Epidemiology of Bacterial Canker of Plum

I 17

EPIDEMIOLOGY OF BACTERIAL CANKER OF PLUM

(PRUNUS DOMESTICA) CAUSED BY PSEUDOMONAS

SYRINGAE PATHOVAR MORSPRUNORUM

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 maintained throughout the growing season. Regression analysis of climatic data revealed a negative correlation between these populations and maximum temperature and sunshine duration.

The pathogen was barely detectable in rain water draining 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!

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 discussed 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. morsprunorum and saprophytic bacterial populations 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. morsprunorum and saprophytic bacterial populations 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 approximately 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. morsprunorum (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 draining 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 draining 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 survival 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. morsprunorum 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 concentrations 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. morsprunorum. Table 4.8 Comparison of mean lesion lengths 110 upwards and downwards from the inoculation 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.. morsprunorum plum strain (D312) to detached plum twigs cv. Victoria and held at C.15°C. for 9 weeks. - Table 4.9 The mean lesion length of plum twigs 115 resulting from . inoculations of a log series of concentrations of Pseudomonas syringae pv. morsprunorum. Table 5.1 Origin and specificity of typing 127 phages. Plate 5.1 Chambers for. incubating leaf petioles 129 at constant humidities controlled by isopiestic equilibration of saturated solutions of inorganic salts. Figure 5.1 Different colony types observed among 131 bacteria isolated from plum trees. Table 5.2 Differential tests of typical Pseudo- 132 monas, syringae pv. morsprunorum and P. syringae pv. syringae (data from Garrett et al., 1966; 1977).

9 Table 5.3 Main categories of bacteriophage 136 sensitivity observed among bacteria associated with plum trees (392 isolates tested).

Table 5.4 Characters of isolates conforming 137 to typical pv. morsprunorum. Table 5.5 Characters of isolates conforming 138 to typical pv. syringae. Table 5.6 Relation of character group (physio- 140 logical, biochemical, bacteriophage sensitivity and colony morphology) with source of isolates in Pseudomonas syringae pv. morsprunorum. Table 5.7 Mean lesion length produced on plum 14 2 branches cv. Victoria following inoculations with Pseudomonas syringae pv. morsprunorum and P^ syringae pv. syringae plum isolates.

Figure 5.2 Growth of Psuedomonas syringae pv. 147 morsprunorum (plum strain) in plum petioles at 98% relative humidity and different temperatures. Figure 5.3 Growth of 4 different plum strains 149 of Pseudomonas syringae pv. morsprunorum in plum and in cherry petioles at 98% relative humidity and 20°C. Figure 5.4 Growth of 4 different cherry strains 150 of Pseudomonas syringae pv. morsprunorum in plum and cherry petioles at 98% relative' humidity and 20°C. Figure 6.1 Comparison of populations of Pseudo- 160 monas syringae pv. morsprunorum from buds or tiny leaves from broken buds of two plum cultivars of non-sprayed (control) trees and of trees sprayed with weak Bordeaux mixture.

Plate 6.1 Inhibition of growth of Pseudomonas 164 syringae pv. morsprunorum and pv. syringae isolates cross-streaked against(A) JA9, wider inhibition zone (B) JA12, narrow inhibition zone (C) JA5, no inhibition. Table 6.1 Mean length of inhibition (mm) induced 165 by four saprophytic bacterial isolates against pathogenic strains in the cross-streaking method.

10 Table 6.2 Mean diameter (mm) of inhibitory 165 zone induced by isolates JA9 and JA15 against pathogenic strains in the poured-plate method. Plate 6.2 Inhibition of growth of Pseudomonas 166 syringae pv. morsprunorum by the antagonistic isolate JA9 in the poured- plate method.

Plate 6.3 Effect of (JA9) large colonies on 168 colony morphology of Pseudomonas syringae pv. morsprunorum plum strain when grown in close proximity (A) normal shining colonies with sharp margins (B) modified dull colonies with diffused margins.

Plate 6.4 (A) Inhibitory action on Pseudomonas 169 syringae pv. morsprunorum plum strain induced by diffusible fraction of dialysis from JA9 culture filtrates (B) no inhibition from the non-diffusible fraction. Table 6.3 Some physiological and biochemical 171 characters of JA9 and JA15 isolates.

11 INTRODUCTION

Bacterial canker of stone-fruit causes considerable damage to plum and cherry trees. Previous studies have concentrated mainly on the disease on cherry, on which host several factors relating to the epidemiology of the disease have been elucidated. Understanding of the disease on plum is less complete. The investigations presented in this thesis are con- cerned with the epidemiology of bacterial canker of plum in South East England. They are directed at providing information on overwintering sites of the pathogen, estimations of leaf surface populations of the pathogen and the influence of climatic factors on them, factors concerning stem infection, bacteriological characters of the bacteria associated with plum trees and finally disease control measures.

12 Historical and Literature Review

Introduction. Symptoms of the disease on plum. Seasonal cycle and epidemiology of the disease. Environmental factors affecting disease development. The causal organism. Host range. Varietal susceptibility of plum to bacterial canker. Control measures.

13 Historical and Literature Review Introduction Smith (1903) first reported a bacterial leaf spot disease on Japanese plum in Michigan, USA. The causal agent was a yellow bacterium which he named Pseudomonas pruni. Jackson (1907) reported plum 'bacteriosis' in which cankers formed in young branches. He reproduced the disease with his isolates from cankers but gave no description of the causal organism. In the next 10 years branch canker and leaf spot diseases of plum and peach were attributed to Pseudomonas pruni in various parts of the USA. In the first monograph of the disease, apricot and nectarine were added as susceptible species (Rolfs, 1915). Griffin (1911) found gummosis and blight of cherry buds in Oregon caused by Pseudomonas cerasus. Later, Barss (1913, 1915) showed that this bacterium caused cankers and gummosis of most stone-fruit trees. These two workers introduced the terms 'bacterial gummosis1 and 'bacterial canker' for the disease. Similar symptoms were reported on apricot in California (Barrett, 1918), on peach in Canada (McCubbin, 1919) and on plum in the Sierra Nevada (Go Ids worthy and Smith, 1930). In Europe Aderhold and Ruhland (1907) attributed a bacterial disease of young cherry trees, characterised by gummosis and death, to Bacillus spongiosus. They suspected that similar symptoms in plum were also caused by a bacterium. In Britain early reports of 'die-back or gurrmosis1 on stone- fruit trees were attributed to various fungi (Cytospora spp., Diaporthe perniciosa and Dermatella prunasti) even though it proved impossible to reproduce the symptoms by inoculations (Dowson, 1913; Belgrave, 1916; Cayley, 1923 and Marsh and Nattrass, 1927). Wiltshire (1920) was the first in this country to associate this disease with a bacterium.

14 He observed rod shaped bodies in sections of diseased plum tissues. Briton-Jones (1925) who also observed bacteria in diseased tissues, failed to isolate them in culture and concluded that there was insufficient evidence to prove the pathological origin of the disease and suggested that excessive or insufficient soil moisture was the main cause. Wormald (1928) demonstrated conclusively that gummosis, die-back and leaf spot of plum and cherry trees in England were caused by bacteria and named the disease 'bacterial canker and leaf spot'. He suggested at least two distinct types of bacteria, both producing similar symptoms, were responsible and he subsequently described in. detail the symptoms and etiology of the disease and the morphological and bacteriological characteristics of the causal organisms which he named Pseudomonas prunicola and Pseudomonas morsprunorum (Wormald, 1931; 1932; 1938).

Symptoms of the disease on plum There are two distinct types of symptoms. Firstly, the canker phase appears in spring when stem cankers are seen, frequently with dark brown gum on their surfaces, and often resulting in the death of young trees. These symptoms may also, but less commonly, occur on branches. Secondly, there is a summer phase on leaves producing reddish- brown leaf spots.

(a) Cankers Cankers arise from infection in autumn or winter with invasion of the cortical and phloem tissues of the stem or branches, possibly through small wounds though the point of infection is usually not discernible. Often the first visible sign of the disease is the pale green or yellowish colour of the foliage in the spring following infection which becomes more apparent in summer. Cankers may become

15 visible as shallow depressions of dead areas surrounded by a ridge of healthy tissues. Brown, dead areas of cortical and phloem tissues can be seen if the outer layers of bark are removed. Dark brown gum also may ooze out of the bark. On older trees or branches, where the bark is thicker, the only visible sign of the canker may be gumming.

Canker length varies from a few centimetres to those that extend the full length of the stem. If a canker girdles a branch, it will cause die-back although the rest of the tree remains healthy. When, however, a trunk is girdled death of the entire tree ensues. In contrast, infections on cherry generally occur during leaf fall through leaf-scars, killing individual buds, fruiting spurs or even branches (Crosse 1955, 1956a). Sometimes cherry branches may die from crotch infections, invariably with gumming, but tree death is less common on this host than on plum.

(b) Leaf spots In late spring and early summer bacteria invade plum leaves via the stomata to form reddish-brown leaf spots, 2-3 rmn. in diameter, more or less circular and often surrounded by a yellow halo. As the leaves mature the necrotic areas become separated from the surrounding healthy tissue and drop out giving a 'shot-hole' appearance. Cherry leaf spots are usually smaller, angular and frequently without a halo.

Seasonal cycle and epidemiology of the disease The most characteristic feature of this disease is the seasonal cycle of a winter canker phase alternating with a summer leaf spot phase. The winter phase begins in autumn and the pathogen initially multiplies very slowly inside the host tissues. Bacterial multiplication increases rapidly in the spring with the invasion of considerable areas of bark,producing cankers. In early summer, with the renewal of secondary

16 growth, the tissues become resistant to further invasion by bacteria, very often progress of cankers is stopped and the bacteria die out. Cessation of canker growth in sunnier was observed by most of the earlier workers (Barss, 1913; Wilson, 1933). Wilson (1939) recorded small lesions from early-autumn and late- spring inoculations but very large cankers from winter inoculations. In early autumn and late spring the host's reaction appeared to be the more important factor. Although Wilson (1939) observed phellogen and periderm formation in late spring and summer wound inoculations he concluded that this did not actually prevent canker activity. Con- trary to this, Erikson (1945b) believed that canker extension might be stopped by periderm formation around the lesion. Shanmuganathan (1962) made extensive studies on the canker phase of the disease. He examined the bacterial growth and periderm development in plum tissues in relation to time of infection. From August inoculations, the pathogen multiplied rapidly for 2-3 weeks but bacterial growth then decreased steadily with the production first of phellogen then of several layers of suberized periderm. This became continuous with the outer periderm, isolating completely the wounded tissues from the surrounding healthy tissues. Bacterial growth declined to undetectable numbers in 2 months. Following inoculations in October, bacterial numbers increased for the first two weeks, then decreased temporarily. They then increased during the following 3 months when the phellogen production was much slower to form a complete barrier. The phellogen failed to lay down suberized layers and was discontinuous with the original outer periderm. There was, however, no evidence of the pathogen breaking through the inconplete barrier. Bacterial growth was slower at first then increased steadily in December inoculations and no phellogen production was observed until after six weeks and the injured

17 tissues remained unbarricaded. Shanmuganathan (1962) concluded that the defensive mechanism of parenchymatons cortical tissues when suberized periderm layers are laid down, is in sharp contrast to that of the tissues of the wood in which barriers are composed of deposits of gum in the vessels. The rate of formation of defensive barriers is maximal during the growing season and declines with the approach of dormancy. Cell death in advance of bacterial penetration, another striking feature of this disease, may be due to a lethal endotoxin produced by autolysis of the bacterial bodies. (Erikson, 1945b; Shanmuganathan, 1962). Barss (1918) believed cankers were non-perennial but Wilson (1939) considered at least a certain proportion could be perennial with P. syringae remaining dormant in the sunnier and becoming active again in autumn. Dye (1954) and Cameron (1962a) also provided evidence that the pathogen persists in cankers during summer. In Britain Wormald (1931, 1937) and Crosse and Garrett (1966) reported that P.morsprunorum could not be isolated from cankers after, early summer. However, there is some evidence of survival eighteen months after inoculation of P. morsprunorum race 2 (Prunier, 1977). Although infection through lenticels was suggested by Wormald (1931), Shanmuganathan (1962) was unable to produce cankers on plum by spraying lenticels with inoculum under pressure, from October to May. Crosse (1955, 1956a, 1956b, 1957, 1959) made intensive studies of branch cankers of cherry and demonstrated that most of these occur from natural leaf-scar infections from early-September to mid-October. The threshold inoculum necessary to establish such infection varied with time of inoculation and degree of resistance of the host.

18 Natural leaf-scar infections of plum are unknown and attempts to induce them experimentally have been generally unsuccessful (Anon., 1961; Shanmuganathan, 1962). The summer phase of the disease begins in late spring with the appearance of leaf spots. Leaf infection occurs at two different times of leaf growth; an early, primary, infection of young developing spur leaves soon after petal fall and secondary infection of the leaves on extension shoots throughout the summer (Crosse, 1954 ; 1963). The exact epidemiological role of the leaf spots is not fully understood. However, Crosse(1954 ) and Crosse and Bennett (1955) discount- ed the hypothesis that leaf spots were the main source of inoculum for cherry branch infection as the bacteria die out of the leaf spots when the necrotic tissues dry up. Crosse (1959) deduced that leaf surface populations of the pathogen, often as high as 10 per leaf, were the main source of leaf-scar infection. This was later confirmed experimentally (Crosse, 1962; 1963). Ereigoun (1974) was unable to detect the pathogen on cherry leaf surfaces until after the first leaf spots appeared. He isolated more P. morsprunorum from the surfaces of leaves with leaf spots than from symptomless leaves and suggested that leaf spots played a major role in 're-priming' the leaf surface populations after they had decreased under adverse conditions.

Environmental factors affecting disease development High incidence of natural cankers on cherry has been attributed to leaf-scar infection during unusually wet weather at leaf fall. Similarly, severe leaf infection in both plum and cherry were observed to follow heavy rainfall in spring when young leaves are developing (Crosse, 1956a; 1956b). However, Shanmuganathan (1962) found no direct relationship between rainfall or leaf surface-wetness and leaf infection.

19 The leaf surface bacterial populations, the main source of inoculum for infection, are subject to continuous leaching by heavy rains. Crosse (1963) suggested that maintenance of epiphytic populations on leaves would probably be by multiplication of the pathogen in dew and static water left after rain. Apart from the leaching effect of rain, he identified ultraviolet rays as another climatic factor responsible for the reduction of epiphytic populations of E\_ morsprunorum on cherry trees. Freigoun (1974) analysed fluctuations of leaf surface bacterial populations on cherry trees with the climatic data and found a strong negative correlation with sunshine duration and vapour pressure deficit. He demonstrated in vitro that populations decreased under drying conditions . In Mediterranean countries, studies of population dynamics on epiphytic phytopathogenic pseudomonads on fruit trees reveal a common pattern of seasonal variation. The numbers tend to be maximal in the spring, minimal in summer and undectable in winter. Undoubtedly environmental factors play a prominent role here. According to Panagopoulos (1966) and Gardan et.al. (1972) decline in leaf surface bacterial populations in summer is due to the prevalence of hot dry weather common in Greece and France respectively. Other workers have recently attempted to correlate fluctuations in epiphytic populations with environmental factors, e.g. Ercolani (1982) identified cambial activity, air temperature, summer rains, winter rains, time of blossoming and warm southerly winds as factors affecting the variability of epiphytic populations of P. syringae pv. savastanoi on olive, which is an evergreen tree. Smitley and McCarter (1982) suggested that the decline of epiphytic populations of P. syringae pv. tomato in the summer was due to high tenperature.

Soil moisture under field conditions had no apparent effect on

20 bacterial canker but canker development has been greatly retarded in experiments under extremely dry soil conditions indicating that soil moisture is an important factor for canker development (Wilson, 1939). Studies on the effect of nutrients on stem- infections have shown that low phosphate levels increased, and high levels decreased, resistance of plum trees to the disease (Beard and Wormald, 1936). Wormald and Garner (1938) observed no significant effect on disease development on young cherry trees with varying amounts of sulphate of ammonia, potash and superphosphate. In California, Wilson (1939) detected that sulphate of ammonia had no effect on the disease on plum trees. Roach (1936) pointed out the importance of available iron as a factor determining resistance to bacterial canker. The resistance to bacterial canker in peach trees in new orchards compared to that in old orchards has been attributed to higher levels of K, Ca, Al, Sr, B and Zn in the host tissues (Weaver, Dowler and Nesmith, 1976).

The causal organism Wormald (1930, 1932, 1938) described two distinct species, Pseudomonas prunicola and P. morsprunorum, as the causal organisms of bacterial canker of stone-fruit trees in the U.K. They were gram negative, non-sporing rods, motile by 1 - 3 flagella and possessed similar colony characteristics but differed in certain biochemical characters (growth in nutrient broth + 5% sucrose and in Uschinsky's solution and in their ability to produce acid from purple lactose agar medium). In Britain, P. morsprunorum was the most widespread organism and responsible for all canker and leaf spot on plum and most of that on cherry. P. prunicola was found occasionally only on cherry and later workers believed that this species was identical with, or a form of, P. syringae.

21 In USA, New Zealand, Holland and some other countries P. syringae, which has a wide host range including weeds, has been recorded as the causal organism of stone-fruit cankers. Apart from Britain P.morsprunorum has been reported as causing bacterial canker in many other countries, e.g. France (Ride and Bulit, 1957); Greece (Zachos and Panagopoulos, 1963); Rumania (Lazar, 1964); Canada (Gourley, 1965); Italy (Ercolani, 1966); Switzerland (Burki, 1968); Germany (Michel, 1969); USA (Jones, 1971); Czechoslovakia (Zacha, 1972); Denmark (Anon. 1974); Poland (Burkowicz, 1975); Norway (Sletten, 1979) and Australia (Foulkes and Lloyd, 1980). The species status of P. morsprunorum has been questioned by Erikson (1945a), Fuchs (1957), Bortels and Gehring (1960) and Cameron (1962a) who asserted that P. morsprunorum should be included in P. syringae because there was insufficient evidence to regard these as distinct species. Lovrekovich et al (1963) found the antigenic properties of P. morsprunorum and P. syringae sufficiently similar to regard them as one species. However, Burki (1968) detected differences between the flagellar antigens of the two organisms. Cameron (1962b) demonstrated that P. syringae had only very little infectivity through leaf-scars of sweet cherry in Oregon. Similarly Crosse and Garrett (1966) found that a North American cherry strain of P. syringae was only weakly infective through leaf-scars, although highly infective through wounds, in contrast to their English cherry strains of P. morsprunorum that were highly infective through both avenues of infection. Burki (1968) and Seemuller and Arnold (1978) also could only successfully infect sweet cherry leaf-scars with cherry < strains of P. morsprunorum again indicating they were pathogenically distinct from P. syringae. Crosse and Garrett (1961, 1963> and Garrett and Crosse (1963)

22 found phage sensitivities of P. syringae and P. morsprunorum to be distinct and further confirmed that they were different organisms. This work was supported by Billing (1963 and 1970). The distinction was further examined ~by Garrett, Panagopoulos and Crosse (1966) in approximately 120 physiological tests using a wide range of isolates of pathogenic fluorescent pseudomonads from plum, cherry and pear in England and fran citrus in Greece. The organisms shared many cornnon characteristics but there were marked consistent differences in others between the stone-fruit (P. morsprunorum) and the remaining isolates (P. ssingae). These included lipolytic, proteolytic, glycosidese and tyrosinase activity; colour of growth and longevity in nutrient broth + 5% sucrose medium; the utilisation of lactic and tartaric acids and use of 1- leucine and 1- tyrosine as sole sources of carbon and nitrogen. Many of these tests have been applied by others who have found both P. morsprunorum and P. syringae to be present in orchards where all infection was previously thought due to P. syringae e.g. Jones (1971) and Foulkes and Lloyd (1980). Grieb et al (1975), on the basis of physiological and biochemical characters of American isolates of the two species, suggested they possessed a species specific reaction spectrum and proposed that they should be designated physiotypes of the same species. Seemuller and Arnold (1978) found that P. syringae isolates were able to produce syringomycin in vitro but P. morsprunorum could not. Therefore, they suggested this as an additional differential character.

The latest revision of the International Code of Nomenclature of Bacteria (Lapage et al., 1975) included some important changes which affected the acceptability of the names of many phytopathogenic bacteria. The ICSB (International Committee on Systematic Bacteriology) was charged with proposing the '1980 Approved Lists of Bacterial names' to the

23 Judicial coirmission. This committee accepted only species or subspecies the names of which had been validly published, that were represented by authentic, extant type or neotype cultures and that have modern descriptions by which they can be differentiated readily from other species or subspecies using practical, laboratory diagnostic methods. The names of many plant pathogenic bacterial species were invalidated by this system. P. syringae is, thus, the only species now regarded as the causal agent of bacterial canker of stone-fruit trees, and also many other diseases of a wide variety of various crops. This nomenclature is not helpful to plant pathologists dealing with the many, largely host related, bacteria pathogenic to plants.

The 'pathovar' system, based on distinctive pathogenicity to one or more plant hosts, was recommended by the committee on Taxonomy of Phytopathogenic Bacteria of the ISPP (International Society for Plant Pathology)to the ICSB to conserve the names of many phytopathogenic bacteria and the ISPP list of acceptable pathovars was compiled (Dye et al, 1980). In this list Pseudomonas syringae pathovar syringae and Pseudomonas syringae pathovar morsprunorum are the new names for those formerly designated Pseudomonas syringae and Pseudomonas morsprumnorum respectively. However, for slnplicity in this thesis these two organisms are subsequently referred to as pv. syringae and pv. morsprunorum.

Within pv. morsprunorum, isolates from plum and cherry appear to . be restricted in the field to their natural hosts, even when the two hosts are planted adjacently. They have distinct phage sensitivities, first detected with the virulent phage A7 and its related phages; cherry strains are sensitive and plum strains insensitive. This was later confirmed by typing with temperate phages * from lysogenic bacteria. (Crosse and Garrett, 1961, 1963; Garrett and Crosse, 1963; Garrett et al., 1966). Crosse and Garrett (1970) demonstrated the pathological

24 differences between plum and cherry strains. Although both strains infected similarly through wounds on branches, they showed a marked host specificity in stomatal and leaf-scar infections. A third host specific pathotype of pv. morsprunorum has been isolated from leaf spots and shoot wilt of myrobalan, Prunus cerasifera (Garrett and Crosse, 1967). Another host specific form is P. syringae pv. persicae (formerly P. morsprunorum persicae) causing serious bacterial canker of peach in Southern France (Prunier et al, 1970). A variant colony type isolated from leaf spots, leaf washings and fruit lesions of cherry (Freigoun, 1974)is characterised by producing on NSA levan colonies with a 'fried-egg' appearance at 48 hours quite distinct from the smooth colonies of typical pv. morsprunorum. They resemble typical pv. morsprunorum more closely after 3-4 days. These isolates formed a fairly homogeneous group that conformed to pv. morsprunorum in most biochemical tests and they were sensitive to phage A7 specific for cherry strains of pv. morsprunorum. They, therefore, were in many respects closer to this pathovar than to pv. syringae. However, they possessed gelatinase activity and utilisation of tartrate was relatively slow. The variant form was less infective through leaf scars but more invasive through wounds than typical cherry strains. These characteristics of the variant isolates were considered sufficient to justify regarding them as distinct organisms and they were designated as race 2 of pv. morsprunorum to distinguish them from the typical race 1, previously described. Later Garrett et al (1977) sub-divided race 2 into 3 groups. Group 1 was the form originally described, group 2 was a non-fluorescent form while in group 3 were placed organisms that hydrolysed arbutin and therefore with gelatinase activity and production of pigment were closer to pv. syringae. This latter group were not sensitive to phage A7 but all three groups were lysed by the

25 race 2 specific phage B1 (Persley and Crosse, 1978).

Host range

In addition to plum (Prunus domestica) and sweet cherry (P. avium), pv. morsprunorum also attacks sour cherry (P. cerasus), apricot (P. armenica), peach and nectarine (P. persica), purple leaved plum (P. pissardii) and almond (P. amygdalus). Further, pv. morsprunorum has been isolated from three weeds under Montmorency sour cherry in Michigan (Lattore and Jones, 1979).

Varietal susceptibility of plum to bacterial canker Almost all varieties of plum grown in England belong to Prunus domestica, a hybrid of P. spinosa (sloe) and P. cerasifera (Myrobalan plum). This largely accounts for the wide range of varieties, which differ considerably in their susceptibility to bacterial canker. Wormald (1934) considered Victoria, Czar, Giant Prune and Prince of Wales as the most susceptible varieties with Utility, President, y r River's Early, Purple P&rshcp, Parshcje Yellow Egg, Dennis ton's Superb and Warwickshire Drooper as resistant. In addition Moore (1946) regarded Pond's seedling as susceptible and Purple Egg and Black Bullace resistant. The Ministry of Agriculture, Fisheries and Food (Great Britain) lists Victoria, Ontario, Early Laxton, Czar and Giant Prune as susceptible with Marjorie's seedling and Warwickshire Drooper as resistant (Garrett, 1980a). The National Fruit Trials catalogue (Smith, 1978) records 18 varieties as susceptible, including most of the above mentioned, and only Utility as resistant. Information on susceptibility to bacterial canker was not available for most of the varieties described. President, reported resistant by early workers, but rated as susceptible in this catalogue may have lost its resistance or new strains of the pathogen

2 6 may have the ability to attack it or early evidence has been based on little data. The factors determining varietal susceptibility are not understood. Harris (1943) reported work which showed no significant growth differences of pv. morsprunorum in bark extracts from plum varieties of varying resistance. Erikson (1945a) thought it unlikely that the resistance of certain plum varieties was due to a deficiency in essential nutrients in the tissues. Erikson and Montgomery (1945) observed greater injury to a susceptible variety than a resistant one when cell-free filtrates of P. morsprunorum and allied species were injected into the bark.

Control measures In Britain Wormald (1937) intensively studied methods for controlling the disease on plum and suggested three ways namely (a) staddle grafting i.e. a resistant rootstock is allowed to branch and the commercial scion variety, usually more susceptible to bacterial canker is budded or grafted to the branches (b) avoiding pruning during autumn and winter when the trees are susceptible and (c) spraying bactericides in spring and autumn, to prevent leaf and stem infection respectively, on the assumption that leaf spot and canker stages were mutually interdependent . Staddle grafting is rarely practiced. However, high working i.e. grafting the scion high on the resistant rootstock was shown to be very effective (Montgomery et al, 1943; Schofield and Clift, 1959). Shanmuganathan (1962) observed interactions between scion and rootstock. Cankers induced on relatively resistant rootstocks when high-worked with the susceptible variety Victoria were longer than when high-worked with the more resistant Early Laxton. Shanmuganathan and Crosse (1963) in a trial in which rootstocks were high-worked with Victoria and Early Laxton found that Belgian Myrobalan,EA16 and Myrobalan

27 B were equally resistant but EC3, Brompton and St. Julien A were all less resistant. Furthermore resistance of the rootstocks was influenced by scion variety but no reciprocal effect of rootstock on scion was detected. However, no stocks were included in these experiments to serve as non-worked controls. Garrett (1979) observed that tree age, soil factors, pathotypes in the inoculum and the season influenced the assessment of rootstock resistance. She, therefore, recommended that in resistance assessment trials trees should be at least three years old, well replicated (to take account of local variation in soil conditions) and that the test inoculum should include all known pathotypes of the pathogen. There was no consistent overall difference between the performance of grafted and non-grafted rootstocks.

No conclusive data is available relating timing of pruning to infection, but in practice growers avoid such treatment in autumn and winter. Moore (1946) could find no evidence that pruning in October or May affected the incidence of bacterial canker. Considerable success has been achieved by spraying chemicals to control the disease in cherry. Wormald's (1937) spray timing in spring and autumn was based on the assumption that the leaf spot and canker stages of the disease were mutually interdependent. Bordeaux mixture controlled the leaf spot and branch cankers on cherry when applied in spring at white-bud stage and in autumn just before leaf fall (Moore, Crosse and Bennett, 1959). In view of Crosse's (1954b) discovery that the epiphytic phase was the source of inoculum for cankers Crosse and Bennett (1959) re-examined the timing of sprays and recommended the applications of Bordeaux mixture in mid-August, early-September and early-October to control leaf scar infection in cherry. They advised the inclusion of cotton seed oil to minimise the phytotoxicity of these sprays; rape seed oil is an effective alternative (Allen and Dirks,

28 1979). Later trials showed that using only two later applications of Bordeaux mixture would normally give a control of leaf scar infection approaching 90% (Crosse, 1967). Crosse concluded that Bordeaux mixture eradicated the leaf surface pathogen population and thus prevented leaf scar infection. Babilas (1970) also recommended two autumn sprays of Bordeaux mixture, 2-3 weeks before and during leaf fall, as preferable to one in spring and one during leaf fall. The recommended standard is however to use three sprays (Garrett, 1980).

The use of streptomycin overseas encouraged investigating on this antibiotic. Streptomycin sprays were inferior to even low-strength Bordeaux mixture in the control of cankers, but superior in control of the less important leaf spot phase (Crosse and Bennett, 1957; Crosse, 1962). Boyd and Paton (1958) applied a bactericidal paint containing streptomycin in late January to the stem and crotch of young trees and claimed reasonable success. However, the agricultural use of streptomycin is not permitted in England as this antibiotic is frequently used in the medical field.

So far, there is no satisfactory spray schedule for control of canker in plum where infections are commonly on the stems of young trees. 'The Grower' (Anon. 1976) reconmended that three winter sprays of Bordeaux mixture from September to January may be helpful to control canker in plum, but this was not based on experimental evidence. In New Zealand, removing the cankered tissues and burning with a hand-held propane burner in early to mid-spring when cankers are developing is reported to be more effective than traditional cutting and wound dressing because of more corrplete eradication of the pathogen and rapid wound healing which prevented reinfection (Hawkins, 1976).

In general, long term control of bacterial diseases is preferable by breeding resistant varieties. In England a plum breeding programme

29 was started in 1970 at Long Ashton Research Station. Resistance to bacterial canker is among the various characteristics of the new selec- tions being assessed.

30 PART II

1. General Methods 1.1 Media 1.2 Isolation and identification of P. syringae pv. morsprunorum. 1.3 Storage and maintenance of cultures 1.4 Bacterial counts 1.5 Preparation of aqueous suspensions of inoculum

31 1. General Methods 1.1 Media Nutrient sucrose agar + crystal violet + cycloheximide (NSA + CVC) This contained (% w/v) nutrient broth, 0.8; sucrose, 5.0; Oxoid agar No.l, 1.5. Crystal violet, 2ppm and cycloheximide, lOOppm were included to suppress gram-positive bacteria and fungi respectively. The medium was prepared in sodium hydroxide and potassium di-hydrogen phosphate buffer (pH6). Cycloheximide was sterilised using a millipore filter and added to the molten agar medium immediately before pouring plates.

Nutrient glycerol agar (NGA) This had the following composition (% w/v); nutrient broth, 0.8; glycerol, 2.0; Oxoid agar No. 1, 1.5. The medium was adjusted to pH7.

Peptone glycerol yeast agar (PGYA) This consisted of (% w/v): peptone, 0.5; glycerol, 2.0; yeast extract (Difco), 0.3; Oxoid agar No.l, 2.0. The medium was adjusted to pH7. All the above media were autoclaved at 121°C for 15 minutes.

1.2 Isolation and identification of P. syringae pv. morsprunorum. Throughout this study pv. morsprunorum was isolated on NSA + CVC medium and plates were incubated at 25 °C. for 2-3 days to allow colony development. Pv. morsprunorum typically forms whitish, shiny, entire, hemispherical

32 colonies with rladial striations. Since variant colony forms have been reported from cherry in recent years (Fr-eigoun, 1974), other similar colony forms were also selected. The presumptive identity of isolates was investigated by test- ing single colony isolates in the- biochemical tests described by Garrett et al. (1966) for the differentiation of stone-fruit pseudomonads.

1.3 Storage and maintenance of cultures All isolates selected were transferred to Bijou bottle

• slopes of NGAfincubated and then stored at 4°C until required. They were subcultured into fresh agar slopes every 6 months.

1.4 Bacterial counts Bacterial suspensions were obtained during the course of this study from leaf washings, crushed buds, petioles, rain water draining down plum trees and diseased tissues. Routinely serial ten-fold dilutions were prepared in sterilised distilled water (SDW) and plated on NSA + CVC medium. After pouring, plates were dried for about 20 minutes in a lamina flow cabinet at room temperature. Then, 0.1 ml. aliquots of the selected dilution were spread with a bent glass rod over the surfaces of three replicate plates. Bacterial colonies were counted after incubation.

1- 5 Preparation of aqueous suspensions of inoculum 24-48 hour bacterial growth of pv. morsprunorum on NGA slopes was quickly suspended in 5 ml. of SDW and transferred into empty sterile Universal (1 "oz.) bottles. The suspensions were standardised to the required concentration

33 (when necessary) using an absorptiometer and reference to a standard curve.

34 2. Overwintering of Pseudomonas syringae pv. morsprunorum 2.1 Introduction 2.2 Materials and Methods 2.2.1 Plant material 2.2.2 Isolation and enumeration of bacteria from whole buds 2.2.3 Test of sample size 2.2.4 Isolation of bacteria from bud surfaces 2.2.5 Efficiency of the impression method to detect bacteria from bud surfaces 2.2.6 Detection of bacteria by the bud impression method in winter 1982/83 2.2.7 Detection of bacteria from bud parts 2.2.8 Detection of bacteria in rain water 2.3 Bacterial populations of whole buds 2.3.1 A preliminary investigation during the winter 1980/81 2.3.2 Fluctuations in the bacterial populations of whole buds during the winter, 1981/82 2.3.2.1 From 6-year-old plum trees 2.3.2.2 From 3-year-old plum trees 2.3.2.3 From 3-year-old cherry trees 2.4 Bacterial populations of bud surfaces 2.5 Bacterial populations of bud parts 2.6 Pathogen populations of dormant buds of nursery trees and mother trees 2.7 Bacterial populations in the rain water draining down plum trees 2.7.1 During the winter of 1980/81

35 . 2.7.2 During the winter of 1982/83 2.7.2.1 Estimations from rain water traps on trunks 2.7.2.2 Estimations from rain water traps on branches 2.8 Discussion

36 2. Overwintering of Pseudomonas syringae £v. morsprunorum

2.1 Introduction Leaf spots and epiphytic populations of Pseudomonas syringae pv. morsprunorum and pv- syringae may be present during the growing season on established stone-fruit trees with no apparent cankers (Crosse, 1954; 1959). Healthy, canker-free nursery material planted in apparently disease- free areas with no nearby sources of infection, often develop leaf spots. Moreover the pathogen has been isolated in large numbers from breaking buds and trees soon after bud break (Shanmuganathan, 1962). It was, therefore, suggested that the winter canker phase was not an obligatory stage in the life-cycle of the pathogen and it could survive the winter in buds and/or on the surface of the trees. Furthermore, plum trees are susceptible to infection, mainly on the trunk, from October to March with December being the most critical month (Shanmuganathan, 1962). For such infection to occur, the pathogen must survive in association with the trees during this period.

Attempts by earlier workers to isolate pv. morsprunorum during the winter season from plum buds by conventional methods were unsuccessful (Anon, 1961, 1962; Shanmuganathan, 1962). Shanmuganathan (1962) adapted a bacteriophage-sensitivity method to detect the pathogen in bud macerates and obtained rapid multiplication of the specific phage in a few samples, providing presumptive evidence of the presence of the pathogen.

In experiments on virus transmission,extracts of dormant

37 cherry buds were found to cause virus-like symptoms in curcurbits and Chenopodium quinoa. However, the lesions were subsequently found to be due to P. s^. pv. syringae which must have originated in the buds (Ramaswamy and Garrett, 1970; Opel et al. 1974). Although Shanmuganathan (1962) could readily isolate the pathogen in rain water draining through plum trees during the growing season, he was unable, using the same trapping technique, to isolate them during winter. Freigoun (1974) using a modified trap detected pv. morsprunorum on cherry trees from November to January but at a very low population level. Although there is circumstantial evidence that the pathogen is introduced into new orchards on the nursery material and can be maintained in orchards in the absence of overt signs of the disease, the pathogen has not been recovered from nursery material and dormant trees. This failure to detect the pathogen in winter could have been due to inadequate sampling size, defects in the isolation method, media etc. Attempts were, therefore, made to isolate the pathogen from dormant nursery and established plum trees by modifying and extending previous isolation methods.

2.2 Materials and Methods 2.2.1 Plant material The plum cultivars Victoria and Ariel low-worked on St. Julien A rootstock, on plots UG123 and UG133 and the cherry cultivar Napoleon, low-worked on Colt rootstock

38 on plots UG124 and UG135 were used. UG123 and UG124 were planted in February 1976, UG133 and UG135 in February 1979.

2.2.2 Isolation and enumeration of bacteria from whole buds In spite of attempts to detect them (Garrett and Crosse, 1963; Garrett et al, 1966; Lazar and Crosse, 1969) there are still no bacteriophages specific for pv. morsprunorum plum strains. Therefore the indirect method employed by Shanmuganathan (1962) to detect the pathogen using bacteriophages could not be used to confirm or extend his observations. It was argued that, since earlier attempts to isolate the pathogen from buds indicated that pv. morsprunorum, if present, was at very low levels the failure to isolate the pathogen may have been due at least in part to small sample size. Isolation was therefore attempted by examining a fairly large sample of buds. One-year-old twigs bearing apparently healthy buds of plum and cherry were taken to the laboratory and the buds carefully removed with sterile forceps. In order to get recoverable numbers of the pathogen, samples of 70 plum buds or 30 cherry buds were taken. Each sample was crushed in 9ml. of SDW in a Stomacher (Colworth, model 80) for about 6 minutes, i.e., until the bud parts separated. After the debris had settled, a series of ten-fold dilutions was made or suspensions were concentrated by centrifuging 5 ml. at 403 relative centrifugal force for 20 minutes and resuspending the bacterial pellets in 1 ml. of SDW. The original suspension, its dilutions or concentrated

39 suspensions were plated on NSA + CVC medium (three replicate plates of each). The resultant bacterial colonies were counted after incubation.

2.2.3 Test of sample size Seven samples, each of 70 healthy plum buds on one- year-old branches (cv. Victoria) from 7 trees (10 bud/tree) and 30 healthy cherry buds on one-year-old branches (cv. Napoleon) from 6 trees (5 bud/tree) were taken in November and crushed separately in 9 ml. of SDW and bacterial counts of the suspensions were made as described earlier. The results are shown in Table 2.1. The standard deviation of log counts was calculated from the following formula

standard deviation = (x± - x) n-1 V n = number of samplings x. = value of each count I x = mean value of all counts

The sample counts gave acceptable agreement. Calculated standard deviations were relatively small compared with the population size of each sample. These sample sizes and procedure were, therefore, routinely adopted.

2.2.4 Isolation of bacteria from bud surfaces The isolation of bacteria from whole buds does not differentiate between those carried externally and those borne internally. Attempts were, therefore, made to investigate these populations separately by use of a sterilant.

40 Table 2.1 Numbers of bacteria (log c.f.u./bud) in 7 samples taken simultaneously from plum and cherry

Plum buds cherry buds pv. other pv. other morsprunorum bacteria morsprunorum bacteria

1 2.70 2.65 3.81 5.02 2 2.47 3.37 4.20 5.00 3 2.51 2.20 4.13 5.03 4 2.34 2.46 4.32 4.97 5 2.32 3.09 4.33 4.82 6 2.68 2.31 3.52 5.18 7 2.44 2.44 3.62 5.13

D. 0.15 0.43 0.34 0.12

S.D. = Standard deviation.

41 Ten one-year-old plum and cherry twigs bearing single buds collected in December were dipped for 2 min. in 70% ethyl alcohol or 5 mi'n. in 2% chloramine-T and then washed in 4 to 5 changes of SDW. Then the treated buds, dry buds and buds washed with 4 to 5 changes of SDW without any previous treatment were pressed onto NSA + CVC medium. From plum bud impressions more bacteria were observed on buds treated with sterilant and the washed buds than dry ones whereas no bacteria resulted from any of the cherry bud impressions.

Failure of these methods to differentiate surface and internal bacteria from plum buds could be due to the following factors. (a) The surface sterilant was inadequate to eliminate bacteria completely from the surface. (b) Bacteria were mobilised on the surface or the internal bacterial populations were moved onto the surfaces by subsequent washing with SDW used to wash out the sterilant. It was felt that stronger sterilants or longer time treatment with sterilants might mean too much penetration and death of internal bacterial populations as the plum buds were slightly open (Plate 2.1). Surface sterilisation was found inadequate to differentiate internal and surface populations of micro-organisms of apple buds (Andrews and Kernerley, 1981). A'bud impression method' was, therefore, investigated as a means of detecting the surface populations.

42 Plate 2.1 Structural differences of plum (cv. 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.

43 2.2.5 Efficiency of the impression method to detect bacteria from bud surfaces A streptomycin resistant strain of pv. morsprunorum was prepared by plating a turbid suspension of a culture (JP 245) isolated from plum buds, on a medium of NSA + lppm streptomycin. After 3-4 days incubation, a few colonies were selected and successively replated on the medium with increasing levels of streptomycin (i.e. 5ppm, lOppm, 30ppm and 50ppm) until a mutant of JP 245 resistant to 50ppm streptomycin, JP 245 SR, was obtained.

Forty eight-hour growth of JP 245 SR on NGA medium was suspended in water and standardised to give a concentra- g tion of 10 c.f.u/ml. using an absorptiometer and reference to a standard curve. Using a finpipette 10 >ul. was carefully deposited on to plum buds (cv. Victoria) on one-year- old twigs held upright by pushing into a lump of plasticine standing in a plastic tray. Twenty twig sections, of 2.5 cm., each bearing a single bud, were removed either immediately, 15 minutes, 1 hour or 3 hours later. Impressions of twig sections dry or immediately after wetting with a few drops of SDW were then made onto the surface of NSA + CVC + 50ppm streptomycin medium. A streptomycin resistant cherry strain JC 30 SR, was similarly prepared and applied in the same manner to buds of cherry. The plates were incubated and the presence of streptomycin resistant colonies of pv. morsprunorum recorded. Up to 1 hour after application, pv. morsprunorum was recovered from 100% of buds in both dry and wet states.

44 After 3 hours, bacteria were recovered from fewer dry buds, 80% of plum and 30% of cherry, while recovery was still 100% from the buds wetted with SDW. Thus, the recovery of bacteria from bud surfaces was facilitated by wetting. These results suggested that a 'bud impression method' might be employed to detect bacteria from bud surfaces although this gives no indication of what proportion of those present is recovered. However, this method provided some information about the relative numbers of buds surface-infested with bacteria at different times during winter.

2.2.6 Detection of bacteria by the bud impression method in winter 1982/83 It was decided to adopt the bud impression method to detect the presence of bacteria on bud surfaces. Samples were taken at intervals from mid-October 1982 to early-April 1983. On each sampling occasion fifty one-year-old twig sections, approximately 2.5 cm. long and each bearing a single bud, were briefly dipped singly in SDW, shaken to remove excess water and impressed upon NSA+CVC plates. The presence of pv. morsprunorum and other bacteria was recorded after incubation.

2.2.7 Detection of bacteria from bud parts* Because of the problem of selectively removing surface bacteria by sterilisation and the inability of staining to differentiate the pathogen from saprophytes, and living cells from dead cells, an attempt was made to determine the presence of bacteria within' the buds by dissecting out each bud part. One-year-old twigs of plum were cut into c. 2.5 cm. lengths each bearing single buds and placed under a dissecting micro-

* Bud parts here refers to bud scales, rudimentary leaves and primordia.. 45 scope. Bud parts were separated using fine sterile forceps. As each part was removed it was placed, inner side down, in sequence on NSA + CVC plates onto c.0.02ml. drops of SDW applied earlier with a multiple applicator. The parts were numbered (1-13) starting from the outer scales (Fig. 2.1). The presence of the pathogen, and other bacteria was recorded after incubation (Plate 2.2).

2.2.8 Rain water samples The source of inoculum for plum stem infection during the winter is obscure as the pathogen has not been isolated from the surfaces of trees during winter. Investigations were, therefore, carried out to try to isolate bacteria from rain water draining down plum trees using modified, purpose- built traps, attached to the trees.

Traps described by Freigoun (1974), consisting of 7 mm. diameter P.V.C. tubing placed spirally around the trunk with the lower end inserted into a collecting bottle, were used during the winter 1980/81. Collections from 12 trees of cv. Victoria with small cankers and 12 trees with large cankers (resulting from inoculations made in 1979) in plot UG123 were pooled separately. To increase the chances of detecting bacteria, rain water samples were concentrated by centrifuging (403 relative centrifugal force) for 20 minutes. As there were generally no pellets of bacteria formed,the supernatants were carefully pipetted out, leaving 3 ml.in the base of each cen- trifuge tube, and 'discarded. These 3 ml. samples were then

46 Plate 2.2 Growth of bacteria from plum bud parts (1-7) on NSA + CVC medium (after 2 days incubation at 25°C. )

Fig.,2.1 Arrangement of bud parts numbered starting from the outermost scale (No.l) to innermost primordia (No. 13) on an isolation plate (inverted)

4 7 pooled and populations were estimated by spreading 0.1 ml. of the suspensions on 3 replicate plates of NSA + CVC medium. In the winter 1982/83, the traps described below were used as these were much easier to fix securely on to the trees. Trunk traps consisted of PVC tubing of 7 mm. diameter into which 9 holes of 7 mm. diameter had been punched at a spacing of approximately 3 cm. The tubes were placed round the trunks c. 60 cm. above ground level in a slanting circle and secured by adhesive tape ensuring that the holes were firmly touching the trunk to catch the water draining down (Plate 2.3). The open ends of the tubes were inserted into sterilised collecting bottles (300 ml. capacity) supported in a plastic basket permanently attached to the tree. The position of the tubes remained unaltered during the winter and the tubes were rinsed with methyl alcohol followed by SDW immediately before placing freshly sterilised bottles in position for each sample. Collections were made from 8 trees each of the cvs. Victoria and Ariel. It was observed that rain water tends to seep directly to the ground from many branches of plum trees without draining down the trunks. Traps similar to those described by Swinburne et al. (1975) were prepared and set therefore, on individual one-year, two-year and three-year old branches on 4 plum trees (cv. Victoria). These traps consisted of a collar of 6 mm. thick double skinned foam neoprene, fixed at the base of a branch and rolled to form a tube at one end into which a polystyrene bottle (30 ml. capacity) was inserted (Plate 2.4).

48 Plate 2.3 (A) A rain water trap attached to the trunk of a plum tree (B) close up view of the trap showing positions of the holes.

49 Plate 2.4 (A) Rain water traps on the branches of a plum

tree (B) close up view of a trap on an one-year- m old branch.

50 Collections were carried out at intervals, and isolations were made as soon as practically possible, always less than 24 hrs. after rain. 5 ml. from the collections of trunk traps of two adjacent trees of the same cultivar were mixed and isolations were made as described earlier. From each collecting bottle of the branch traps, isolations were made individually as the numbers of bacteria were expected to be very low in water collected from these traps.

2.3 Bacterial populations of whole buds 2.3.1 A preliminary investigation of enumeration of bacterial populations from whole buds during the winter 1980/81 Estimations of bacterial populations were carried out at approximately 3-weekly intervals taking a random sample of 70 plum buds or 30 cherry buds from plots UG123 and UG124. Typical pv. morsprunorum types, together with other bacteria , were isolated throughout the winter from both plum and cherry buds (Table 2.2). In plum buds the numbers of pv. morsprunorum decreased et from November to January, becoming virtually undatable by the end of January.

Numbers showed an increase in late February3 and a rapid rise was observed in March to reach over 3 x 10 c.f.u./bud towards bud-break. In cherry buds, the numbers of pv. morsprunorum always higher than 10^ c.f.u./bud, increased gradually during the winter (except in late-February and mid-March when numbers dropped slightly) and reached a very high level (>• 10^ c.f.u./bud) in April just before bud-break. Bacterial numbers of cherry buds were consistently much higher than

51 Table 2.2 Bacteria (c.f.u./bud) isolated from whole buds of plum and cherry/ winter 1980/81

Plum (7° *ud, Cherry <30 samples) 1 samples) Date of ; sampling pv. pv. morsprunorum saprophytes morsprunorum saprophytes

2 3 14. 11.80 2.5 x io * 2.0 X 10 * 3 4 16. 12.80 30.6 1.7 7.3 X io 1.3 X 10 3 4 6.1.8 1 9.0 12.2 1.3 X io 1.8 X 10 4 4 27. 1.81 0.47 22.5 3.2 X io 3.2 X io 4 4 11. 2.81 3.3 3.3 4.0 X io 5.0 X io 2 4 3 24. 2.81 41.0 1.9 X 10 1.4 X io 2.7 X io 3 2 5 3 12. 3.81 3.6 x io 1.1 X 10 7.1 X io 7.0 X 10 3 2 5 5 26. 3.81 1.4 x 10 1.1 X io 1.9 X io 1.2 X io 3 2 6 4 8. 4.81 2.2 x 10 1.3 X io 1.2 X io 3.8 X io

* not counted

5 2 those of plum buds.

2.3.2 Fluctuations in the bacterial populations of whole buds during the winter, 1981/82 Bacterial populations were* estimated from plum cv. Victoria (plots UG133 and UG123) and cherry cv. Napoleon (plot UG135), at approximately weekly intervals to compare the population fluctuation in 3-year-old plum trees (when trees are more susceptible to bacterial canker) with 6-year- old plum trees (when they show some resistance) with the population of 3-year-old cherry trees.

2.3.2.1 From 6-year old plum trees Pv. morsprunorum was isolated throughout the winter together with other bacteria (Fig. 2.2.A). Pv. morsprunorum populations maintained a comparatively constant, but low 2 level with a mean value of 10 c.f.u./bud, and increased

3 rapidly to 10 c.f.u./bud in April just before bud-burst at which time an almost pure population of the pathogen was present on the isolation plates. Saprophytic bacteria fluctuated at a similar level to the pathogen but on some occasions, in February and March, populations were at very low levels, c.5 c.f.u./bud. Contrary to the pathogen, their numbers declined to an undetectable level just before bud burst. 2.3.2.2 From 3-year old plum trees Pathogen populations (Fig. 2.2.B) fluctuated at a similar level to that from the 6-year-old plum trees (10^ c.f.u./ bud) until January but the numbers dropped slightly in February

53 5 TD o- JO Z> 4 ll (J -= 3

u D JQ oc cn O _J O Nov Dec Jan " Feb Mar Apr

B 5 r T3 D JQ 3 LLT U' -O 3 L V Uo jQ o C o» o -I O Nov Dec Jan Feb Mar Apr 1981 T982

pv. morsprunorum ---- saprophytic bacteria

Fig. 2.2 Fluctuations from November to April of Pseudomonas syringae pv. morsprunorum and saprophytic bacterial populations from crushed whole buds' of (A) 6- year-old (B) 3-year-old plum trees.

5 4 and March (c.50 -15 c.f.u/bud). An increase up to £.6 x 10 c.f.u./bud was observed in April just before bud burst. An almost pure population of pv. morsprunorum was isolated on some occasions in January and throughout March and April. Saprophytic bacterial populations were much lower than the pathogen populations (mean value of 10 c.f.u/bud) throughout the sampling period. Numbers declined to an undectable level on some occasions in January and were not detected from early March onwards.

2.3.2.3 From 3-year-old cherry trees Bacterial populations (Fig. 2.3 ) were higher in buds of this host than in plum buds throughout the season. Pathogen populations were fluctuating c.10 c.f.u./bud at the beginning of the winter and some increase was observed 4 after January when numbers of c.10 c.f.u./bud were detected. Saprophytes fluctuated at a similar level to the pathogen but their numbers decreased on one occasion in February and showed a declining trend from late March as bud break approached.

2.4 Bacterial populations of bud surfaces The frequency (%) of buds surface-infested with the pathogen and saprophytes during the winter 1982/83 as detected by the bud impression method is shown in Fig. 2.4. In plum, 65% of buds yielded the pathogen in October but thereafter the percentage gradually decreased to less than 10% by April. The percentage of buds from which surface- infesting saprophytic bacteria were recovered was much higher than that from which the pathogen was recovered.

55 5

O Nov Dec Jan Feb Mar Apr 1981 1982

pv. morsprunorum saprophytic bacteria

Fig. 2.3 Fluctuations from November to April of Pseudomonas syringae pv. morsprunorum and saprophytic bacterial populations from crushed whole buds of 3-year- old cherry trees.

56 lOOr

"D D A

13/10 30/II 13/1 23/2 1982 1983 Date of sampling

Plum: Cherry: • pv. morsprunorum O pv. morsprunorum • saprophytes A saprophytes

Fig. 2.4 Frequency (%) of detection of Pseudomonas syringae pv. morsprunorum and saprophytic bacteria on the surfaces of plum and cherry buds at approximately six weekly intervals from October to April.

57 A similar trend to the pathogen was observed in that the percentage of buds infested with saprophytes decreased gradually from 100% in October to about 30% in April. Detection of the pathogen by this method on cherry bud surfaces proved very difficult. The highest frequency (only 12%) was in November and declined to an undectable level after January. Most of the colonies recovered from cherry were of pv. morsprunorum, race 2 type. A very high percentage of buds were surface-infested with saprophytic bacteria in early winter. However, from January to March the numbers of buds surface-infested with saprophytes

/ remained fairly constant at c. 60%.

2.5 Bacterial populations of bud parts Fifteen twig pieces bearing single buds were taken randomly from plum cv. Victoria (plot UG133) at approximately monthly intervals during the winter 1982/83. The frequency (%) of bud parts from which pathogenic and saprophytic bacteria were recovered is shown in Fig. 2.5.

Bacteria, both pathogenic and saprophytic types were found more frequently on the outer scales and parts than on the few innermost parts and primordia. The four innermost parts (10th - 13th) were almost free from a recoverable population of the pathogen except for a very few from 10th and 11th parts in November. The percentage of outer bud parts (1 - 9) infested with the pathogen was highest in November and fluctuated between c. 20 - 60%, but was lower in December and February, never exceeding 25%. In January only parts 2 and 6 yielded > 10% from which the pathogen was detected.

58 pv. morsprunorum A

saprophytes B.

Sampling dates: • 3/11/82 A 1/12/82 O 25/1/83 • 16/2/83

Fig. 2.5 Frequency of infestation of plum 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.

59 The pattern of frequency of bud parts infested with saprophytes was very different. The saprophytes were readily recovered from the majority of parts and only the two innermost were 100% free from recoverable saprophytes. In December and January a high percentage (60 - 90%) of outer bud parts infested with saprophytes was obtained but it fluctuated between 30 and 70% in November and February..

2.6 Pathogen populations of the dormant buds of nursery trees and mother-trees Bacterial canker appears in new orchards and may kill trees in the first few years. There are two possible sources of inoculum, either a) introduction with the planting material from the nursery or b) it spreads into orchards from an outside source eg:- an adjacent contaminated orchard. Since many orchards are established at some distance from another then planting material from the nursery is the most likely possibility. The possible ways of introducing the pathogen to nursery trees could be either a) budwood from the mother-trees used for budding/grafting scions to rootstocks is infested with the pathogen or b) inoculum is spread from other plum trees to the developing trees in the nursery. To determine if maiden trees are in fact harbouring the pathogen, buds of nursery trees of a range of plum cultivars and the cherry cv. Napoleon were examined in March i.e., shortly before the trees would be planted out to establish new orchards. Samples consisted of 100 plum buds or 50 cherry buds per variety. They were crushed in 5 ml. of SDW and isolation of the pathogen was as previously

60 described (p. 39). The pathogen was isolated, together with saprophytic bacteria. The population levels of the pathogen recovered from these buds are given in Table 2.3. Pv. morsprunorum was also recovered from buds of the cultivars Victoria, Marjorie's Seedling and Cambridge Gage. However, the pathogen population could not be enumerated in these cultivars because fast growing colonies of saprophytic bacteria quickly coalesced and overran the colonies of pv. morsprunorum. Nevertheless, it was clear that numbers of the pathogen in cv. Victoria were relatively high and in Marjorie's Seedling and Cambridge Gage were low. These results show that pv. morsprunorum is surviving the dormant season in association with buds of nursery trees of all the plum cultivars tested and the cherry (cv. Napoleon) trees. The population was far higher from cherry (cv. Napoleon) than from any plum cultivar. The inoculum could have been introduced during growth in the nursery or on budwood. The possible introduction on scion budwood from mother-trees was tested by examining 13 cultivars of plum mother-trees during the time of budding, i.e., in early-August. Samples of 100 buds (50 from each of two trees) of each cultivar were crushed in 5 ml. of SDW and bacteria were isolated as previously described. The pathogen was recovered from 8 cultivars (Table 2.4). There was clearly a difference in the numbers of pv. morsprunorum harboured in different cultivars. Merton Gem had very high populations Ariel, Czar, Grove's Late Victoria and Giant Prune moderate numbers Ontario, Marjorie's Seedling and Laxton Cropper very low populations and Cambridge Gage, Anna Spath, Bonne de Bry, Early Laxton and Opal none (or

61 Table 2.3 Pathogen populations from buds of nursery trees in March

pv. morsprunorum Host Cultivar (c.f.u./bud) (100 bud samples)

Plum Ontario 100 Czar 52 Opal 46 Anna Spath 15 Grove1 s Late Victoria 15 Cherry Napoleon 3,660

* 50 bud sample

Table 2.. 4 Estimated pathogen populations from buds of different cultivars of plum mother-trees in August

pv. morsprunorum Relative numbers of Cultivar (c.f.u./bud) leaf spots observed

1. Merton Gem 7400 few 2. Ar^el 270 many Czar 100 few Grove1s Late Victoria 98 many Giant Prune 79 many Ontario 6 none Marjorie's Seedling 4 none 8. Laxton Cropper 2 none 9. Cambridge Gage 0 none 10. Anna Spath 0 none 11. Bonne de Bry 0 none 12. Early Laxton 0 none 13. Opal 0 none

62 the population levels were undetectable). It was observed that there were some differences in the levels of leaf infection between cultivars of mother- trees. A rough estimate of the numbers of leaf spots, made to see if there was any correspondence between this and the bud populations suggested that cultivars with leaf spots harboured more bacteria in buds than those free from leaf spots. It appeared that leaf infection and bud infestation could be correlated. Thus, there is no doubt that the pathogen can be introduced into new orchards with the plant material from the nursery.

2.7 Bacterial populations in rain water draining down plum trees 2.7.1 During the winter of 1980/81 Collections were begun at the end of November and carried out at intervals until the end of March. Amounts collected were inconsistent both between dates and bottles throughout the sampling period. This may have been due to inefficiency of the traps as they were easily dislodged and/or differences in intensity and direction of the rain. The numbers of pv. morsprunorum (c.f.u./ml.) isolated from trees with small and large cankers are given in Table 2.5. It can be seen that pv. morsprunorum was not isolated during the period from November to February. The first appearance of the pathogen on isolation plates was on March 3rd, i.e., at bud swelling, when numbers were very low (1-10 c.f.u. ml.-l ). A relatively high population was isolated in mid-March (301 and 4400 c.f.u. ml."1) but numbers had

63 Table 2.5 Amount of water collected and numbers of Pseudomonas syringae pv. morsprunorum (c.f.u. ml.~"^~) from rain water draining down plum trees (winter 1980/81)

Trees with small cankers Trees with large cankers Date No. of pv. Total amount No. of pv. Total amount morsprunorum of water morsprunorum of water (c.f.u. ml.-l) collected (c.f.u. ml.) collected (ml.) (ml.)

28.11.80 0 530 0 390 2.12.80 0 225 0 70 14.12.80 0 610 0 390 20.12.80 0 710 0 410 21. 1.81 0 330 0 120 10. 2.81 0 225 0 135 10. 3.81 1 580 10 120 14. 3.81 301 620 4400 760 26. 3.81 6 315 200 190

Table 2.6 Mean numbers of bacteria (c.f.u. ml. ) isolated from rain water draining down plum trees collected in trunk traps during winter 1982/83.

Victoria Ariel Date of pv. pv. Sampling morprunorum saprophytes morsprunorum saprophytes

9.11.82 7 384 IS 229 16.11.82 8 435 6 345 24.11.82 68 1338 93 1197 20.12.82 12 646 81 1037 13. 1.83 3 127 6 . 182 24. 3.83 47 273 192 231 11. 4.83 2100 425 780 13

64 decreased again in the next collection at the end of March. Pathogen numbers isolated were slightly higher in water collected from the trees with larger cankers than from those with smaller cankers but the amounts of water collected were with one exception invariably less. The numbers of bacteria recovered were not related to the amount of water collected.

2.7.2 During the winter of 1982/83 2.7.2.1 Estimations from rain water traps on trunks In the winter 1982/83 the modified collecting traps and improved sampling techniques described were used. Collec- tions were made at intervals from November to April.Table 2.6 shows the fluctuations of mean bacterial concentrations in rain water where it can be seen pv. morsprunorum was isolated from November 1982 to January 1983 albeit at very low levels. Pathogen numbers from both cultivars fluctuated similarly although they were higher from cv. Ariel until March. Numbers were low in January (mean value of c.3 c.f.u. ml. * from Victoria and c.6 c.f.u. ml."•'- from Ariel) and a steep rise was observed in March (mean c. 50 c.f.u. ml. ^ from Victoria and c.2 x 10z c.f.u. ml."1 from Ariel) when the buds were swelling. A slower increase in the pathogen numbers after March from cv. Ariel coincided with its blossom period which occurred earlier in cv. Ariel than cv. Victoria. How- ever, pv. morsprunorum colonies which were in the minority during winter (^.90 c.f.u. ml. ), dominated isolation plates from both cultivars in March and April i.e., just before bud-break (c. 103 c.f.u. ml.- 1 ).

65 Levels of saprophytic bacteria were much higher than 2 3 -1 pathogen populations (between 10 - 10 c.f.u. ml. ) from the two cultivars and populations fluctuated in a similar way to those of the pathogen until March. In contrast to the cv. Victoria where there was a slight increase, a rapid decline in numbers (c. 10 c.f.u. ml. * ) of saprophytes was observed in April in cv. Ariel.

2.7.2.2. Estimations from rain water traps on branches The size and position of branches, as might be expected, influenced the amount of rain water collected from them. Thus, the mean amount of water collected per branch from 3 year-oldbranches was the greatest with less from 2-year- old branches and the least amount from 1-year-old branches (Table 2.7)

Fluctuations in the bacterial populations isolated from these sources are shown in Table 2.8. The pattern of fluctuation was broadly similar for all three ages of branches and also to that on the trunks except that there was a less marked increase of the pathogen after March. Branch age and size influenced the amount of water collected, however the mean bacterial numbers (c.f.u. ml. ^ ) isolated from the three types of branches were similar. Saprophytic bacteria were c. 10 times higher than the pathogen population until January; thereafter they declined to levels similar to the pathogen.

2.8 Discussion The successful isolation of the pathogen from dormant

66 Table 2.7 Mean amount of rain water per branch collected from the traps on 1, 2 and 3-year-old branches on plum (cv. Victoria) trees during winter 1982/83

Mean amount of rain water (ml./branch)

Age of the branch

Date of collection 1 year 2 year 3 year

24.11.82 5.8 12.4 24.8 20.12.82 5.3 15.9 18.5 13. 1.83 2.5 9.3 13.3 24. 3.83 3.1 8.7 18.4 11. 4.83 4.4 8.3 18.1

Table 2.8 Mean numbers of bacteria (c.f.u. ml.~ ) isolated from rain water draining down plum trees collected in traps on 1, 2 and 3-year-old branches during winter 1982/83

Date of pv. morsprunorum Saprophytes sampling 1-year 2-year 3-year 1-year 2-year 3-year

24.11.82 34 53 67 664 737 878 20.12.82 15 15 9 1119 318 306 13. 1.83 3 4 7 77 98 83 24. 3.83 16 56 100 34 77 63 11. 4.83 27 89 120 19 109 119

67 buds of plum and cherry trees proves that the pathogen overwinters in association with its host in symptomless apparently healthy dormant buds of field and nursery trees. Therefore, a pathogenic phase (canker phase) is not obligatory for its life cycle. Buds of some other perennial trees are also known to harbour phytopathogenic bacteria during the winter, e.g.:- Erwinia amylovora (Baldwin and Goodman, 1963) P. syringae pv. syringae and pv. papulans (Burr and Katz, 1982) in apple buds and Xanthomonas campestris pv. juglandis in walnut buds (Mulrean and Schroth, 1981). Lower numbers of the pathogen per bud were isolated from plum buds than from cherry. This could be partly due to the fact that plum buds are much smaller than cherry. Bacterial numbers on both hosts were fairly constant and low throughout the winter. Regression analysis of meteorological data (rainfall, maximum and minimum temperature, sunshine hours, vapour pressure deficit) with the fluctuations of pathogenic or saprophytic bacterial populations showed no significant correlation. This is not surprising since climatic conditions during the winter are less variable than in summer. The fact that the detection of constant numbers suggests that the overwintering bacteria can survive protected in buds without drastic changes in the numbers. The ambient conditions may be different from the microclimate in buds which may provide more favourable conditions for bacterial survival during the periods of adverse climatic conditions. Even from this protected environment the numbers recovered were low; this would not necessarily indicate the exact numbers of bacteria surviving in association with

68 buds since no attempt was made to relate numbers obtained to actual numbers. Nevertheless, it can be assumed that having employed a standard isolation method throughout the sampling period, the fluctuations recorded indicate true variation and were not simply due to sampling error.

The pathogen numbers started to increase in April with the increased temperature and physiological changes of the trees at bud-break. In deciduous perennial trees, at the time of breaking dormancy, respiratory activity and phosphoril: ation of stored material would be increased, as a result, the level of soluble sugars tends to increase (Priestley, 1981). There is a suggestion that at bud-break, with greatly altered metabolism of host tissues, there is some energy waste (C.A. Priestley, personal communication) and this may be available for bacterial multiplication and it may have resulted in the rapid increase of bacterial numbers in breaking buds.

The sharp rise of the pathogen populations and decline of the other bacteria at bud-break could be due to the recognition reaction by plants. Leben (1971) stressed the importance of the recognition reaction of the plants which permits the growth and survival of 'residents' and rejects other microflora. Moreover, the rapid multiplication of the pathogen may have suppressed the growth of saprophytic bacteria in a direct competition.

External surfaces of buds (especially of plurnj) supported the pathogen and some saprophytes. The bacteria from the surfaces and inside plum buds cannot be differentiated altogether as the slightly open form of the buds would help

69 the bacteria to move inside and on to the surfaces of buds in free water. This may have partly contributed to more plum buds being apparently infested with pv. morsprunorum on their surfaces than cherry. Further, plum bud surfaces are pubescent but the tightly closed cherry buds are glabrous and waxy (Plate 2.1). As the bud scales on plum were not tightly closed the hairs on plum buds may give added protection for primordia and other young tissues from damage by low temperatures or other adverse climatic conditions during the winter. The decrease in numbers of both plum and cherry buds surface-infested with pv. morsprunorum as the season progressed ties up with the low temperatures during that period. The fact that the surfaces of more buds from both hosts were infested with saprophytic bacterial populations than with the pathogen throughout the winter suggests that phytopathogenic bacteria were more sensitive to winter conditions than saprophytes or saprophytic bacteria removed more readily from bud surfaces.

Bacteria live closer to the outer bud parts, which consist of loosely arranged bud scales on plum buds, than the inner parts where rudimentary leaves are packed closely together and harbour few or no pv. morsprunorum or other gram-negative bacteria. Scanning electron microscopic studies in apple buds have proved that external surfaces and outer- scales supported large numbers of organisms while only a few micro-organisms were associated with floral and leaf primordia (Andrews and Kernerley, 1981). The spaces between the inner parts would be suitable for the organisms that can tolerate reduced oxygen tension (Leben, 1971). This could be the reason for the inability to detect pv. morsprun- 70 orum from the innermost parts of the buds, as pv.morsprunorum normally would not live under reduced oxygen tension.

Although the time that buds become infested has not been experimentally determined it can be assumed that the developing buds become infested during the growing season from the large numbers of bacteria that live epiphytically. Some preliminary investigations showed that immature plum buds were already infested with the pathogen in July. The source of inoculum in new orchards planted with apparently healthy material can now be clearly attributed to introduction with the plant material in or on the bud- wood from mother trees or associated with maiden trees at planting. This finding is similar to that of Dowler and Petersen (19 67) in U.S.A. who reported symptomless bud- wood was a carrier of peach bacterial canker to new plantings. Very low numbers of pv. morsprunorum were isolated from rain water draining down plum trees during winter. This could either be due to the actual numbers of the pathogen surviving on the trunks being low or the rain may not have washed off the bacteria as readily from the surfaces of branches and trunks as from leaf surfaces. The bacteria may survive in lenticels and other cavities on the trunk from where wash out would be difficult. The intensity of rain probably affects the ease with which bacteria are washed from tree surfaces. The numbers of bacteria isolated were too low to be able to relate population fluctuations with the intensity of rain or climatic conditions. Further study using simulated rain would give more information about these aspects. It can be reasonably assumed that very low levels

71 of plum stem infection in these plots could be a result of the very low inoculum levels mobilised on to tree surfaces (see Section 4) when the trunks were most susceptible to infection. The absence of wounds or lack of rain to mobilise and wash in bacteria soon enough after the wounds have been made (see Section 4) may be other contributory factors. In current nursery practice the copper bactericide Wetcol is applied only to maiden trees in October to remove epiphytic bacteria. However, these studies suggest that nursery practice should be modified. Mother trees should be examined for any signs of infection, e.g.:- leaf spots and cankers and care taken to avoid obtaining bud-wood from those trees. The growers should be encouraged to grow cultivars that harbour no or few bacteria in buds. The most effective method in preventing the transmission of the pathogen to new orchards would be the use of pathogen-free bud-wood. Micro-propagation of plants (which is currently one aspect under investigation at East Mailing Research Station) using tissue culture methods should give promising results in achieving the production of healthy pathogen-free plants. This might enable the young trees to escape infection for the first few critical years after planting.

10 1 3. Epiphytic bacterial populations on leaves 3.1 Introduction 3.2 Materials and Methods 3.3 Results 3.3.1 Bacterial populations on the leaves of 3-year- old plum trees 3.3.2 Bacterial populations on the leaves of 6-year- old plum trees 3.3.3 Bacterial populations on jthe leaves of 6-year- old cherry trees 3.4 Fluctuation of leaf surface bacterial populations in relation to meteorological conditions

3.5 Discussion

73 3. Epiphytic bacterial populations on leaves

3.1 Introduction The presence of high populations of phytopathogenic bacteria on the leaves and other aerial surfaces of susceptible stone-fruit hosts, frequently without showing disease symptoms, is a well established fact (Crosse, 1954, 1959, 1963; English and Davis, 1960; Panagapoulos and Crosse, 1964;- Luisetti and Paulin, 1972; Gardan et al. , 1972). Crosse (1954) first discovered the presence of large numbers of pv. morsprunorum on the surfaces of cherry leaves. He demonstrated a relationship between the epiphytic pathogenic population and the incidence of leaf-scar infection suggesting that the. epiphytic population was the main source of inoculum for infection (Crosse, 1959). Although he could not conclusively prove the presence of pv. morsprunorum on trees in winter, Shanmuganathan (19 62) found large epiphytic populations of the pathogen on plum trees, mobilised and dispersed during rain, in the growing season and -he .stressed their importance in leaf and stem infection. However, although these populations are clearly important in leaf infection, their role in the stem infection is more difficult to interpret since this normally occurs after leaf fall, during late autumn or winter.

Crosse (1963) found that the amount of leaf surface inoculum increased with tree age in the cherry cultivars Napoleon and Roundel and attributed this to the differences in the micro-climate of the leaf canopy.

In this study, leaf surface populations of pv. morsprunom

74 on young, susceptible 3-year-old trees were compared with those on 6-year-old plum trees, with a denser leaf canopy, and also with those on 6-year-old cherry trees during the growing season "to determine the inoculum levels during this period and their relationship with populations in buds of hosts of the same age and type. The influence of climatic factors on the fluctuation of these bacterial populations was also studied using regression analysis after the manner of Freigoun (1974) to determine which of these factors were the more important.

3.2 Materials and Methods Ten trees of plum cv. Victoria and cherry cv. Napoleon were selected for this study from plots UG123, UG133 and UG124. Samples of 200 disease-free leaves, 20 from each tree, were collected, usually at about 0930 hours when the leaves . were fully dry from overnight dew. Sampling began immediately after the leaves - emerged in late-April, 1981 and continued, at approximately weekly intervals, until late-September,1981 when the leaves were yellowing. The total leaf area of the samples was calculated by measuring the area of twenty randomly selected leaves using a leaf area meter (LI COR model L1-3000, Lambda Instruments .Cor- poration).

The sampling procedure and methods were similar to i those originally used by Crosse (1959). Leaf samples were • washed in 41. of SDW (into which the wetting agent (Manoxol OT) was incorporated at 0.0025% concentration) for 4 hours and shaken periodically. During the early part of the

75 growing season less water was used, adjusted according to the mean leaf size. Serial ten-fold dilutions were prepared and 0.1 ml. aliquots were plated onto 3 replicate NSA + CVC plates. After incubating at 25 °C for 2-3 days, pv. morsprunorum- like colony types and other bacterial colonies (saprophytes) were recorded.

3.3 Results Contrary to Freigoun's (1974) results, a pure population of the pathogen was isolated from both plum and cherry leaves from the time sampling bagan until the end of May, i.e. before the appearance of leaf spots. The presence of gram-negative (for which the isolation medium was selective) saprophytic bacteria on isolation plates coincided with the .first appearance of leaf spots.

3.3.1 Bacterial populations on the leaves of 3-year-old plum trees Fluctuations in bacterial populations throughout the growing season are shown in Fig. 3.1.A. Very high populations of pv. morsprunorum were isolated until the end of July. Populations increased from bud burst until May when they reached c. 8 x 10 5 .c.f.u. cm- 2 leaf and 5 -2 from then onwards maintained a level of c. 10 c.f.u. cm leaf until they declined in August. Populations were lower during3 August an-d2 early-September when they fluctuate4 d at —c-. 10 c.f.u. cm leaf. An increase** of up to —c . 10 c.f.u. cm leaf was observed in the last estimation in the latter part of September. Saprophytic bacterial populations first appeared on 76 pv. morsprunorum saprophytic bacteria

1981

Fig. 3.1 Fluctuations of Pseudomonas syringae pv. morsprunorum and saprophytic bacterial populations on leaves of (A) 3-year-old plum trees (B) 6- year-old plum trees (C) 6-year-old cherry trees (D) weekly mean maximum temperature over the sampling period.

77 isolation plates at the end of May and fluctuated around a lower level (c. 10^ c.f.u. cm"2 leaf) than that of the pathogen throughout the sampling period, except on some occasions in July and September when the numbers dropped to c. 102 c.f .u. cm-2 leaf.

3.3.2 Bacterial populations on the leaves of 6-year- old plum trees Pv. morsprunorum populations fluctuated on the trees ' of this age in a similar manner (Fig. 3.I.B.) to that on the leaves of 3-year-old trees, with the highest level being observed in May (c. 4 x 10^ c.f.u. cm"2 leaf) but the decline began much earlier, in mid-July. Numbers of the pathogen population remained around 102 c.f . u. cm leaf during August and early-September until they increased up to c. 6 x 10* c.f .u. cm z leaf, in late September. Saphrophytes were first detected in early-June. They fluctuated at a roughly constant, but lower, level than that of the pathogen throughout the season (c. 5 x 102 c.f .u. cm leaf).

3.3.3 Bacterial populations on the leaves of 6-year- old cherry trees On the leaves of this host, populations fluctuated in a similar manner to the populations on plum leaves (Fig. 3.1.C) but they were at. a . lower level in the early 4 -2 part of the growing season (c. 10 c.f.u. cm leaf). The highest levels estimated were on two occasions in May and June (c. 5 x 104 c.f.u. cm""2 leaf); they declined in July to (c. 5 x 102 c.f.u. cm"2 )leaf and fluctuated at around that level thereafter except in mid-September when they 7 8 -2 dropped to their lowest level (c. 70 c.f.u. cm leaf).

Saprophytes appeared in June. Numbers were consistently low and fluctuated between c. 102 and 50 c.f.u. cm- 2 leaf throughout the isolation period except in mid-July when the numbers dropped to an undectable level. Throughout the growing season, pathogen numbers were highest on the leaves of 3-year-old plum trees (mean 4 -2 of 4 x 10 cm leaf) lower on 6-year-old plum trees (mean .of 5.5 x 103 cm~^ leaf) and lowest on 6-year-old cherry trees (mean of 1.8 x 103 cm leaf). Fluctuations of the pathogen population followed a similar pattern on leaves of all three host types suggesting the influence of a common factor/s. The influence of the climate, a well known factor responsible for the fluctuation of epiphytic bacteria was therefore investigated using meteorological data. 3.3.4 Fluctuation of leaf surface bacterial populations in relation to meteorological conditions. Attempts were made to relate the fluctuation of the pathogenic and saprophytic bacterial populations to meteorological conditions. Data were obtained from the weather station at East Mailing Research Station. The climatic factors used 'as independent variables in the regression analyses were: rainfall (mm.) sunshine duration, (hours), maximum and minimum temperature (degrees centigrade), vapour pressure deficit (mb at 0930 hours) and the previous bacterial count. Due to the slight yariation in the time interval between successive observations, mean values

79 of meteorological data were used. The correlation matrix of the analysis is given in Table 3.1. The model used in this analysis was y = a + bx^ + CX2 + dx^ + ex^ + fx,. + gx^ where y is bacterial population x^ = sunshine duration X2 = rainfall x^ = maximum temperature X4 = minimum temperature X5 = VPD xg = previous count. There was a negative significant (P ^ 0.05) correlation with sunshine hours and the populations of 3 year-old plum trees whereas the correlation was not significant for populations on 6-year-old plum and cherry trees. Maximum temperature was another factor which showed significant, negative correlation with the leaf surface populations of 3-year-old and - 6-year-old plum (P<0.01) and cherry (P<0.05). The count of the' previous sample also had a very high significant correlation on the pathogen population at a given time. The other meteorological data tested i.e., rainfall, minimum temperature and vapour pressure deficit were not significantly correlated with the leaf surface populations.

Correlation coefficient values for saprophytic bacterial populations on plum and cherry leaves were not significant when analysed with the same variables as used in the analysis of pathogen populations i.e., there was no

80 Table 3.1. Correlation matrix for the pathogen population.

Correlation coefficients

Variable Plum Plum Cherry

(3yr.old) (6yr.old) (6yr.old) 1. Sunshine duration -0.5442* -0.4262 -0.4232 2. Rainfall 0.0479 0.1015 -0.0090 3. Maximum temperature -0.6137** -0.6637** -0.5411* 4. Minimum temperature -0.2893 -0.3825 -0.3508 5. Vapour pressure deficit -0.0464 -0.0403 -0.0114 6. Previous count 0.7913*** 0.8196*** 0.6342** Degrees of freedom 19 19 19

***r **t * values significant (PC0.001, P<0.01) and (P<0.05) respectively..

81 correlation between the meteorological data or previous count and the saprophytic bacterial population.

3.5 Discussion The experiments reported in- this section confirm the presence of large numbers of typical pv. morsprunorum on the surfaces of plum and cherry leaves. As soon as buds had burst to produce green leaf tissues the pathogen was recovered in pure populations. This was not surprising in view of the fact (Section 2) that there was a big increase in the pathogenic and a concurrent decrease in saprophytic populations immediately prior to bud burst. The overwintering pathogen populations would disperse on to leaves to form the initial leaf-surface populations. Thus, the non-pathogenic survival chain (bud leaf surfaces) throughout the year is established. This suggests that on plum and cherry trees, pathogenic phases occur only when the conditions are favourable for infection. Shanmuganathan (1962) also pointed out that the behaviour of pv. morsprunorum is similar in that sense to certain fungi like Botrytis cinerea which is normally a saprophyte having the ability to infect plant tissues.

In this study the numbers of pv. morsprunorum isolated from leaf surfaces of plum and cherry trees were much higher than the earlier experiences on cherry (Crosse 1963; Freigoun, 1974). This may be partly due to the differences of climatic conditions. The detection of higher bacterial populations from the leaf surfaces of younger plum trees than older trees throughout the growing season differs from Crosse's (1963) observations of an increase of pv. morsprunorum leaf

82 surface populations on cherry with increasing tree age which he attributed to differences in the micro-climate resulting from denser or thinner leaf canopies on older and younger trees respectively. The more rapid increase of pathogen populations in breaking buds of younger plum trees compared with older trees (Section 2) agrees with the higher numbers detected from the leaf surfaces of younger plum trees. The higher initial population level appears to have been maintained throughout the sampling period. The pv. morsprunorum populations on plum trees were higher than those on cherry trees of the same age. Shanmuganathan (1962) also isolated greater numbers of pv. morsprunorum from rain water (derived mainly from leaf surface populations) draining down plum trees than from cherry trees during the peak period of summer. It has been further possible to detect a seasonal trend in the leaf surface inoculum on both plum and cherry. The pathogen populations fluctuated but showed a gradual increase before May and a declining trend afterwards. The sharp decline of populations in July and August, when the weekly mean maximum temperature was above 20°C, showed a strong negative correlation with the temperature. Similar observations were made on epiphytic populations of various crops by Panagopoulos (1966), Gardan et al. (1972), Ercolani (1982) and Smitley and McCarter (1982). The sunshine hours showed a significant negative correlation with the leaf surface populations of younger plum trees only, where the leaves were more exposed to ultra violet rays of sun light than older plum and cherry with

83 denser leaf canopies. In spite of this, the numbers on the leaves of young plum trees maintained a higher level compared to older trees as they were already at a higher level at the beginning of the season. Freigoun (1974) also found a negative correlation between leaf surface populations on cherry and sunshine hours. Erwinia aroidae on tobacco leaves is also reported to decrease with the increase in exposure to sunlight (Tsuyama, 1971).

The other climatic factors analysed, i.e. rainfall, minimum temperature and vapour pressure deficit (VPD) had no significant correlation with the pathogen populations. Heavy rains may leach some of the bacteria from the leaf surfaces but the populations may have built up quickly in free water left after rain or dew. To determine any effect of rainfall it would be necessary to enumerate the bacterial populations immediately before and after the rain. The populations may decrease in extremely dry conditions (Freigoun, 1974). Similarly, the minimum temperature and VPD may show negative correlations only when the temperatures are very low or VPD very high. Controlled environment studies are needed to evaluate the effect of these factors.

The effect of climatic factors may be more readily established if the counts could be made more frequently. Freigoun (1974) using daily fluctuations of the populations during September and October found that VPD between 1000 hours and 14 00 hours showed a negative correlation with the population at 0900 hours. The previous counts of the pathogen populations on both plum and cherry had a highly significant correlation with the bacterial counts as expected.

84 Saprophytic bacteria appeared on isolation plates at the end of May when the pathogen population started to decline probably due to higher maximum temperatures. The saprophytic bacterial population remained at nearly constant and low levels in all three hosts. The fact that none of the climatic factors correlated with saprophytic populations suggests they are less sensitive to climatic factors.

The effect of ambient climatic conditions may be useful for predicting the fluctuations of populations but for a better understanding of how epiphytic bacteria react to prevailing conditions it would be necessary to consider the microclimate at the leaf surfaces.

Although the importance of epiphytic pathogen populations on leaf-scar infection in cherry is strongly established, they are not of such direct importance in the life cycle of the disease on plum. On this host, leaf fall usually occurs before the stems become susceptible to infection. However, these epiphytic populations provide the inoculum for infestation of buds and tree surfaces during the summer and these are the origin of the overwintering populations which may be the source of stem infection in winter and are certainly the means whereby the pathogen is maintained on the host.

85 4. Studies on factors relating to the establishment of plum stem infection 4.1 Introduction 4.2 Materials and methods 4.2.1 Inoculation of field trees 4.2.2 Inoculation of detached twigs 4.2.3 Recording of results and reisolation of the pathogen 4.3 Effect of wound size and position on infection - field studies 4.4 Effect of wound size and duration of its susceptibility to infection - controlled environment studies 4.4.1 Effect on the frequency of infection 4.4.2 Effect on lesion length 4.4.3 Effect on survival of the pathogen in the lesions 4.5 Effect of temperature on infection - controlled environment studies at 3.5° C, 10°C and 15°C 4.6 Effect of inoculum concentration on infection 4.6.1 Field studies 4.6.1.1 Effect on the frequency (%) of infection 4.6.1.2 Effect on lesion length 4.6.1.3 Comparison of lesion length upwards and downwards from the inoculation point 4.6.2 Controlled environment studies 4.6.2.1 Effect on the frequency (%) of infection 4.6.2.2 Effect on lesion length 4.7 Discussion

86 4. Studies on the factors relating to the establishment of plum stem infection and the development of the disease 4.1 Introduction Wounds are the only experimentally proven avenue of plum stem infection. Various types of wounds ranging from large wounds resulting from mechanical damage, pruning of suckers or damage by wild animals such as hare or rabbits to minute injuries caused by insects are common on the trunks. Shaftmuganathan (19 62) working on a limited number of trees tested infection through relatively large wounds (3mm. discs) and small wounds (needle prick). He made wounds of either size at several places on the trunk and sprayed them with inoculum. He found no difference in the number of cankered trees resulting from infection through the two sizes of wounds. The real effect of wound size would have been masked by the multiple infection that occurred through several wounds. Further, this work did not give any indication of the effect of the position (height above ground level) of the wound on infection. Because of the incomplete knowledge on this aspect, it was decided to investigate the importance of the size of wounds and their height above ground level on the trunk. Crosse (1956a) demonstrated that the leaf scars, i.e. the main natural avenue of infection on cherry, remained susceptible for about 6 days. Nothing is known about the duration of susceptibility of stem wounds to infection on pliim. Infection by wild strains through open wounds in a field experiment cannot be ruled out altogether but if the wounds were protected it could create a changed environment surrounding the wounds. Therefore because of

87 this and because of the shortage of field trees it was decided to study the duration of susceptibility of wounds to infection using detached twigs. Although plum stems are susceptible to infection at any time during winter, canker development is largely inhibited until March. The reasons for this are still not clearly understood. Some workers have suggested that higher temperatures in early spring would enhance the progress of cankers (Wilson, 1939; Dye, 1957; Davis and English, 1959). Shanmuganathan (1962) attributed this to the physiological changes occurring in the tissues of plum trees. He also pointed out that conditions in early-spring were probably more favourable for rapid diffusion of any bacterial toxin. In the field, fluctuations in temperature will have some influence on the physiological state of the tissues and it is impossible to entirely isolate temperature and host physiology. Therefore, it was decided to take the twigs from dormant field trees and compare the effect of temperature on infection in controlled environment rooms. Natural infections of plum trees are believed to origin- ate during the winter season when the pathogen population is paradoxically apparently at its lowest level. However, the level of natural infection varies greatly between orchards. The amount of inoculum prevailing in an orchard is probably one contributory factor. No experiments have yet been done on the threshold inoculum level required to initiate infection or the effect of inoculum concentration on infection. These factors were, therefore, investigated to determine the level of population that might be expected to cause an unacceptable

88 level of disease.

4.2 Materials and methods 4.2.1 Inoculation of field trees The inoculation site was wiped with cotton wool soaked in methyl alcohol. After the alcohol had evaporated, either a A -shaped cut (c. 10 mm. long) was made in the bark with a sterile scalpel or a disc (4 mm. in diameter) was punched out using a sterile cork borer or a puncture deep enough to penetrate the wood was made with a sterile needle. 30 ^jul. of an aqueous suspension of isolate D 312 was introduced into the wound with a finpipette. SDW was introduced into some wounds to act as controls. All wounds were bound with polythene tape to prevent rapid drying of host tissues (which would prevent infection) and to prevent the wash out of inoculum or introduction of wild inoculum during rain.

4.2.2 Inoculation of detached twigs In experiments where field trees could not be used, material was detached and brought to the laboratory. The inoculation method was based on that of Dr. D.C. Harris (personal communication) which he used for inoculation of apple twigs with Phytophthora spp. Plastic trays (size 21 x 36 cm.) were surface sterilised by wiping with methyl alcohol. The bottom of the tray was lined with cotton wool moistened with SDW to maintain a high level of humidity. Two wooden dowels (30 cm.) were dotted with vaseline to keep the twigs in position and placed about 12 cm. apart on the cotton wool layer. Twigs from 2—year- old branches of plum trees (cv. Victoria) were prepared for inoculation as follows. They were cut into 16 cm. lengths

89 and the buds were removed. The twigs were dipped in 70% alcohol for 2 min. and washed with 4 changes of SDW. The cut ends of the twigs were sealed with molten wax to reduce water loss. The twigs were inoculated centrally either by punching out a 4 mm. disc with a cork borer or by pricking once with a needle. Unless otherwise stated, 30 yul. of a 10® c.f.u. mirl bacterial suspension was introduced into the wound with a finpipette. SDW was introduced to some wounds to act as controls. The inoculated twigs (14 - 16/tra^ were, then placed on the wooden dowels in the plastic trays, (Plate 4.1), the trays were enclosed in polythene bags misted with SDW, sealed and held at constant temperature in controlled environment rooms of 3.5°C, 10°C or 15°C as these temperatures were around mean minimum and mean maximum during winter and mean maximum during spring respectively.

4.2.3 Recording of results and reisolation of the pathogen Field trees were examined in mid-May, i.e. before canker margins were visible or any other symptoms had appeared. Cortical tissues of the bark were removed with a sterile scalpel at and near the inoculation site of field trees and detached shoots to expose the extent of necrosis and the length of necrosis measured both up and down from the inoculation court. The inoculated bacterium was reisolated from randomly selected cankers. Small pieces of tissue from the leading edges of lesions were removed aseptically and crushed in a few drops of SDW. After c. 15 - 30 minutes the "suspensions were streaked on NSA + CVC plates and colonies were observed after incubation.

do Plate 4.1. A tray lined with moistened cotton wool used to incubate inoculated plum twigs showing the arrangement of twigs on wooden dowels.

91 4.3 Effect of wound size and position on infection - field studies Four-year-old plum trees cv. Ariel low-worked on St. Julien A rootstock (plot UG133) were inoculated on 16 December 1982 at two places on the trunk, c. 100 cm. (high) and c. 50 cm. (low) above ground level, either by punching out a disc (large wound) or by making a needle prick (small wound). The four treatments were fully randomised in 8 replicate blocks. After wounding, an aqueous suspension of D312 (concentration 3.4 x 10 7 c.f.u. ml -1 ) was introduced. At the time of recording results, in May," all inoculations with the pathogen through large and small wounds resulted in infection, with one exception. This exception was one small wound in the higher position (replicate 1) which developed a very small lesion (4 mm.). The trunk of this tree was found to be damaged and the growth was retarded which probably prevented the infection from developing as in the other replicates. These results show that there was no difference between the two sizes of wounds on the frequency of infected trees.

However, some differences were observed in the lesion lengths resulting from inoculations of the two sizes of wounds. Mean canker lengths of 8 replicates were 47.6 mm., 152.9 mm., 245.6 mm. and 220.6 mm. from small wounds at high and low positions and large wounds at high and low positions respectively. The striking feature of this experiment was the difference in mean lesion length produced at higher and lower positions from inoculation of small wounds. The lesions at the higher positions were much smaller than those at the lower inoculation position. Cankers at both higher and lower positions

92 resulting from inoculation of large wounds were both much higher than even the low position inoculation of small wounds. There was a good agreement between the control inoculations. Necrosis resulting from control wounds remained confined to the area of damaged tissues with no significant extension beyond them indicating that they had remained free from infection. For analysis, the size of the wound response (2 mm. and 10 mm. for small and large wounds respectively) was deducted from the total canker length to give a more accurate figure for the actual progression of infection. Analysis of variance

of the log trrins^ormRd . " ' canker length (Table 4.1) showed that there was a very highly significant inter- action (P< 0.001) between the position on the trunk and size of the wound. The size of the wound is significant (P<0.001) for inoculations made higher up on the trunk (at c. 100 cm. from ground level) but not for those at the lower level. The position is significant (P<0.001) only for small wounds but not for large wounds. This suggests that the lower part of the trunk may be slightly more susceptible especially to small wounds.

4.4 Effect of wound size and the duration of its susceptibility on infection—controlled environmental studies Plum twigs were taken from field trees on 8th December 1982 and prepared for inoculation as described earlier. Each replicate consisted of 28 twigs. All twigs were wounded with either a large wound (discs of 4 mm. in diameter) or small wound (needle prick) as in the field experiment. Three repli-

93 from small and large wounds in high and low

positions (field trees) •

Position of wound small large on the stem wound wound

High (100 cm.) 1.225 2.360 Low ( 50 cm.) 2.148 2.291

Standard error of difference (S.e.d.) = 0.180

94 8 — 1 cate twigs were inoculated v/ith a 10 c.f.u. ml. suspension of pv. morsprunorum strain D312 and one treated with SDW to act as control immediately, 1 day, 2 days, 5 days, 7 days, 14 days and 21 days after wounding. The treatments were replicated 8 times and twigs inoculated at different times placed in random order in trays. The trays were kept in a constant temperature room at 10°C. Resultant lesion lengths were recorded on 10th March 1983 and reisolation of the inoculated bacterial strain was attempted from all twigs. Dark brown gum (normally associated with infection) or clear gum (normally a wound reaction) was observed from some shoots especially around the inoculation site. Brown lesions oft the bark, sometimes with brown stained streaks in the wood that extended beyond the bark lesion, were also recorded (Plate 4.2). 4.4.1 Effect on the frequency of infection In control inoculations the wound response varied from 6 to 11 mm. for large wounds and for small wounds from 1 to 3 mm. Therefore, lesions longer than 11 mm. and 3 mm. from large and small wounds respectively were • taken as indicating progressive infection. The percentage of infected twigs resulting from inoculations through the two wound sizes for the different intervals between wounding and inoculation are shown in Fig. 4.1. A. Infection was 100% when wounds, large or small, were inoculated immediately. The same results were observed in the field experiment when inoculations were made immediately after wounding the plum stems. For large wounds infection remained at 100% inoculating one day after

95 Plate 4.2 Lesion development in plum twigs 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 the twig.

96 wounding but steadily declined thereafter to 83% in 2 to 5 days, 75% after 7 days and 67% after 14 and 21 days. In contrast, the rate of infection decreased sharply for inoculations through small wounds. When inoculation followed one day after wounding, infection was only 17% and had fallen to 8.3% when inoculations were 14 to 21 days after wounding.

4.4.2 Effect on lesion length There was a marked difference between the lengths of lesions resulting from inoculation of the two sizes of wounds. In both, there was a big reduction in lesion length between inoculation immediately after wounding and one day later. Inoculation from 1 day or any period up to 21 days after wounding had no great effect on lesion length (Fig. 4.I.B.).

Analysis of variance was carried out on data for wound size and duration of susceptibility of wounds to infection. 5 mm. was deducted from the data for large wounds but no such deduction was made from small wounds. The data were transformed to log values.

The effect of wound size on lesion length was very highly significant (P<0.001), i.e. inoculations through the large wounds resulted in significantly larger lesions than those resulting from inoculations of small wounds (Table 4.2). The effect of the interval between wounding and the time of inoculation showed a highly significant difference (P.<0.001) between twigs inoculated immediately and 1 day after wounding. Thereafter, from 1-21 days there was no difference in susceptibility. Therefore, the results for all inoculations made after 1 day or more are combined in Table 4.3. There was a highly significant difference (P<0.001)

98 Table 4.2 Effect of wound size on lesion length on plum twigs inoculated with Pseudomonas syringae pv, morsprunorum (isolate D312)

Wound size Log. (Lesion extension beyond wound (.mm))

Large 0.877 Small 0.415

S.e.d. = 0.050 Inoculations were made in early-December and twigs held at 10°C. for 13 weeks.

99 Table 4.3 Effect of the duration of wound susceptibility on lesion length on plum twigs inoculated with Pseudomonas syringae JDV. morsprunorum (isolate D312 * Log (lesion length beyond wound (mm.))

S. e. d. Immediately 1-21 days (Horizontal Treatment after after comparisons)

Inoculated+with ) bacterial suspension) 1.177 0.632 0.047 Control 0.564 0.438 0.082

S. e. d. 0.088 0.036 (vertical comparisons)

* data combined from the two wound sizes + inoculated early-December and held at 10°C for 13 weeks

Table 4.4 Effect of interval between wounding and time of inoculation on the survival* of inoculated Pseudomonas syringae £v. morsprunorum (isolate D312) from plum twigs

Interval between % of twigs from which wounding and D312 reisolated inoculation Immediately after 95.2 1 day 82.4 2 days 74.0 5 days 77.2 7 days 71.5 14 days 36.2 21 days 54.3

* twigs were held at 10°C for 13 weeks

10 0 between the wound response of control twigs and progressive lesions on the inoculated twigs.

4.4.3 Effect on survival of the pathogen in the lesions The presence of the inoculated bacteria in bark tissues at the time of recording the results was determined by attempts to reisolate the pathogen from the advancing edges of the lesions and also from the tissues at the inoculation court when no lesion extension was observed. The data were taken as presence or absence of the pathogen and the analysis was carried out using a logistic model in regression analysis for both wound size and duration. There was no significant effect of wound size on the frequency of reisolation of the pathogen. There was a significant (P <0.01) difference between the survival of the pathogen in lesions resulting from inoculations made at different intervals after wounding (Table 4.4). Thus, the frequency of reisolation of the pathogen was very high when it was inoculated immediately after wounding and decreased with the time of inoculation.

4.5 Effect of temperature on infection-controlled environment at 3.5°C, 10 and 15°C Plum twigs were taken from field trees on 11.11.82, i.e., at the beginning of the dormant season, and inoculated with pv. morsprunorum plum strain D312 (typical strain) or D505 (atypical virulent strain) by punching out 4 mm. diameter discs from the bark. Fifteen twigs, 5per strain and 5 controls were placed randomly in a tray and kept in constant temperature rooms of 3.5°C, 10°C or 15°C. The treatments were replicated

10 1 4 times. The experiment was repeated on 14.1.83, i.e. in the middle of dormancy and at the time when plum stems are known to be most susceptible. Thus, in the two experiments twigs were different in physiology. Lesion lengths resulting from the November inoculations were recorded on 22.12.82, i.e. after 6 weeks and from January inoculations on 22.3.83, i.e. after 9 weeks. At the time of recording, apart from gum, some callus formation was evident around the wounds of some twigs that had been inoculated in November. This was more prominent in the control twigs kept at 10°C or 15°C. Wood staining was also observed, particularly at 10°C and 15°C. Figs. 4.2. A and 4.2.B. show the effect of temperature on lesion development when twigs were inoculated in November and January respectively. The lesions produced by the two pathogenic strains were longer than those from controls. The lesion length from the two strains increased with the temperature. Lesions resulting from the bacterial strains D312 or D505 normally extended from 5 mm. to 31 mm. In the controls necrosis was confined to the wounds (4 mm.) or extended only upto 7 mm. except in one wound where 3 mm. necrosis was observed due to wound healing by callus formation in a November inoculation. Therefore, minimum wound response, i.e. 3 mm. was deducted from the recorded lesion length to give the values of lesion extensions beyond wounds. These figures were used in an analysis of variance to examine the effect of temperature on lesion length in the two experiments separately and then the combined data were analysed to find out the effect of the two inoculation times on lesion length.

102 A B

November inoculation January inoculation 20

.e 16

%e 12 c o« 8 ±

s 4 2

O 3-5 IO 15 3-5 IO 15 Temp °C Temp °C

• Control, o D505, a D3I2

Fig. 4.2 Mean lesion length caused by 30^ul. from a suspension of 108 c.f.u. ml.~l of Pseudomonas syringae pv. morsprunorum 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.

103 For analysis 0.1 mm. was added to all values as for the single 3 mm. lesion, the extension was zero. Table 4.5.A summarises the analysis of log (lesion extension beyond wound + 0.1 mm.) at different temperatures resulting from the November inoculation. The lesion length produced by the two strains increased significantly (P<0.01) with the temperature. Lesions produced by the two pathogenic strains were significantly (P<0.001) different from the wound response of controls. However, the differences between the lesion lengths from the two strains were not significant.

The analysis of January inoculations (Table 4.5.B.) shows that among the three temperatures investigated, variations were significant at P<0.05 while the variations among the controls and the strains were significant at P< 0.001. The differences between the two strains were not significant. To compare the November and January inoculations, lesion length was considered as lesion extension beyond wound per day as the periods from inoculation to recording varied. Once again 0.1 was added to all values and multiplied by 100 to make the logarithms positive. The analysis of log (100 x /lesion extension beyond wound per day + 0.1 mm._7) is summarised in Table 4.6. There was a significant difference between the November and January inoculations (P<0.05). Interactions between temperatures and time of inoculation were significant at P<0.001.

The inoculated bacterial strains were readily reisolated both from the advancing edges of the bark lesions and also from stained parts of the wood.

104 Table 4.5 Log mean lesion lengths resulting from inoculation of twigs with Pseudomonas syringae pv. morsprunorum and held at constant temperatures

S.e.d. for A. November inoculation B. January inoculation horizontal comparison Inoculum

0 3 . 5 C 10°C 15 °C 3.5 °C 10°C 15 ° C B

Control 0.39 0.21 0.46 0.83 0.86 0.80 pv. morsprunorum 0.08 0.05 « D312 0.61 0.86 0.95 0.89 1.00 1.15 O D505 0.69 0.87 0.98 0.94 0.95 1.09

S.e.d. for vertical comparison 0.07 0.03 I

Table 4.6 Log mean lesion length/day resulting from inoculation of twigs with Pseudomonas syringae pv. morsprunorum and held at constant temperatures

S.e.d. for A. November inoculation B. January inoculation horizontal comparison Inoculum 3.5 °C 10°C 15 ° C 3 . 5 ° C 10°C 15 ° C

Control 0.77 0.57 0.84 1.01 1.03 0.98 pv. morsprunorum CD 0.068 O D312 0.99 1.25 1.33 1.07 1.18 1.33 D505 1.07 1.26 1.36 1.12 1.13 1.27

S.e.d. for vertical comparison 0.057 00 10 2-0 30 40 50 60 7-0 Log inoculum concentration Cc.f.u.)

Fig. 4.3 A) The frequency of infection and B) mean lesion length resulting from inoculation of a log series of concentrations of Pseudomonas syringae pv. morsprunorum plum strain (D312) to 4 to 5-year- old field trees of plum cv. Victoria in December. It 0 7 4.6 Effect of inoculum concentration on infection 4.6.1 Field studies In order to have sufficient replicates and because of tree shortage, trees from two experimental plots at East Mailing Research Station were selected. 35 five-year-old plum trees, cv. Victoria low-worked on St. Julien-A rootstock on plot UG133 and 21 four-year-old plum trees, cv. Victoria low-worked on Pixy rootstock on plot SP127 were chosen. Seven serial dilutions of culture D312 ranging from 4 x 108 to 4 x 102 c.f.u. ml.— 1 were prepared and 30 ;ul. of an appropriate concentration, inserted into a A-shape cut on the trunk c. 70 cm. above ground level. Inoculations were made on 15.12.82. The treatments were randomised in seven replicate blocks each of eight trees. Blocks 1 to 4 and three trees from block 5 were in plot UG133 while blocks 6, 7 and five trees from block 5 were in plot SP127.

4.6.1.1 Effect on the frequency of infection Lesion lengths were recorded on 11.5.83. The A -shaped scalpel wounds tended to vary slightly in size and wound response varied from 10 - 16 mm. Therefore, lesions longer than 16 mm. were taken as indicating progressive infection. The percentage of infected trees that resulted from different inoculum concentrations (Fig. 4.3.A.) shows there was no infection at the lowest inoculum concentration employed (c. 10 c.f.u.), but 14.3% of trees were infected with a concentration of c. 102 c.f.u. The threshold concentration for infection, therefore, was between c.10 c.f.u. and c.lO^ c.f.u. The infection rate increased sharply in the successive

108 inoculum concentrations and reached 100% at inoculum levels 4 of c. 10 c.f.u. or more, showing a sigmoid response. The median inoculum concentration (TC 50) defined as the concentration required to give 50% infected trees was around 800 c.f.u.

4.6.1.2 Effect on lesion length The total lesion lengths and the lesion extension upwards and downwards from the inoculation courts were recorded in May. The trees were further examined in July when it was apparent that further lesion extension had resulted and was causing death of some trees. Lesion lengths resulting from the log series of inoculum concentrations are shown in Fig. 4.3.B. Lesion lengths increased as inoculum concentration 3 increased from c. 10 c.f.u. to c. 10 c.f.u. Sharp increases in lesion length were observed from successive higher inoculum concentrations. The highest mean lesion length was obtained from the concentration of c. 10 c.f.u. A sigmoid dosage response relationship was observed over the range of inoculum concentrations used. Wound response in controls, i.e. 9mm. was subtracted from lesion lengths to obtain the lesion lengths beyond wounds and the corrected data transformed to log values. It was decided to analyse the data first to find whether there were any effects of the two different plots used, i.e., different rootstocks, tree age and possibly soil factors, on lesion length . The analysis showed that there was no significant difference between the plots. Further, analysis of variance carried out to find the effect of inoculum concentration on lesion length showed a highly significant (P-C0.001) difference between concentrations (Table 4.7.) 109 Table 4.7 Mean lesion lengths on the trunks of plum trees resulting from a log series of inoculum concentrations of Pseudomonas syringae pv. morsprunorum

Inoculum concentration Mean log lesion extension (c.f.u.) beyond wound (mm)

Control (SDW) 0.222 12 0.488 1.2 x 102 0.531 1.2 x 103 1.173 1.2 x 104 1.963 1.2 x 105 2.355 1.2 x 106 2.511 1.2 x 107 2.429

Standard error of difference = 0.196

Table 4.8 Comparison of mean lesion lengths upwards and downwards from the inoculation point on plum trunks (means of 7 replicates)

Inoculum concentration Mean lesion length (mm.) (c.f.u.) upwards downwards

1.2 x 104 74.3 86.6 1.2 x 105 118.6 139.3 1.2 x 106 117.9 232.1 1.2 x 107 135.7 150.0

110 Reisolation of the pathogen was attempted from 2 replicate blocks and the pathogen was isolated from all inoculum concentrations except from the highest. The records of tree death at the end of July, showed 4 5 that one tree from each of c. 120, 10 and 10 c.f.u., 2 trees from c. 10 3 c.f.u. and 3 trees from c. 107 c.f.u. (the highest inoculum concentration) were dead. The highest number of dead trees (5) were observed from the inoculum concentration of c. 10^ c.f.u. 2 3 The three dead trees from c. 10 and 10 c.f.u. had small lesions (<50 mm.) when recorded in May. Of the other dead trees 2 had lesions of ^ 250 mm. and 8 trees ^ 300 mm. in May. 4.6.1.3 Comparison of lesion length upwards and downwards frqm the inoculation point The record of lesion length indicated that most lesions extended further in a downward direction than upwards from the inoculation point. To determine whether this observed difference between the upward and downward extension of the lesion was significant, the data from inoculating higher 4 7 concentrations, i.e. from c. 10 to c. 10 c.f.u. were re- examined by taking the upwards and downwards lesion length from the point of inoculation separately (Table 4.8). Analysis of variance of the data was carried out taking log (upwards or downwards lesion length - 4.5 mm.), i.e. deducting half the wound size to give a truer value for progressive infection. This showed no significant difference between upwards and downwards extension of the lesion. The

111 only significant effect was of inoculum concentration confirm- ing the results above.

4.6.2 Controlled environment studies Plum twigs taken from field trees on 19th November 1982 were prepared for inoculation by punching out 4 mm. diameter discs. A log series of dilutions of culture D312 from 102 to 108 c.f.u. ml."11 was prepared and 30 /il. were inoculated to each twig as described earlier (p. 89) in a tray such that sixteen twigs, i.e 2 shoots per inoculum concentration and 2 controls were placed in each tray and randomised. The treatments were replicated 8 times. The treated twigs were subjected to 15°C. constant temperature. Lesion lengths were recorded on 21st of January 1983 and reisolations were attempted from half the replicates (from randomly selected trays).

4.6.2.1 Effect on the frequency (%) of infection Lesions from the control inoculations extended from 6 to 10 mm. due to the wound response. Any lesion greater than 10 mm. was, therefore, regarded as being a true infection capable of producing a progressive lesion. Fig. 4.4.A. shows the increase in infection with increasing inoculum concentration. The curve fitted was % infection = 103.48-89.58 (0.6424X) where x is log (inoculum concentration, c.f.u.). On this basis the lowest inoculum concentration employed (c.3 c.f.u.) produced a low incidence of infection (14.3%). The infection increased with inoculum concentration and reached a maximum c. 95% from c. 3 x 10 c.f.u.

112 A 120

Ou I I I I I I I .« • i_ OO 1-2 2-4 3-6 48 60 Log inoculum concentration (c.f.u./3)

0 1 2 3 4 5 6 7

Log inoculum concentration (c.f.u./3)

Fig. 4.4 A) The frequency of infection and B) mean lesion lengths resulting from inoculation of a log series of concentrations of Pseudomonas syringae pv. morsprunorum plum strain (D312) to detached plum twigs cv. Victoria and held at c. 15°C. for 9 weeks.

113 4.6.2.2 Effect on lesion length Lesion length increased markedly with the increasing inoculum concentration and a linear relationship was observed within the range of concentrations employed (Fig. 4.4.B.). The line fitted here was y = 10.64 + 0.77 x where y is lesion length, mm. and x is log (inoculum concentration, c.f.u.). Lesion extension beyond the wound was calculated by subtracting 5 mm. from lesion length as the minimum wound response in controls was 6 mm. Analysis of variance of the data showed that inoculum concentration had a very highly significant (P <0.001) effect on resultant lesion length (Table 4.9. ) . The pathogen was recovered from 37% of the samples taken of twigs inoculated with the lowest concentration of inoculum. The percentage recovery from samples increased 3 until the maximum (100%) at c. 10 c.f.u.; but at higher concentrations the pathogen was not isolated so readily.

4.7 Discussion The results indicated that wound size had no effect on the frequency of infection when inoculated immediately after wounding on either the trunks of field trees or on detached twigs. Even through minute wounds of the size of a needle prick,100% infection occurred. The remarkable feature of the results was the effect of position of small wounds on the trunks of field trees on the lesion lengths resulting from their inoculation. Luisetti et a_l. (1976) obtained longer cankers from inoculation of P. morsprunorum f. sp. persicae on

114 Table 4.9 The mean lesion length of plum twigs resulting from inoculations of a log series of concentrations of Psuedomonas syringae pv. morsprunorum

Inoculum concentration Mean log extension beyond (c.f.u.) wound (mm.)

Control (SDW) 0.499 3 0.657 30 0.829 3 x 102 0.816 3 x 103 0.922 4 0.937 3 x 10* 0.956 3 x 105 0.972 3 x 106

Standard error of difference = 0.059

Inoculated in mid-November and held at 15 °C for 9 weeks.

115 peach branches at 75 cm. than 175 cm. above ground and attributed that to the temperature differences between the two positions. This may also be true for the infection on plum trunks. However, some influence of rootstock on differences in susceptibility of the tissues at the two heights, cannot be ruled out. This experiment, however, was carried out on the cv. Ariel low-worked on St. Julien A. rootstock. Therefore, further experiments are needed using a wider range of cultivars/rootstock to determine whether there is any such influence of rootstock on severity of infection through small wounds. Furthermore, records of temperature at the heights of inoculation courts would help to establish any effect of temperature. Alternatively, a controlled environment study would help to resolve the effect of temperature. During this study, a few natural infections were observed in some orchards in Kent, however, it was not possible to locate the infection court as there were no visible wounds on the lesions and diseased trees were detected only at the later stages of infection. Therefore, it is suggested that the entry points were very small wounds, most probably in the lower part of the trunks of the trees. The differences between the lesion lengths resulting from inoculations through large and small wounds on detached twigs were notable and comparable to the results from inoculations in the higher position on the trunks of field trees. Previous experiences (Dr. C.M.E. Garrett, personal communication) and field inoculations in this study had indicated that branches were more resistant to infection than trunks.

116 The reasons for this are not clearly understood. However, because of that the effect of the size of wounds may have been more marked on the twigs than on the trunks. The duration of the susceptibility of wounds to infection was very much dependent on the size of wound. The lesser susceptibility of small wounds as compared Vltth large wounds may reflect the fact that small wounds take less time to heal. A large wound (c. 4 mm. in diameter) may remain a potential entry point for at least 21 days even though the rate of infection and ultimate lesion length were reduced as the time between wounding and inoculation increased. Therefore, prevention of wounds by implements or animals and early coverage of any wounds that do occur, is important especially during December and January when the trees are fully dormant and most susceptible to infection.

The significant differences in canker lengths between November and January inoculations on detached twigs agreed with Shanmuganathan's (1962) assertion of the greater susceptibility of field trees in December and January. Lesion development was temperature dependent and progressed more rapidly at higher temperatures and less rapidly at lower temperatures. Dye (1957), Davis and English (1969) also reported similar effects of temperature on canker development on peach trees caused by Pseudomonas syringae (syn. P. syringae pv. syringae). Therefore, it is suggested that the retardation of lesion development in field inoculations made in winter is due to the low temperatures prevailing at that time and lesions will extend more readily when temperature rises in the spring. Physiological changes of the trees in the spring, especially

117 rapid translocation of stored material and energy waste (Priestley, 1981) may also enhance the lesion development as Shanmuganathan (1962) suggested. When isolates D505 and D312 were inoculated in 1979/80 to the trunks of plum trees in the field, isolate D505 was much more virulent and produced much longer cankers (c.500 mm.) than D312 (c. ' 75 mm.) (Dr. C.M.E. Garrett, personal communication). However, the results of the twig inoculations in which there was no significant difference between the lesions from the two isolates were recorded only a few weeks after inoculations; differences may have shown up if they had had a longer period of development. The typical sigmoid relationships between inoculum concentration on both frequency of infection and lesion length in the field trees were similar to the observations of Crosse (1957) and Crosse and Garrett (1970) on leaf-scar infection in cherry with pv. morsprunorum. The threshold inoculum level 9 for plum trunks was very low (<10 c.f.u.). The fact that 100% infection resulted from inoculations of c. 10^ c.f.u. and above indicates the necessity of reducing the natural inoculum level.

Maximum lesion £lengt h in the field trees was achieved from inoculum of c. 10 c.f.u. Although the decrease in lesion 7 length at c.10 c.f.u. was not significant, this, combined with the failure to reisolate bacteria and the lower rate of tree death ultimately recorded from the highest inoculum concentration, seems to indicate that c. 10 c.f.u. was the optimum concentration for infection. Other workers have suggested that host cells are killed in advance of penetration by toxins

118 produced by the autolysis of bacterial bodies (Erikson, 1945b; Shanmuganathan, 1962). Possibly more rapid killing of host cells resulted from the highest inoculum concentration (c. 7 10 c.f.u.) and stimulated a host defence reaction and bacterial death and thus resulted in less tree death. Determination of the effect of inoculum concentration was obtained using the highly susceptible cv. Victoria inoculated in December when the trees were most susceptible. Results will undoubtedly vary according to cultivar used and the time of inoculation in a similar way to that reported by Crosse (1957) for leaf-scar infection on cherry. As tree death often ensues from field inoculation experiments and well replicated field trials are expensive, one purpose in repeating the experiments using detached twigs in controlled environment studies was to determine how far these results related to those on trunks of field trees. In the detached twigs, subjected to more favourable conditions for disease development in the controlled environment than field trees, the threshold inoculum level was much lower than 4tha t on field trees. Nearly 100% infection resulted from c^ 10 c.f.u., i.e. similar to the result of field inoculations. Lesion length increased linearly with the increased inoculum concentration and maximum length was achieved with the highest concentration used, i.e. 10 c.f.u. Thus, the results from twig inoculations resembled the results from the field trees fairly well. The variations noted were probably due to differences between controlled environment and variable field conditions, differences in susceptibility of twigs and trunks, and differences arising from use of detached

119 portions and growing trees. However, it can be concluded that it is feasible to use twigs in a controlled environment instead of trunks of field trees as results were reasonably comparable. By this means more uniform material and Conditions can be selected and more experiments performed in a year, at relatively low cost. Nevertheless, it is necessary to take selected studies to the field for confirmation of results.

120 5. Some bacteriological characters, phage sensitivities and in vivo growth of the bacteria associated with plum trees 5.1 Introduction 5.2 Materials and Methods 5.2.1 Cultures 5.2.2 Chemicals 5.2.3 Standard differential physiological and biochemical tests 5.2.4 Colony characteristics 5.2.5 Bacteriophage-sensitivity tests 5.2.6 Pathogenic potential 5.2.7 Inoculation of field trees 5.2.8 Leaf petioles as in vivo growth medium 5.3 Some bacteriological characters and phage sensitivities of the bacteria associated with plum trees 5.3.1 Colony morphology 5.3.2 Differential physiological, biochemical reactions and phage sensitivities of isolates 5.3.2.1 Isolates typical of Pseudomonas syringae pv. morsprunorum or pv. syringae in standard differential tests 5.3.2.1.1 Characters of the typical Pseudomonas syringae pv. morsprunorum isolates 5.3.2.1.2 Characters of the typical Pseudomonas syringae pv. syringae isolates 5.3.2.1.3 Relationship of character group with source 5.3.2.1.4 Pathogenicity of typical pv. morsprunorum and typical pv. syringae isolates 5.3.2.2 Some characters of the isolates that differ from typical Pseudomonas syringae pv. morsprunorum and pv. syringae 5.4 In vivo growth of plum and cherry strains 5.4.1 Preliminary investigation 5.4.2 Determination of optimum growth conditions for Pseudo- monas syringae pv. morsprunorum inside petioles 5.4.3 Growth differences of plum and cherry strains in petioles of both hosts 5.5 Discussion

121 5. Some bacteriological characters,phage sensitivities and in vivo growth of the bacteria associated with plum trees 5.1 Introduction Pseudomonads attacking stone-fruit trees are, in most countries, regarded as P. syringae (syn. P. syringae pv. syringae). In the United Kingdom the separate identity of pseudomonads on plum and cherry trees as pv. morsprunorum is well substantiated (Crosse and Garrett, 1963; Garrett et al. , 1966). From studies involving selection of typical colony forms, it was believed that pv. morsprunorum was homogeneous (Garrett et al. , 1966). The two host - adapted types reacted similarly in biochemical tests except that most plum strains were fluorescent in proline medium and most cherry strains were non-fluorescent. However, they differ in bacteriophage sensitivity, i.e. cherry strains are sensitive to phages of the A7 group but plum strains are insensitive (Crosse and Garrett, 1961, 1963; Garrett and Crosse, 1963). No specific phages for plum strains have yet been detected in spite of attempts to do so (Garrett and Crosse, 1963; Garrett et al., 1966, and Lazar and Crosse, 1969). Therefore, it has been possible only to distinguish plum strains from cherry strains of pv. morsprunorum by phage reactions, and all pv. morsprunorum strains from pv. syringae by biochemical characters. Phage reaction does not differentiate pv. morsprunorum plum strains from pv. syringae.

As recently as 1974 Freigoun identified a variant cherry strain (race 2) that differed from typical pv. morsprunorum in colony form and gelatine hydrolysis. As a result

12 2 of further work, groups 1, 2 and 3 were recognised among race 2 cherry strains (Garrett et al., 1977). Other studies on fruit tree pseudomonads have revealed the presence of variant/intermediate strains between pv. syringae and pv. morsprunorum mainly from cherry and peach (Crosse and Garrett, 1963; Prunier et al. , 1970; Seemuller and Arnold, 1978; Lattore and Jones, 1979; Martins, 1979; Burkowicz, 1981). However, in England, the organism associated with plum trees has been characterised as one homogeneous group, pv. morsprunorum, except for Wormald's (1932) report on Pseudomonas prunicola (now known to be pv. syringae) and Garrett's (1980b) work on biochemical variants and pv. syringae detected mainly from necrotic tissues from a wide range of plum cultivars and localities.

In this study, bacteria from a variety of healthy and infected plant organs mostly from cv. Victoria at East Mailing Research Station were isolated onto NSA + CVC medium, i.e. selective for gram-negative bacteria and on which medium typical pv. morsprunorum colonies are readily recognisable. Such colonies were selected at random for confirmation of their identity. In addition, other colony types having a close resemblance to typical plum strains, cherry strain race 1, race 2 or pv. syringae were selected for investigation. Most of these isolates were compared with authentic pv. morsprunorum and pv. syringae isolates in the standard differential physiological and biochemical tests described by Garrett et al. , (1966). They were also examined for their phage sensitivity using the cherry strain specific phages of the A7 group and B1 (race 2 specific) and also some of

123 wider host range that attack pv. morsprunorum, pv. syringae and related pseudomonads. In addition, the pathogenic potential was examined by the hypersensitive reaction in tobacco leaves (Klement, 1963). The pathogenicity to plum trees was examined with selected isolates on branches only. Al- though plum and cherry strains show marked host specificity in leaf-scar inoculations they will cross infect through wound inoculations in winter (Crosse and Garrett, 1970). Determination of pathogenicity of plum and cherry strains, or mutant forms of either, in the two hosts by this field method is not suitable for screening large numbers of isolates being expensive in land, tree- material and duration of experiment time.

A preliminary investigation was made to examine the in vivo growth of plum and cherry strains in host tissue in the growing season (when trees are normally immune to routine inoculation procedures) in the hope that this might prove a useful way of distinguishing pathogenicity of strains. In vivo growth in petioles, i.e. in a small defined area of host tissue, was therefore investigated.

5.2 Materials and Methods 5.2.1 Cultures Isolates were collected from various organs of different plum cultivars grown on plots at East Mailing Research Station, the National Fruit Trials at Brogdale, Faversham, Kent and orchards at' Toat Farm, Pulborough and Elvingstone Farm during the period November, 1980 to April,1983. Colonies typical of pv. morsprunorum and similar types were selected

124 at random from the isolation plates (NSA + CVC). A few authentic strains of pv. morsprunorum and pv. syringae (supplied by Dr. C.M.E. Garrett) were included in the tests for comparison .

5.2.2. Chemicals Bacto nutrient broth and yeast extract (Difco Laborat- ories, Detroit, Michigan, U.S.A.), Oxoid agar No. 1 or 2 and Oxoid bacteriological peptone (Oxoid Ltd., Southwark Bridge Road, London, SE1) and Analar or similar grades of chemicals were used throughout. All media were autoclaved at 121°C. for 15 min.

5.2.3. Standard differential physiological and biochemical tests The methods for the following tests were those described for the differentiation of pv. morsprunorum and pv. syringae by Garrett et al. (1966):- oxidase activity, liquefaction of gelatin, arbutin hydrolysis, growth characteristics in nutrient sucrose broth, tyrosinase activity, and the utilisation of lactic and tartaric acids as sole carbon sources. Pigment production was examined on proline medium (Crosse et al., 1968) in preference to King's B (King et al. , 1954). Arginine metabolism and oxidative and fermentative metabolism of carbohydrates were tested in Thornley's medium 2A(Thornley, 1960) and Hugh and Leifson medium (Hugh and Leifson, 1953) respectively.

5.2.4. Colony characteristics Colony morphology was examined, first on NSA + CVC

125 and then NSA after 2 days' incubation.

5.2.5 Bacteriophate-sensitivity tests Bacteriophage methods described by Crosse and Garrett (1963) and Garrett and Crosse (1963) were adopted using PGYA (see p. 32) medium. Isolates were typed with 11 phages supplied by Dr. C.M.E. Garrett. The origin and specificity of these are given in Table 5.1. Phage typing was at routine test dilution (R.T.D.), i.e. at the highest dilution producing confluent lysis on the appropriate indicator strain. The dilutions were prepared from high titre stocks(maintained at 0°C. ) as required.

5.2.6 Pathogenic potential The pathogenic potential of the isolates was estimated by their ability to induce hypersensitivity in tobacco leaves. Heavy suspensions, from 48 hour growth on NGA in SDW, of 8 — 1 representative isolates (c. 10 c.f.u. ml. ). were infiltrated into tobacco (cv. White Burley) leaves using sterilised disposable syringes as described by Klement et al. (1964). Two replicate infiltrations were made from each isolate and examined for hypersensitive reaction at 48 hours. 5.2.7 Inoculation of field trees Three-year-old branches of 6-7 year-old trees (cv. Victoria and Grove's Late Victoria) on plots UG123 and UG125 were inoculated with selected cultures by making a A -shape cut as described in Section 4 (p.89). Branches on ten replicate trees were inoculated per isolate in December and lesion lengths were recorded in the following June.

12 6 Table 5.1 Origin and specificity of typing phages

Phage Host bacterium (origin) Specificity

A9 pv. morsprunorum pv. morsprunorum cherry strain race 1 cherry strain

A12 ) pv. morsprunorum, ) pv. morsprunorum pv. syringae, some A23 ) A26 cherry strain race 1 other pathogens and some saprophytes

A15 ) pv. syringae host range as S9t ) A12 group )

'012 ) pv. syringae pv. morsprunorum S3C ) cherry and plum strains, pv. syringae

B1 pv. morsprunorum pv. morsprunorum cherry strain race 2 race 2

C9/C22 temperate phage from pv. morsprunorum pv. morsprunorum cherry strains cherry strain C9

N5/A5 temperate phage from pv. morsprunorum pv. morsprunorum cherry strains cherry strain A5

127 5.2.8 Leaf petioles as in vivo growth medium Constant humidity chambers were prepared by using transparent plastic boxes (size 13.5 x 7.5 x 6 cm.) containing saturated salt solutions to maintain constant relative humidities (Winston and Bates, 1960). In a preliminary investigation, 10 leaf petioles were taken either a) from field or b) potted greenhouse grown plum trees and incubated at 20°C. inside humid chambers for 7 days. The petioles were then crushed individually in 5 ml. of SDW in a stomacher (Colworth model 80) and 0.1 ml. samples of the suspensions, or ten-fold dilutions of them, were plated on three replicate plates of NSA + CVC medium. Large numbers of pv. morsprunorum (c. 10 c.f.u./petiole) were isolated from the leaf petioles from field trees whereas the petioles from greenhouse grown trees yielded no bacteria.

In all later investigations, therefore, leaf petioles were taken from greenhouse grown trees. Using a suction pump the bacterial suspensions were infiltrated into 2 cm. lengths of leaf petioles of approximately equal thickness on fully expanded leaves from potted greenhouse grown trees of 2-year-old plum (cv. Victoria) and one-year-old cherry (cv. Napoleon). Then the petioles were placed on double- sided adhesive tape (2.5 x 5cm) on a platform of a plastic mesh (mesh size 1.5 x 1.5 cm.) placed inside the humid chamber. The lids were then sealed with adhesive tape (Plate 5.1) and the chambers incubated at constant temperatures. Bacteria were recovered by crushing individual leaf petioles as described earlier.

128 Plate 5.1 Chamber for incubating leaf petioles at constant humidities controlled by isopiestic equilibration of saturated solutions of inorganic salts.

1 2 9 5.3 Some bacteriological characters and phage sensitivities of bacteria associated with plum trees 5.3.1 Colony morphology Twelve colony types with some similarity to typical plum strains or to the colony types described for the different races of cherry strains were observed among the 526 isolates examined and they were broadly grouped into 4 categories (Fig. 5.1). Group 1 comprises smooth, round, levan, radially striated colonies. This was divided into 3 sub- groups according to the amounts of crystal violet accumulation. Light coloured colonies with little or no crystal violet accumulation typical of pv. morsprunorum were placed in sub-group 1.1. These were designated as round striated typical (RST). Colonies similar to these but surrounded with a smooth band (some cherry strains of pv. morsprunorum are known to have similar colony characters) (RSSB)or narrow frill (RSF) were also included in this group. Sub-group 1.2, consisting of colonies that accumulated slightly greater amounts of crystal violet and therefore appeared a little darker but were .otherwise of the 3 variations in sub-group 1.1., were designated RSD,SDSB and RSDF. Some colonies had a more granular appearance, accumulated an intermediate amount of crystal violet and were frilled (RSGF) and were therefore placed separately in sub-group 1.3. All colony types in these three sub-groups were readily distinguished in NSA in which medium colony characters were studied subsequently .Group 2 comprised colonies similar to pv.morsprunorum race 2 cherry strains ("fried-egg" colonies) with a wider (FEW) or narrower (FEN) margin. Some colonies were striated

130 1. Round radially striated (RS) 1.1 Lighter colonies

RST RSSB RSF (typical) (Smooth band) (Frilled) 1.2 darker colonies (D)

RSD SDSB RSDF (Smooth band) (Frilled) 1.3 Granular frilled (GF)

RSGF

2.• Fried egg (FE)

FEW (wide) (narrow) 3. Striated wavy edged (SW)

SWG (granular)

Watery

Fig. 5.1 Different colony types observed among bacteria isolated from plum trees

12 2 but also wavy edged (SW) and they were placed in group 3

together with similar colonies but which had a more granular

appearance (SWG) whereas watery, glistening, lighter colonies

(WAT) were separated into group 4.

5.3.2 Differential physiological, biochemical reactions and phage sensitivities of isolates 526 isolates were first tested in the differential physiological and biochemical tests of Garrett et aJL. , 1966 (Table 5.2). The overall result was that the largest single group of isolates (234, 44.5%) conformed to pv.morsprunorum while only 40 isolates (7.6%) were typical of pv. syringae. The remaining 252 (47.5%) were a mixture of types that differed in one or more characters from either pathovar.

Some 392, comprising 179 (or 76%) of isolates similar to pv. morsprunorum, 36 (or 90%) of pv. syringae types, and 177 (or 70%) of the types that differed from them in physiological and biochemical tests and, representative of all sources, were additionally tested for phage sensitivity. As the standard pv. morsprunorum plum strain (D312) and pv. syringae (W50) were sensitive to phages A15, A12, A23, A26 and S9t, the isolates sensitive to these phages (with biochemical characters corresponding to the two pathovars) were regarded as typical. On this basis 154/179 (86%) were confirmed as typical of pv. morsprunorum and 23/36 (64%) as typical of pv. syringae in biochemical characters and phage sensitivity.

Selection of isolates from plates varied. In some cases the emphasis was on typical pv. morsprunorum colonies

132 Table 5.2 Differential tests of typical Pseudomonas syringae py. morsprunorum and P. syringae pv. syringae (data from Garrett et al., 1966; 1977) .

pv. morsprunorum

Cherry strains Test Plum pv. Strain syringae Race Race 2

1 Group 1 Group2 Group 3

1. Gelatin liquefaction (GL) 2. Arbutin hydrolysis (AR) 3. Pigment production in CO proline-medium (PR) CO 4. Growth in nutrient sucrose White White White White White Yellow broth (NSB) Tyrosinase activity (TY) 6. Utilisation of tartaric acid (TA) 7. Utilisation of lactic acid (LA) 8. Bacteriophage sensitivity a) A7 group + + + + + b) B1 + + + c) Syringae group + + RST RST 9. Colony morphology* FEN FEW FEW RST/RSD

* as described in Fig. 5.1 + , positive reaction - , negative reaction for confirmation of identity. On other occasions there was

a deliberate attempt to isolate into culture representatives

of the less typical but similar colony forms. Thus, no abso-

lute figures of the relative frequencies of different types

can be given. However, when the 392 isolates were grouped

according to their source, it was seen that of 184 bud

isolates, > 50% were pv. morsprunorum and <^5% pv.syringae.

In contrast, only 13% of all rain water isolates conformed

to either pv. morsprunorum (c. 6%) or pv. syringae (c.6%).

Nearly half of leaf washings isolates were pv. morsprunorum

but none was pv. syringae. The numbers of isolates tested

from other sources were generally small but it would appear

that pv. morsprunorum was dominant in leaf petioles and

leaf spots, and pv. syringae on trunk surfaces. Although

the selection from plates was not truly random and, therefore,

these figures are biased, nevertheless the relative propor-

tions of pv. morsprunorum and pv. syringae from these sources

are a fair reflection of their frequencies as observed by

their colony form in isolation plates.

5.3.2.1 Isolates typical of Pseudomonas syringae pv. mors-

prunorum or pv. syringae in standard differential

tests.

5.3.2.1.1. Characters of the typical Pseudomonas syringae

pv. morsprunorum isolates.

The isolates were identical with each other and with the standard D312 plum strain of pv. morsprunorum in all physiological and biochemical tests. Almost all pv. morsprunorum plum isolates produced a fluorescent pigment and

932 only a few (3/154) lacked that on proline medium. According

to phage sensitivities 4 main groups were identified among

/ these isolates, based on lysis of phage A15 or the A12

group phages and the production, or not, of a halo surround-

ing the lysed area. These were designated as phage-types

A, B, C and D (Table 5.3).

The characteristics of these isolates are summarised

in Table 5.4. Over 80% (groups 1A and IB) were sensitive to phage A15. There were 16 isolates which differed from the majority' of their group in being sensitive to one or more cherry strain specific phages and this group included a single example of the colony type WAT. The 4 other colony variants (all RSSB) were otherwise identical the majority of their phage group. No isolates were sensitive to the race 2 specific phage Bl.

5.3.2.1.2. Characters of the typical Pseudomonas syringae

pv. syringae isolates *

Pv. syringae isolates differed markedly from pv. morsprunorum in the physiological and biochemical tests examined but all isolates in this group were homogeneous in those tests. However, the four main phage-types identical

Wtlh those from the plum isolates of pv. morsprunorum were also observed among pv. syringae. Equal numbers of isolates were from groups A, C and D and only 2 isolates were from group B (Table 5.5). One isolate, with RST colony morphology, was sensitive to the A7 group and other cherry strain specific phages. The colony morphologies of pv. syringae isolates were somewhat less homogeneous and included all 7 colony

13 5 Table 5.3 Main categories of bacteriophage sensitivity observed among bacteria associated with plum trees (392 isolates tested).

Bacteriophage Phage type* A7 Group C9/C22 N5/A5 B1 A12 A23 A26 S9t A15

A - + + + + +

B - +H +H +H +H +

C - +H +H +H +H -

D - + + + + _

+ = confluent lysis

+H = confluent lysis with a surrounding halo

= no lysis

* a very few isolates showed additional sensitivity

to one or more of A7 group, C9/C22 and N5/A5

phages

13 6 Table 5.4 Characters of isolates conforming to typical pv.morsprunorum

Biochemical tests Phages (at RTD)

Numbers of A7 C9/ N5/ Group isolates GL AR PR NSB TY TA LA Group C22 A5 B1 A12 A23 A26 S9t A15 Colony on NSA

l.A. 1 60 + W + + — — — + + + + + RST 2 3 + W + + — + + + + + + + RST(2) + WAT (1)

3 1 + w + + - + - + + + + + RST

l.A. a 3 _ w + + - - - + + + + + RST

l.B. 1 43 - - + w + + _ - +H +H +H +H + RST(40)+ i RSSB (3) 2 4 + w + + - + + - +H +H +H +H + RST 00 3 1 - - + w + + +H + + - +H +H +H +H + RST

4 1 - - + w + + - +H +H - +H +H +H +H + RST

i.e. 1 23 + w + + _ _ - +H +H +H +H RST(22)+ * RSSB(1) 2 5 + w + + - + + - +H +H +H +H - RST

3 1 + w + + + - - - +H +H +H +H - RST

l.D 9 - - + w + + - - - + + + + - RST Standard ) pv. morsprunorum ) - - + w + + - - - + + + + + RST (D312) )

+ , Confluent lysis Note: A further 22 isolates showed very minor differences in phage +H , Confluent lysis with a halo sensitivity patterns and 3 isolates that were insensitive , No reaction to all phages employed, but with RST colony form and identical biochemical characters were also observed. Table 5.5 Characters of isolates conforming to typical pv. syringae

Biochemical tests Phages (at RTD)

Numbers of A7 C9 / N5/ Group isolates GL AR PR NSB TY TA LA Group C22 A5 B1 A12 A23 A26 S9t A15 Colony on NSA

2. A 7 + + + Y + - - - - + + + + + RSSB (6) RSD (1)

2.B. 1 + + + Y + - -• - - +H +H +H +H - RSD

1 + + + Y - - + + + + - +H +H +H +H - RST

+ 2.C 7 + + Y + \ - +H +H +H +H RSD (3) SDSB (2) RST (1) RSGF (1)

2 . D 7 + + + Y + + + + + RSD (3) RSDF (2) RSSB (1) RSF (1) Standard ) pv. Syringae ) W50 ) + + + Y + - + + + + + + + RST

+ , confluent lysis Note: Another 7 isolates showed minor differences in patterns +H, confluent lysis with a halo of phage sensitivities and 6 isolates of colony form - , no reaction group 1 (RS) identical in biochemical characters were phage insensitive. types in sub-groups 1.1, 1.2 and 1.3.

5.3.2.1.3 Relationship of character group with source

Of the wholly typical pv. morsprunorum isolates from

buds, 62% were in Group 1A and 25% in IB but only 3% and

8%were in 1C and ID. However, of the leaf isolates (i.e.

from leaf washings, leaf spots and petioles) the majority

were in group 1C (51%), 37% in IB but only 9% and 2% from

1A and ID (Table 5.6).

As there were very few pv. syringae isolates no clear

relationship could be seen between character group and source,

although 6 out of 7 group A isolates originated from trunk

surfaces and 4 out of 7 group D isolates were from buds.

5.3.2.1.4. Pathogenicity of typical pv. morsprunorum and

typical pv. syringae isolates

Pathogenic potential

76 isolates, representing the 4 phage-types, were

selected from the 154 isolates designated as typical pv. morsprunorum on the basis of biochemical tests and phage- typing to test their ability to induce an hypersensitive reaction in tobacco leaves. All these isolates,and 23 isolates typical of pv. syringae,induced hypersensitivity.

Pathogenicity in plum branches

The screening of isolates on plum in the field was limited to selected isolates because of availability of host suitable^ material. Thus only representatives of the groups

IB and ID of pv. morsprunorum were used.

More pv. syringae isolates were examined because though Wormald (1932) reported pv. syringae types on plum,

13 9 Table 5.6 Relation of character group (physiological, biochemical, bacteriophage sensitivity and colony morphology) with source of isolates in Pseudomonas syringae pv. morsprunoum

Distribution of isolates according to their source Total Character No. Host buds leaf leaf petioles rain Group isolates cultivar wash spots water

1. A 64 Vic 50 3 1 0 0 Ariel 3 0 0 0 0 Others 7 0 0 0 0

1. Aa 3 Vic 2 1 0 0 0 l.B 49 Vic 21 13 0 6 1 Ariel 3 0 0 0 5 l.C 29 Vic 3 16 4 1" 0 Ariel 0 0 1 0 0 Others 0 0 4 . 0 0 l.D 9 Vic 8 1 0 0 0

Total 154 97 34 10 7 6 it has since rarely been isolated or the disease attributed

to it in South East England. Therefore, representatives

of all groups A, B, C and D were inoculated.

Mean canker lengths resulting from inoculations are shown in Table 5.7. The two isolates representing pv. morsprunorum, JP666 and JP354 from character groups IB and ID produced cankers of similar length to the standard (D312) pv. morsprunorum isolate of character group 1A.

Of the six pv. syringae isolates, four were weak patho-

gens producing cankers of c. 13 - 17 mm. but two isolates

(from groups 2C and 2D)were non-pathogenic.

5.3.2.2 Some characters of the isolates that differ from typical Pseudomonas syringae pv. morsprunorum and pv. syringae

Four isolates (2 from buds and 2 from rain water) of RST(3) and RSD(l) colony forms were identical to pv. morsprunorum in all differential tests, but,with the exception of lack of tyrosinase activity, were of phage-type B and gave an hypersensitive reaction in tobacco leaves. They could probably, therefore, be correctly assigned to pv. morsprunorum as such variants have previously been reported.

There were 63 isolates, all of which produced an hypersensitive reaction in tobacco leaves, (the majority from buds (49) and the rest from leaf washings, rain water and trunk surfaces) that liquefied gelatin, hydrolysed arbutin, produced fluorescent pigment in proline medium and yellow growth in nutrient sucrose broth but that differed from typical isolates of pv. syringae in remaining biochemical tests in that they possessed tyrosinase activity (4) or

141 Table 5.7 Mean lesion length produced on plum branches cv. Victoria following inoculations with Pseudomonas syringae pv. morsprunorum and P. syringae pv. syringae plum isolates

Phage- Mean (of 10 Culture Pathovar type Source replicates) canker length (mm. )

JP666 ) 1.B Ariel bud 35.8 JP354 j pv. morsprunorum l.D Vic bud 32.4 JP349 ) resistant Vic bud 26.6

JP408 ) 2. A Vic TPS 17.6 ) JP670 j 2.B Ariel RAW 13.6 JP657 ) 2. C Vic RAW 14.9 ) pv. syringae JP 40 2. C Vic bud 11.6 JP211 2. D Vic bud 13.4 JP415 2. D Vic TRS 11.8

D312 Standard pv. morsprunorum 1. A Vic LSP 28.8

Control (SDW) 11.3

RAW = rain water TRS = trunk surface LSP = leaf spots SDW = sterile distilled water

142 failed to utilise lactate (38) or had both these characters (21).

Among the 4 isolates showing tyrosinase activity,

2 phage-sensitivity patterns were observed. The only RSDF

colony form was insensitive to all test phages and the remain-

ing 3 isolates were of SDSB colony form. Of the 38 isolates

that failed to utilise lactate, most of them (26) from buds,

were c.40% of phage-type C, 4% phage-resistant and the rest were of phage-type B and D. The most frequent (35) colony

forms were of group 1 (RS) types, and the others were of

FEW. There was no correlation between phage-type and source

or colony form. The isolates having tyrosinase activity

and lacking utilisation of lactate (21) all, with one exception, from buds, were distributed among phage-types C and

D, except for a very few that were sensitive only to phage

A12 or were phage-resistant. The failure to use lactate and non-specific nature of the phages, makes the relation of these intermediates/variants to pv. syringae uncertain.

48 isolates, nearly half of which came from necrotic tissues and half from leaf washings, produced white growth in nutrient sucrose broth but were otherwise very unreactive in differential tests, i.e. were oxidase negative, neither produced fluorescent pigment in proline medium nor hydrolysed arbutin, showed no tyrosinase activity and did not utilise tartrate or lactate. Of these, 33 isolates liquefied gelatin and they were all from leaves, either washings or leaf spots, of the cv. Victoria at East Mailing Research Station (EMRS).

The 14 isolates that showed no liquefaction of gelatin were all, except one, from necrotic tissues, either leaf spots or cankers, from various plum cultivars at Brogdale. All

143 colony forms were of group 1 (RS) types except a few FEW.

A group of representative isolates were shown to have oxida-

tive metabolism in the Hugh and Leifson test and possessed no arginine hydrolase. One isolate, of phage-type C, was also sensitive to the A7 group of phages and c.50% showed sensitivity to some phages and c.50% were insensitive fo the test phages. They produced an hypersensitive reaction in tobacco leaves. Therefore, these isolates resembled in certain characters isolates from Prunus cerasifera (Garrett and Crosse, 1967) and P. morsprunorum f. sp. persicae (Gardan et al., 1972).

Three of these isolates JP53 and JP54 from trunk cankers and JP103 (with the ability to liquefy gelatin) from leaf spots were selected for field inoculation to plum branches to confirm their pathogenicity. JP53 and JP54 produced cankers

(mean canker lengths 36 and 24 mm.) similar to pv.morsprunorum standard isolate while JP103 was highly virulent producing very long cankers extending to the main trunk (mean canker length 696 mm) . All figures are mean of 10 replicate inoculations .

A group of 68 isolates (c. 75% from rain water and the rest from leaf washings) showed no hypersensitive reaction on tobacco leaves. Of these, 41 were fluorescent pseudomonads that produced yellow growth on NSB and of which 31 isolates were also oxidase positive. None was of colony form RST.

Most oxidase positive isolates were of colony sub-groups

1.2 and 1.3 with one exception of FEW while the oxidase negative isolates were mostly of colony type RSD with a few exceptions of RSGF.

13 942 The 27 non-fluorescent isolates (equal numbers from

rain water and leaf washings) were,with 2 exceptions, oxidase

positive and of various colony types.

Because these isolates were mainly oxidase positive,

non pathogenic and of the less typical colony forms, they

were not investigated further.

The rest of the isolates could not be grouped into

distinct categories as they gave a variety of reactions

in the differential tests.

5.4 In vivo growth of plum and cherry strains

5.4.1 Preliminary investigation

Suspensions in SDW of plum strain JP206 or cherry 4 -1 strain JC51 at 10 c.f.u. ml. were infiltrated into 50

plum or cherry petioles each of 2 cm. long. Immediately

after infiltration, plum or cherry petioles were crushed

in 5 ml. of SDW and suspensions were plated (0.2 ml./plate)

on NSA medium.

Mean numbers of pv. morsprunorum isolated (c.f.u./

petiole) were : for plum strain in plum, 10.4 and in cherry

13.3 and for cherry strain in plum 11.7 and in cherry 13.8

for each 50 petiole sample tested.

Thus, with a large sample of petioles (50) only a very low bacterial count was obtained. Therefore, with a

small sample, it was very difficult to detect the pathogen

just after infiltration. As it was necessary to get bacteria-

free petioles from greenhouse-grown trees and because of the shortage of trees in the greenhouse, it was decided to try smaller samples (single petioles) and growth determin- ations only after 2 days.

145 5.4.2 Determination of optimum growth conditions for pv.

morsprunorum inside petioles

The plum strain JP206 was infiltrated as a SDW suspen- 3 -1 sion of 8.3 x 10 c.f.u. ml. into 5 replicate petioles

and these were then kept inside the humid chambers. Relative

humidities (RH) of 98%, 75% and 33% were maintained inside

the chambers using I^SO^ or I^C^O^, NaCl and MgCl2,6H20 res-

pectively. These were then subjected to temperatures of

4,10,20,25 and 30°C. Bacterial numbers were estimated after

2, 5 and 9 days. (It was not possible to reisolate countable

numbers before 2 days).

No bacteria were isolated from lower relative humid-

ities, i.e. 75% and 33%, at any temperature. Recovery of

bacteria was achieved only at 98% RH. The estimations of

bacterial growth at 98% RH and at different temperatures

are shown in Fig. 5.2. Up to 5 days, bacterial numbers

increased at all temperatures tested; they reached the highest 7 level c. 10 c.f .u./petiole at 20°C. and 25°C. in 5 days.

The population was maintained at that' level at 20°C. but

dropped slightly at 25°C. in 9 days. At both lower (4°C,

10°C.) and the highest (30°C.) temperatures, growth was

slower. However, at 10°C., in 7 9 days the numbers reached a similar maximum level (c. 10 c.f.u/petiole) to that at 5 25°C. while the numbers at 30°C decreased (c. 10 c.f.u./ petiole). The growth rate was slowest at 4°C. and the maximum 5 -1 reached in 9 days, was c. 10 c.f.u. ml. , the same level

as that at 30°C.

Optimum conditions, therefore, were 98% RH and 20°C.

and these were the conditions employed in the following experiment. 146 ft) o 8 a. ir U

c-f O CI co_ Ql

Q. C

o c o 8 5 3 4 5 6 Days after inoculation

Fig.5.2 Growth of Pseudomonas syringae pv. morsprunorm (plum strain) in plum petioles at 98% relative humidity and different temperatures.

147 5.4.3 Growth differences of plum and cherry strains inside

petioles from the two hosts 4 -1 10 c.f.u. ml. suspensions of JP74 plum strain and

JC72 cherry strain isolated from leaf spots were infiltrated into plum and cherry petioles (5 petioles/treatment). These were then kept inside humid chambers at 98% RH and incubated at 20°C. Bacterial numbers were estimated after 2, 5 and

7 days. The experiment was repeated with 3 more plum strains

JP206, JP245, JP256 from petioles, buds and leaf washings and cherry strains JC30, from leaf spots and JC51, and HI 11 from leaf washings.

The growth rates of plum and cherry strains inside petioles of the two hosts are shown in Figs. 5.3 and 5.4.

All four plum strains behaved similarly showing very high 7 growth rates in seven days (reaching c. 10 c.f.u./petiole).

However, the growth rate was relatively low in cherry 4petiole s reaching a maximum of only between c. 250 to c. 10 c.f.u. in 7 days. The growth rates of cherry strains in petioles of both hosts were moderate. However, they grew slightly better in plum petioles than in cherry. \ Thus, there appeared to be some difference in growth rate in the 2 hosts associated with host type of strains.

It is therefore possible that such a method might be useful in pathogenicity determination but further work needs to be done with other organisms, including saprophytes, to see if it is sufficiently specific to be of value.

148 Days after inoculation

5.3 Growth of 4 different plum strains of Pseudomonas syringae pv. morsprunorum in plum and in cherry petioles at 98% relative humidity and 20°C.

149 Fig. 5.4 Growth of 4 different cherry strains of Pseudomonas syringae pv. morsprunorum in plum and cherry petioles at 98% relative humidity and 20°C.

150 5.5 Discussion

Typical pv. morsprunorum isolates - were homogeneous

in colony morphology, physiological and biochemical charac-

ters. The fact that almost all isolates possessed the light

coloured RST colony forms indicates that selection on RST

colony form on NSA, with or without CVC, is a reliable method

for distinguishing typical pv. morsprunorum from other bac-

teria associated with plum trees. However, it is not in-

fallible as a few RST colonies were found in pv. syringae

and a few RSSB in pv. morsprunorum. Therefore, to distinguish

pv. morsprunorum from pv. syringae, it is necessary to

examine the isolates in the differential biochemical charac-

ters .

In contrast to pv. morsprunorum, colony morphology

of typical pv. syringae isolates was variable; the most

common types were RSD and RSSB. These and other colony types

found in pv. syringae were not specific for this pathovar.

They were found occasionally among pv. morsprunorum, and

in other types. Therefore, colony form alone in NSA or NSA +

CVC medium is not reliable for pv. syringae when it is present

together with similar types on isolation plates.

Four main phage-types were identified among typical

isolates of pathogens on the basis of sensitivity to phage

A15 and production of a halo surrounding the lytic area.

Resistance to phage A15 has also been observed in some pv. morsprunorum cherry strains (Crosse and Garrett, 1970).

As there are no specific phages for pv. morsprunorum plum

strains and pv. syringae, the same four phage-types were observed in pv. syringae isolates. Furthermore, isolates

13 949 identical to pv. morsprunorum or pv. syringae in differential biochemical tests but showing different phage sensitivity patterns, and also some that were insensitive to all phages employed, were detected. Phage resistant isolates of the pathogens may be lysogenic strains, isolates lacking receptor sites for phages or mutants insensitive to the phages used.

However, although lysogenic strains were frequent among cherry strains, they have rarely been found in plum strains

(Garrett and Crosse, 1963; Lazar and Crosse, 1969).

In addition, a few isolates showed sensitivity to the cherry strain specific phages of the A7 group. Lazar and

Crosse (1969), when attempting to detect lysogeny in plum strains, found an occasional isolate from plum was sensitive to the A7 group phages. These isolates may have been transmitted from the adjacent cherry trees or they were perhaps genuine host-adapted plum strains having sensitivity to the A7 group phages. If so the specificity of A7 phages for cherry strains is not absolute.

Typical pv. morsprunorum dominated buds and leaves.

However, some isolates of pv. syringae occurred on trunk surfaces, in rain water collections and associated with buds. Therefore, in South East England pv. syringae does occur on plum even though it is not frequent.

Although the division of isolates into four main phage- types was somewhat arbitrary, it appears however, to have some epidemiological significance. Isolates from buds consisted mainly of phage-types A and B but phage-types C and

D were rarely detected. In contrast, isolates from leaves were mainly of phage-types B and C but phage-types A and

152 D were seldom found. This differentiation between source

and phage-type may be related to the fact that bud isolates

were obtained during winter and leaf isolates during summer.

Phage-type B was common to both sources having the ability

to survive throughout the year.

Phenotype variability of P. syringae pv. savastanoi

isolates on olive trees at different times of the year was

reported by Ercolani ( 1983). He suggested that alternation

of heterogeneous populations rather than recurrent modifica-

tion of homogeneous populations was responsible for the

observed phenotype fluctuations. The factors responsible

for the observed fluctuations of phage-type A (sensitive

to phage A15 and producing no halo) and C (insensitive to

phage A15 and halo producing) on plum trees is not clear.

However, plum strain mutants resistant to phage A15 after

passage through plum leaves, have been reported by Crosse

and Garrett (1970), indicating that sensitivity to A15 was

not stable. Nevertheless, the stability of halo production

surrounding the lytic area of the isolates after passage

through host tissues is yet to be tested. Therefore, it

is not possible to suggest at this stage that the presence

of phage-types A and C in different sources during different

•seasons on plum trees is due either to alternation or modi-

fication of the plum isolates.

All typical pv. morsprunorum and pv. syringae isolates

tested were potentially pathogenic (capable of inducing

hypersensitivity in tobacco leaves) but the pv. syringae

isolates tested showed only weak pathogenicity on plum

branches. Therefore, they are probably of little epidemio-

logical significance.

153 The bacterial colonies with some similarity to the pathogenic colonies associated with plum-trees were investigated in differential tests (Garrett et al., 1966, 1977) to see how they differed from the pathogen, and were grouped according to certain character differences but no attempt was made to identify them completely. Among these isolates a considerable number of intermediate/variant isolates having pathogenic potential were detected in this study. A few representative isolates from necrotic tissues, of a group of isolates similar to the isolates from Prunus cerasifera (Garrett and Crosse, 1967) and P. morsprunorum f.sp. persicae (Gardan et al. , 1972) proved pathogenic to plum branches and included one highly virulent isolate. Therefore, there are other organisms among plum isolates that could have epidemiological significance if they were to be of widespread occurence. However, no survey has yet been carried out to see how they were distributed in orchards. It is also necessary to test the pathogenicity of representative isolates of the different colony, biochemical and phage- types in order to determine any significance in the epidemiology of the disease. This could economically be done using twig inoculations in controlled environment as described in Section 4.

Although Barclay (1968) found that oxidase positive fluorescent pseudomonads were mainly confined to roots and rhizospheres of cherry and to a lesser degree apple, but oxidase negative organisms were isolated mainly from aerial surfaces of the plants, in this study some oxidase positive leaf surface isolates of the P. fluorescens type were found.

Oxidase positive isolates are, therefore, not limited to 13 952 the root zone of established stone-fruit trees.

Investigations on growth rates in vivo between plum and cherry strains showed marked differences. The difference was much clearer with plum strains inside the two hosts.

Some preliminary investigations showed that there was a lag phase of more than 20 hours in plum strains before they multiplied after insertion into cherry petioles. As plum strains grow better in plum than in cherry petioles it suggests that some factors inhibit the growth of plum strains inside cherry petioles. Therefore, leaf petioles may provide an in vivo growth medium for distinguishing between the pathogenicity of plum and cherry strains. However, growth of more isolates, including saprophytes, needs to be tested before this method can be properly evaluated.

Plum strains of pv. morsprunorum have been reported ineffective through cherry leaf scars and they inhibited infection by cherry strains when present in the same inoculum

(Crosse and Garrett, 1970). It has been suggested that this may be due to plum strains causing an hypersensitive reaction in the host that prevents infection. The growth inhibition of plum strains in cherry petioles suggests that a similar mechanism may be involved. This area needs further investigation with single and mixed inocula; in vivo growth in petioles might be a useful avenue to explore host-parasite relations.

155 6. Prospects for control

6.1 Introduction

6.2 Effect of Bordeaux mixture (winter sprays) on the pathogen

population associated with plum buds

6.2.1 Materials and methods

6.2.2 Results

6.3 Detection of antagonistic organisms

6.3.1 Materials and methods

6.3.1.1 Cross-streaking method

6.3.1.2 Poured-plate method

6.3.1.3 Examination of culture filtrates

6.3.2 Results

6.3.2.1 By cross-streaking

6.3.2.2 By poured-plates

6.3.2.3 Effect of antagonistic bacteria on colony form of

the pathogen

6.3.2.4 Antagonistic properties of the culture filtrates

6.3.2.5 Characters of antagonistic isolates

6.4 Discussion

13 954 6. Prospects for control

6.1 Introduction

Most bacterial diseases have proved to be extremely

difficult to control. Bacterial canker of stone-fruit trees

is no exception. One major obstacle is the lack of suitable

bactericides. Bordeaux mixture is the most effective bacter-

icide so far tested against cherry canker (Moore, Crosse

and Bennett, 1959; Crosse, 1962, 1967). Routine autumn spray

schedules of Bordeaux mixture effective on cherry, have virtually no effect on the disease on plum because they are aimed at preventing leaf scar infection. On plum, infection usually occurs on the main trunk sometime during the dormant season. Plum buds have now been shown to harbour overwintering populations of the pathogen. Low numbers of the pathogen were isolated in rain water draining down plum trees during winter suggesting the mobilisation of the pathogen on the surface of the trees (Section 2). These inocula could be sources of stem infection. The generally low level of natural infection of bacterial canker on plum in South

East England makes assessment of sprays in canker control difficult. However, following a year in which 6 trees died from natural infection in the experimental plot, an experiment was designed to determine the effectiveness of Bordeaux mixture on the control of canker and more particularly to assess its effect on the winter bud population of pv. morsprunorum.

The use of antagonistic micro-organisms is another method currently being explored in the control of plant diseases. However, only a limited number of studies have

157 been made in respect of controlling plant bacterial diseases

using other organisms. Such work was given impetus by the

successful biological control of crown gall caused by Agro-

bacterium tumefaciens on peach and almond that has been

developed in Australia using the bacteriocinogenic A.

radiobacter strain 84 (New and Kerr, 1972 and Htay and Kerr,

1974 ). This is now widely used in Australia, New Zealand,

U.S.A., Canada and some European countries against crown

gall of a range of stone-fruit trees, and also roses, and

is the first and only commercial application of biological

control of a bacterial plant disease. Some studies were

carried out to explore the possibility for biological control

of plum bacterial canker by screening organisms from the

tree surfaces for antagonism to the pathogen.

6.2 Effect of Bordeaux mixture (winter sprays) on the pathogen

population associated with plum buds

6.2.1 Materials and methods

Four-year-old plum cvs. Victoria 28 trees (14 each

sprayed and non-sprayed control) and Ariel 24 trees (12 each sprayed and non-sprayed) were used in a fully randomised design as single tree plots. Wetcol was sprayed 3 times i.e., on 7.1.82, 5.3.82 and 14.4.82 on the trees covering the surfaces, of branches and trunk using a knapsack sprayer.

The rates of wetcol applied were 75 1./ 1000 1. of water in the first spray and 35 1./1000 1. of water in the 2nd and 3rd sprays. In the last spray, cotton seed oil (7.5 1./

1000 1. of water) was included to reduce phytotoxicity as the buds were opening at that time.

16 6 The bacterial populations of both sprayed and unsprayed trees were assessed (when the numbers of bacteria in buds were generally . more detectable) 10 days after the 2nd spray, A the just before and again 6 days after 3rd spray, by crushing A 70-bud samples (7 buds/tree) from 10 trees of each variety, in 9 ml. of SDW and plating the suspensions. The last assessment of cv. Ariel was on the surface population of the young leaves as on this variety, buds had leafed out at the time of sampling. These leaf populations were estimated by washing

200 leaves (20 leaves from each of 10 trees) in 1 1 .of SDW as described in Section 3 (p.75).

6.2.2 Results

The results of the assessment of pathogen populations of the sprayed and non-sprayed trees are shown in Fig. 6.1.

Ten days after the 2nd spray, from the buds of sprayed and non-sprayed trees of cv. Victoria, no great difference was observed in the pathogen populations. At the time of the 3rd spray in April, buds were swelling and the pathogen populations were expected to have increased sharply. Estima- tion of pathogen populations just before the 3rd spray, showed that they were then at similar levels in sprayed and non-sprayed trees. Six days after spray application, 3 the pathogen population had increased from c. 10 to c.

5 x 10 c.f.u./bud in non-sprayed trees but in sprayed trees 3 4 the increase was smaller from c. 10 to 5 x 10 c.f.u./bud).

In cv. Ariel, ten days after 2nd spray a remarkable decrease in the pathogen population (from c. 3 x 10^ c.f.u./ bud in non-sprayed to 8.6 c.f.u./bud in sprayed trees) was

159 7

6 Sprayed Control T3 5 n 4 Sprays applied: £ u 3 7/1/82 cO 3 5/3/82 2 14/4/82

C7» Victoria Ariel Victoria Ariel Victoria Ariel O 15/3/82 14/4/82 20/4/82

post spray 2 pre spray 3 post spray 3

Fig. 6.1 Comparison of populations of Pseudomonas syringae pv. morsprunorum from buds or tiny leaves from broken buds of two plum cultivars of non-sprayed (control) trees and of trees sprayed with weak Bordeaux mixture.

160 observed. The probable reason for this is that the bud

swelling started earlier in the cv. Ariel making the bud

tissues more penetrable to Bordeaux mixture thereby facili-

tating closer contact with the bacteria. However, the popula-

tion had built up from ten days after the 2nd spray to the

time of 3rd spray in the sprayed trees. Just before the

3rd spray, only a- slight difference was observed between 3 the populations on the leaves of sprayed (c. 10 c.f.u. -2 4 -2 cm. leaf) and non-sprayed- (c. 10 c.f.u. cm. leaf) trees.

Again 6 days after 3rd spray, marked reductions of the patho- 5 -2 gen populations were observed (c. 3 x 10 c.f.u. cm. leaf _2

from non-sprayed trees to only-c.45 c.f.u. cm. leaf from

sprayed trees).

The effect of the spray on natural trunk infection

could not be assessed because in this experiment, during

the summer after spray application, there was no natural

infection observed except on one sprayed tree of cv. Ariel. 6.3 Detection of antagonistic organisms

6.3.1. Materials and methods

6.3.1.1. Cross-streaking method

Saprophytic bacteria isolated from crushed buds and

leaf washings of plum were streaked diametrically on NSA plates and allowed to grow for 4-5 days at 25°C. Growth was then removed with a sterile glass slide and the plates were inverted over chloroform - impregnated filter papers

for about 15 minutes to kill residual bacteria. Plates were ventilated for about 10 min. to allow the chloroform to evaporate and 4 pathogenic isolates per plate were then

13 959 cross-streaked at right angles to the original culture.

Plates were examined after 2-3 days and any inhibition of

growth was recorded.

6.3.1.2. Poured-plate method

Selected test bacteria were inoculated into the centre of NSA plates and incubated for 4-5 days at 25°C. The growth was then removed as described above. 6 ml. of PGYA containing a test isolate of pv. morsprunorum at c. 10 c.f.u. /ml. was poured over the plates to form a thin layer. Plates were incubated at 25°C. After 1-2 days growth inhibition was recorded.

6.3.1.3. Examination of culture filtrates

Selected bacterial cultures (which showed some antagonism to the pathogen in plate tests) were grown in the following liquid medium viz. NH4H2PC>4, . 0.1%; KC1, 0.02%; MgSC>4.

7H20, 0.02%; Sucrose, 0.3% and pH was adjusted to 7.2. The medium was autoclaved for 15 minutes at 121°C. The cultures were shaken (100 strokes/minute) in an orbital shaker (MKV) at 18°C for 5-6 days. They were then centrifuged (403 relative centrifugal force) for 20 minutes in a MSE super minor centri- fuge. The supernatants were filtered through size 0.22 /im. millipore filters. The filtrates were then placed in dialysis tubing (diameter 1.5 cm.) secured by double knots at the ends, immersed into a glass tube (size 6 x 30 cm.) filled with SDW and dial-ysed on a rocker for 24 hrs. The fractions of dialysis were concentrated to about 5 ml. by evaporating under vacuum at 37°C. The antagonistic properties of these

162 6.3.2. Results

6.3.2.1. By cross-streaking

29 isolates which showed colony forms different from

pv. morsprunorum on NSA were tested for antagonistic action

against representative cultures of pv. morsprunorum plum

strains, cherry strains races 1 and 2 and pv. syringae

employing the cross-streaking method. The test was replicated

4 times.

Two isolates (JA 9 and JA15) showed strong antagonistic

action while another two (JA10 and JA12) were observed to

give weak antagonistic action. The growth inhibition of

isolates JA 9, the weakly antagonistic JA12 and a non-antagon-

istic isolate are shown in Plate 6.1. The mean lengths of

inhibition (Table 6.1) show that JA 9 was very strongly antagonistic (c. 47 mm. inhibition) to all three forms of pv. morsprunorum and only slightly less so to the pv.syringae tested. The action of JA15 (inhibition c. 27 mm.) was weaker and again with this isolate there was a difference between sensitivity to pv. morsprunorum and pv. syringae. JA12 was only slightly weaker than JA15 but isolate JA10 was much less antagonistic to pv. morsprunorum (inhibition c. 8 mm.) and showed no antagonism to pv. syringae.

6.3.2.2. By poured-plates

Isolates JA 9 and JA15 were, tested in duplicate in the poured-plate test. The results (Table 6.2) confirm the stronger activity of JA 9 and JA15 against all forms of pv. morsprunorum compared to pv. syringae and the greater antagonism of JA 9 over JA15. Plate 6.2 shows the zone of

163 Plate 6.1 Inhibition of growth of Pseudomonas syringae pv. morsprunorum and pv. syringae isolates cross- streaked against (A) JA 9, wider inhibition zone (B) JA 12, narrow inhibition zone (C)' JA 5, no inhibition.

164 Table 6.1 Mean length of inhibition (mm) induced by four saprophytic bacterial isolates against pathogenic strains in the cross-streaking method

pv. morsprunorum pv. morsprunorum pv. morsprunorum pv. syringae Test culture plum strain cherry strain cherry strain cherry strain race 1 race 2

JA 9 48.0 46.3 47.3 33.3 JA 10 9.3 7.8 8.8 0 JA 12 22.5 17.0 20.8 13.8 JA 15 28.5 26.8 27.8 14.0 CO •H

Table 6.2 Mean diameter (mm) of inhibitory zone induced by isolates JA 9 and JA 15 against pathogenic strains in the poured-plate method

pv. morsprunorum pv. morsprunorum pv. morsprunorum pv. syringae Test culture plum strain cherry strain cherry strain cherry strain race 1 race 2

JA 9 36 50 56.5 10.5 JA 15 32 21 29.5 12.5 Plate 6.2 Inhibit ion of growth of Pseudomonas syringae pv. morsprunorum by the antagonistic isolate JA 9 in the poured-plate method.

1 6 6 inhibition produced by JA 9 against pv. morsprunorum plum

strain.

6.3.2.3. Effect of antagonistic bacteria on colony form

of the pathogen

Turbid aqueous suspensions of pv. morsprunorum plum

strain, JP 74 and antagonistic isolates JA 9 and JA15 were prepared and 1 ml. of JP 74 was mixed with either 1 ml. of JA 9 or JA15. Suspensions were diluted serially and plated on 3 replicate plates of NSA (0.1 ml./plate).

JA 9 was observed to change the colony morphology of pv. morsprunorum when the two bacterial types were growing closely together on the plates. The round striated, raised, typical plum strain colonies (Plate 6.3.A.) changed to dif- fuse edged, flatter colonies (Plate 6.3.B.) that looked more similar to race 2 cherry strains of pv. morsprunorum.

The changed colonies reverted to typical colonies when restreaked alone on NSA. JA15 which had shown weaker antagonistic activity did not change the colony morphology of the pathogen when grown on the same medium together.

6.3.2.4. Antagonistic properties of culture filtrates

50 /il. of either the concentrated diffusible fraction or the non-diffusible fraction of the dialysis from isolate

JA 9 was placed in the centre of three replicate PGYA plates previously seeded with pv. morsprunorum plum strain, JP74, and incubated at 25°C. for 24 hrs. A clear growth inhibition from the diffusible fraction was observed but no inhibition was seen from the non-diffusible fraction (Plate 6.4). The experiment was repeated with fractions of dialysis from

13 965 Plate 6.3 Effect of (JA 9) large colonies on colony morphology of Pseudomonas syringae pv. morsprunorum plum strain when grown in close proximity (A) normal shining colonies with sharp margins (B) modified dull colonies with diffused margins.

1 (5 8 Plate 6.4 (A) Inhibitory action on Pseudomonas syringae pv. morsprunorum plum strain induced by diffusible fraction of dialysis from JA 9 culture filtrate (B) no inhibition from the non-diffusible fraction.

169 JA15 on JP74 but no inhibition of JP74 was observed with

either fraction.

6.3.2.5 Characters of antagonistic isolates

On the basis of Bradbury1 s . key to assist in screening

bacteria isolated from plants (Bradbury, 1970), these isolates

were investigated in a limited number of tests to broadly

determine to what genera they belonged (Table 6.3.).

The results revealed that JA15 was an oxidase positive,

fluorescent pseudomonad, probably within the group Pseudomonas

fluorescens. It was then tested for phage sensitivity using

the phages employed in Section 5 and for the hypersensitive

reaction in tobacco leaves. It was insensitive to all phages

and produced no hypersensitive reaction.

JA9 showed both an oxidative and a fermentive reaction

in glucose metabolism and also because of having other characters described in Table 6.3. it appears that this isolate may belong to Azotomonas. Further tests are needed to confirm the identity. This isolate also did not produce an hypersensitive reaction in tobacco leaves.

6.4 Discussion

Bordeaux mixture apparently had only a small effect on the populations in dormant plum buds in spite of their slightly open form. As no systemic bactericide is available it is clearly extremely difficult to control the pathogen in dormant buds. However, a strong bactericidal effect was observed when the buds were swelling and at bud-break. This fact accounted for the more effective control in the cv.

Ariel where buds were swelling at the time of the 2nd spray.

13 968 Table 6.3 Some physiological and 'biochemical characters of JA9 and JA15 isolates

Character JA9 JA15

1. Colony morphology pearly white, white, on N.S.A. round, smooth, frilly edged and slightly and flat raised 2. Gram stain reaction 3. Oxidase activity 4. Growth in sucrose broth yellow yellow Growth at 41°C. * Growth at 4°C. * Pigment production in proline medium 8. Acid from glucose (Hugh-Leifson test) (1) Oxidative (2) Fermentative 9. Arginine hydrolase 10. Gelatin liquifaction 11. Arbutin hydrolysis 12. Tyrosinase activity 13. Utilisation of (a) tartaric acid (b) lactic acid

* Growth in nutrient sucrose broth in water baths at constant temperatures.

171 Similar results were obtained in the cv. Victoria (later flowering than cv. Ariel) from the 3rd spray and a more effective control was observed in the opened buds in cv. Ariel.

These results encourage one to hope that the life cycle of pv. morsprunorum on plum can be broken and the populations on young leaves reduced or even eliminated. If the residual population was kept at a low level throughout the growing season with a weak Bordeaux mixture (strong Bordeaux mixture is known to be phytotoxic to plum) it should reduce infestation of buds and, thus, the overwintering populations associated with plum trees in the subsequent dormant season and thereby reduce the inoculum level for stem infection. This can be tested experimentally easily.

Furthermore, the application of Bordeaux mixture (with cotton seed oil as adjuvant to reduce phytotoxicity) to nursery trees before planting out (or, if planted early in winter, in the field at bud swelling) and again at bud-break, should reduce the initial bacterial populations to a very low level in a new orchard. This aspect should be given much emphasis as the inoculum concentration required for infection is very low (Section 4). A spray programme on mother trees in the growing season also may reduce the level of inoculum that would carry over to budwood.

In the absence of a good bactericide, an alternative, biological control using antagonistic organisms would be very useful to keep the pathogen at a very low level. The results of the experiments in vitro are encouraging.

The fact that both whole cells of isolate JA9 and the diffusible fraction of its culture filtrate showed strong

13 970 antagonistic action suggests that this isolate produced a

low molecular weight (<5 to 10f000) bactericidal substance

active against pv. morsprunorum. Further studies are needed to determine its exact chemical nature. The antagonistic action of this culture is further proved by its ability to change the colonial appearance of the pathogen. A similar phenomenon has been observed with Pseudomonas solanacearum on a selective medium. When certain antagonists were present,

P. solanacearum colonies changed from normal, round, pulvinate, fluidal, tan colour to flat, small, lavender coloured colonies

(Granada and Sequeira, 1981).

The prospects for the use of the pseudomonad JA15 cannot be disregarded merely because the antagonistic action of this isolate is weaker. It's use together with JA9, and perhaps with other antagonists, might increase their effectiveness.

One difficulty of using isolates for biological control is that of establishment especially when using single antagonists active against pathogens in vitro but derived from other sources. If a combination of two or more antagonists from the same ecological habitat can be used a more effective antagonism might result.

JA15 showed closer similarity to pv. morsprunorum than

JA9. One might speculate, therefore, that since JA15 required similar growth conditions to pv. morsprunorum it would more readily establish in the ecological niche of pv. morsprunorum than would JA9.

The performance of these isolates in the field is yet to be evaluated. Clearly much work has to be done before there would be any prospect that such control could become commercial.

173 General conclusions

The experiments described in this thesis lead to the conclusion that Pseudomonas syringae pv. morsprunorum overwinters protectively during adverse climatic conditions, in association with apparently healthy plum buds, at nearly constant but very low numbers even in very young orchard trees. Pv. morsprunorum tends to colonise mostly on the outer bud parts and the surfaces of plum buds together with saprophytes.

The infestation of developing buds by the pathogen during the growing season provides a means of transmission of the pathogen to nurseries in bud-wood from mother trees and subsequently to new orchards in the young trees. It is, therefore, most important to obtain pathogen-free bud- wood to prevent the pathogen being transmitted to nurseries and new orchards.

The numbers of the pathogen in buds increase during bud-break and they spread onto leaves to initiate leaf surface populations. Thus, it is clear that pv. morsprunorum could survive as a non-pathogen throughout the year and that pathogenic phases are not obligatory for its life cycle. The pathogenic populations on plum leaves were higher than on cherry and younger plum trees, contrary to expectation, harboured more pathogenic populations than older trees.

Saprophytic populations appeared later in spring and maintain a constant, lower level than the pathogen. The pathogenic populations were more sensitive to climatic factors, especially to higher temperatures and sunshine duration, than saprophytes .

13 972 Attempts to determine the inoculum potential during

the winter revealed that only very low numbers of the pathogen

could readily be isolated from the trunks of plum trees.

In experimental plots, there was a very low level of natural

infection. Higher inoculum levels may prevail in areas where

the disease incidence is high.

The threshold inoculum level for infection of freshly

made wounds on cv. Victoria plum trees in December was very

low. Inspection of cankers in experimental plots and other

orchards in Kent did not reveal the avenue of infection

as there were no visible wounds in cankered areas. Therefore,

it was not possible to conclude the nature of the wounds

that were the avenues for infection.

Studies on the canker phase revealed that wound size,

within the limits of pin prick and discs of 4 mm. in diameter,

had no effect on the frequency of infection. Minute wounds,

therefore, also should be regarded as entry points. How-

ever, their duration of susceptibility seemed to be low.

Although inoculation through small wounds at higher positions

above ground level on the trunks of field trees developed

small cankers, those on lower parts of the trunks provided

entry points for inoculum and caused slightly larger cankers.

Therefore, if some form of disease control is to be approached by protecting the trunks from wounds, emphasis should be

given to protection of the lower part of the trunk. Further- more, the methods of preventing infection depend on maintain-

ing very low inoculum levels and covering wounds as soon

as possible.

Experiments on temperature in controlled environments

175 lead to the conclusion that temperature influences the development ofcankers. It is possible, therefore, that the retardation of canker development during the winter season could be partly due to the low temperatures prevailing at that time. The effect of the physiological state of the trees on canker development was also evident from inoculations at two different times during winter.

Studies on the pathogen have thrown more light on the nature of the organisms associated with plum trees.

Typical pv. morsprunorum types dominated the bacteria isolated.

The vast majority of pv. morsprunorum were of RST colony form and selection on this basis on isolation plates was quite reliable and adequate for estimation of populations by colony counts. The occurrence of different phage-types in buds and leaf surfaces needs further studies to determine whether there is a modification or alternation in the pathogen population with the host organ infested and/or the season.

Low numbers of pv. syringae were identified on the > basis of characters in differential biochemical tests and phage sensitivity patterns; but the colony form was not consistent in this pathovar. Nevertheless, pv. syringae was certainly present on plum trees even though it was not frequent. Isolates of this pathovar were much less virulent than pv. morsprunorum.

Among other organisms with superficial similarity to these two pathovars, was a group of isolates that reacted like isolates from Prunus cerasifera and" P. morsprunorum f. sp. persicae and they seemed to have epidemiological oh significance producing cankers when inoculated . to plum

176 branches. This type could have been overlooked on isolation plates, nevertheless, further studies should be carried

out to find out their distribution among orchards.

The role of other pseudomonads, some of them oxidase negative, having pathogenic potential and some others oxidase positive with no pathogenic potential has not been determined, but, it is unlikely they play a significant role in the epidemiology of bacterial canker.

In vivo growth rate measurements in petioles of plum and cherry showed differences between plum and cherry strains.

It is suggested that, subject to more complete investigation of the mechanism involved, this might be a useful laboratory system for investigating host-parasite relationships.

The findings in this thesis lead to some suggestions for improved control of the disease or at least indicate in what areas further research is needed to achieve this aim.

In the establishment of new orchards, it is necessary to improve nursery practice to prevent introduction of the pathogen by a chemical spray schedule for mother-trees during the growing season to prevent infestation of bud-wood and for nursery trees during bud-break.

To prevent disease in established orchards, a systemic bactericide would be ideal. However, as no systemic bactericide is available at present, the pathogen population in dormant buds cannot be controlled. Even though the Bordeaux mixture re'duces the pathogen population in breaking buds at a time when populations would otherwise increase, copper sprays are often phytotoxic to young tissues and can be

177 used only at low concentrations. If bacteria are not eliminated or inoculum comes from outside orchards, it is necessary to maintain leaf surface populations at a low level throughout the growing season by further sprays to reduce the possibility of bud infestation which would carry over the pathogen to next season. This aspect needs further investigation to evaluate the efficiency of available bactericides in preventing bud infestation and timing of sprays. This would be more profitably carried out in areas where natural infection is more serious.

In plum breeding programmes for resistance to bacterial canker, emphasis should be given to selection of cultivars which harbour less bacteria in buds as parent lines.

Pending the introduction of a satisfactory chemical control and breeding of resistant cultivars, it would be valuable to explore the possibilities of the application of a biological control agent. Preliminary investigations on organisms antagonistic to the pathogen in vitro showed that it is not difficult to discover antagonistic organisms.

Nevertheless, their performance in the field depends on their ability to establish and survive in the field and then to produce the antagonistic product in sufficient amount to give good control. The antagonistic effect might be increased if a combination of two or more antagonists could be used. It would be a more fruitful line of investigation if this study also could be carried out in areas where natural infection is higher than at East Mailing.

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