I 17 EPIDEMIOLOGY OF BACTERIAL CANKER OF PLUM (PRUNUS DOMESTICA) CAUSED BY PSEUDOMONAS SYRINGAE PATHOVAR MORSPRUNORUM BY KALUDURA INDRA JINADARIE GUNAWARDENA EAST MALLING RESEARCH STATION MAIDSTONE, KENT, U.K. A thesis presented for the degree of Doctor of Philosophy in the University of London, September, 1983 1 ABSTRACT Investigations on overwintering sites proved that the pathogen overwinters in association with apparently healthy plum buds with no evidence that climatic factors affect the low numbers present. Pv. morsprunorum colonises the surfaces and outer bud parts but not the innermost primordia. The likelihood of transmission of the pathogen to new orchards was demonstrated by detection of the pathogen from bud-wood and buds of nursery trees. Pathogen populations increased sharply at bud-break and initiated the leaf surface populations which were main- tained throughout the growing season. Regression analysis of climatic data revealed a negative correlation between these populations and maximum temperature and sunshine dura- tion. The pathogen was barely detectable in rain water drain- ing down plum trees during winter. However, the threshold inoculum level for infection was very low during winter and a sigmoid relationship was demonstrated between inoculum concentration and both frequency of infection and lesion length. Wound size did not affect the frequency of stem infection but there was a significant difference between the lesion 1 enrt.hs rr r)\--r-rmt height? rn the trun!<r follow- ing inoculation through small wounds. A relation between time of inoculation and temperature with lesion length was also demonstrated. Pv. morsprunorum dominated isolates from buds and leaves. 2 The epidemiological significance of the four phage-types detected among these isolates is discussed. A few isolates conformed to pv. syringae but were only weakly pathogenic. Some oxidase positive isolates,more comparable to Pseudomonas fluorescens, especially from leaf washings and rain water were detected. Bordeaux mixture had little effect on the pathogen population in dormant buds, but controlled the pathogen in breaking buds. In preliminary investigations,two bacterial isolates antagonistic to pv. morsprunorum in vitro showed some promise for biological control. The epidemiological significance of findings is dis- cussed in relation to possible control measures of the disease in the field. 3 ACKNOWLEDGEMENTS I wish to thank the Director, Dr. I.J. Graham-Bryce, for providing the facilities at East Mailing Research Station. I gratefully acknowledge the financial support provided by the Food and Agriculture Organisation of the United Nations. I record my sincere gratitude to my supervisor, Dr. C.M.E. Garrett, for her constant and valuable guidance, constructive criticism and sustaining support throughout this study. I sincerely thank Dr. S.A. Archer, my Director of Studies and Dr. S.N. de S. Seneviratne, Department of Agriculture, Sri Lanka for their encouragement and support. I am also indebted to Mr. G.P. Barlow and Mr. K.J. Martin for the statistical analyses, Miss M.K.Davis for preparation of diagrams and the members of the photo- graphic studio. Many others have given their time generously and provided valuable help including particularly Mrs. D.A. Fletcher, Mrs. R. Gough and Dr. R.C. Hignett. Finally, I would like to thank Mrs. J. Allen-King for typing the manuscript. 4 CONTENTS Part I Introduction 12 Historical and literature review 13 Part IE Experimental 1. General methods 31 2. Overwintering of Pseudomonas syringae pv. 35 morsprunorum 3. Epiphytic bacterial populations on leaves 73 4. Studies on factors relating to the establish- 86 ment of plum stem infection Some bacteriological characters, phage sensi- 121 tivities and in vivo growth of bacteria associated with plum trees 6. Prospects for control 156 Part IH General conclusions 174 References 179 5 LIST OF TABLES, PLATES AND FIGURES Table 2.1 Numbers of bacteria (log c.f.u./bud) 41 in 7 samples taken simultaneously from plum and cherry Plate 2.1 Structural differences of plum (cv. 43 Victoria) and cherry (cv. Napoleon) (A) plum bud surfaces - pubescent, (B) cherry bud surfaces - glabrous, (C) Longitudinal section of a plum bud showing loosely bound bud parts and (D) Longitudinal section of a cherry bud with tightly closed bud parts. Figure 2.1 Arrangement of bud parts numbered 47 starting from the outermost scale (No.l) to- innermost primordia (No.13) on an isolation plate (inverted). Plate 2.2 Growth of bacteria from plum bud 47 parts (1-7) on NSA + CVC medium (after 2 days incubation at 25°C.). Plate 2.3 (A) A rain water trap attached to 49 the trunk of a plum tree (B) close up view of the trap showing positions of the holes. Plate 2.4 (A) Rain water traps on the branches 50 of a plum tree (B) close up view of a trap on an one-year-old branch. Table 2.2 Bacteria (c.f.u./bud) isolated from 52 whole buds of plum and cherry, winter 1980/81. Figure 2.2 Fluctuations from November to April 54 of Pseudomonas syringae pv. morsprun- orum and saprophytic bacterial popu- lations from crushed whole buds of (A) 6-year-old (B) 3-year-old plum trees. Figure 2.3 Fluctuations from November to April 56 of Pseudomonas syringae pv. morsprun- orum and saprophytic bacterial popu- lations from crushed whole buds of 3-year-old cherry trees. Figure 2.4 Frequency (%) of detection of Pseudo- 57 monas syringae pv. morsprunorum and saprophytic bacteria on the surfaces of plum and cherry buds at approx- imately six weekly intervals from October to April. 6 Figure 2.5 Frequency of infestation of plum 59 bud parts with (A) Pseudomonas syringae pv. morsprunorum (B) saprophytic bacteria (15 bud samples). Where frequencies for different sampling dates converged a single value for these is denoted by a large circle. Table 2.3 Pathogen, populations from buds of 62 nursery trees in March. Table 2.4 Estimated pathogen populations from 62 buds of different cultivars of plum mother-trees in August. Table 2.5 Amount of water collected and numbers 64 of Pseudomonas syringae pv. morsprun- orum (c.f.u. ml. * ) from rain water draining down plum trees (winter 1980/81. Table 2.6 Mean numbers of bacteria (c.f.u. 64 ml."*) isolated from rain water drain- ing down plum trees collected in trunk traps during winter 1982/83. Table 2.7 Mean amount of rain water per branch 67 collected from the traps on 1, 2 and 3-year-old branches on plum (cv. Victoria) trees during winter 1982/83. Table 2.8 Mean numbers of bacteria (c.f.u. 67 ml.""l) isolated from rain water drain- ing down plum trees collected in traps on 1, 2 and 3-year-old branches during winter 1982/83. Figure 3.1 Fluctuations of Pseudomonas syringae 77 pv. morsprunorum and saprophytic bacterial populations on leaves of (A) 3-year-crld plum trees (B) 6- year-old plum trees (C) 6-year-old cherry trees (D) weekly mean maximum temperature over the sampling period. Table 3.1 Correlation matrix for the pathogen gl population. Plate 4.1 A tray lined with moistened cotton 91 wool used to incubate inoculated plum twigs showing the arrangement of twigs on wooden dowels. Table 4.1 Mean log lesion length beyond wound 94 resulting from small and large wounds in high and low positions (field trees). 7 Plate 4.2 Lesion development in plum twigs 96 inoculated with Pseudomonas syringae pv. morsprunorum (A) water control (B) a lesion with diffused edges (C) a lesion with sharp edges (D) a lesion and staining in the wood extending the length of twig. Figure 4.1 Effect of wound size and duration 97 of susceptibility of wounds on the A) frequency of infection and B) mean lesion length, resulting from inoculations wit^j 30 >ul. f^onj a suspension of 10 c.f.u. ml. of Pseudomonas syringae pv. morsprunorum plum strain (D312) in plum twigs on 8.12.1982 held at 10°C. for 13 weeks. • large wound, 0 small wounds. Table 4.2 Effect of wound size on lesion length 99 on plum twigs inoculated with Pseudo- monas syringae pv. morsprunorum (isolate D312). Table 4.3 Effect of the duration of wound 100 susceptibility on lesion length on plum twigs inoculated with Pseudomonas syringae pv. morsprunorum (isolate D312) . Table 4.4 Effect of interval between wounding 100 and time of inoculation on the sur- vival of inoculated Pseudomonas syringae pv. morsprunorum (isolate D312) from plum twigs Figure 4.2 Mean lesion length caused by 30 ,ul. 103 from a suspension of 10® c.f.u. ml.-1 of Pseudomonas syringae pv. morsprun- orum plum strains D312 and D505 and water controls (wound response) in detached plum twigs when inoculated in A) November B) January, and held at different temperatures. Table 4.5 Log mean lesion lengths resulting 105 from inoculation of twigs with Pseudomonas syringae pv. morsprunorum and held at constant temperatures. 8 Table 4.6 Log mean lesion length/day resulting 106 from inoculation of twigs with Pseudomonas syringae pv. morsprunorum and held at constant temperatures. Figure 4.3 A) The frequency of infection and 107 B) mean lesion length resulting from inoculation of a log series of concen- trations of Pseudomonas syringae pv. morsprunorum plum strain (D312) to 4 to 5-year-old field trees of plum cv. Victoria in December. Table 4.7 Mean lesion lengths on the trunks 110 of plum trees resulting from a log series of inoculum concentrations of Pseudomonas syringae pv. morsprun- orum. Table 4.8 Comparison of mean lesion lengths 110 upwards and downwards from the inocu- lation point on plum trunks (means of 7 replicates). Figure 4.4 A) The frequency of infection and 113 B) mean lesion lengths resulting from inoculation of a log series of concentrations of Pseudomonas syringae pv.