documented (12, 13). Since the early 1950‘s aspen has been the largest single source of pulpwood in the Lake States. In addition, the importance of aspen in wildlife management has been recognized (6, ll). Aspen is well suited to 10 to 20 year rotations (9), with rapid, natural regeneration after clearcutting. Aspen regenerating in this manner forms natural clones, which may cover up to 35 acres (3, 17). Clones often intermingle in areas that have been continually cut and regenerated for long periods. Isolated clones may form by the produc- tion of suckers from the expanding root system of a single seedling growing in the open. Isolated, naturally occurring clones are easily recognized on the basis of homogeneity of sex, fall coloration, time of leaf abscission, leaf shape and size, and bark appearance, among other characteristics (4). Clones of E, tremuloides and H, grandidentata can be established artificially by use of adventitious shoots from root cuttings of clones or from individual trees (18). The impact of Hypoxylon canker, caused by Hypoxylon mammatum (Nahl.) Miller (syn. H, pruinatum , on the aspen resource is substantial (1, 14). In Michigan, Wisconsin and Minnesota, the annual loss of 8.4 x 106 m3 (300 x 106 ft3) of aspen per year to Hypoxylon canker is greater than 2 the desirable level of harvest (16). Control of the disease would be of obvious benefit in expanding the production of aspen, but currently there are no practical control measures. "Improved" aspen plantations are being established (5), and there is some evidence that genetic resistance to Hypoxylon canker could be incorporated into stock for these programs (15). There appear to be differences in susceptibility of clones to natural infection (8). Significant differences were detected in amounts of natural infection among clones growing in areas of homogeneous envi- ronment and inocul um density. Nhi 1e identification of resistant geno- types may be practical and easily performed under such conditions, methods of identifying resistance independent of the vagaries of climate and inoculum density might find more widespread application. Two such methods were attempted in the following experiments: cl ones were inoculated _'i_11_ sjtg with living mycelium of H. manmatun; excised leaves from aspen clones were tested for their relative sensitivity to a host- selective toxin produced by the fungus in culture. There are indications that clones of H. tremuloides will differ in Tength of cankers following inoculation with H. mamnatum (10). Clone Variability was masked, however, because of the extreme variability in pathogenicity of H. manmatum isolates (2, 10), and the apparent clone- isOlate interactions with some isolates (10). Therefore, H. mamatum isolates with high pathogenicity were identified in these experiments Wh‘i ch could be relied upon to cause large cankers on many aspen geno- 11.)!13es. These isolates were used to test for differences in rate of Canker enlargement among clones, with the premise that clones producing the smallest cankers may be most resistant to the disease. Attempts 3 were made to correlate the measured length of cankers with amount of natural infection in such clones. Recent reports (19, 20) of a host-selective toxin produced in culture by H, mammatum indicated its possible use in identifying resistance to the fungus among genotypes of H, tremuloides. Host- selective toxins are used in screening agricultural crops for disease resistance (7, 21, 22). The objectives of this work were 1) to verify reports of the occurrence of a host-selective toxin from H, mammatum and 2) to attempt to determine if relative sensivity of aspen to the toxin is correlated with actual differences in susceptibility to Hypoxylon canker. 10. ll. l2. l3. LITERATURE CITED -- GENERAL INTRODUCTION ANDERSON, R. L. 1964. Hypoxylon canker impact on aspen. Phyto- pathology 54: 253-257. BAGGA, D. K., and E. B. SMALLEY. 1974. Variation of H ox lon pruinatum in cultural morphology and virulence. Phytopathology 64: 663-667. BARNES, B. V. 1966. The clonal growth of habit of American aspens. Ecology 47: 439-447. BARNES, B. V. 1969. Natural variation and delineation of clones of Populus tremuloides and E, grandidentata in northern lower Michigan. Silvae Genet. 18: 130-142. BENSON, M. K. 1972. Breeding and establishment--and promising hybrids. In Aspen: Symposium Proceedings. USDA Forest Service Gen. Tech._Report NC-l. pp. 88-96. BYELICH, J. D., J. L. COOK, and R. I. BLOUCH. 1972. Management for deer. IH_Aspen: Symposium Proceedings. USDA Forest Service Gen. Tech. Report NC-l. pp. 120-125. BYTHER, R. S., and G. H. STEINER. 1972. Use of helminthosporoside to select sugarcane seedlings resistant to eye spot disease. Phytopathology 62: 466-470. COPONY, J. A., and B. V. BARNES. 1974. Clonal variation in the incidence of Hypoxylon canker on trembling aspen. Can. J. Bot. 52: 1475-1481. EINSPAHR, D. H., and M. K. BENSON. 1968. Management of aspen on 10 to 20 year rotations. J. For. 66: 557-562. FRENCH, J. 2., and P. D. MANION. 1975. Variability of host and pathogen in Hypoxylon canker of aspen. Can. J. Bot. 53: 2740—2744. GULLION, G. N., and F. J. SVOBODA. 1972. The basic habitat resource for ruffed grouse. IH_Aspen: Symposium Proceedings. USDA Forest Service Gen. Tech. Report NC-l. pp. 113-119. HUGHES, J. M., and J. D. BRODIE. 1972. Selected economic aspects of management. In Aspen: Symposium Proceedings. USDA Forest Service Gen. Tech? Report NC-l. pp. 27-34. MAINI, J. S., and J. H. CAYFORD. 1968. Growth and utilization of poplars in Canada. Can. Dept. For. and Rural Development. Dept. Publ. no. 1205. 257 p. 4 14. 15. 16. 17. 18. 19. 20. 21. 22. 5 MANION, P. 0., and F. A. VALENTINE. 1971. Diseases of trembling aspen in the Adirondack region of New York. Plant Dis. Rptr. 55: 662-665. MANION, P. D., and F. A. VALENTINE. 1974. Quantitative inheritance of tolerance to Hypoxylon mammatum in aspens. American Phytopatho- logical Society, Proceedings 1: 60-61 (abstract). MARTY, R. 1972. The economic impact of Hypoxylon canker on the Lake States resource. IH_Aspen: Symposium Proceedings. USDA Forest Service Gen. Tech. Report NC-l. pp. 21-26. PERALA, D. A. 1972. Regeneration: Biotic and sylvicultural factors. In Aspen: Symposium Proceedings. USDA Forest Service Gen. Tech. Report NC-l. pp. 97-102. SCHIER, G. A. 1973. Origin and development of aspen root suckers. Can. J. For. Res. 3: 45-53. SCHIPPER, A. L. 1973. Mammatoxin, a Hypoxylon mammatum metabolite responsible for canker development in aspen. Second Int. Cong. of Plant Pathology. Abstracts of Papers (abstract no. 0677). American Phytopathological Society, Inc. St. Paul, MN. SCHIPPER, A. L. 1975. Hypoxylon pathotoxin necessary to the infec- tion of aspen by Hypoxylon mammatum. American Phytopathological Society, Proceedings 2: 46-47. WHEELER, H. E., and H. H. LUKE. 1955. Mass screening for disease- resistant mutants in oats. Science 122: 1229. WHEELER, H. E., A. S. WILLIAMS, and L. D. YOUNG. 1971. Helmintho- s rium ma dis T-toxin as an indicator of resistance to southern corn ea b Tght. Plant Dis. Rptr. 55: 667-671. PART I Inoculation of Aspen with Hypg§ylon mammatum as a Possible Method of Identi ying esistant Genotypes INTRODUCTION Aspen and other poplars are increasingly important raw materials in wood fiber industries. Quaking aspen (Populus tremuloides Michx.) pro- vides 45% by volume of paper and boxboard fiber in the Lake States (5),N and is used to a large extent in Canada (1079 Hypoxylon canker, caused by Hypoxylon mammatum (Wahl.) Miller (syn. H, pruinatum), is a limiting factor in growth of aspen (l, 12). Genetic improvement of the species may be possible, especially for use in stands with rotation periods of ten to twenty years (4, 7), and such improvement could include resistance to Hypoxylon canker (13). Bigtooth aspen (H, grandidentata Michx.) is virtually immune to natural infection by the fungus, but evidence of resistance in quaking aspen is lacking. The clonal growth of aspen in nature was described by Barnes (3). Copony (6) indicated that clones differ in amounts of natural infection, but the meaning of this variability in terms of genetic resistance is not clear. The following experiments tested inoculation with living fungus mycelium as a possible technique for screening clones j__§jtg_ for resistance to H, mammatum, and'attempts were made to correlate the size of the resulting cankers with amount of natural infection in the clones. MATERIALS AND METHODS Preparation gf_inocu1um. Single ascospore isolates of H, mammatum were obtained from perithecial stromata on a naturally-infected tree in Ingham County, Michigan. All eight spores were removed from a single ascus in serial order, and cultured on 2% malt extract agar. Nine additional single spore isolates with known pathogenicity were obtained from New York State. These latter isolates had been used in previous tests of artificial inoculation (9), and exhibited wide variability in morphology and virulence. All cultures were maintained separately on 2% malt extract agar. Three weeks before use for inoculation, a mycelial suspension was prepared from each isolate culture by aseptically blending a portion of the culture in sterile water. One ml of each suspension was introduced aseptically into 20 ml screw-cap vials half filled with moist, sterile wheat. Wheat grains were colonized rapidly, and were shaken periodically to insure complete colonization. Preliminary tests using both infested wheat grains and infested agar plugs as inoculum indicated that infested grain produced more and larger cankers than did agar plugs. Inoculations 1H_l974.-Four E, tremuloides stands near East Lansing, Michigan were selected. These were determined to be natural clones based on root continuity between ramets, homogeneity of leaf morphology, fall coloration, and other similarities (3). Two of the clones were growing on well-drained sites, and two on poorly drained sites. All four clones were gynecious. 9 During June, July and August, a total of 39 trees (4-10 cm DBH) on the periphery of each clone, were incoulated with 12 H. mamnatum i so- lates as described in previous work (9). Thirteen trees were inocu- lated during each month. Each tree was inoculated on four individual branches with one of four isolates (or 3 isolates plus a control wound). Branches were about 2 m from the ground, and inoculations were made 6-10 cm from the main stem at the side of each branch. A 4 mm diam wound was cut through the bark which exposed the xylem. Each wound was inoculated with a single wheat grain infested with the appropriate isolate. Control wounds received a moist, sterile wheat grain. Wounds were covered with 2 cm wide masking tape, wrapped twice around the branch. Isolates were grouped in incomplete block fashion, using trees as blocks. Special care was taken to distribute the four replicate inoculations evenly around the somewhat circular area occupied by each clone. Canker lengths were measured 70 days after inoculation, and data were subjected to analysis of variance with canker length as the depen- dent variable. Inoculations jH_l975.-0ne-hundred natural clones of E, tremuloides, occurring in 12 geographic areas of Michigan (Fig. l), and 13 clones of E, grgndidentata in 7 of these areas, were inoculated as described above in late April through early May, except that wounds were covered with 3 cm wide Parafilm M (American Can Co.) in 1975 tests, instead of 2 cm wide masking tape as in previous experiments (9, 15). Clones were inoculated from south to north, coincident with spring leaf emergence, to reduce variability caused by differing phenology of the plants. Two isolates with high virulence and one isolate with low virulence in 1974 10 OWNED @ (6) ® 6) ———— Figure 1. Locations of 12 geographical areas of Michigan wherein aspen clones were studied during 1975. Each area is approximately 20 km in diameter. Seven to 10 clones were located in each area. 11 tests, and one control inoculation, were used on 4 separate branches of 5 replicate trees in each clone. Canker were measured 70 days after inoculation. §ng 1 Qf_natural infection.-The number of naturally infected trees in each of the clones inoculated in 1975 was counted at the same time that cankers were measured. All trees examined were 4 cm DBH or greater, and were termed infected if one or more cankers on the main stem were visible from the ground. All trees in most clones were examined. In clones with more than 100 trees, a transect 4 m wide was made through the center of each clone so that the ortet was included. Dead trees were counted as infected if H, mammatum stromal masses were clearly visible. RESULTS Inoculations 13.1225, Cankers resulting from artificial inoculation became visible outside their masking tape covers in about 6 weeks. Cankers extended rapidly in baSal and apical directions from the wound. By 10 weeks after inoculation cankered bark was wrinkled and cracked, with a typically yellow-orange mottled surface. Excision of outer bark indicated more extensive necrosis and discoloration beneath the surface which included the xylem near the inoculation wound. Some branches were completely girdled by necrotic tissue. The lengths of cankers were measured from apical to basal ends, after removal of outer bark. Mean lengths of cankers caused by inoculation with 12 isolates during June, July, and August are listed in Appendix A. Four of the 12 iSolates were generally non-pathogenic, producing short cankers (21 mm or less) on 0-8% of inoculated branches. Five isolates produced cankers after inoculation in all 3 months of the experiment, while 3 isolates produced cankers only following June and July inoculations. Success of inoculations in August was poor and the smallest cankers were observed after August inoculations. Only 6 of the 12 isolates produced cankers on all 4 clones during June and July. ‘ Statistical analysis was restricted to data obtained with these 6 isolates, from inoculations made during June and July. Analysis of variance of canker length produced by the six isolates indicated a significant effect of time of inoculation (Table l). The 12 uummem ucoovmrcmwm mmpmuvucv « uumemm acmovmvcmpm xpzmv; mounowucr «e .se o.o~ u m mmcopo m cpgppz sumcm— cmxcmu we :orumw>wu ucmvcmpm u m mews: . m u mgmaom come Leggy \m 13 N \a mm.~o¢ «PP Loccu nee. ma.mw~ mp gaze: x mcopu x manpomm um“. mm.oa~ mp mcopu x weapon" omw. m¢.mmF m case: x oompomm map. om.mmm m case: x mcopo “mo. op.amw m m = = mcopu Pmo. om.m~m m m = .. mcopu a mac. mm.mmpp m o = = wcopu 5 one. ~o.oopp m o = = mcopu « mmo. mm.om- m m = = mcoFu «« moo. oa.~mmp m < weapomp cmgpvz mcopu «s Poo. m¢.ommm m mcopu «« Poo. me.¢_w~ m mumpomm ee moo. o~.memm F coco: u a an _ mcmzam new: sovmmgm uomemm ucmopmvcmFm a mo momgmmo I. .enmp .zpac new menu mcvgsc Enumesos .m.mo moumpom. m saw: mmcopo mmuvopaemgp .a e we covpmpaoocw an vomsoo spacey cmxcmo co mocnwcm> mo mvmzpn=< .p mpnme l4 longest cankers resulted from June inoculations. Clone and isolate effects on canker length were also significant. No two-way or three- way effects were significant at P<.l. Additional partitioning of the clone sum of squares within isolates identified those isolates which produced the significant clone effect. Of these, the isolates producing the most significant clone effects (isolates A and B, Table 1) also produced the most cankers (41 and 44 cankers respectively on 48 total inoculated branches), and the longest cankers (mean length of 92 mm and 72 mm respectively, on clone RL ll inoculated in June). The isolate with lowest virulence caused one short canker on 48 inoculated branches in 1974. This isolate (Appendix A, isolate 605-6) had caused few cankers in previous tests (9). Iso- lates A and B were selected as having the highest virulence, and isolate 605-6 having the lowest virulence; hence they were used in the 1975 tests. Inoculation Qf_clones jg_l975.- Cankers appeared sooner in 1975 tests, emerging from the paraffin film cover after about 4 weeks. The two pathogenic isolates caused cankers on 465 (94.5%) and 472 (96.1%) of 492 and 491 branches of E, tremuloides, respectively. Cankers ranged from 12 to 114 mm in length 70 days after inoculation. The isolates with low virulence caused 13 cankers on 565 inoculated branches (2.3%), while control wounds produced 4 typical Hypoxylon cankers on 520 branches of E, tremuloides. Mean lengths of cankers produced by the two virulent isolates on 100 clones of P, tremuloides are listed in Appendix B. Canker lengths produced by the two highly virulent isolates were subjected to analysis of variance, using the sum of canker length pro- duced by the two isolates on each tree as the dependent variable 15 (Appendix 8). Among the E, tremuloides clones, a significant area effect was indicated (Table 2). Clones in northern areas (Fig. 1, areas 11 and 12) had significantly shorter cankers than those in area 8. Clones within areas were also significantly different, with some clones produc- ing longer cankers than others. Additional partitioning of sums of squares identified those areas in which clones were significantly different. Clones within 10 of the 12 areas produced cankers of different lengths after 70 days (error probability <.05). The two highly virulent isolates produced cankers of different size, indicated by the significant isolate effect. Isolate A usually produced larger cankers than isolate 8 (overall mean length of 58.5 mm and 45.7 mm respectively). This difference was statistically variable among regions, however, as the isolate X region effect was significant (Table 2). Amount of natural infection in clones of H, tremuloides ranged from 0% of the trees infected within 14 clones, to 58% in the clone with heaviest infection. Regression analysis was applied to 99 of the 100 clones, with 20 to 100 trees in each. Percent of naturally infected trees in each clone was plotted as the dependent variable against each of three independent variables: 1) mean canker length (MCL) produced by isolate A on the clone; 2) MCL produced by isolate B on the clone; 3) MCL produced by isolate A plus MCL produced by isolate B on the clone. Significant linear correlations between lengths of cankers re- sulting from inoculations and amount of natural infection were not observed in 11 of the 12 areas, using any of the 3 variables. Branches of H, grandidentata inoculated with isolates A and B became infected, and produced cankers that were 13 to 99 mm long 70 days 16 as o.mp u m mmcopu m cpcpwz gumcmp cmxceo mo cowumw>mu cgmucaam u m mean: . «compo ucmuvmpcmwm mmpmopucp « Soweto «cauwcaeucm seems; manageaer .. m u mgmacm cams Locsm \n N ma.~mm men “League HAJMMMKW monmwwwomu mpm. mN.amm mm Ammmc mo armapmc< .N m—nme 17 after inoculation. Isolates A and B produced cankers on 59 (95%) and 60 (97%) of 62 and 63 inoculated branches respectively. Cankers in some clones were surrounded by large amounts of callus tissue. Analysis of variance of canker length among clones, disregarding possible geographic area effects, indicated significant clone differences in reaction to both highly virulent isolates (Table 3). At least 15 trees in each clone of H, grandidentata were examined, and only one naturally infected tree was found. 18 Table 3. Analysis of variance of canker length caused by inoculation of 13 E, grandidentata clones with 2 isolates of H. mammatum. Clones were located in 7 geographic areas of Micthan, But were analyzed disregarding possible area effects. Degrees of F significant Effect Freedom Mean Square at P less than Isolate A: Clone 12 12518.99 .01 Error 46 8041.50 Isolate 8: Clone 12 69860.70 .01 Error 47 3188.30 DISCUSSION Previous work with H, mammatum demonstrated extreme variability in virulence of isolates (2, 9). Genetic variation in susceptibility of the host was also suggested among clones opr, tremuloides (6, 9), and among interspecific hybrids of H, tremuloides and E, grandidentata (13, 15). Infection in nature is most often associated with young lateral branches near the main stem (8, 11), or with insect galls on branches (ll, 14). Therefore, lateral branches of young trees were inoculated in an attempt to measure the variation in susceptibility among clones. The experiment in 1974 demonstrated that inoculations in June produced more and larger cankers than later inoculations. In addition, use of H, mammatum isolates with high virulence contributed to produc- tion of cankers, with successful infections on 95-96% of inoculated branches in these tests. A single ascospore isolate, representing a single genome of the fungus, may be more reliable for producing cankers on many host genotypes than isolates from margins of natural cankers ("mass" isolates) which are of unknown nuclear condition (16). Differences among clones in length of cankers 70 days after inoculation were observed in 1974 and 1975. The concurrent observation of differences among geographical areas might indicate a macroclimatic control of canker size. However, the significant differences among clones within 10 of the 12 areas (Table 2) would more clearly indicate 19 20 either microclimatic or genetic control of canker length. In addition, within-clone variance of canker length was sufficiently small (5 20.0 mm in 1974; s = 19.6 mm in 1975, Tables 1 and 2), to indicate highly significant clone differences, with probability of error less than .01 in most areas. The meaning of this variability among clones as it relates to actual resistancecncsusceptibility to H, mammatum could be questioned. Length of cankers following artificial inoculation was generally not correlated with amount of natural infection in the clones. However, infection in nature is subject to external variables which include inoculum density and environmental factors. For example, among the seven clones in area 9, 0% to 1% of the trees were naturally infected. This low level of infection may have been the result of low incidence of E, tremuloides, and therefore low amounts of inoculum. Artificial inoculation of these clones, however, produced cankers in frequency and size comparable to those observed in regions with greater amounts of natural infection. Amount of natural infection in a clone, therefore, may be a poor in- dicator of genetic potential for resistance or susceptibility when deter- mined in areas of differing inoculum potential. 1 Infection of E, grandidentata branches was observed after inocula- tion with H, mammatum. Rogers (15) also reported infection of H. grandidentata main stems by similar methods. Concurrent observations in this test of large amounts of callus tissue around the diseased area in branches of some clones may explain the low natural incidence of Hypoxylon canker among bigtooth aspen. The pathogen could be excluded from healthy tissue by this mechanism (15). Clones within this species may differ also in canker length after 70 days, but verification of this 21 must await further tests on a larger sample of clones under a conrnon environment. H, tremuloides clones used in these tests were on "average" sites, that is, not in standing water or on particularly dry sites. However, microclimatic and nutritional differences among clones within geographic areas might directly affect length of cankers on these clones. There- fore, artificial inoculation tests such as these may be most useful for identifying potentially desirable host genotypes in the field. Such genotypes could be screened more intensively under a common environment after vegetative propagation. Such propagation is time consuming and expensive, and therefore preliminary screening of clones 1H_§itg_by inoculation or other means would be economically desirable. 10. 11. 12. LITERATURE CITED ANDERSON, R. L. 1964. Hypoxylon canker impact on aspen. Phyto- pathology 54: 253-257. BAGGA, D. K., and E. B. SMALLEY. 1974. Variation of H ox lon pruinatum in cultural morphology and virulence. Phytopathology 64: 663-667. BARNES, B. V. 1969. Natural variation and delineation of clones of Populus tremuloides and H, grandidentata in northern lower Michigan. 51 vae Genet. 18: 130-142. BENSON, M. K. 1972. Breeding and establishment--and promising hybrids. IH_Aspen: Symposium Proceedings. USDA Forest Service Gen. Tech. Report NC-l. pp. 27-34. BLYTH, J. E., and J. T. HAHN. 1974. Pulpwood production in the lake states by county. USDA Forest Service Res. Note NC-l75. 4 p. COPONY, J. A., and B. V. BARNES. 1974. Clonal variation in the incidence of Hypoxylon canker on trembling aspen. Can. J. Bot. 52: 1475-1481. EINSPAHR, D. W., and M. K. BENSON. 1968. Management of aspen on 10 to 20 year rotations. J. For. 66: 557-562. FRENCH, D. W., and N. OSHIMA. 1959. Host bark characteristics and infection by Hypoxylon pruinatum (Klot.) Cke. For. Sci. 5: 255-258. FRENCH, J. R., and P. D. MANION. 1975. Variability of host and pathogen in Hypoxylon canker of aspen. Can. J. Bot. 53: 2740-2744. MAINI, J. S., and J. H. CAYFORD. 1968. Growth and utilization of poplars in Canada. Can. Dept. For. and Rural Development. Dept. Publ. no. 1205. 257 p. MANION, P. D. 1975. Two infection sites of H ox lon mammatum (Wahl.) Mill. in trembling aspen (Populus tremulo1des Michx.). Can. J. Bot. 53: 2621-2624. MANION, P. D., and F. A. VALENTINE. 1971. Diseases of trembling aspen in the Adirondack region of New York. Plant Dis. Rptr. 55: 662-665. 22 13. 14. 15. 16. 23 MANION, P. 0., and F. A. VALENTINE. 1974. Quantitative inheritance of tolerance to H o lon mammatum in aspens. American Phyto- pathological Society, Proceedings 1: 60-61. NORD, J. C., and F. B. KNIGHT. 1972. The importance of Sa erda inornata and Oberea schaumii (Coleoptera: Cerambicidae) gaileries as infection courts of Hypoxylon pruinatum in trembling aspen, Populus tremuloides. Great Lakes Entomologist 5: 87-92. ROGERS, J. D. 1963. Hypoxylon canker of aspen: 1. Screening for disease resistance. II. Developmental morphology and cytology of H ox lon pruinatum. Ph.D. Thesis. The University of Wisconsin. 9 p. VALENTINE, F. A., P. D. MANION, and K. E. MOORE. 1975. Genetic control of resistance to Hypoxylon infection and canker development in Populus tremuloides. North Central Forest Tree Improvement Conference, Proceedings. in press. PART II Variation of Aspen in Sensitivity to a Host-selective Toxin Produced by Hypoxylon mammatum . 24 INTRODUCTION In 1973 Schipper (15) reported a host-selective toxin produced by Hypoxylon mammatum (Wahl.) Miller. This fungal metabolite had its greatest effect on the major host of the fungus, Populus tremuloides Michx. (trembling aspen), and a lesser effect on H, grandidentata (big- tooth aspen) and H, balsamifera (balsam poplar), which are infrequently attacked species. Low sensitivity was also reported for E, 2123. (European white poplar) and H, deltoides (eastern cottonwood), which are not infected in nature. Substances toxic to aspen leaves were also found in bark of cankered aspen. Other host-selective toxins have been used to screen agricultural crops for resistance to the fungi which produce them (4, 18, 19). There- fore, an attempt was made to produce the toxin reported by Schipper, to delineate conditions for its production and purification, and to evaluate its usefulness in screening aspen for resistance to H ypoxylon canker. Clonally-propagated E, tremuloides was used to evaluate differential sensitivity of aspen clones to H, mammatum toxin. 25 MATERIALS AND METHODS Host material.- Five clones of H, tremuloides were propagated from root cuttings of natural clones, which were located near East Lansing, Michigan (clones no. 1 - 5). Four clones were gynecious and one was androecious. In addition, four clones of H, deltoides were established from branch cuttings of four trees growing on the property of Michigan State University at East Lansing. Trees were maintained for three years in pots in the greenhouse, and leaf and stem material from these trees were used routinely throughout the present experiments. In addition, 24 H, tremuloides and 3 H, grandidentata clones, propagated indepen- dently by Dr. J. W. Hanover, Department of Forestry at Michigan State University, were assayed for their sensitivity to H, mammatum toxin and for their susceptibility to the fungus. Additional leaves collected in the field from H, tremuloides, H, deltoides, g, grandidentata, and H, ngg_were used. Pathogen isolates.- A single ascus of H, mammatum was dissected and all eight spores were cultured independently (isolates RL5-l through RL5-8). Ten additional single ascospore isolates were obtained from New York State. Three ”mass" isolates from margins of natural cankers were used in some tests. All isolates had been used previously (6, 7), and hence their pathogenic capabilities were known. In most assays of toxin activity, culture filtrates were used from the isolate with the highest pathogenicity in field tests [isolate RL5-2 (ref. 5, isolate A)]. 26 27 Production Qf_toxin.- Isolates of H, mammatum were grown in liquid culture media of two types. Fries' liquid medium with yeast extract, used by previous workers (9, 13) was not used due to its toxicity to H, tremuloides tissue. The toxicity was observed after autoclaving the medium. The medium (1) used most often consisted of the following in- gredients (in g/liter): glucose 10.0, KZHPO4 0.75, KH2P04 0.75, MgSO4-7 H20 0.5, CaCl2 0.1; also present were 1.0 m1 of a vitamin stock solution per liter (thiamine, 10.0 mg, plus biotin, 0.5 mg in 100 ml water), and 1.0 ml of a trace element stock solution per liter (FeC6H507~3 H20, 214.3 mg; ZnS04-7 H20, 158.4 mg; CuS04-5 H20, 31.6 mg; MnSO4-4 H20, 11.4 mg; M00 16.2 mg: H380 7.0 mg; all in 400 ml water). The medium 3’ 3’ was adjusted to pH 6.0 with 20% phosphoric acid before autoclaving in l-liter Roux bottles with plastic foam stoppers, using either 200 or 400 ml of medium per bottle. After autoclaving, a 19.8% solution of L- asparagine was added aseptically to the medium by filtering through a membrane (0.22 um pores), to provide a final concentration of 0.5 g/liter. Nutritional requirements of H, mammatum for asparagine, thiamine, and biotin were established by Oshima (12). This medium showed no toxicity to leaves of either 3, tremuloides, E, deltoides, or E, grandidentata. 4 hyphal fragments/ One ml of a mycelial suspension (containing 3 - 5 x 10 ml) of H, mammatum was added to each bottle. Cultures were maintained for varying time periods under low light intensity and without agitation in a 27 C room during most tests. Tests of growth and toxin production at different temperatures were performed in incubators, in darkness. Cultures were filtered through tared, oven-dry, glass-fiber filter paper after incubation. Mycelial dry weight was determined for each culture after oven-drying the residue (90 C, 24 hr) and subtracting the tare weight of the paper. 28 Purification and extraction 9j_toxin.- After vacuum distillation to 10% or 1% of the original volume, compounds of high molecular weight (above ca. 1000 d) were routinely removed from culture filtrates. Re- moval was accomplished either by adding an equal volume of cold (-10 C), anhydrous methanol to the concentrated filtrate (14), or by Sephadex G-15 dextran gel filtration with removal of compounds eluting in the void volume, or by a combination of both methods. Fractions containing these large compounds were not toxic to either aspen or cottonwood leaves. In some instances toxic metabolites were purified further by partitioning into organic solvents, followed by vacuum removal of the solvent and re-dissolution into water. All vacuum evaporation was per- formed at less than 60 C. Toxin activity was assayed using either crude culture filtrates, deproteinized filtrates, fractions from dextran gel columns, or organic solvent extracts of deproteinized culture filtrates. Bioassay.- Toxin preparations were assayed by a leaf puncture tech- nique, similar to that used by Hiroe, et al. (8). Excised leaves were punctured with a l6-gauge hypodermic needle, and a 20 uliter drop of test solution was immediately placed on the puncture. Leaves often were treated with four test solutions placed individually in the four quadrants of each leaf. Normally, two to four replicate leaves were used in each assay of any toxin preparation. Leaves were maintained in enclosed chambers (Figure 1), containing distilled water to maintain high relative humidity. Lesions developing from toxic solutions typically radiated from the point of toxin application, producing a discrete black necrotic spot, clearly delineated from the remaining 29 .um>oEwe Lm>oo cow; 3mw> now Am .mcomumcmamca :wxop cur: umpemcp mew: mmumpn vamp use .prmz umppwpmwu mcwcwmpcoo mrmw> cw ummcmeew mew: mmFowuwa New; .mpmra cwmrmocoq mew zeros Lopez cm__wumwc mchwmucoo .Lopmoowmmu umpcm>cou m we 3mw> wuwm A< .cwxou Ezumesme corxxomxz mo mcovumcmnmca gov: umemmm mew; mw>umF cows: cw mcmnemzo teas: .Am use cwmno cmsm ace mcoam> ergoEEm on ammoaxm mam: o: mmumowucw Auv .cm>smmno meme: umumowucv msm mcopou \n .AmcowmmF o: mmuamwucw . mumscom meowmmp mmumowucw +v Lmumz cw poem comm scam acronym muczoqsoo cum: m mcopo mmuwo—35mcu .m mo mm>mmp mo ucmspmmcp An vmcwsgmpmu mm; app>vuo< \m 38 umc mare xcpm cm» new» xcau :mmcm + om.oupm.o m 2.3. u u - xcmu mzpn + Pm.o-¢m.o c Amgmmmqmmwuv ma—n um; can - Name 1 . mm.o-~¢.o m u - i - mz—n . p¢.o-mm.o N i u i . mapn - om.o-mN.o F aza Ioeiz 2 >3 \. sum>wuu< em mean + .A>u>u> .Pupum~,cmumzuuwom ovumomrpocmuzn ;u_3 umpzpm ecu mmpmpa cmzmp ems» me moppvm co omppomm asumEEmE copzxoqx: sage cmxop umNPppcaozp mo asamcmopmsocgu Lmzmp avg» cmpmm mpnrmr> mucaanou .N mpnmh 39 umumm>cms _Pm mcmz mmpmcp_Pm mcamppp .mmczupau mumowpamc N Eocm nmcwmuno m=Fm> cams mucmmmca -mc maven gmmm .mewp mEmm mg» on umxmmmm cam .mzmu Ne cu m: umpmnzocw mmczppzo cw Espmesms .1 mo In new > D ..r .. .... .... as”... . .. W I.» ...?.t:..:. ......... Cittt’: u ..\ ..\. L w... .. . ... We... .8.“ m .. m0. ’...:....... m W. 1.. m. w ...... \l. .\ a. m. . ....+ ... I .\ .\\..3 >50 m. .\.\ .I:Ou>z r08 \ l .mwxm PeacoNPLo; mg» mo cowumscowmcmcu cmmzwp an macwom on umupwe mm: mm>mmp mmcwop -:Emcu .m mo mmcoammc mcwpmomvcp mcmp :ommmmcmmc cmmcwp mze .mmwumam comm mo mm>mmp mpmowfimmc e co mcowmmp mo Lmumemwc cams mzp mucmmmcmmc ucwoq :omm .cwxop Enumssme am umNVchaozp mo compmcucmo -coo mcmxcm> op mmcwoppmu .m we can m mcopo mmuwop iasmsp mspamdm $0 mm>mmp we hpw>wummcwm .v mszmmm ... «_Exuhv co_5=coucououmxoh 8.9. 1* I l... O 322.... ... i v m l i Q "o f m. 1 9% m . * . 2a m. 3 23.9. m 1 330.382. ... I call ri _ p h b 'N 40 pH level, except at pH 4.0. The activity of toxin was greatest at pH 4.0, when filtrates from cultures initiated at pH 6.0 were altered in pH from 3.0 to 10.0. Cultures were incubated at several temperatures from 15 C to 35 C, using media containing glucose at either 2.5 g/liter or 5.0 g/liter. The fungus did not grow at temperatures above 30 C, and growth at 15 C was barely detectable (Figure 7). Optimum growth for H, mammatum isolate RL5-2 was at 25 and 27.5 C in medium containing 5.0 g glucose/liter. Approximately equal amounts of toxin were produced under all conditions which allowed fungal growth. Comparative toxin production Hy fggggl.isolates.- Five isolates were grown in medium containing 5.0 g glucose/liter. Two highly virulent isolates (RL5-2, RL5-7) one with intermediate virulence (211-8), and two with low virulence (605-6 and RL5-8) (5), were chosen. Inoculum was 4 hyphal fragments per standardized for each isolate culture to 3 x 10 culture bottle. Four replicate cultures of each isolate were incubated for 28 days at 27 C. Each culture was filtered and the residue was weighed; all filtrates from the same isolate were then pooled. To compensate for slow growth and low levels of toxin production by the isolates with low virulence, the volume of pooled filtrate from the 4 slowest growing isolates was concentrated to equal volume per mg dry weight of mycelium. The mean weight of mycelium produced by isolate RL5-7 (the most rapidly growing isolate) was used as a standard. Pooled filtrates were then adjusted to pH 5.0, and assayed on leaves of H. tremuloides clone 5. Isolates of H, mammatum varied in ability to pro- duce toxin in culture (Figure 8). To determine if toxin production by different isolates was related 41 .mmumuNucw mm oLmN ems» memmcm mpcwom acoEm .mo.v a pm Amva mocmcmmmvm pcmommwcmwm pmmmm whmnmcm cmaaav m mcopo mo mmmcp m Eocm mm>mmp mmmwoF35mcu .m.mmmowpamc e :o zpwowxom com mmAmmmm mcm mmFooa mcmz mmumcmpwm mc=m_=o mumDNFQmm .mmcmupzo mumo -mpamc m mo cmme mgp mucmmmcmmc gamma cmzop mg» c? “swam nmmm .mamm FN com o mm1mp um mmummzmcw mmmz mmmaupzo .mmooapm mep_mm ohm Lo m.N mcwcwmpcoo mpmms cw EsmmEEme .: Am Agamcm Lmzopv cuzccm mepmoze mam Acamcm Lmamsv cowmozmoca :_xoh .N mczmwm «UV 229.2...3... c8313:— . 12:3. ooo SOUS—o . n." I o.“ ‘OOIOIIn (But) an Ma IDII'MW AMMHVHVQ. Pfl GROW (tutu rurogq uogso'. .mmumowmcw we oLmN cmcu memmcm mpcwom mcosm .mo.v a pm Ammpv mocmcmm term ucmowmmcmmm pmmmm .Azamcm Lmaaav m mcopo mo mmmcu as» some mm>mm— mmmmopzemcp am mpmuwpamc o3» co xuwomxou com mmAmmmm mcm .o.m 1; cu mmmmanmm cmcm mam: mmmmcm rpm; .mmczupmo mpmowpmmc m mo :mme mew mucmmmcamc gmmcm cmzop mg» cw ucwom nomu .msmm mN tom mmcmmpcmme mam .o.e om o.m zolpm mmummppcw mcmz mmcaupzu .Espmssms .: Am Acumen Lmzopv guzocm mepmoxs mcm Acnmcm Lmaasv composmocq :Pxo» .o mcammm 23:5 .o ....o .83:- D.» D m D m 06 o.” W O O O O O I!) 0) 5 (6w) 'rM Ara loggoalw NO (mm) mom uouso'] uoow :Aungpv ugxol O‘DQ 42 to their virulence, the same five isolates were inoculated into three- year-old main stems of ramets of H, tremuloides clones 2, 3, and 5, using previously described techniques (6, 7). Trees were 1 to 1.5 m tall, and were maintained in 14 cm pots in the greenhouse under continuous light (13 - 14 hr daylight, supplemented with 12 hr fluorescent light). Trees were watered daily, fertilized weekly with Plant Marvel (20-20-20, wtzwtzwt, of available N, P, and K, respectively) in water solution, and supplemented during the second week of the experiment with MgPO4 in a soil drench. Each tree was inoculated 20 cm from the soil line with a single H, mammatum isolate growing on wheat grain. Six replications of each clone - isolate combination were used. Cankers were measured 56 days after inoculation from apical to basal ends after removal of outer bark (5). Cankers developed as described in previous tests (5) from inoculations with isolates RL5-2, RL5-7, and 211-8, while isolates 605-6 and RL5-8 were low in virulence. Isolate 605-6 caused two small cankers on stems of trees in clone 5. Mean canker length produced by each isolate on clones 2, 3, and 5 was plotted against the mean diameter of lesions produced by culture filtrates of the same isolates when assayed on leaves of clones 2, 3, and 5 (Figure 9). Significant linear correlations were observed using stems and leaves of clones 3 and 5. Clone 2 was sensitive to toxin from only one isolate in leaf assays, but developed cankers from inoculations with all three isolates of H. manmatum with highest virulence. Eighteen single spore isolates and three mass isolates of H, mammatum all produced toxin in culture. Eighteen of these isolates were pathogenic in previous inoculation tests (6, 7), and the remaining three isolates were generally non-pathogenic on most inoculated clones of E, 43 .Amo.v av memomemgmwm mmmz m mam m mm:o_o Low mmcwp cowmmmcmmm .m mzm .m .N mmcopo mmmwopssmcp .m :o mmummu cmzz mumF -omw :omm Am mmmsmo Amsmpm mpmoNFch my :pmcmp mecmu :mms umcvmmm mmupopa Amm>mmp mumowpmmm my mmumcupwm mm:m_=o mo.meo_xou mmcmmmcmmm pcvom comm .EzmeEme .: mo mmmmromw m Am mmmzmo cumcmF mecmo .m> cowuozcocq sexOH .m mczmwm «SE. 593.. Loxcou .522 an 9N =— I‘lljlilllllfilll! .\+ N ”to.” eeeoeeh aurora uogse1 uoew mu m o co. 0 ...”.m: V 1 GD :Aungpv u l on L: (“E“) to; .Amo.v av mcmtmmcmm s_mcmumm uwcmwm mo: mam mepmp :oEEoo m An mmmczoeczm mcmmmEmwm :mmz .m mcopo mmmworssmcm am mo mm>mmF mmmowramc e co mmzmmmm :mzp .o.m In op cam .ugmwmz act _mwpmoae we as cma mE:_o> szcm op mmmmmnmm mcmz menu—mo mum om, omm some mmpmcupww mmpoom .EzmeEmE coFNmomN: mo mmpm—omw m a; compommocm :wxoh .m mesmmm 0.0—02 53.05.50:— . I m-nm¢ o-noo .-.—N N-m.¢ «.nua N (uuu) JOJBWDEQ uogse1 uoew IPV umu ID IA Q A: a .— 44 tremuloides. Isolates differed, however, in size of lesions which were induced by their culture filtrates. Non-pathogenic isolates or those of low virulence produced the smallest amounts of toxin. Variability 9f host response tg_toxin.- Lyophilized toxin was dis- solved in water (8.0 mg/ml), diluted serially to 1.0 mg/ml, and assayed on leaves of E, tremuloides clones l, 2, 3, 4, and 5. Leaves from clones .3 and 5 were highly sensitive to toxin, and produced significantly larger E lesions than leaves from clones l, 2, and 4 (Figure 10 and Table 3). ; Leaves from 24 additional clones were tested with concentrated, 1 deproteinized culture filtrate of isolate RL5-2. Again, significant : differences in toxin sensitivity among clones were observed (Table 4). 9 To determine if varying sensitivity to toxin among clones was re- lated to their susceptibility to H, mammatum, four three-year-old stems of H, tremuloides clones l, 2, 3, 4, and 5, and six one-year-old stems of each of 24 clones (represented in Table 4), were inoculated with H, mammatum isolate RL5-2. The twenty three-year-old stems were inoculated in mid-March in the greenhouse at the time of leaf flush after a two- month period of dormancy in outdoor cold frames. Trees were maintained as above under continous light in 30 cm diameter pots. One-year-old stems were inoculated at the same time, but these plants had been held in a state of continous growth in the greenhouse since their propagation during the preceding summer. Stems were maintained in two-inch square Plant Band containers (Monarch Mfg. Co., Salida, Colo.), under the same lighting, watering, and fertilization regimes described above. Cankers developed on 162 of 163 inoculated stems. One-year-old stems were harvested 24 days after inoculation, three-year-old stems 56 days after inoculation. Cankers were examined in the laboratory under 7x 45 T \ \ I "I l 1 Clone: 4 Mean lesion Diameter (mm) 0 2 4 ‘ s a Toxin Concentration (mg/ml) Figure 10. Sensitivity of 5 Populus tremuloides clones to lyophilized H, mammatum toxin. Each point represents the mean diameter of lesions on 4 replicate leaves of each clone. Confidence intervals (95%) around points indicating expo- sure of clones 3 and 5 to 8 mg/ml toxin are delineated. ‘ ‘11.. . - ‘u._‘_. - l i- 46 Table 3. Response of 5 genotypes of Populus tremuloides to inoculations with Hypoxylon mammatum isolate RL5-2, and to its host- selective toxin. Means in each column followed by a common letter are not significantly different (P <.05). Mean Lesion Mean Canker Clone Diameter (mm) Length (mm) 5 18.0 a/ A 35.0 b’2 3 11.3 B 66.0 X 4 1.8 C 60.0 XY 2 1.8 C 32.8 Z 1 0.9 C 39.8 YZ a/ Mean diameter of lesions caused by exposure of 4 replicate leaves to 8.0 mg/ml lyophilized toxin (see Fig. 10). b/ Mean length of cankers among 4 replicate three-year-old stems. 47 Table 4. Response of 24 genotypes of Populus tremuloides to inoculations with Hypoxylon mammatum isolate RL5-2, and to its host-selective toxin. Means in each column followed by a common letter are not significantly different (P <.01). Mean Lesion Mean Canker Clone Diameter (mm) Length (mm) 281 20.8“ A 41 .2b/ wxvz 201 19.5 A 39.8 VWXYZ 71 15.5 A 52.2 UVW 242 9.4 B 37.0 XYZ 32 9.1 B 31.5 WXYZ 62 8.0 DC 19.0 Z 203 7.9 BC 71.3 U 404 6.8 BCD 32.8 WXYZ 64 6.0 BCDE 39.5 WXYZ 97 5.9 BCDE 51.3 UVNX 94 5.4 BCDEF 34.2 WXYZ 243 5.0 BCDEF 33.2 WXYZ 34 4.9 BCDEF 56.4 UVW 9 4.9 BCDEF 47.5 UVWXY 8 4.6 CDEF 41.3 VWXYZ 63 4.4 CDEFG 37.5 WXYZ 66 3.3 DEFG 31.8 WXYZ 33 2.8 DEFG 66.0 UV 6 2.8 DEFG 21.5 YZ 74 2.3 DEFG 37.7 WXYZ 69 2.3 DEFG 22.8 YZ 405 1.4 EFG 42.2 VWXYZ 406 1.1 FG 42.0 VWXYZ 412 0.0 G 25.7 XYZ a/ Mean diameter of 8 lesions caused by exposure of 2 replicate leaves to concentrated, deproteinized culture filtrate. b/ Mean length of cankers among 6 replicate one-year-old stems. 48 magnification, and measured from apical to basal ends after removal of outer bark. Canker lengths varied significantly among clones of both three- year-old and one-year-old stems (Tables 3 and 4). Mean canker length observed on each clone was plotted against sensitivity to toxin in assays of leaves from the same clone. Among the three-year-old stems of clones l, 2, 3, 4, and 5, mean canker length was plotted against the mean diameter of lesions on leaves exposed to 8.0 mg lyophilized toxin per m1 (Table 3, from Figure 10). No significant correlation between sensitivity to toxin and size of cankers was observed among these five clones. Among the one-year-old stems of 24 E, tremuloides clones, mean canker length was plotted against the mean diameter of lesions on leaves exposed to concentrated, deproteinized culture fil- trate of isolate RL5-2 (Table 4). Again, no significant correlation between lesion size and size of cankers was observed. The 24 clones were then statistically grouped according to their toxin sensitivity. For example, clones 281, 201, and 71, which were lnot significantly different in their sensitivity to toxin, comprised group A. Group G included clones 63 through 412 (see Table 4). The mean toxin sensitivity of all clones in each group was then calculated, as well as the mean length of all cankers on all clones in each group (Table 5). Linear regression analysis between the two values thus obtained for groups A through G, indicated a significant, positive relationship between toxin sensitivity and mean canker length (r = 0.87). ‘W AJ.’ m'i Wyfl 49 Table 5. Response of 24 clones of Populus tremuloides to inoculations with Hypoxylon mammatum isolate RL5-2, and to its host- selective t0x1n. Clones are ranked in groups (A through G) not significantly different in toxin sensitivity (P <.01). Clone Mean Lesion Mean Canker Group Diameter (mm) Length (mm) A 18.60 a/ 44.40 b/ B 6.66 41.25 C 6.08 41.29 D 4.39 39.54 E 4.00 40.21 F 3.48 39.55 G 2.27 36.36 r = 0.87 V a/ Mean diameter of lesions caused by exposure of 2 replicate leaves 2f all clones in each group to concentrated, deproteinized iltrate. 0] Mean length of cankers among 6 replicate one-year-old stems of all clones in each group. c/ Correlation coefficient (r) between the two indices is significant at 1% error probability. lm‘t? 4! u five-....- I- a DISCUSSION These experiments corroborate earlier evidence (15, 16) of pro- duction of host-selective metabolites by H, mammatum in culture. Such compounds were regular metabolic products of the fungus, produced under all conditions tested that allowed growth of mycelium. Toxic meta- bolites were active only on certain Populus species, and produced spreading, black necrotic lesions only on leaves of H, tremuloides, which is the major host of H, mammatum. The effectiveness of leaf assays in determining toxin concentra- tion or activity was demonstrated by use of lyophilized, partially purified toxin supplied to leaves in various concentrations. The linear dosage response relationship between lesion size and toxin con- centration (plotted logarithmically) indicates that lesion size is related to the concentration of toxin applied to the leaf. Leaves of E, deltoides, which is not infected with H, mammatum in nature, were not affected by toxin, either in the form of crude culture filtrate, con- centrated, deproteinized culture filtrate, or when lyophilized toxin was supplied to the leaf. Schipper also reported low sensitivity of H, deltoides leaves to H, mammatum toxin (15). Chromatography of toxic components on silica gel plates indicated that at least two toxic compounds may be produced by H, mammatum. This is in agreement with results obtained by Schipper (17) which indicated that up to four host-selective compounds could be extracted from rye grain cultures of the fungus. In the case of H, mammatum toxin(s) it 50 51 is not known whether all or indeed any of the compounds indicated here- in are produced during actual infection of stems. However, host- selective compounds have been isolated from bark infected with H, mammatum (15). The toxic components identified in these experiments are compounds of low molecular weight (less than 1000 d). Toxic activity of culture filtrates was not lost by removal of large compounds either by methanol precipitation of proteins and polysaccharides, or by their separation on dextran molecular sizing gels. Toxin was partitioned into n-butanol, leaving salts and yellow-colored compounds in the aqueous phase. These procedures could be useful for further purification and characterization of toxic compounds. Also, the thermal stability of toxic components may facilitate their characterization via gas chromatography and mass spectrometry. Toxin production by different isolates of H, mammatum varied significantly. The ability to produce toxin by five isolates in culture was directly related to their ability to produce cankers when inoculated into stems of E, tremuloides clones 3 and 5. In tests of 21 isolates of H, mammatum, all isolates produced toxin, but the lowest amounts were produced by isolates which were essentially non-pathogenic [isolate 605-6 (6), and isolates RL5-4, RL5-8]. These three non-pathogenic isolates all demonstrated the 'conidial' growth form in vitro (2), which has been associated with low pathogenicity (2, 6). It seems, therefore, that a threshold level of toxin-production may be necessary for infection of E, tremuloides stems. Previous evidence also indicated that presence of toxin was necessary for infection by H, mammatum (16). Sensitivity to H, mammatum toxin(s) may be related to he: 3- 52 susceptibility of aspens to infection by the fungus. Leaves from clones of H, grandidentata, which is rarely infected in nature, devel- oped small, brown lesions after treatment with toxin. In contrast, large, black lesions were observed on E, tremuloides leaves. Toxin completely blackened the leaves of some clones after exposure of up to 48 hr. The significant variation among 3, tremuloides clones in sensitivity to toxin in leaf assays might indicate their variation in susceptibility to H, mammatum. Attempts to prove this relationship by inoculating young stems in the greenhouse were largely unsuccessful, however. Sensitivity to toxin in leaf assays was generally not correlated with size of cankers produced by inoculation of stems with the fungus. Lack of correlation between the two values was attributed to clones such as 33 and 34 (Table 4), which produced 1009 cankers after inoculation with H, mammatum, but were low in sensitivity to toxin. Alternatively, clone 63 was highly sensitive to toxin, but produced short cankers after inoculation. When clones were grouped statistically according to their toxin sensitivity, however, a direct relationship of toxin sensitivity with canker length was indicated (Table 5). This type of grouping may compensate for spurious results which might be produced by any single clone in either toxin sensitivity 0r rate of canker development after inoculation. Also, inoculation of young main stems of E, tremuloides clones may not be an optimal procedure for determining susceptibility to Hypoxylon canker. Infection in nature is most commonly associated with lateral branches of sapling-sized trees following insect attack or branch death (10, 11). The relationship between toxin sensitivity and susceptibility of clones to infection by H, mammatum must be evaluated 53 more rigorously before attempting to use H, mammatum host-selective metabolites as a tool for screening for resistance. At present, it is recommended that procedures for identifying resistance to Hypoxylon canker include both inoculation with the fungus and tests with its host-selective toxin. Genotypes which emerge as desirable in both respects could be used for establishing plantations. These could then be evaluated for any long-term gain in disease resistance after exposure to conditions of natural inoculation and infection. ‘C'_““""j 10. 11. LITERATURE CITED BAGGA, D. K. 1969. Physiology of H ox lon pruinatum and its pathogenesis on quaking aspen. Ph.D. hesis, University of Wisconsin, Madison. 165 p. BAGGA, D. K., and E. B. SMALLEY. 1974. Variation of H ox lon pruinatum in cultural morphology and virulence. Phytopatfiglogy 64: 663-667. BROWNING, B. L. 1967. Methods of Head Chemistry. Phenolic Substances, pp. 225-230. J. Wiley 8 Sons. 384 p. BYTHER, R. S., and G. W. STEINER. 1972. Use of helminthosporoside to select sugarcane seedlings resistant to eye spot disease. Phytopathology 62: 466-470. FRENCH, J. R. 1976. This Thesis, Part I. FRENCH, J. R., and P. D. MANION. 1975. Variability of host and pathogen in Hypoxylon canker of aspen. Can. J. Bot. 53: 2740- 2744. FRENCH, J. R., and J. H. HART. 1976. Variability of canker length on aspen clones following inoculation with H o lon mammatum. American Phytopathological Society, Proceedings I: 97 (afistact). HIROE, I., and S. AGE. 1954. Phytopathological studies on black spot disease of Japanese pear, caused by Alternaria kikuchiana Tanaka (English Series 1) Pathochemical studies (1). on a new phytotoxin, phyto-alternarin produced by the fungus. Jour. Fac. Agric. Tottori University 2: 1-26. LUKE, H. H., and H. E. WHEELER. 1955. Toxin production by Helminthosporium victoriae. Phytopathology 45: 453-458. MANION, P. D. 1975. Two infection sites of H ox lon mammatum in trembling aspen (Pppulus tremuloides). Can. J. Bot. 53: 2621- 2624. NORD, J. C., and F. B. KNIGHT. 1972. The importance of Saperda inornata and Oberea schaumii (Coleoptera: Cerambycidae) galleries as infection courts of Hypoxylon pruinatum in trembling aspen, Populus tremuloides. Great Lakes EntomOTDgist 5: 87-92. 54 12. 13. 14. 15. 16. 17. 18. 19. 55 OSHIMA, N. 1957. Physiology of Hypoxylon pruinatum (Klot.) Cke. Ph.D. Thesis, University of Minnesota, St.*Paul. 61 p. PRINGLE, R. B., and A. C. BRAUN. 1957. The isolation of the toxin of Helminthosporium victoriae. Phytopathology 47: 369-371. PRINGLE, R. B., and R. P. SCHEFFER. 1967. Isolation of the host- specific toxin and a related substance with nonspecific toxicity from Helminthosporium carbonum. Phytopathology 57: 1169-1172. SCHIPPER, A. L. 1973. Mammatoxin, a Hypoxylon mammatum metabolite responsible for canker development in aspen. Second Int. Cong. of Plant Pathology, Abstracts of Papers (abstract no. 0677). American Phytopathological Society, Inc., St. Paul. SCHIPPER, A. L. 1975. Hypoxylon pathotoxin necessary to the infection of aspen by Hypoxylon mammatum. American Phytopatholo- gical Society, Proceedings 2: 46-47 (abstract). SCHIPPER, A. L. 1976. personal communication. WHEELER, H. E., and H. H. LUKE. 1955. Mass screening for disease- resistant mutants in oats. Science 122: 1229. WHEELER, H. E., A. S. WILLIAMS, and L. D. YOUNG. 1971. Helmin- thosporium maydis T-toxin as an indicator of resistance to southern cornlleaf blight. Plant Dis. Rptr. 55: 667-671. SUMMARY Identifying aspen genetically resistant to Hypoxylon canker and establishing such stock in plantations may be valuable contributions toward increasing the production of raw materials for forest based "“7 L. “Q‘s. L :fir'nn industries. Losses to the disease could be reduced in this manner, allowing a greater portion of the aspen resource to be converted to usable cellulose. :.<.:- | Identification of resistance within Populus tremuloides may be accomplished through study of naturally existing clones, selecting genotypes either low in amounts of natural infection, or which produce short cankers after inoculation with Hypoxylon mammatum. The present experiments indicate that inoculation with the fungus may be generally applied over wide geographical areas, and that clone differences in canker length may be readily detected, provided that standardized inoculum types and techniques are used. Moreover, this technique may be useful in areas where natural incidence of Hypoxylon canker is low, and where susceptibility among the host population may not be expressed due to environmental factors. Resistance may also be expressed among aspen genotypes by low sensitivity to host-specific metabolites produced by H, mammatum in culture. 3, grandidentata is virtually immune to natural infection by H, mammatum, and leaves from this species exhibited low sensitivity to toxic substances from culture filtrates of the fungus. P, tremuloides 56 57 leaves exhibited wide variability in sensitivity to the same meta- bolites, indicating that resistance may be present within this species also. Extreme caution must be exercised in the application of these techniques, however. It is recommended that procedures for selection of resistant genotypes include both inoculation with the fungus under standardized conditions, and tests with its host-specific toxin(s). Neither technique alone is a proven indicator of resistance to Hypoxylon canker. The procedures outlined in the present experiments may be useful since they are cheap and easily accomplished. The alternatives, which would normally include establishment of a random sample of aspen geno- types in a common plantation, followed by long-term evaluation of disease incidence, would involve unnecessary expenditure of time and space on genotypes with little promise of desirable qualities. Pre- liminary screeing of E, tremuloides clones jg_§jtg_by inoculation with fungus mycelium, or by use of excised leaves in assays of toxin sensitivity, would allow a proper focus on genotypes which are potentially desirable from both aspects. Such genotypes could then be used for plantation or for hybridization with E, grandidentata. Hybrids between these two species have demonstrated advantages in resistance to canker enlargement; use of higher levels of resistance in H, tremuloides parental lines may provide even greater gains in this respect. APPENDIX fin-Full. t .. . .Ir.uti.-lt1ra.u:.l 58 Appendix A. Mean canker lengths caused by inoculation of four clones of Pppulus tremuloides with 12 isolates of Hypoxylon mammatum, during June, July, and August, 1974. 59 June Clone RL 11 RL 1 RL 8 RL 4 no. mean no. mean no. mean no. mean Isolate cankers length cankers length cankers length cankers length RL5-2(A) 4 92 3 64 3 71 3 60 RL5-6(B) 4 72 4 73 3 61 4 44 RL5-3(C) 4 84 3 62 3 51 3 55 RL5-7(D) 4 75 4 53 4 46 4 46 211-2(E) 2 99 4 65 3 82 2 74 211-8(F) 3 56 4 34 4 43 2 57 RL5-5 4 46 0 - 1 47 2 50 208-6 4 77 0 - 0 - 0 - RL5-8 0 - 1 21 0 - 0 - RL5-l l 16 0 - 0 - 0 - 605-6 0 - 0 - 0 - o - RL5-4 0 - 0 - 0 - 0 - control 0 - 0 - 0 - 0 - All Isolates 30 72 23 56 21 57 20 51 All no. cankers = 94 Clones mean canker length = 60.8 60 Appendix A, continued July Clone RL ll . RL 1 RL 8 RL 4 __ no. mean no. mean no. mean no. mean Isolate cankers length cankers length cankers length cankers length RL5-2(A) 3 80 4 47 3 52 3 38 RL5-6(B) 4 62 4 63 4 52 3 36 RL5-3(C) 2 61 4 60 3 45 4 41 RL5-7(D) 4 64 4 58 2 42 1 18 211-2(E) 2 78 4 82 4 71 2 30 211-8(F) 4 61 4 35 4 38 3 24 RL5-5 l 44 2 29 0 - 3 32 208-6 0 - 0 - 0 - 0 - RL5-8 0 - 0 - 0 - 0 - RL5-l 0 - 0 - 0 - 0 - 605-6 0 - 0 - 0 - 1 21 RL5-4 0 - 0 - 0 - 0 - control 0 - 0 - 0 - 0 - All Isolates 20 65 26 55 20 51 20 33 All no. cankers = 86 Clones mean canker length = 51.3 61 Appendix A, continued August Clone RL ll . RL 1 RL 8 RL 4 no. mean no. mean no. mean no. mean Isolate cankers length cankers length cankers length cankers length RL5-2(A) 4 37 4 14 4 l4 3 28' RL5-6(B) 3 23 4 24 4 21 3 l6 RL5-3(C) 2 23 4 18 4 19 4 15 RL5-7(D) l 35 0 - 2 21 2 13 211-2(E) 0 - 0 - 2 32 0 - 211-8(F) 0 - 0 - 0 - 0 - RL5-5 0 - 0 - 0 - 0 - 208-6 0 - 0 - 0 - 0 - RL5-8 2 12 0 - l 12 0 - RL5-l 0 - 0 - 0 - 0 - 605-6 0 - 0 - 0 - 0 - RL5-4 0 - 0 - 0 - o - control 0 - 0 - 0 - o - All Isolates 12 27 12 18 17 20 12 18 All no. cankers = 53 Clones mean canker length = 20.6 62 Appendix A, continued All Months Clone RL 11 RL 1 HL___§__ RL 4 éilnes Isolate 3 =- 3 =- 3 :- 3 =- 3 :- RL5-2(A) 11 69 ll 40 10 43 9 42 41 48.6 RL5-6(B) ll 55 12 53 ll 44 10 33 44 46.6 RL5-3(C) 8 63 ll 45 10 36 ll 35 40 43.7 RL5-7(D) 9 66 8 55 8 38 7 32 32 48.9 211-2(E) 4 88 8 74 9 66 4 52 25 69.6 211-8(F) 7 59 8 35 8 40 5 37 28 42.7 RL5-5 5 46 2 29 1 47 5 39 13 40.8 208-6 4 77 0 - 0 - 0 - 4 76.5 RL5-8 2 12 1 21 l 12 0 - 4 14.3 RL5-l 1 l6 0 - 0 - 0 - 1 16.0 605-6 0 - 0 - 0 - l 21 1 21.0 RL5-4 0 - 0 - 0 - 0 - 0 - control 0 - 0 - 0 - 0 - 0 - All Isolates 62 61 61 48 58 44 52 37 233 48.2 63 Appendix 8. Mean canker lengths (MCL) caused by inoculation of 100 clones of Pppulus tremuloides with two highly virulent isolates of Hypoxylon mammatum, during 1975. 64 natural infection MCL combined RL5-6 + sum of MCL no. % no. MCL MCL MCL all clones, trees in- area clone cankers RL5-2 RL5-6 RL5-2 each area tallied fected l 1 5 67 55 123 21 33 2 5 72 50 123 a/ 28 43 3 5 64 54 118 AB 31 23 4 4 53 45 98 105.5 29 28 5 4 51 39 90 21 38 6 5 47 31 78 22 0 2 l 5 73 52 126 38 3 2 5 67 56 123 26 58 3 5 73 43 116 AB 20 15 4 5 60 42 102 101.8 41 2 5 5 60 35 96 23 0 6 5 48 47 95 24 17 7 5 55 36 91 20 42 8 5 46 21 67 66 0 3 1 5 78 51 129 20 10 2 5 71 57 129 20 15 3 5 66 57 123 38 26 4 5 71 51 122 35 40 5 5 73 48 122 AB 23 0 6 5 77 44 121 117.0 34 24 7 5 57 53 109 33 18 8 5 59 45 105 65 14 9 4 59 46 104 20 6 10 2 48 37 85 20 0 4 l 4 79 58 138 20 6 2 4 73 47 120 79 9 3 5 63 48 111 41 7 4 5 56 52 108 AB 45 29 5 4 62 42 104 110.5 35 0 6 5 63 41 104 40 23 7 4 51 40 92 64 11 5 1 5 81 74 155 43 5 2 4 78 69 148 54 14 3 5 61 62 123 42 12 4 5 66 54 120 AB 51 20 5 5 65 51 116 118.1 81 6 6 5 61 42 103 62 10 7 4 47 47 94 75 36 8 5 56 32 87 74 8 65 Appendix 8, continued 6 1 5 82 60 142 2 5 80 61 141 3 5 63 66 130 4 4 69 47 116 5 5 55 50 104 AB 6 4 50 51 102 113.0 7 5 68 32 100 8 5 63 35 98 9 5 57 38 95 10 2 33 49 82 7 1 3 66 77 143 2 5 71 70 140 3 3 72 55 127 4 3 54 56 110 5 4 51 50 101 AB 6 2 52 46 98 113.8 7 4 46 50 96 8 2 49 43 92 9 2 41 48 88 8 1 4 78 74 151 2 5 81 61 141 3 5 73 61 134 4 5 65 67 132 A 5 5 75 52 127 124.9 6 5 68 47 115 7 5 66 45 111 8 5 52 41 93 9 1 5 96 59 115 2 4 68 65 133 3 5 74 52 125 AB 4 5 66 46 113 122.1 5 5 59 48 108 6 5 56 45 101 10 1 5 66 53 119 2 5 64 47 110 3 4 62 44 105 4 5 54 46 100 5 5 56 40 97 AB 6 5 53 39 92 95.4 7 4 49 42 91 8 5 49 39 88 9 5 37 40 77 10 5 50 27 76 N \IO-thUTKO-th OO-—"CDO--J _ul 66 Appendix 8, continued 11 1 3 51 43 94 48 2 4 36 33 69 25 3 1 44 24 68 32 4 5 31 34 65 34 5 4 34 29 63 B 41 6 5 35 -27 62 63 6 83 7 5 38 23 62 52 8 4 33 28 61 50 9 5 32 26 58 78 10 3 23 20 43 63 12 1 5 52 44 95 32 2 2 53 4O 93 25 3 5 43 35 77 52 4 5 44 31 75 B 100 5 5 38 26 64 68 7 74 6 5 32 26 57 75 7 4 24 29 53 48 8 4 25 15 40 20 8] Among areas, combined sums of mean canker length surmounted by a common letter are not significantly different (P <.05). 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