Pathological Studies on Bacterial Canker Disease on

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and Environmental Affairs,Faculty of Agriculture at Moshtohor, ... Dr. Nawal. Abdel-Moneim Eisa, Professor of Plant Pathology, Faculty of. Agriculture at ...... samples were transferred to the laboratory in plastic bags, kept in refrigerator at 7°C, ...
Pathological Studies on Bacterial Canker Disease on Some Fruit Trees

By Ahmed Abd El-Hady El-Siesy B.Sc. Agricultural Sciences (Plant Pathology), Fac. Agric. Moshtohor, Zagazig Univ. Benha Branch, 2003

Thesis Submitted in partial fulfillment in the requirement for the degree of Master of Science In Plant Pathology

Agriculture Botany Department Faculty of Agriculture Benha University 2007

SUPERVISION COMMITTEE Pathological Studies on Bacterial Canker Disease on Some Fruit Trees By Ahmed Abd El-Hady El-Siesy B.Sc. Agricultural Sciences (Plant Pathology), Fac. Agric. Moshtohor, Zagazig Univ. Benha Branch, 2003

This thesis for M.Sc. degree in Plant Pathology under the supervision of:

1. Prof. Dr. Abdou-Mahdy Mohamed Mahdy Vice Dean for Community Services and Environmental affairs , Professor of Plant Pathology, Agric. Botany Dept., Fac. Agric., Moshtohor Benha University

2. Prof. Dr. Nawal Abdel-Moneim Eisa Professor of Plant Pathology, Agric. Botany Dept., Fac.,Agric., Moshtohor,Benha University

3. Prof. Dr. Gehad Mohamed Dessouky EL- Habbaa Professor of Plant Pathology, Agric. Botany Dept., Fac.,Agric., Moshtohor,Benha University.

4. Prof. Dr. Nagy Yassin Abd El- Ghafar Professor of Plant Pathology, Department of Plant Pathology, Fac., of Agriculture, Ain Shams University.

ACKNOWLEDGEMENT First at all, great thanks and gratitude be to "Allah", who guide me to this way and assist me in all my life. All words, all feelings and all praise will not be enough to thank "Allah". A word of gratitude is not enough towards the great effort and help that Prof. Dr. Abdou-Mahdy Mohamed Mahdy, Professor of Plant Pathology Vice-Dean of Faculty for Community Development and Environmental Affairs,Faculty of Agriculture at Moshtohor, Benha University, did in the whole work. He has been always patient, helpful and kind hearted. His advices are my guide in work. He gave me his time and effort to introduce this thesis in the best form and it was a pleasure to work under his supervision. I would like to express my gratitude towards Prof. Dr. Nawal Abdel-Moneim Eisa, Professor of Plant Pathology, Faculty of Agriculture at Moshtohor, Benha University. She offered me a lot of here experience in my work. Here expert advises, bounding patience and constant support with here lovely soul were the major factors behind my work and it was an honor for me to work under here supervision. A word of love and gratitude to Prof. Dr. Gehad Mohamed Dessouky EL- Habbaa, Professor of Plant Pathology, Faculty of Agriculture at Moshtohor, Benha University, for suggesting the subject of this study and preparing the manuscript. He has been a great support by his clinical observations and effort which were very helpful throughout the work. He was kind enough to share me my problems during this work. Many thanks are also offered to Prof. Dr. Nagy Yassin Abd El- Ghafar, Professor of Plant Pathology, Department of Plant Pathology, Fac., of Agriculture, Ain Shams University. for his valuable and gracious help at the beginning of this investigation. At last but not least, I am indebted to all staff members and my colleagues at Plant Pathology Branch, Department of Botany Faculty of Agriculture at Moshtohor, Benha University, for their help and encouragement and to everyone helped this work to arise.

Contents INTRODUCTION…………………………………………..………………

1

REVIEW OF LITERATURE ……………………………..……………….

3

MATERIALS and METHODS

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EXPERIMENTAL RESULTS ……………………………………….………

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I. Isolation of bacterial canker ……………………..……….……

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II- Pathogenic reaction on different hosts ……………………….

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46 III. Identification of isolated bacteria…………………………. 46 1.Using traditional techniques………………………………… 46 1.a- Morphological and cultural characters…………………… 47 1.b- Biochemical and physiological characteristics……………. 2. Verification the identification using PCR (Polymerase Chain Reaction) 50 techniques………………………………. 51 V. Factors affecting the growth of P. syringae in vitro 51 1. Effect of Temperature…………………………..……………..

2. Effect of pH values……………………………………………… 3. Effect of Relative humidity (RH)……………….………………

53 54

56 VI. Disease control………………………………………………… 1.Effect of some chemical compounds on growth of Pseudomonas 56 syringae………...……………………………. 2.Effect of some antibiotics on growth of Pseudomonas 61 syringae……………………………………………………...

3. Effect of bioagents on growth of bacterial pathogens………. 4.Effect of bactericides on controlling canker disease on immature peach fruits………………………………….. 5.Effect of some bioagents on controlling canker disease on immature peach fruits…………………………………… 6. Effect of bactericides on bacterial canker disease, under artificial inoculation conditions ……………………………. 7. Effect of bioagents on bacterial canker disease, under artificial inoculation conditions …………………………

66

DISCUSSION…………………………………………………

79

SUMMARY ……………………………………………………

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REFERENCES …………………………………………………

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ARABIC SUMMARY …………………………………………

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67 72 74 77

INTRODUCTION Stone and pome fruit trees are very important trees in the world, they including a large number of fruit trees like apricot (Prunus armeniaca L.), Peach (Prunus persica L.), Apple (Malus domestica L.) and Pear (Pyrus communis L.) which consider a very important nutritional fruit trees in the world where they are rich in carbohydrates, sugars, vitamins (B and C), enzymes, fats and protein. In Egypt, the total cultivated area of these fruit trees reached about 168,900 feddan yielded about 1,021,540 tons where the total yield of apricot is about 73000 tons from cultivated area 18,500 feddan while, the yield of peach is about 360000 tons from cultivated area 79,180 feddan. Also, the yield of pear is 39,000 tons from cultivated area 7,200 feddan while, the total yield of apple is about 550000 tons from cultivated area 64,200 feddan (FAO Stat., database, 2005). The most important economical diseases of stone fruits are listed as brown rot (Monilia spp.), leaf curl (Taphrina deformans), rust(Tranzschelia discolor), scab (Cladosporium carpophilum), bacterial spot (Xanthomonas campestris) and bacterial canker (Pseudomonas syringae ) as recorded by Penrose, (1998). Bacterial canker of stone fruits caused by P. syringae Van Hall has become a serious problem in many parts of the world (Cameron, 1962 and Mohammadi et al., 2001). Also, the bacterial canker has implicated in the problem known as "peach- tree short- life" in the southeastern United States where it caused a great loss of peach trees in the central valley of California (English and Davis, 1964, Dowler and Petersen, 1966 and Weaver et al. 1974). Pseudomonas syringae causes many important and common diseases including bacterial canker of stone fruit, blossom blight or blast of pear, brown spot of bean, citrus blast and black pit, and blights and leaf spots of pea, cowpea and lilac (Elliott 1951, Stapp 1961 and Hayward and Waterston,1969).

Introduction 1

The disease occurs on the aboveground parts of the trees, and may resulted in localized canker or death of entire limbs or trees. Symptoms of bacterial canker appear on branches, twigs, buds, leaves, and fruits. The most conspicuous symptoms are the cankers that exude gum during late spring and summer. Gumming is common on stone fruit trees, whether on trunks, twigs, or fruit when injuries occur. Thus, the name gummosis does not define a cause, only a response. Cankers on the twigs are darkened areas often at the base of buds. On trunks they are often darker than the normal bark, sunken in their centers, and they may extend for a considerable distance. Leaves and shoots growth beyond the canker may wilt and die during the growing season (English and Davis,1960 and Hetherington, 2005). Many factors including soil texture, low soil pH, soil depth, tree nutrition, tree age, nematode parasitism, rootstock selection , cultural practices such as early fully pruning and environmental factors such as freezing temperatures and rain can influence severity of bacterial canker of stone fruits (English et al., 1980 and Lownsbery et al., 1977). The current study aimed to throw the light on bacterial canker disease which appeared in the last few years in stone and pome trees orchards in Egypt. Surveying and isolation of the bacterial canker pathogens from different localities of Egypt. Identification of the isolated pathogenic bacteria using traditional and PCR techniques. Also, studying some environmental factors affecting growth of isolated bacterial pathogens like temperature degrees, pH values and relative humidity. Controlling the pathogenic bacteria using some chemical compounds, antibiotics and some bacterial antagonists.

Introduction

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REVIEW OF LITERATURE Isolation and pathogenicity of canker bacteria: Burki (1968) investigated Pseudomonas syringae isolates of cherry, plum, apricot and pear pathologically, serologically, physiologically and biochemically and compared them with authentic strs. of Pseudomonas morsprunorum. He found that the bacterial canker of sweet cherry and leaf spot of plum and apricot were caused by P. morsprunorum. While, the pear blossom blight pathogen and isolates from sour cherry were P. syringae. Isolates causing sweet cherry canker and those causing leaf spot of plum seemed to be highly host specific. Except for one isolate from apricot, leaf scar infection of fruiting spurs of sweet cherry was successful only with isolates from sweet cherry. Pear and sour cherry isolates had a similar pathogenicity and could infect pear blossom. Most P. syringae strs. induced hypersensitive necroses in tobacco leaves at an inoculum conc. of 107 cells/ml, but for P. morsprunorum 108 was required. In agglutination tests, the flagellar (H) antigens were highly specific in distinguishing the 2 spp. whereas the 0 antigens showed little specificity. Cancino et al. (1974) stated that physiological and pathogenicity tests revealed that Pseudomonas syringae is the causal agent of pear blast in Chile. The disease has apparently been present for many years, but confused with fire blight showed physiological disorders, or phytotoxicity . P. syringae has not been reported previously to affect pear blossoms, in Chile. In certain years the disease has been severe and it; characterized by blasting of flowers, leaf necrosis, and cankers of fruit spurs, and small branches. No exudates have been observed on lesion. The identification of the causal organism was based on physiological tests, oxidase reaction, serology, pathogenicity tests with peach seedlings, and the hypersensitive reaction of tobacco leaf tissue.

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Dowler and Weaver (1974) isolated pathogenic and nonpathogenic fluorescent Pseudomonads from apparently healthy peach twig and trunk tissue samples collected monthly in Georgia and South Carolina. No. pathogenic bacteria were isolated during the summer months. Morphological and biochemical tests showed that the pathogenic isolates were closely related to Pseudomonas syringae, but about 50% of the fluorescent isolates were nonpathogenic. Inoculation of mature trees in three fields with these isolates during early fall pruning resulted in death of trees by the following March. Heterogeneous populations of Pseudomonas exist in apparently healthy peach orchards in the southeastern United States. French and Miller (1974) described the disease symptoms caused by Pseudomonas syringae and reported the recommended control measures.Pseudomonas syringae was newly recorded on peach in Florida. Sands and Kolias (1974) observed symptoms typical of pear blast on all commercial varieties of pears in Connecticut. The disease appeared after mutually moist weather conditions around the time of pear bloom in 1972 and 1973. Pseudomonas syringae was isolated from the diseased pears and the isolates produced symptoms when inoculated into pears. Dorozhkin and Grigortsevich (1976) found that Pseudomonas syringae is wide spread in Belarussia where it attacks pear and cherry, then apple and plum. Symptoms are bark cracking and leaf curl; wilted leaves remain on the tree for a long time. Trees may die in the 1st yr or, in the chronic form infection, after several years. Allen and Dirks (1978) found that biochemical tests indicated that isolates of Pseudomonas from the Niagara Peninsula were similar to English isolates of P. morsprunorum and P. syringae. Pathogenicity tests on nine sweet-cherry varieties, Prunus mahaleb, the P. avium

Review of Literature

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variety Mazzard, apple, pear and peach indicated that the Ontario isolates were pathologically distinct. Comparative tests of isolates from England and Ontario revealed close similarities between isolates of P. morsprunorum, whereas isolates of P. syringae appeared to belong to distinct races. Trials indicated that sweet-cherry varieties grown in Ontario are sufficiently susceptible to both bacterial species consider the disease as a serious problem. It is considered that the existence of two species of Pseudomonas in Ontario will make breeding for resistance difficult. Burkowicz et al. (1978) recorded that cankers, die-back of branches and, in severe cases, tree "apoplexy" are caused by the disease. Isolates from diseased twigs gave reactions typical of P. syringae in physiological and biochemical tests. On inoculating apricot leaves, in an orchard, typical symptoms were produced and the bacterium was re- isolated. Roos and Hattingh (1983a) reported that distinctive physiotypes of pathogenic Pseudomonas to attack stone fruit and occasionally apples, causing blister bark in South Africa. They included fluorescent Pseudomonas syringae pv. syringae, races 1 and 2 of P. syringae pv. morsprunorum, and intermediate forms as well as non-fluorescent strains. Details are given of the symptoms of bacterial canker infection and the epidemiology of the disease. Some advice is given on reducing the spread of infection, but satisfactory control measures in South Africa have not yet been developed. Roos and Hattingh (1983b) reported that Oxidase-negative, green fluorescent Pseudomonas isolates (403) from cankers, symptomless branches and symptomless buds on plum, apricot, peach and nectarine trees and from healthy leaves of the first 2 hosts were characterized by GATTa tests for gelatin liquefaction, aesculin hydrolysis, tyrosinase activity and tartrate utilization. Most isolates were assigned to P. syringae pv. syringae (Pss) but P. syringae pv.

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morsprunorum (Psm)and intermediate forms were also identified. The hypersensitive reaction on tobacco leaves was a reliable criterion for establishing pathogenicity to plum and apricot host plants. A resident phase of Pss was found on symptomless leaves and buds. Pss appeared to be the major pathogen causing bacterial canker of stone fruit in Cape Province, South Africa. Tominaga, et al. (1983) identified 31 isolates from shoots and fruits of cankered trees which were short, rod-shaped, aerobic, Gram (-) and motile and could be divided into 3 groups (A, B and C) by differential tests for distinguishing Pseudomonas syringae pv. syringae and P. syringae pv. morsprunorum. On the basis of bacteriological characters and pathogenicity, group C bacteria was identified as P. syringae pv. morsprunorum while groups A and B were regarded as strains of the same bacterium. Ercolani and Ghaffer (1985) found that bacterial canker and gummosis on apricot and peach trees in the Kabul area were caused by Pseudomonas syringae pv. syringae and the bacterium causing similar diseases on almond was identified as P. amygdali. Roos and Hattingh (1986a) found that pathogenic isolates of Pseudomonas syringae pv. syringae (inducing a hypersensitive reaction in tobacco and infecting plum leaves) were obtained at intervals, 10 Oct. 1981-15 June 1982, from weeds in apricot and plum orchards in the SW Cape Province (South Africa). Roos and Hattingh (1986b) isolated pathogenic Pseudomonas spp. from many apparently healthy buds of stone fruit trees, with higher percentages of active, expanding buds than dormant buds containing the pathogens. This indicated a resident phase in buds, which can be a potential source of inoculum for bacterial canker of stone fruit in South Africa

Review of Literature

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Severin et al. (1986) noted an apoplectic dieback on young trees in some orchards. At the beginning of winter, lesions with necrotic tissue, sometimes with exudates, around petiole wounds were observed. The disease developed up to the start of vegetative growth. Cankers formed on older branches where extensive led to wilt and death of the distal part. On the basis of cultural characters, the pathogen was identified as Pseudomonas syringae pv. syringae. Shane and Baumer (1987) monitored that population of P. syringae pv. syringae and symptom developed after introduction of bacterial suspensions (5 X I06 cfu/ ml-1) into wheat leaf intercellular spaces and incubate it at 18-20° C under light mist. Spray, wound, or vacuum infiltration were inappropriate inoculation methods. P. syringae pv. syringae is a weak pathogen that requires moist conditions during the incubation period for significant infection. Foliar symptoms and login bacterial populations 3 days after inoculation were positively. Wimalajeewa (1987) reported that infection of apricot trees with Pseudomonas syringae pv. syringae (bacterial canker) occurred through buds, flowers, leaves, fruit and stems but not leaf scars through which natural infection can occur. Only stem and bud inoculations consistently led to the establishment of cankers. Isabel et al. (1988) found that cherry blossoms inoculated with a rifampicin-resistant strain of Pseudomonas syringae pv. morsprunorum died or gave rise to fruits containing necrotic spots at or near the blossom ends. Scanning electron microscopy of developing fruits indicated that the pathogen had invaded the entire pericarp, including the endocarp. Bacteria also spread to the fruit stalk and, to a lesser extent, to the spurs. Mesocarp cells below the lesion collapsed. Infected fruits, stalks, and spurs contained, respectively, calculated 109, 107, and 102 colony forming units of P. syringae pv. morsprunorum as determined by a dilution plate method on an agar

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medium supplemented with 50 µg/ml rifampicin. This is the first report of systemic spread of P. syringae from blossoms to developing fruit of a deciduous crop. Takanashi (1988) detected in 1980 a new bacterial disease of Prunus salicina in Japan. On the basis of bacteriological tests against a reference culture the pathogen was identified as Pseudomonas syringae pv. morsprunorum. Hattingh et al. (1989) studied symptoms of Pseudomonas syringae pv. syringae infection of stone and poem fruit trees in South Africa. Infection mechanisms via natural openings in leaves, blossoms and seed by electron microscopic (EM). Systemic spread in shoots was observed. A modified life cycle of bacterial canker on stone fruit trees caused by P. syringae is proposed and problems and prospects for disease control discussed. Takikawa et al.(1989) identified on the basis of lab. the causal organism of bacterial canker of kiwi fruits in Japan as P. syringae tests. The pathogen is similar to P. syringae pv. morsprunorum. The pathogen reproduced characteristic canker and leaf spot symptoms in inoculated kiwi fruit. The bacterium was pathogenic to inoculated kiwi fruit and weakly pathogenic to peach and Japanese apricot but was not pathogenic to 24 other plant species tested. The pathogen is thought to be a pathovar of P. syringae. Shams-Bakhsh and Rahimian (1997) noted bacterial canker of stone fruits in most regions of the Mazandaran province. The bacterial strains isolated from stone fruit trees were fluorescent oxidase negative, had more than one polar flagella, produced acid and levan from sucrose and did not rot potato tuber slices but were variable in production of syringomycin. They were identified as P. syringae pv. syringae.

Review of Literature

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Weingart and Volksch (1997) investigated polymerase chain reaction (PCR) fingerprinting using primers corresponding to repetitive (ERIC and REP) and insertion sequences (IS50) as a method to distinguish the pathovars of P. syringae. After amplification of total DNA with the ERIC-, REP- and IS50-PCR followed by agarose gel electrophoresis, most of the tested pathovars showed specific patterns of PCR products. The differences between the fingerprints among strains within a pathovar were small, with the exception of pathovars syringae, aptata and atrofaciens. The fingerprints of the related pathovars savastanoi, phaseolicola, glycinea, morsprunorum, tabaci, lachrymans and mori generated with the ERIC- and REP-primers were found to be very similar, showing the potential of this technique for taxonomical studies. In contrast, the IS50-PCR fingerprints of these pathovars were clearly distinguishable. The fingerprint patterns of a strain were highly reproducible with all 3 tested primer sets and also when whole cells were added to the reaction mixture. It is concluded that this PCR technique with the ERIC-, REP- and IS50primers is a rapid, simple, reproducible and low cost method to identify and classify strains of the Pseudomonas syringae pathovars. Little et al. (1998) isolated strains of P. syringae pv. syringae from healthy and diseased stone fruit tissues from 43 orchard sites in California, USA, in 1995 and 1996. These strains, together with P. syringae strains from other hosts and pathovars, were tested for pathogenicity and were genetically characterized by using enterobacterial repetitive intergenic consensus (ERIC) primers and PCR. All 89 strains of P. syringae pv. syringae tested were moderately to highly pathogenic on Lovell peach seedlings regardless of the host of origin, while strains of other pathovars exhibited low or no pathogenicity. Scortichini et al. (1999) evaluated apricot genotypes grated on various rootstocks for susceptibility to natural infection by

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Pseudomonas syringae pv. syringae in Piedmont (north-west Italy) during the period 1993-96.The presence of the pathogen was confirmed by biochemical and pathogenicity tests, as well as by comparing whole-cell protein profiles of isolates with type-strains of the pathogen. Abu-Ashraf et al. (2000) studied the differentiation of pathovars of Pseudomonas syringae and Xanthomonas campestris which was conducted by analysis with polymerase chain reaction (PCR) of topoisomerase genes. Differences among the pathovars on the migration patterns of the PCR products on agarose gel. Banding patterns of respective strains were pathovar specific with some exceptions. The technique is rapid, simple and reproductive to identify and classify phytopathogenic P. syringae and X. campestris at pathovar level, and it may be a useful diagnostic tool for these important plant pathogens. Guevara et al. (2000) observed the symptoms of dieback disease on branches of peach (Prunus persica) in Trujillo, Aragua and Miranda, Venezuela. Disease was appeared as cankers with gum exudates between healthy and diseased areas and red spots with yellow halos on leaves. The causal agent was identified using biochemical and physiological tests as Pseudomonas syringae pv. syringae. Mohammadi et al. (2001) isolated a total of 27 bacterial strains from cankerous tissues of apricot, nectarine, peach, plum, sour cherry and sweet cherry trees in Tehran province and identified as Pseudomonas syringae pv. syringae (Pss), the causal agent of the bacterial canker disease, based on the levan production, oxidase test, potato rot, arginine dihydrolase and tobacco hypersensitive reaction (LOPAT), and gelatin liquefaction, aesculin hydrolysis, tyrosinase activity and Na-tartrate utilization (GATTa's) group tests. Pss strains showed slight differences in morphology, phenotypic (biochemical Review of Literature

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and physiological) characteristics, serological properties, plasmid DNA, cellular protein profiles and antibiogram. They were divided into three distinct groups based on hippurate and formate utilization, which was correlated with protein profile in SDS-PAGE. The virulence of Pss was significantly associated with the degree of necrosis on immature sweet cherry fruits and the rate of in vitro syringomycin production. Kotan and Sahin (2002) observed in the spring and summer of 1999 and 2001, a serious disease with typical bacterial canker symptoms on nearly 80% of apricot trees grown in commercial orchards and home gardens in Erzurum, Erzincan and Artvin, Turkey. The causal organism was isolated and identified as Pseudomonas syringae pv. syringae, and its pathogenicity was confirmed. This was thought to be the first record of occurrence and outbreak of a bacterial canker disease on apricot trees in Turkey. Fiori et al. (2003) found in surveys carried out since the end of 1998 in Sardinia, Italy, allowed to ascertain severe die-backs in hazelnut (Corylus avellana) orchards. Longitudinal cankers along the twigs and the main branches. Sometimes, the death of the whole tree was also observed. The hazelnut cultivars recently introduced from the Italian peninsula (Piedmont) such as Tonda Gentile delle Langhe was attacked more than the local cultivars which showed only cankers. Isolations were performed in spring and autumn from symptomatic tissues. Fluorescent colonies on King's medium B revealed their pathogenicity to hazelnut, pepper, tomato and pear seedlings and, to a lesser extent, apricot, peach and lemon fruits. The isolates did not incite any disease in lilac and apple. Biochemical and physiological tests allocated the isolates to the Pseudomonas syringae group Ia. Slide agglutination and ELISA tests carried out using an antiserum toward P. syringae pv. syringae gave positive results. Strains were also compared with other Pseudomonas associated with hazelnut

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decline by repetitive PCR using BOX primers. The comparison revealed that the isolates obtained in Sardinia were different from P. avellanae, the causative agent of hazelnut decline in northern Greece and central Italy, and from the P. syringae pv. syringae strains previously isolated in Sardinia from local C. avellana cultivars. Scortichini et al. (2003) assessed that total of 101 Pseudomonas syringae pv. syringae strains, from international culture collections or isolated from diseased tissues of herbaceous and woody plant species, by repetitive PCR using the BOX primer, and for the presence of the syrB gene. Representative strains were also tested for pathogenicity to lilac, pear, peach, corn and bean, as well as for virulence to lemon and zucchini fruits. The unweighted pair-group method using arithmetic averages analysis (UPGMA) of genomic fingerprints revealed 17 different patterns which grouped into three major clusters, A, B and C. Most of the strains (52.4%) were included in patterns 1-4 of group A. These patterns comprised strains obtained from either herbaceous or woody species, and showed four fragments of similar mobility. Genetic variability was ascertained for strains isolated from apple, pear, apricot, citrus species. and cereals. No clear relationship was observed between host plant and bacterial genomic fingerprint. Variability was also observed in pathogenicity and virulence tests. The inoculation of pear leaves discriminated strains isolated from pear as well as the very aggressive strains, whereas inoculation of lilac, peach and corn did not discriminate the host plant from which the strains were originally isolated. Lemon fruit inoculation proved very effective for P. syringae pv. syringae virulence assessment. The syrB gene was present in almost all strains. Vasinauskiene and Baranauskaite (2003) reported blossom infection, shoot dieback and blight similar to fire blight on pear trees in Lithuania. Morphological, biochemical and serological analysis identified the causal organism as P. syringae pv. syringae.

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Berger (2004) found that plum decline was associated with Pseudomonas syringae pathovars syringae and morsprunorum. The trunks of affected plum trees (Prunus domestica) were girdled by bacterial cankers resulting in sudden death of infected trees. Invasions through blossoms, leaves and wounds during the vegetation period were limited to the infection sites and, plum trees coped effectively with both P. syringae pathovars eliminating them eventually. Vicente et al. (2004) isolated fifty-four Pseudomonas syringae isolates from wild cherry (Prunus avium) together with 22 representative isolates from sweet cherry and 13 isolates from other Prunus spp., pear and lilac were characterized by physiological, biochemical, serological and pathogenicity tests. Isolates from wild cherry were predominantly P. syringae pv. syringae (Pss), but P. syringae pv. morsprunorum (Psm) races 1 and 2 were also found. Physiological and biochemical tests discriminated (Psm) races 1 and 2 from other P. syringae isolates. Agglutination and indirectenzyme-linked immunosorbent assay tests with three different antisera showed that (Psm) race 1 and race 2 were very uniform and indicated high variability amongst other P. syringae isolates. However, pathogenic Pss isolates could not be distinguished from nonpathogenic isolates of P. syringae on the basis of physiological, biochemical or serological tests. Pathogenicity tests on rooted lilac plants and on micropropagated plantlets of lilac and two wild cherry clones differentiated Pss. and Psm. isolates and demonstrated a range of aggressiveness among Pss. isolates. Serological tests could be used as an alternative to the classical physiological and biochemical tests to increase the speed of detection and discrimination of isolates, but pathogenicity tests are still necessary to discriminate the pathogenic Pss. isolates.

Factors affecting growth of canker bacteria:

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Daniell and Chandler (1974) found that peach seedlings were grown for 7 months in containers with soil from old and new peach sites, inoculated with either of 2 isolates of Pseudomonas syringae and held at 3, 8 and 23°C and variable (outdoor, -17 to 14 deg ), neither the soil nor isolate differentially affected seedling growth, canker length or seedling mortality. Plants kept at variable temps., mean 3.3 deg and mean max. 8.5 deg , developed longer cankers than those at 8 deg Prunier et al. (1976) found that Infection by Pseudomonas morsprunorum f. sp. persicae led to the death of over 100, 000 peach trees in France during (1967-1969). The disease remained localized during several years with mild winters but spread rapidly with exceptional spring frosts in 1975. At the micro-climate level, affected areas are on high ground, or facing north, and the infection spreads most rapidly on parts of the tree near the soils, which are subject to greater temperature fluctuations. Klement (1977) reported that bacterial canker disease is widespread in Europe except for the Mediterranean areas. It usually develop at pruning wounds or other points of injury. Phloem and cambium become susceptible just after leaf drop until budding. If the phloem necrosis does not girdle the branch or trunk, cankers develop by the middle or end of summer. In early summer the bacterium dies out in infected tissue, and lives epiphytically on leaf surfaces, without causing any symptoms. The extent of bacterial necrosis of the phloem depends on the severity of winter frost. Necrosis is found only if P. syringae has time to proliferate before the onset of frost. The most effective method of control is to prune in spring rather than in winter. Wimalajeewa and Flett (1985) recorded that in a survey of the major nurseries during winter 1978 and 1979, the bacterial canker pathogen occurred on most of the stone fruit material in all nurseries especially on apricot. The epiphytic populations of Pseudomonas Review of Literature

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syringae pv. syringae on leaves, buds and shoots of apricot and cherry were assessed periodically (1979-1983) by determining the proportion of trees bearing the bacterium or by counting numbers of bacteria. Populations consistently reached peak levels during spring (highest) and late autumn. Populations were lowest during mid- to late summer. High proportions of tree contamination and high populations coincided with periods when max. temps. were (19 – 25)°C, and when rainfall was moderately high. Wimalajeewa (1987) observed that infection of apricot trees with Pseudomonas syringae pv. syringae (bacterial canker) through buds was highest with inoculations in late autumn and winter (MayJul.) and lowest with inoculations in summer (Dec.-Feb.). The number of stem inoculations, resulting in extensive cankers, was highest in late winter and spring (Aug.-Nov.) and lowest in summer and early autumn (Dec.-Mar.). Leaves and fruits were susceptible only during spring (Sep.-Nov.), when they were immature. The importance of these findings in relation to epidemiology and control of bacterial canker is discussed. Bordjiba and Prunier (1991) inoculated apricot trees with P. syringae pv. syringae, P. syringae pv. morsprunorum and P. viridiflava in the field by spraying with a bacterial suspension twice at the beginning of spring. The bacteria, all associated with bacterial canker of apricot in France, established epiphytic populations that persisted throughout the growing season (Apr.-Nov.). With the exception of some leaf spot damage in May, no disease symptoms developed. Under appropriate environmental conditions, epiphytic populations could provide an important source of inoculum for disease development in winter or in early spring of the following year. Sobtczewski and Jones (1992) inoculated dormant 1-year-old shoots of two sweet cherry (Prunus avium) cultivars with Pseudomonas syringae pv. syringae or with Pseudomonas syringae

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pv. morsprunorum and then incubated sequentally at 15, -10, and 15° C for 7, 1.5, and 10 days, respectively. Dark-stained necrotic tissue extended downward from the point of inoculation at the tip of the shoots. Inoculations with P. s. syringae resulted in significantly greater necrosis than inoculations with P. s. morsprunorum. The population and the distribution of bacteria in shoots just before exposure to freezing temperatures were greater for P. s. syringae than for P. s. morsprunorum. Spotts and Cervantes (1995a) studied several factors affecting the severity of bacterial canker of pear. In the orchard, in Oregon, USA, infection of shoots by Pseudomonas syringae pv. syringae occurred only when the inoculum dose exceeded 106 CFU/shoot. However, under favorable conditions in a growth chamber, cankers formed on detached shoots inoculated with 105 CFU/shoot. In orchard and growth chamber experiments, shoots were susceptible from time of bud swell until after fruit harvest. The severity of Pseudomonas canker of detached shoots increased if they were frozen at -10 degrees C for 24 h before inoculation. Shoots were most susceptible when inoculated immediately after wounding, and no cankers developed in the orchard when 3-day-old wounds were inoculated. Additionally, no cankers resulted from inoculation of leaf scars at leaf drop. Actively growing, current-season shoots were more susceptible than shoots that had set a terminal bud. Cao et al., (1999) reported that in excised dormant stems of peach (Prunus persica), prune (Prunus domestica), and almond (Prunus dulcis), stem diameter, stem hydration, and freezing-thawing influenced the extent of infection caused by Pseudomonas syringae pv. syringae. Bacterial lesion length increased with increasing stem diameter, demonstrating the need to account for the effects of stem diameter when lesion length data are analyzed. Lesion length increased or decreased with stem hydration or dehydration,

Review of Literature

16

respectively. However, tissue water content was not a good indicator of tissue susceptibility to infection by P. syringae pv. syringae, as larger diameter stems had larger lesions and lower water content than did smaller diameter stems.

Disease control : Heimann, (1973) mentioned symptoms caused by Pseudomonas syringae on the bark and leaves of bacterial canker are described in German. Climatic, cultural and nutritional factors favoring the disease, and control with copper oxychloride preparations. Gaignard et al. (1976) reported that in 3 trials peach trees at leaf fall were heavily artificially contaminated with Pseudomonas morsprunorum pv. persicae and sprayed with various copper compounds and antibiotics. Oxytetracycline gave a high degree of control with the minimum of infected leaf scars and numbers of bacteria/leaf in the following spring. Bordeaux mixture and copper oxychloride reduced infection by half but were somewhat phytotoxic. Wimalajeewa et al., (1991) conducted field trials during 198285, to develop a comprehensive spray programme for the control of P. s. pv. syringae on apricot and cherry trees. Five spray schedules were evaluated: 3 sprays in autumn + 1 winter + 2 spring; 2 autumn + 1 winter + 2 spring; 3 autumn; 2 winter; and 2 spring. Apricot was sprayed with copper hydroxide (2.5 g/litre in water) and cherry with Bordeaux mixture. Levels of canker on apricot were significantly reduced in all spray schedules, except the 3 autumn sprays or 2 winter sprays. None of the spray schedules differed significantly from each other. All treatments reduced canker on cherry and differences occurred between treatments. A reduction (94%) in epiphytic populations of the pathogen on apricot was observed following application of the Copper hydroxide. From the trials, a schedule consisting of 2 sprays in autumn (at 50-70% and 90-100% leaf fall), 1

17

Review of Literature

in winter and 2 in spring (pre-bloom) is recommended for control of P. s. pv. syringae on apricots and cherries. Jindal and Rana (1992) found that spraying apricot trees with streptocycline (200 micro g/ml) + Blitox [copper oxychloride] (0.2%) immediately after disease appearance and again 3 times at 10-d intervals gave 74.83% reduction in leaf infection and 80% in fruit infection due to Pseudomonas syringae pv. syringae. Spotts and Cervantes (1995b) collected a total of 323 strains of Pseudomonas syringae pv. syringae from six pear orchards in the Mid-Columbia region of Oregon and Washington from 1989 through 1992. Of all strains of P. s. pv. syringae, 8, 25, 75, and 99% did not grow on modified casitone-yeast extract-glycerol medium amended with 0.25, 0.5, 1.0, and 2.0 mM CuSO4, respectively. Over 70 and 90% of the strains in all six orchards were sensitive to 50 and 250 µg of oxytetracycline per ml, respectively. Strains of P. s. pv. syringae resistant to 50 and 500 µg of streptomycin per ml were found in six and four of six orchards, respectively. Twenty-five strains were resistant to both copper (1 mM) and streptomycin (100 µg /ml), and three of those were resistant to copper, streptomycin, and oxytetracycline (250 µg/ml). To their knowledge, resistance is correlated positively to the antibiotic spray programs in the orchards. Hickey and Zwet (1995) & Vanneste and Yu (1996) found that fire blight can be reduced by spraying with Pseudomonas fluorescens or Erwinia herbicola. The control of fire blight can be on pear and apple when the population of antagonistic bacteria were higher than the pathogen on stigmas flower. On apple, spraying of Pseudomonas fluorescens and Erwinia herbicola separately or together significantly reduced infection by Erwinia amylovora. There was no evidence of synergy between two bio-agents, but a mixture of them may have the potential to control fire blight more consistently over a wider range of

Review of Literature

18

climatic conditions. However, treatment with streptomycin controlled infection better than any bio-control agent Lindow et al. (1996) reported the efficacy of antagonistic bacteria Pseudomonas fluorescens strain A 506 and the antibiotics streptomycin and oxytetracycline, to which this antagonists is resistant individually and in combination in the control of fire blight in field trials conducted in orchards in which natural fire blight epidemics occurred. They found that the incidence of fire blight in trees treated with both strain A 506 and antibiotics was reduced to 27% of that in trees treated with antibiotics alone. Pabitra-Kalita et al. (1996) used four species of bacteria (Bacillus subtilis, B. polymyxa, Pseudomonas fluorescens and Serratia marcescens) and 3 species of fungi (Aspergillus terreus, Trichoderma viride and Trichoderma harzianum) isolated from the phylloplane of lemon cv. Assam lemon, inhibited in vitro growth of Xanthomonas campestris pv. citri [X. axonopodis pv. citri], the incitant of citrus canker. When the antagonists were tested for their efficacy in the control of citrus canker by applying them over crop foliage of Assam lemon, they also reduced citrus canker incidence under field conditions. B. subtilis was the most effective antagonist exhibiting max. (14.7 mm) inhibition of the pathogen and reducing the disease incidence by 61.9%. Sobiczewski (2001) mentioned that the protection of orchards and nurseries against plant diseases caused by bacteria constitute one of the most important and difficult to solve problems in fruit production. In Poland, there are almost exclusively copper compounds, which act only as protectants and can cause, especially on apples, phytotoxic effect. In some countries, copper antibiotics are used for control of plant diseases caused by bacteria. Recently such a product (Hortocyna) was registered in Poland against fire blight. New perspectives of fruit trees protection create inducers of resistance to

19

Review of Literature

plant diseases caused by bacteria (Bion, Regalis), breeding of resistant cultivars using both conventional methods and genetic engineering and introduction of products based on biological control agents. Schoofs et al. (2002) found that the use of Serratine-P, a phage tail-like bacteriocin, produced by Serratia plymiticum, shows an interesting antibacterial activity against E. amylovora. Its mode of action consists in the perforation of the cytoplasmic membrane of the target cell, inducing perturbations in cellular exchanges and a final lysis of the bacterial cell. Tawfik et al. (2002) investigated the bactericides Agrimycin 17 at rate 120 ppm, kasugamycine at rate 0.65% and starner (oxolinic acid 20%) at rate 0.15% spraying on blossoms when weather conditions are favorable for fire blight disease. They found that all treatments controlled the fire blight disease, but starner was more effective in comparison to untreated pear trees. As well as, screening of rest-breaking agents revealed that mineral oil at 3%, thiourea at 1.5%, urea at 3% and KNO3 at 1.5% combined with Borax at 5 ppm were effective for enhancing bud burst, early flowering when used at late January. Berger (2004) reported that plum decline was associated with Pseudomonas syringae pathovars syringae and morsprunorum in Baden-Württemberg. Copper compounds that were applied extensively during leaf fall and bud burst, were not effective. A minority of P. syringae strains isolated from cankers on plum trees were moderately resistant, while most strains were sensitive to cupric ions. Pajk (2004) reported that potential infection of host plants with bacteria Erwinia amylovora is reduced by the application of some biotic methods. Biological products include those based on microorganisms (Erwinia herbicola [Pantoea agglomerans], Bacillus

Review of Literature

20

subtilis, Bacillus subtilis var. niger, Pantoea ananatis pv. uredovora, Pseudomonas fluorescens, Pseudomonas syringae (strain A 506), Rahnella aquatilis, Serratia plymuthica) and those based on plant extracts. Edgecomb and Manker (2006) reported that B. subtilis QST 713 provides control of key bacterial pathogens such as Erwinia amylovora (fire blight of pome fruit), Xanthomonas campestris (bacterial spot of tomato and pepper) and Pseudomonas and Xanthomonas spp. (bacterial spots of ornamentals). B. subtilis QST 713 works through novel, multiple modes of action that involve the biological action of B. subtilis competing for nutrients on the host surface in addition to the antimicrobial activity of lipopeptide metabolites produced by B. subtilis causing permeability changes of the cytoplasmic membrane and subsequent disintegration of the pathogen cells. As determined by US-Environmental Protection Agency and International Regulatory Authorities, B. subtilis QST 713 is exempt from the requirement of a tolerance because there are no synthetic chemical residues, and it is safe to workers and the environment. As a result, treated fruits and vegetables can be exported throughout the world without restrictions. B. subtilis QST 713 is also safe to non target; beneficial organisms have been shown to be an effective tool for disease control in organic crop production and in integrated disease control programs contributing to resistance management and reduction in the use of synthetic fungicides.

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Review of Literature

MATERIALS & METHODS I- Sampling and isolation of the canker bacteria: Diseased samples of pear, apple, apricot and peach trees (i.e. flowers, leaves, branches, stems, buds and fruits) as clear in Figs. (1, 2 & 3) with typical bacterial canker and shot hole symptoms were collected from various localities of Daqahliya, Qalyubiya and Beheira governorates, in the spring and summer of 2004 seasons. Collected samples were transferred to the laboratory in plastic bags, kept in refrigerator at 7°C, where each sample was kept alone for further studies. Isolation procedure was carried out on all infected samples after brought from orchards. Diseased samples were washed in current water then in sterile distilled water (SDW), pieces of diseased tissues were macerated in 2 ml of sterile saline solution (0.85 NaCl) in Petridishs (7cm) and left for 30 min. A loopful of the resulting suspension was streaked on the surface of nutrient agar (NA) and King’s B media. These plates were incubated at 28 ± 2°C for 2-3 days. Observations were daily recorded and any emerged colony was picked up and transferred to nutrient glucose agar slant medium for maintenance till use in subsequent tests. All picked colonies were purified using the single colony technique (Fahy and Persley, 1983).

Materials and Methods

22

Fig.(1): Symptoms of bacterial canker on apricot trees: (A) depressed canker on branches (B) oozes and gumming on branches (C) shot hall on leaves (D) The whole diseased tree.

Fig. (2): Symptoms of bacterial canker on peach trees: (A) shot hall on leaf (B) infected peach tree (C) healthy peach tree (D) bacterial spots on peach fruit.

23

Materials and Methods

Fig.(3): Symptoms of bacterial canker on pear trees (A) Infected leaves (B) Infected shoot (C) canker on trunk.

The used media: 1-King's B (KB) medium (King et al., 1954) used for the nonselective isolation, cultivation and pigments production of Pseudomonas. (20g Difco proteose peptone, 1.5g K2HPO4, 1.5g MgSO4. 7H2O, 15 ml glycerol, 15g agar, 1000 ml distilled water). Medium was adjusted to pH 7.2. 2- Nutrient agar (NA) medium (Jacobs and Gerstein, 1960) used for the cultivation of a wide variety of bacteria (3g beef extract, 5g bactopeptone (Difco), 15g agar, 1000 ml distilled water). Medium was adjusted to pH 7.2. 3 Nutrient broth medium (Jacobs and Gerstein, 1960) used for the cultivation of a wide variety of bacteria for different testes (3g beef extract, 5g bactopeptone (Difco), 1000 ml distilled water). Medium was adjusted to pH 7.2.

Materials and Methods

24

4- Nutrient glucose agar medium (Ronald, 1946) used for the cultivation and maintenance of

Pseudomonas and Xanthomonas

species.(15g agar, 5g pancreatic digest of gelatin, 3g beef extract,10g glucose, 1000 ml distilled water). Medium was adjusted to pH 7.2. 5-Nutrient yeast extract glucose agar (NYGA) medium (Lelliott and Stead, 1987) used for the cultivation and maintenance of Erwinia spp.(3g beef extract, 5g bactopeptone (Difco), 5g glucose, 1g yeast extracts, 15g agar, 1000 ml distilled water). Medium was adjusted to pH 7.2. 6-Xanthomonas Agar medium (Lelliott and Stead, 1987) used for the cultivation and maintenance of Xanthomonas species (15g agar, 10g pancreatic digest of gelatin, 10g sucrose, 6g beef extract, 1000 ml distilled water). Medium was adjusted to pH 7.2. 7-Yeast extract peptone dextrose agar (YPDA) medium (Lelliott and Stead, 1987) used for

cultivation and propagation of tested

antagonists (5g yeast extract, 10g peptone, 5g glucose, 20g agar, 1000 ml distilled water). Medium was adjusted to pH 7.2. 8- Yeast extract dextrose calcium carbonate agar (YDC) medium (Lelliott, and Stead, 1987) used for cultivation and propagation of tested pathogenic bacteria (10g yeast extract, 20g finely precipitated CaCO3, 20g glucose, 15g agar, 1000 ml distilled water). glucose was autoclaved individually. Medium was adjusted to pH 7.2.

II- Pathogenic reaction on different hosts: Bacterial suspension was prepared by removing the bacterial growth from two days old cultures in sterile saline solution (0.85 NaCl) to give a concentration 107 CFU (colony forming units/ml).

25

Materials and Methods

Four different methods of inoculation were used according to Fahy and Persley, (1983) and Lelliott & Stead, (1987) as follows : (A) Spraying and injection of seedling: one year-old seedling of apricot, peach and pear were inoculated with bacterial suspension using two methods. The first method was achieved by spraying the bacterial suspension on seedlings using a hand atomizer. The second method was injection with bacterial suspension in the tip of growing shoots using a fine hypodermic syringe. These inoculated seedlings were incubated in moist chamber in the experimental farm, Faculty of Agriculture at Moshtohor for 2 days before and after inoculation at 28 ± 2°C. Development of symptoms on leaves and shoots were recorded for up to ten days. Re-isolation as previously mentioned was performed from plants showing disease symptoms. (B) Inoculation of detached fruits: small immature peach fruits were surface sterilized with sodium hypochlorite solution 0.5 % for 2 min., then rinsed with sterile distilled water (SDW). These fruits were inoculated with a drop of bacterial suspension using a sterile needle. The inoculated fruits were kept in moist chamber at 28 ± 2°C. The symptoms were recorded after 5 days from inoculation. (C) Hypersensitivity test (inoculation of tobacco seedlings): The seedlings of tobacco (Nicotiana tabaccum L.) were inoculated with bacterial suspension using a fine syringe into the intercellular spaces of the lower side of the leaves. Inoculated seedlings were incubated in a moist chamber for 48 h before and after inoculation. The symptoms were recorded after 5 days from inoculation.

Materials and Methods

26

(D) Inoculation of germinated beans: The bean seeds were surface sterilized with sodium hypochlorite 0.5% for 2 min, then washed by distilled water. These seeds were germinated in Petri-dish at moist chamber for 3-5 days. Germinated seeds were inoculated with direct pricking with sterilized needle charged with inoculum and planted in pots (15 cm ф filled with about 300 g peat moss). These pots were kept in moist chamber for 3 days after inoculation at 28 ± 2◦C. Development of symptoms were recorded after 5 days from planting. Symptoms on the tested hosts were recorded clearly as following: (1) on tobacco seedlings, appeared as water-soaking of inoculated tissue with 48 hrs then dryness, light-brown localized necrosis with 3 days. (2) on beans seedlings, appeared as yellow-brown discoloration on inoculated cotyledons. (3) on detached fruits of peach, appeared as black localized area with bacterial ooze in inoculated area. (4) on peach, apricot and pear seedlings, appeared as brown spots on inoculated leaves and small dark-green lesion at entry and exhibited points on inoculated branches.

III- Identification of isolated bacteria 1- Using the traditional techniques: Identification of the bacterial isolates was conducted on the bases of their morphological, cultural and physiological characteristics according to schemes suggested by Schaad, (1980); Fahy and Persley (1983); Krieg and Holt (1984) and Lelliott & Stead (1987). Identification tests were carried out on pathogenic bacteria, which verified their infection abilities as mentioned above as following:

27

Materials and Methods

Morphological characteristics Different morphological characteristics of the subjected bacterial isolates i.e. cell shape, Gram stain, and spore formation was carried out. Cultural characteristics Various cultural properties of the examined isolates, i.e. the colonies characteristics on different media, oxygen requirements and growth at different temperatures were also studied. Biochemical and physiological characteristics: The following physiological characters and biochemical activities were used as bases for bacterial classifications: -Reducing compounds from sucrose. -Degradation of macromolecules: -Gelatin hydrolysis test. -Starch hydrolysis test. -Other tests: - Catalase test. - Salt tolerance test. - Pigment production. - Relation to free O2. - Hydrogen sulfide production (H2S). - Levan formation. - Growth on Potato dextrose agar (PDA). - Growth on Yeast extract dextrose CaCO2 (YDC). - Nutrient-broth yeast extract agar (NBY). - King’s medium B agar (KB).

Materials and Methods

28

- Peptone yeast extract agar (PYEA). - Pectate degradation. - Indole production. - KOH 3%. - Oxidase reaction. - Hypersensitivity reaction 2-Verification the identification using PCR (Polymerase Chain Reaction) technique: The RAPD-PCR technique (Random amplified polymorphic DNA) was used as described by (Little et al, 1998) using 4 primers as listed in Table (1) to confirm the traditional identification of three isolates of those identified as Pseudomonas syringe. Table (1) The used primers and their sequences Primer Name OPERON A-06 OPERON A-11 OPERON D-12 OPERON J-08

Nucleotide sequence 5'-GGTCCCTGAC-3' 5'-CAATCGCCGT-3' 5'-CACCGTATCC-3' 5'-CATACCGTGG-3'

DNA preparation: Total genomic DNA was extracted from 10 ml of 24-h shake cultures of bacterial cells. After centrifugation at 10,000 × g for 10 min, the bacterial pellet was resuspended in 1.5 ml of buffer (100 mM Tris-HCl [pH 7.5], 100 mM EDTA [pH 8.0]). The pellet was rinsed twice with cold 70% ethanol, dried in vacuum, and dissolved in 0.5 ml of TE (Tris-HCl + EDTA) buffer. One microliter of ribonuclease at 10 mg/ml was added (final concentration 20 µg/ml)

29

Materials and Methods

and kept at 4oC overnight to completely digest the DNA. The DNA was re-precipitated, rinsed with cold 70% ethanol, dried, and dissolved in 40 µl of TE. The DNA was quantified by the minigel method. After quantification, the DNA was dissolved in 200 µl of TE and kept at –20oC for later use. DNA concentration by UV spectroscopy: A dilution of DNA by adding 20 µl of the refrigerated DNA solution to 0.98 ml of distilled water in a micro-centrifuge tube, was prepared and mixed well. The UV lamp tin the spectrophotometer was warmed up for 20 min. and wavelength of the spectrophotometer was set to 260 nm. Distilled water was added to one curette used the distilled water as a blank and set the absorbance to zero. The absorbance of the diluted DNA was measured. The concentration of DNA was calculated, according to Sambrook et al. (1989), assuming that DNA at a concentration of 50 µg/ml had an OD of 1 at 260 nm as follows: DNA concentration (µg/µl) =

OD 260 x dilution factor x 50 µ g/ml 100

After quantification, the DNA was dissolved in 200 µl of TE and kept at –20oC for later use. RAPD-PCR amplification A working DNA solution was made by diluting the stock DNA solution to about 0.1 ng/µl. Each amplification reaction was performed in a 13-µl volume consisting of 0.2 mM each of dATP, dCTP, dGTP, and TTP (Sigma Chemical Co., St. Louis, MO); 2 mM MgCl2; 0.3 units of Taq DNA Polymerase (Promega, Madison, WI); 4

Materials and Methods

30

µM primer, 0.2 ng of DNA template; 1.25 µl of 10x Taq polymerase buffer (Promega); and sterile water added to a final volume of 13 µl. Sterile distilled H2O was used in place of DNA template as a control to ensure that there was no contamination. The solution was overlaid with mineral oil. Amplification was carried out in a Perkin-Elmer model 480 thermal cycler programmed for 10 min at 94oC for initial denaturation and 30 cycles that consisted of 3 min at 94oC, 1 min at 50oC, and 1 min at 72oC, followed by a final 10 min extention at 72oC. The fastest ramp time was used for temperature transition. After amplification, 5µl of the solution for each sample was electrophoresed in a 1.2% agarose gel in 1X TBE buffer (0.089 M Tris-borate, 0.089 M boric acid, and 0.002 M EDTA). A 1-kb DNA ladder (0.15 µg) (Gibco BRL, Bethesda, MD) was used to estimate the size of each amplified DNA fragment. The gel was run for 1-2 hours at 100 volts, stained with ethidium bromide (1mg/ml) for 15 min, and photographed under ultraviolet light. The test of each primer was repeated at least twice to ensure the consistency of each RAPD band (Kearns et al., 1998)

V- Factors affecting the growth of tested pathogenic bacteria in vitro: 1- Effect of temperature The data of bacterial growth were recorded as follows:A- Determination of optical density (OD.): This test was carried out by inoculation conical flasks (250 ml) containing 50 ml nutrient broth (NB) with 0.5 ml (107 cfu/ml) from 24- hrs- old culture of

31

Materials and Methods

isolated Pseudomonas syringe bacteria (Pb-6, Al-8 and Pb-14). The inoculated flasks were set on a shaker (200 rpm) and incubated at different temperatures i.e. 15, 20, 25, 30,35and 40ْ C for 48 hrs. Three flasks were used as replicates. All readings data of OD were measured for the diluted cultures at 10-7 and the optical density of the turbid liquid cultures were measured calorimetrically at 525 nm using a Spectrophotometer (SPECTRONIC 20-D) (Abd El-Ghafar, 1988). Standard curve : Serial dilutions ranged between 10-1 to 10-7 of tested cankered bacteria grown on nutrient broth (two days old cultures) were done. The prepared nutrient plates were inoculated with 10-7 dilution by spreading 0.1 ml on the surface of the plate then the plates were incubated at 25 ± 2ْ C for 48 h. Three plates were made for the tested dilution as replicates. The total bacterial counts of each of the tested cankered bacterial colonies were recorded after two days from inoculation. On the other hand, the same tested dilution (10-7) of cultured bacteria was measured for its optical density calorimetrically at 525 nm using a Spectrophotometer (SPECTRONIC 20-D) and the determined bacterial count was compared with OD reading in each case of tested bacteria to know approximately the count of bacterial colonies for the tested pathogenic bacteria.

Materials and Methods

32

2.5

200 180 160 140 120 100 80 60 40 20 0

OD.

2 1.5 1 0.5 0 -10 -9 -8 -7 -6 -5 -4 -3 -2 -1

CFU

Standard curve

0

log of dilution OD.

CFU

Fig.(4) Standard curve show the relationship between OD at 525 nm and CFU count of different dilutions. B- Determination the number of bacterial colonies: Nutrient agar (NA) medium was used in this study. This medium was inoculated by a liquate 0.1 ml of dilution 10-7 of 24- hrs-old culture of pathogenic bacteria and distributed using L-shaped glass-rod. Inoculated plates were incubated at different temperatures which previously mentioned for 48 hrs. The total counts of bacterial colonies were recorded for each temperature degree and calculated the concentration (Sobtczewski and Jones, 1992 and Berger, 2004). 2- Effect of pH values: The data of bacterial growth were recorded as follows:A-Determination the optical density was done as previously mentioned. where, nutrient broth (NB) medium was adjusted to obtain different values of pH 6, 6.5, 7, 7.5 and 8 using pH meter by adding drops of N/10 HCl or N/10 NaOH before autoclaving. These flasks were inoculated with pathogenic bacteria as previously mentioned and

33

Materials and Methods

incubated at 28ْ C for 48 hrs .The results were recorded as mentioned before (Abd El-Ghafar, 1988). B- Determination the number of bacterial colonies was done as previously mentioned where, the pH values of nutrient agar media were adjusted before autoclaving. This medium was poured in Petridishes and inoculated with bacterial suspension as mentioned before. Concentrations of bacterial cells were estimated as previously mentioned Ju-Luric (1978). 3- Effect of relative humidity (RH): Saturated salt solution in different concentration was used to make certain relative humidity degrees in the air space in plates containing nutrient agar (NA) medium Shurtlett and Averre 1997). Saturated solutions were prepared to produce different degrees of RH i.e. 55, 60, 65,70,75,80,85,90,95 and 100% saturated solutions were poured in the lids of each plate containing NA medium and inoculated with 0.1 ml of bacterial suspension as previously mentioned .Bacterial growth was measured as mentioned before.

VI- Disease control: 1- Effect of some chemical formula and commercial antibiotics under lab conditions: a- On growth of tested pathogenic bacteria: Five chemical compounds namely Champion 77% (Copper oxychloride 77%), Copper oxychloride 50% (Copper oxychloride 50%), Kocide 2000 (Copper hydroxide 53.8%), Anti–shot (trail bactericide) and Starner 20% (oxalinic acid 20%) as clear in Table (2). In addition to seven antibiotics namely Erythromycin, Ofloxine,

Materials and Methods

34

Cefoperazone, Penicillin, Tetracycline, Cevoran and Chloramphenicol as clear in Table (3) were tested for their effect on growth of tested pathogenic bacteria and disease severity on immature peach fruits under artificial inoculation conditions. The tested concentrations of different chemical compounds were 200,400,600 and 800 mg/l and all antibiotics were applied at concentration of 50, 100, 150 and 200 ppm. The effect of chemical compounds and antibiotics were studied by two methods. A. Determination of inhibition zone: The plate method were applied for studying sensitivity of tested bacteria to chemical compounds and antibiotics, nutrient agar medium was used. The inhibitory effect of the chemicals and antibiotics under investigation was evaluated using the filter paper disc method (Thornberry, 1950). Filter paper discs (Whatman No.1, 7mm in diameter) were saturated with the different concentration of used chemicals and antibiotics. The discs were then placed on the surface of the medium previously inoculated with tested pathogenic bacterium, using appropriate amounts of 24 hrs old broth culture as inoculum. The plates were incubated at 28°C for 72 hrs. Four replicates for each concentration were used. The degree of inhibitory action was estimated by measuring the diameter of the zone of inhibited growth surrounding the disc. B. Determination of the effective concentration: a liquates (0.1 ml) of bacterial suspension (107 dilutions of 24 hrs-old cultures) were spread onto nutrient agar medium amended with different tested chemical compounds or amended with concentrations of antibiotics.

35

Materials and Methods

Inoculated plates (four plates for each treatment) were incubated at 28°C for 3 days. Bacterial colonies were counted to determine the count of resistant bacterial colonies. Untreated medium with chemical compound and antibiotics was used as control. Table (2):Common name, active ingredient and chemical formula of tested compounds as bactericides. Active

Trade name

ingredient

Champion

Copper

(WP)

hydroxide

Copper oxychloride (WP)

Copper oxychloride

kocide 2000

Copper

(WP)

hydroxide

Chemical name

Cu (OH)2 77%

3Cu (OH)2.CuCl2 50%

Cu (OH)2 53.8 % 5-ethyl-5,8-dihydro-8-

Starner (WP)

Oxalinic

oxo-1,3-dioxolo(4,5)

Acid

aminoline-7-arboxylic acid.

Anti-shot

Trial bactericide

(WP) WP: Wettable powder

Materials and Methods

36

Table (3): List of tested antibiotics, their active ingredients and chemical formula. Trade name &

Active ingredient

molecular weight

(%)

Erythromycin

Erythromycin

(M.W. 733.94)

(80%)

Tetracycline

Tetracycline

(M.W. 480.90)

(83.3%)

Penicillin

Penicillin

(M.W. 356.37)

(83.3%)

Ofloxacin

Ofloxine

(M.W. 361.368)

(83.3%)

Chloramphenicol

Chemical formula

Pharco

C22H24N2O8.XH2O

Edco

C16H17N2O4SNa

Cid

C18H20FN3O4

Sedico

Chloramphenicol (83.3%)

Ceforan

Cefuroxime

(M.W. 424.386)

(83.3%)

Cefoperazone

Cefoperazone

(M.W. 667.65)

(80%)

company

C37H67NO13

C11H12N2CL2O5 (M.W. 323.132)

Producing

Arabic Co. Pharmaceuticals

C16H16N4O8S

Aventis

C25H26N9NaO8S2

Pfizer

b- On disease percentage (immature peach fruits): Disease percentage on immature peach fruits (cv. Mit-Ghamer) was carried out under artificial inoculation conditions. These fruits were surface sterilized by dipping in sodium hypochlorite solution (0.5%) for 2 min., and then rinsed several times in sterile distilled

37

Materials and Methods

water (SDW). Sterilized fruits were distributed in sterilized plastic boxes and pricked using sterile needle. Each prick was inoculated by 0.05 ml of chemical compounds or antibiotics and at the same, the prick was inoculated by 0.05 ml of tested pathogenic bacterial suspension (107 cfu/ml) of 24 hrs old culture. Also, the second treatment, some sterilized fruits were treated with bactericides after 24-hrs from inoculation with suspension of pathogen. The third treatment, some sterilized fruits were inoculated with suspension of pathogen after 24-hrs from treatment with bactericides. The last treatment, some sterilized fruits were inoculated with suspension of pathogen as control treatment. Approximately 10 ml of sterilized distilled water (SDW) added in each box as humidity source (saturated filter paper " Whatman No.1" with 10 ml of SDW) and three treated fruits were placed for each plastic box. Four plastic boxes were used as replicates for each concentration/ treatment. After inoculation for 5 days at 28°C, disease reduction was estimated using the following equation: Dd C - Dd t Dd C

Disease reduction =

X 100

Where, Dd C = Disease diameter (%) in check Ddt = Disease diameter (%) in treatment 2- Effect of some antagonists: a- On growth of tested pathogenic bacteria Five

isolates

of

tested

antagonistic

bacteria

namely

Pseudomonas fluorescence (Pf-1), Pseudomonas fluorescence (Pf-5)

Materials and Methods

38

Pseudomonas putida (Pt-13), Bacillus subtilis (Bs-3) and isolate of Serratia marcensens (Sm-1) were obtained kindly from Plant Pathology Department, Faculty of Agriculture, Ain Shams University, Egypt. Pseudomonas spp isolates were cultured on King's B medium for 24 h meawhile, Bacillus subtilis and Serratia marcescens were cultured on nutrient agar medium only. These isolates were tested for their ability to inhibit the growth of tested pathogenic bacteria on nutrient agar. A loopful of each antagonistic bacteria (24 hrs-old culture) was placed at the center of the plates containing media previously inoculated superficially by spreading the tested pathogenic bacteria (24 hrs-old culture) on the poured nutrient media. The plates were incubated at 28°C for 72 hrs. Diameter of resulted inhibition zone was measured after incubation period (Raaijmakers et al., 2002). Three replicates were used for each bio-agent isolate. b- On infection with cankered bacteria on immature peach fruits All used antagonists were tested again to study their effect on cankered bacteria infected immature peach fruits under artificial inoculation conditions. Disease reduction was estimated as previously mentioned. 2- Effect of some chemical formula and commercial antibiotics on bacterial canker disease under artificial conditions in the greenhouse: Starner 20% , Champion 77% and Copper oxychloride 50% as chemical compounds and Chloramphenicol and Cefoperazone as antibiotics were applied as spray treatment on the foliage of apricot seedlings (one-year-old).The chemical compounds were used at dose

39

Materials and Methods

800 mg/l and antibiotics were used at dose 200 ppm. Seedlings of apricot (cv. canino) were treated with bactericides before the treatment with the pathogen (spray on the foliage or injection in the tip of branches) by 24 h. Treated seedlings were maintained in humid chamber for 24 h before and after treatment with the pathogen. Disease severity was recorded after 10 days from pathogen inoculation. 3-Effect of tested antagonists on bacterial canker disease under artificial conditions in the greenhouse: Isolates of Bacillus subtilis (Bs-3), Pseudomonas fluorescens (Pf-5) and Serratia marcensens (Sm-1) were grown on yeast extract peptone dextrose agar (YPDA) medium for 24 h at 28°C and suspended in saline solution (0.85% NaCl). Bacterial suspensions were adjusted at the concentration of 107 cfu/ml as determined from a standard curve based on absorbance at 525 nm. Bacterial suspension of bioagents was applied as spray treatment on the foliage apricot seedlings. Bioagents were sprayed before the treatment with the pathogen (spray on the foliage or injection in the tip of branches) by 24h. Treated seedlings were maintained in humid chamber for 24-h before and after treatment (Claflin, 2003). Percentage of disease reduction (PDR) was calculated also for leaves and shoots treated with antagonists, in presence of tested pathogenic bacteria. After 10 days from pathogen inoculation as previously mentioned.

Disease assessment: The number of bacterial spots was estimated as mean number of spots per leaf, where five leaves were randomly selected from each

Materials and Methods

40

seedling. Also, the number of infected leaves/branch was estimated where four branches were chosen for each treatment on the plant as replicates. Then the infection percentage of infected leaves/plant were determined. On the other hand, The length of infected (cankered) and un-infected part (cm) on the same shoot inoculated by injection with tested pathogenic bacteria was measured in order to estimate the percentage of infected part to the whole shoot where four branches were selected as replicates (Endert and Ritchie, 1984). Percentage of disease reduction (PDR) was calculated also for leaves and shoots treated with antagonists, chemical formula and antibiotics in presence of tested pathogenic bacteria as mentioned above.

Statistical analysis: Data were statistically analyzed using the (F) test and the value of LSD (at 5 %) according to (Gomez and Gomez, 1984).

41

Materials and Methods

EXPERIMENTAL RESULTS I. Isolation of bacterial canker: The bacterial canker disease occurs on branches, twigs, buds, leaves, and fruits. The most conspicuous symptoms are the cankers that exude gum during late spring and summer as clear in Figs. (1, 2 & 3)on apricot, peach and pear trees. Gumming is common on stone fruit trees, whether on trunks, limbs, twigs or fruits when injuries occur. Thus, the name gummosis does not define a cause, only a response. Cankers on the twigs are darkened areas often at the base of buds. On limbs or trunks, they are often darker than the normal bark, sunken in their centers, and they may extend for a considerable distance. Moreover, the grown leaves and shoots may be wilted, cankered and died during the growing season. In contrast, leaves and flowers from the other infected buds may remain symptomless. Leaf infections appear as water-soaked spots to become brown and dry. Leaf samples used for isolation was collected only from ElNobaria, Experimental Farm, Faculty of Agriculture at Moshtohor and El-Amar localities. While, the fruit samples were collected only from Mit-Ghamer localities. Also, the bud samples were collected from MitGhamer, Kafer-El-Gamaal, Toukh and El-Nobaria localities. As well as, stem samples were collected from Mit-Ghamer, Toukh and Kafer-ElAmar localities while, the flower samples were collected from ElNobaria and Toukh localities. All tested samples revealed bacterial infections. Data in Table (4) show that fifteen bacterial isolates were isolated from different parts of peach, apricot, pear and apple which were collected from different localities of Egypt. In this respect, the bacterial isolates coded as Pb-1,Ps-2 and Pf-4 were isolated from buds, stems and fruits respectively of peach in Daqahlyia (Mit-Ghamr). Meanwhile, the isolates coded as Rs-3 and Lb-11 were isolated from stem and bud of pear and apple respectively in the same

Experimental Results

42

governorate (Mit-Ghamr). On the other hand, the bacterial isolates coded as Pb-5, Pb-6, Ps-13 and Pl-15 were isolated from peach in Qualubia governorate while, Al-7 and As-12 were isolated from apricot in the same governorate. Meanwhile, the isolate coded as was isolated from pear flower in Qualubia governorate (Moshtohor), while, the isolates coded as Pw-9 and Pb-14 were isolated from flower and buds of peach respectively in Beheira governorate. The isolate Al8 was isolated from leaf of apricot in the same governorate. Table (4): Source of bacterial isolates collected from different hosts and localities at different governorates during 2004 growing season.

Host

Peach

Apricot

Pear Apple

Source of Sample bud stem fruit bud bud stem leaf flower bud leaf stem leaf stem flower bud

Mit-Ghamr Moshtohor

Code of isolate Pb-1 Ps-2 Pf-4 Pb-5 Pb-6 Ps-13 Pl-15 Pw-9 Pb-14 Al-7 As-12 Al-8 Rs-3 Rw-10

Mit-Ghamr

Lb-11

Governorate

Locality

Daqahliya Daqahliya Daqahliya Qalyubiya Qalyubiya Qalyubiya Qalyubiya Behera Behera Qalyubiya Qalyubiya Behera Daqahliya Qalyubiya

Mit-Ghamr

Daqahliya

43

Mit-Ghamr Mit- Ghamr Kafr-El-Gamal Toukh Toukh Moshtohor El-Nobaria El-Nobaria El-Amar Kafr-El-Amar El-Nobaria

Experimental Results

II- Pathogenic reaction on different hosts: In this experiment, fifteen bacterial isolates were examined for their reaction on different hosts (tobacco, beans, apricot, peach and pear). In this respect, data in Table (5) and Fig. (5) reveal that Al-8, Pb-6 and Pb-14 isolates were pathogenic on tobacco, beans, apricot, peach and pear, Rs-3, Al-7 and Lb-11 isolates were pathogenic only on tobacco plants, but As-12 isolate was less pathogenic on tobacco plants. Meantime, Rs-3 and Lb-11 isolates were pathogenic on pear plants and Pb-1, Ps-2, Pf-4, Pb-5, Pw-9, Rw-10, Ps-13 and Pl-15 isolates were not pathogenic on all tested hosts. Non pathogenic isolates were eliminated and the pathogenic ones (Pb-6, Al-8 and Pb14) were used in further studies. Symptoms on the tested hosts were recorded clearly as following: (1) on tobacco seedlings, appeared as water-soaking of inoculated tissue with 48 hrs then dryness, light-brown localized necrosis with 3 days. (2) on beans seedlings, appeared as yellowbrown discoloration on inoculated cotyledons. (3) on detached fruits of peach, appeared as black localized area with bacterial ooze in inoculated area. (4) on peach, apricot and pear seedlings, appeared as brown spots on inoculated leaves and small dark-green lesion at entry and exhibited points on inoculated branches.

Experimental Results

44

Table (5): Pathogenicity test and virulence of isolates on some differential hosts. Code of isolate Pb-1

Tobacco -

Beans -

Reaction Apricot* -

Peach* -

Pear* -

-

-

-

-

Rs-3

++

-

-

+

Pf-4

-

-

-

-

Pb-5

++

++

-

Pb-6

++

++

++

Al-7

++

-

Al-8

++

++

++

++

++

Pw-9

-

-

-

-

-

Rw-10

-

-

-

Lb-11

++

-

-

-

+

As-12

+

-

-

-

-

Ps-13

-

-

Pb-14

++

++

++

++

++

Pl-15

-

-

-

-

-

Ps-2

-

* Two methods of inoculation (spray and injection ) - = Nonpathogenic + = virulence ++ = highly virulence

45

Experimental Results

Fig. (5):Reaction of tested isolates: (A) On tobacco plants (B) On bean seedlings, (C) On peach fruits and (D) On apricot seedlings as injection of branch.

III. Identification of isolated bacteria 1.Using traditional techniques 1.a-Morphological and cultural characters: All tested isolates were short rods and non spore formers except As-12 isolate was formed spores. All isolates were Gram negative while, the isolate As-12 was Gram positive. On common medium like yeast extract dextrose calcium carbonate agar (YDC) medium, all isolates grow with different colour, where, they are white in three isolates (Rs-3, Lb-11 and As-12), translucence in three isolates (Pb-6, Al-8 and Pb-14) and yellow in one isolate (Al-7). Meanwhile, on king's B medium (K.B), three isolates produced florescent pigments i.e. Pb-6, Al-8 and Pb-14), but the rest of isolates didn't (Table 6).

Experimental Results

46

After these traditional tests, these isolates may be belongs to four genera Erwinia, Bacillus, Xanthomonas and Pseudomonas according to their inspected morphological and cultural characteristics. Table (6): Identification of the isolated cankered bacteria which reacted positively on different hosts using the morphological and cultural characters. Test Growth and colour on common media Gram reaction Pigments (K.B) Spore production Bacterial genera * *

Bacterial isolates No. LbAl-7 Al-8 11

As12

Pb-14

+ white

+ white

+ translucence

-

-

+

-

-

+ fluorescent

-

-

+ fluorescent

-

-

-

-

+

-

[P]

[X]

[P]

[E]

[B]

[P]

Rs-3

Pb-6

+ white

+ translucence

+ yellow

+ translucence

-

-

-

-

+ fluorescent

[E]

** E = Erwinia spp.; X = Xanthomonas spp ;

P = Pseudomonas spp.; B = Bacillus spp.

1.b-Biochemical and physiological characteristics: Data in Table (7) show that the two isolates i.e. Rs-3 and Lb-11 gave positive reaction with gelatin liquefaction, KOH (3%), Levan test, tobacco hypersensitivity and reducing substance from sucrose and growth on NaCl (5%). Meantime, they gave negative reaction with starch hydrolysis, pectate degradation, fats hydrolysis, methyl red test (M.R.), production of H2S, production of indole, urease production, nitrate reduction, production of pigments, growth at 50°C, oxidase reaction, potato rot, arginine dihydrolase and fermentative metabolism in o/f test. On the other hand, these isolates gave different reaction in utilizing of different carbon sources. Meanwhile, the three isolates i.e. Pb-6, Al-8 and Pb-14 gave positive reaction with pectate degradation, production of

47

Experimental Results

pigments, KOH 3%, Levan test, tobacco hypersensitivity and reducing substance from sucrose and growth on NaCl (5%). Meantime, they gave negative reaction with starch hydrolysis, gelatin liquefaction, fats hydrolysis, methyl red test (M.R.), H2S production , production of indole, urease production, nitrate reduction, growth at 50°C, oxidase reaction, potato rot, arginine dihydrolase and oxidative metabolism in o/f test. These isolates were able to utilize L (+) lactose, dextrose, sorbitol, mannose and sucsenic as carbon source. Also, the isolate Al-7 gave positive reaction with the tests, starch hydrolysis, H2S production, production of pigments, KOH (3%), tobacco hypersensitivity and reducing substance from sucrose and growth on 5% NaCl. Meantime, these isolates gave negative reaction with gelatin liquefaction, pectate degradation, fats hydrolysis, methyl red test (M.R.), production of indole, urease production, nitrate reduction, growth at 50°C, Levan test, oxidase reaction, potato rot, arginine dihydrolase and oxidative metabolism in o/f test. This isolate gave different reaction in utilizing of carbon sources. The last one isolate As-12 appeared positive reaction with starch hydrolysis, gelatin liquefaction, pectate degradation, fats hydrolysis, nitrate reduction, reducing substance from sucrose and growth on NaCl (5%), growth at 50°C, potato rot, tobacco hypersensitivity. Also, it gave negative reaction with the tests of methyl red test (M.R.), H2S production, production of indole, urease production, production of pigments, KOH (3%), Levan test, oxidase reaction, arginine dihydrolase and fermentative metabolism in o/f test. it gave different reaction in utilizing of carbon sources. Finally, the aforementioned testes and their result revealed that the two isolates i.e. Rs-3 and Lb-11could be identified as Erwinia amylovora, while, the isolates i.e. Pb-6,Al-8 and Pb-14 could be identified as Pseudomonas syringae. Meanwhile, the isolate Al-7 could be identified as Xanthomonas campestris but the isolate As-12 is Bacillus polymyxa.

Experimental Results

48

Table (7): Identification of isolated bacteria from infected hosts, to determine species using physiological and biochemical tests. Test Starch hydrolysis

Reaction Rs-3 Pb-6 -

Al-7 +

Al-8 -

Lb-11 -

As-12 +

Pb-14 -

Gelatin liquefaction

+

-

-

-

+

+

-

Pectate degradation

-

+

-

+

-

+

+

Fats hydrolysis

-

-

-

-

-

+

-

Methyl red test (M.R.)

-

-

-

-

-

-

-

Production of H2S

-

-

+

-

-

-

-

Production of indole

-

-

-

-

-

-

-

Urease production

-

-

-

-

-

-

-

Nitrate reduction

-

-

-

-

-

+

-

Growth In 5% NaCl

+

+

+

+

+

+

+

Production of pigments

-

+

+

+

-

-

+

KOH 3 %

+

+

+

+

+

-

+

Growth at 50°°C

-

-

-

-

-

+

-

Levan test

+

+

-

+

+

-

+

Oxidase reaction

-

-

-

-

-

-

-

Potato rot

-

-

-

-

-

+

-

Arginine dihydrolase

-

-

-

-

-

-

-

Tobacco hypersensitivity o/f test Utilization from : Lactose

+

+

+

+

+

+

f

o

o

o

f

+ f

-

+

d

+

-

-

+

Dextrose

-

+

d

+

-

+

+

Sorbitol

d

+

-

+

d

d

+

Mannose

-

+

+

+

-

-

+

Sucsenic Bacterial isolate identified

+

+

+

+

+

-

+

Ea.

Ps.

Xc.

Ps.

Ea

Bp.

Ps.

o

**Ea = E. amylovora , Ps = Pseudomonas syringae Xc = Xanthomonas campestris , Bp = Bacillus polymyxa *F = fermentative metabolism ; O =oxidative metabolism d = different reaction

49

Experimental Results

2. Verification the identification using PCR (Polymerase Chain Reaction) techniques: From the above results, the three isolates i.e. Pb-6, Al-8 and Pb14 were identified as P. syringae and used only in this test. The traditional identification of these isolates was confirmed using PCRRAPD amplification of DNA with four random primers as clear in Fig.(6). In this respect, the four tested primers revealed the similarity and diversity between the three tested isolates with superiority of the primer OP-A-11 (5'-CAATCGCCGT-3') in revealing high similarity between the three tested P. syringe isolates where, the fractionated DNA bands with this primer were clear typical in size and appearance of the three isolates. These fractionated DNA bands ranged between more than 1000 to 200 bp and all the resulted DNA bands were 10 bands of the three isolates. The developed bands of the three isolates showed very close similarity with the primer OP-A-11 and confirmed that this primer could be useful in identification the P. syringe isolates. This result also confirmed the previously mentioned traditional identification which revealed that the three tested isolates are identified as P. syringe. On the other hand, the other tested primers i.e. OP-D-12, OPA-06 and OP-J-08 revealed also high similarity but with clear diversity between the three tested isolates and this result might be mean that these three isolates are P. syringe but different isolates.

Experimental Results

50

Fig.(6): RAPD-PCR amplification of three P. syringae isolates using 4 primer (OPERON - A-06, A-11, D-12 and J-08)

V. Factors affecting the growth of P. syringae in vitro: 1. Effect of temperature: Data in Table (8) and Fig (7) indicate clearly that the temperature degree affects greatly on growth of P. syringae isolates on solid media. In this respect, the best temperature degrees were 25 and 30oC respectively for the three tested P. syringae isolates where, the highest CFU were recorded at those degrees. Meanwhile, the growth on 20 oC was in the second rank for the isolates Pb-6 and Al-8 where the growth of them followed those on 25oC. It is clear also from the obtained results that lowest growth of tested P. syringae isolates was at 15 and 35oC. Meanwhile, the degree 40oC was not favorable for growing P. syringae isolates where no growth was recorded for the three tested isolates.

51

Experimental Results

Table (8): Effect of different temperature degrees on growth of Pseudomonas syringae, in vitro. Temperature (ºC) 15 20 25 30 35 40 Mean



CFU* solid media

Optical density (O.D.) Liquid media

Isolates Pb-6 Al-8 Pb-14 Mean Pb-6 10 8 4 7.3 0.25 32 30 18 26.6 0.59 52 40 41 44.3 1.31 34 32 29 31.6 0.89 25 27 23 25.0 0.72 0.0 0.0 0.0 0.0 0.00 25.5 22.8 19.1 22.4 0.62

Al-8 Pb-14 Mean 0.20 0.15 0.20 0.47 0.35 0.40 1.22 1.19 1.24 0.78 0.77 0.81 0.74 0.68 0.71 0.00 0.00 0.00 0.56 0.52 0.56

Colony forming unit/plate

O .D.

LSD at 5 % for

Isolates Temperature Interaction

0.015 0.021 0.036

1.4 1.2 1 0.8 0.6 0.4 0.2 0 15

20

25

30

35

40

Temperature Pb-6

Al-8

Pb-14

Fig. (7): Relationship between different temperature degrees and growth of Pseudomonas syringae isolates, in vitro. On the other hand, data of optical density determination of inoculated K.B medium with P. syringae isolates indicate also that the best growth of tested P. syringae was at 25oC for the three isolates followed by 30oC for Pb-6 and Al-8 isolates and 20oC for Pb-14 isolate. Whereas, the lowest growth was recorded on 15oC for the three tested

Experimental Results

52

isolates. Also, data of 40oC confirmed that this degree was not suitable for growing of P. syringae where the growth of them were zero. 2. Effect of pH values: Data in Table (9) and Fig (8) indicate that in solid media the best growth of tested P. syringae was recorded at pH 6.5 and pH 7 where pH 6.5 was the optimum for isolates Al-8 and Pb-14. While, pH 7 was the best for Pb-6 isolate. On the other hand, the pH values i.e. 6,7.5 and 8 were not favorable for growing the tested pathogenic P. syringae. The obtained data of optical density determination indicate that the optimum pH value for growing all three tested P. syringae is pH 6.5 followed by pH 7. Meanwhile, the rest tested pH values i.e. 6,7.5 and 8 were not suitable for growing the tested pathogenic P. syringae.

Table (9): Effect of different pH values on growth of pseudomonas syringae isolates, in vitro.

pH values 6 6.5 7 7.5 8 Mean

CFU* solid media

Optical density (O.D.) Liquid media Isolates Pb-6 Al-8 Pb-14 Mean Pb-6 Al-8 Pb-14 Mean 1 2 3 2.0 0.11 0.10 0.11 0.10 16 21 18 18.3 1.03 0.99 1.01 1.01 13 23 15 17.0 0.52 0.45 0.50 0.49 2 2 3 2.3 0.12 0.10 0.11 0.11 1 1 2 1.3 0.05 0.05 0.07 0.05 6.6 9.8 8.2 8.2 0.36 0.66 0.38 0.46

* Colony forming unit/ plate LSD at 5 % for

Isolates pH Interaction

0.012 0.015 0.026

53

Experimental Results

1.2 1 O.D.

0.8 0.6 0.4 0.2 0 6

6.5

7

7.5

8

pH PB6

AL8

PB14

Fig. (8): Relationship between different pH values and growth of Pseudomonas syringae isolates, in vitro using OD. 3. Effect of relative humidity (RH): Data in Table (10) and Fig. (9) indicate that the best growth of tested P. syringae was increased with increasing relative humidity (RH) levels: In this respect, the best relative humidity degrees were 100% and 95% respectively for the three tested P. syringae isolates. The highest CFU were recorded at those degrees. The CFU at RH 100% was ranged between 83 – 92 cfu/plate. Growth of P. syringae was less developed on 50% relative humidity, where colony forming unit (cfu) ranged from 21–30 cfu/ plate.

Experimental Results

54

Table (10): Effect of relative humidity (R.H) on growth of Pseudomonas syringae isolates, in vitro. CFU* Isolates Al-8 Pb-14 29 30 30 35 34 40 54 61 55 63 86 81 92 86 54.2 56.5

RH % Pb-6 21 29 31 58 59 80 83 51.7

50 65 73 80 84 95 100 Mean

Mean 26.6 31.3 35.0 57.6 59.0 82.3 87.0 54.1

*Colony forming unit/ plate LSD at 5 % for

Isolates RH Interaction

0.171 0.261 0.453

No. bacteriacells/plate

10 9 8 7 6 5 4 3 2 1 0 50

65

73

80

84

95

100

RH % Pb-6

Al-8

Pb-14

Fig.(9):Relationship between relative humidity (R.H) and growth of Pseudomonas syringae isolates, in vitro.

55

Experimental Results

VI- Disease control: 1. Effect of some chemical compounds on growth of Pseudomonas syringae. Effect of different rates of five chemical compounds were tested on growth of Pseudomonas syringae isolates. Results in Tables (11&12) and Figs. (10&11) show that all tested chemical compounds were effective to reduce the growth of P. syringae compared with the control. Growth reduction was increased with increasing the rates of chemical compounds. Anti–shot was the most effective compound in reducing the growth of P. syringae, followed by Copper oxychloride and Champion where bacterial cells count were 7.8 x 107, 9.5 x 107 and 10.3 x 107 ml respectively. On the other hand, Kocide 2000 was the lesser effective one in reducing the growth of the tested pathogenic bacteria where bacterial cells count was 18 x 107 ml. In the same direction, Anti–shot was the most effective compound to inhibit the growth of P. syringae followed by Copper oxychloride and Champion where their inhibition zone were ranged between 32.5-34.0, 21.2-24.0 and 17.7-18.7 mm, respectively (Table, 12). While, Kocide 2000 was the lesser effective one in inhibiting the growth of tested pathogenic bacteria where, the inhibition zone was ranged between 9.2-11.0 mm.

Experimental Results

56

Table (11): Effect of different chemical compounds, at different rates on growth (count of bacterial cells) of Pseudomonas syringae isolates , in vitro. Bacterial cells count Chemical Rate Isolates compound (mg/l) Pb-6 Al-8 Pb-14 Mean 200 18 17 16 17.0 400 16 15 14 15.0 600 14 13 13 13.3 Starner 800 9 10 9 9.3 Mean 14.3 13.8 13.0 13.7 200 11 12 12 11.6 400 11 11 11 11.0 600 10 10 10 10.0 Champion 800 9 8 8 8.3 Mean 10.3 10.3 10.3 10.3 200 20 18 19 19.0 400 18 17 18 17.6 Kocide 2000 600 18 16 16 16.3 800 16 15 14 15.0 Mean 18.0 16.5 16.8 14.4 200 11 11 11 11.0 400 10 10 10 10.0 Copper 600 9 9 9 9.0 oxychloride 800 8 8 8 8.0 9.5 Mean 9.5 9.5 9.5 200 10 12 11 11.0 400 9 8 9 8.6 Anti - shot 600 7 6 6 6.3 800 5 4 5 4.6 Mean 7.8 7.5 7.8 7.7 0.0 30 28 25 27.6 Control LSD at 5 % for

Chemical Rate Isolates Interaction

0.037 0.037 0.030 N.S.

57

Experimental Results

Starner

1.4 1.2 1 0.8 0.6 0.4 0.2 0

Bcterial cells count

Bacterial cells count

Champion

0

200

400

600

2 1.5 1 0.5 0 0

800

200

Rate PB6

AL8

PB14

PB6

1.2 1 0.8 0.6 0.4 0.2 0 200

400

AL8

800

PB14

600

2.5 2 1.5 1 0.5 0 0

800

200

400

600

800

Rate

Rate PB6

600

Kocide 2000 Bacterial cells count

Bacterial cells count

Copper oxychloride

0

400 Rate

AL8

PB6

PB14

AL8

PB14

Bacterial cells count

Anti - shot 1.5 1 0.5 0 0

200

400

600

800

Rate PB6

AL8

PB14

Fig. (10): Relationship between growth of Pseudomonas syringae isolates as count of bacterial cells and different rates of chemical compounds, in vitro.

Experimental Results

58

Table (12): Effect of different chemical compounds at different rates on growth (inhibition zone (mm)) of Pseudomonas syringae isolates, in vitro. Inhibition zone (mm) Chemical Rate Isolates compound (mg/l) Pb-6 Al-8 Pb-14 Mean 200 8 12 16 12.0 400 12 14 18 14.6 600 12 15 20 15.6 Starner 800 18 16 21 18.3 Mean 12.5 14.2 18.7 15.1 200 12 10 6 9.3 400 18 18 22 19.3 600 19 19 22 20.0 Champion 800 22 24 25 23.6 Mean 17.7 17.8 18.7 18.0 200 5 11 4 6.6 400 8 14 11 11 Kocide 2000 600 10 19 13 14 800 14 22 16 17.3 Mean 9.2 16.5 11.0 12.2 200 18 15 17 16.6 400 23 21 20 21.3 Copper 600 26 23 28 25.6 oxychloride 800 28 26 31 28.3 Mean 23.7 21.2 24.0 22.9 200 25 24 30 26.3 400 32 32 31 31.6 Anti - shot 600 35 34 35 34.6 800 40 40 40 40 Mean 33.0 32.5 34.0 33.1 0.0 0.0 0.0 0.0 0.0 Control LSD at 5 % for

Chemical Rate Isolates Interaction

0.043 0.043 0.033 0.165

59

Experimental Results

Cham pion

Starner

Inhibition zone (mm)

Inhibition zone (mm)

3 2.5 2 1.5 1 0.5

2.5 2 1.5 1 0.5 0

0

0

0

200

400

600

200

800

400

600

800

Rate

Rate Pb-6

Pb-6

Al-8

Copper oxychloride

Pb-14

Kocide 2000

4

2.5 Inhibition zone (mm)

Inhibition zone (mm)

Al-8

Pb-14

3 2 1 0 0

200

400

600

2 1.5 1 0.5 0

800

0

200

Rate Pb-6

400

600

800

Rate

Al-8

Pb-14

Pb-6

Al-8

Pb-14

Inhibition zone (mm)

Anti - shot 6 4 2 0 0

200

400

600

800

Rate Pb-6

Al-8

Pb-14

Fig. (11): Relationship between growth of Pseudomonas syringae isolates as inhibition zone (mm) and different rates of chemical compounds, in vitro.

Experimental Results

60

2. Effect of some antibiotics on growth of Pseudomonas syringae. Effect of different rates of seven antibiotics were tested on growth of Pseudomonas syringae isolates. Results in Tables (13&14) and Figs. (12&13) show that all tested antibiotics were effective in reducing the growth of P. syringae compared with the control. This reduction was increased with increasing the rates of antibiotics. Ofloxine and Cefoperazone were the most effective in reducing the growth of P. syringae, where bacterial cells count were ranged between 11.5 – 12.0 x 107 and 12.0-13.3 x 107 ml, respectively. Meantime, Cevoran and Chloramphenicol were moderately effective, where bacterial cells count were ranged between 16.0–17.0 x 107 and 15.5-17.8 x 107 ml. respectively. On the other hand, the recorded results with Penicillin exhibited that it was the lesser effective one in reducing the growth of tested pathogenic bacteria, where bacterial cells count was 20.0-21.8 x 107 ml. In the same direction, Ofloxine and Cefoperazone were the most effective to inhibit the growth of P. syringae, where the inhibition zone, ranged between 18.5-20.3and 12. 0-12.8 mm, respectively

(Table, 14). Meantime, Chloramphenicol and Cevoran were moderately effective, where bacterial cells count were ranged between 2.5-3.0 and 1.6-1.7, respectively. Meanwhile, the inhibition zones were ranged between 15.8-16.8 and 15.316.0 mm, respectively. In contrary, Penicillin was the lesser effective one in reducing the growth of tested pathogenic bacteria where inhibition zone was 3.8 mm.

61

Experimental Results

Table (13): Effect of different antibiotics, at different rates on growth (count of bacterial cells) of Pseudomonas syringae isolates , in vitro. Bacterial cells count Rate Antibiotic Isolates (ppm) Pb-6 Al-8 Pb-14 Mean Erythromycin

Ofloxine

Cefoperazone

Penicillin

Tetracycline

Cevoran

Chloramphenicol Control LSD at 5 % for

Experimental Results

50 100 150 200 Mean 50 100 150 200 Mean 50 100 150 200 Mean 50 100 150 200 Mean 50 100 150 200 Mean 50 100 150 200 Mean 50 100 150 200 Mean 0.0

23 21 20 18 20.5 18 12 11 7 12.0 19 16 11 7 13.3 25 24 21 17 21.8 22 20 19 15 19.0 20 18 15 14 16.8 21 20 16 14 17.8 30

Isolates Antibiotics Rate Interaction

23 21 20 16 20.0 17 13 10 6 11.5 18 14 10 6 12.0 23 22 19 16 20.0 22 19 18 16 18.8 21 17 16 14 17.0 21 19 15 13 17.0 28

23 20 19 17 19.8 17 12 10 7 11.5 18 16 12 7 13.3 24 23 21 16 21.0 21 18 17 15 17.8 19 17 15 13 16.0 19 16 15 12 15.5 25

0.024 0.037 0.032 N.S.

62

23.0 20.6 19.6 17.0 20.1 17.3 12.3 10.3 6.6 11.6 18.3 14.6 11.0 6.6 12.8 24.0 23.0 20.0 16.3 20.9 21.6 19.0 18.0 15.3 18.5 20.0 17.3 15.3 13.6 16.6 20.3 18.3 15.3 13.0 16.7 27.6

Erythromycin Bacterial cells count

Bacterial cells count

Ofloxine 2 1.5 1 0.5 0 0

50

100

150

2.5 2 1.5 1 0.5 0 0

200

50

100

Rate PB6

AL8

PB6

PB14

AL8

100

150

2 1 0.5 0 0

200

50

AL8

PB6

PB14

2.5 2 1.5 1 0.5 0 50

200

AL8

PB14

100

150

2.5 2 1.5 1 0.5 0 0

200

50

100

150

200

Rate

Rate PB6

150

Te tracycline Bacterial cells count

Bacterial cells count

Ce voran

0

100 Rate

Rate PB6

PB14

1.5

count

3 2.5 2 1.5 1 0.5 0 50

200

Cefope razone Bacterial cells

Bacterial cells count

penicillin

0

150

Rate

AL8

PB6

PB14

AL8

PB14

Bacterial cells count

Chloramphenicol 2.5 2 1.5 1 0.5 0 0

50

100

150

200

Rate PB6

AL8

PB14

Fig. (12): Relationship between growth of Pseudomonas syringae isolates as count of bacterial cells and different rates of antibiotics, in vitro.

63

Experimental Results

Table (14): Effect of different antibiotics, at different rates on growth (inhibition zone (mm)) of Pseudomonas syringae isolates, in vitro. Inhibition zone (mm) Rate Antibiotic Isolates (ppm) Pb-6 Al-8 Pb-14 Mean Erythromycin

Ofloxine

Cefoperazone

Penicillin

Tetracycline

Cevoran

Chloramphenicol Control LSD at 5 % for

Experimental Results

50 100 150 200 Mean 50 100 150 200 Mean 50 100 150 200 Mean 50 100 150 200 Mean 50 100 150 200 Mean 50 100 150 200 Mean 50 100 150 200 Mean 0.0

4 7 8 11 7.5 8 16 22 28 18.5 7 9 14 18 12.0 2 3 4 6 3.8 8 9 10 11 9.5 11 13 18 19 15.3 11 15 17 20 15.8 0.0

Isolates Antibiotics Rate Interaction

5 7 10 13 8.8 13 18 23 27 20.3 7 10 16 18 12.8 2 3 4 6 3.8 8 9 10 11 9.5 13 15 17 19 16.0 10 17 19 20 16.5 0.0

4 6 8 11 7.3 10 17 22 27 19.0 8 10 14 17 12.3 2 3 4 6 3.8 9 10 11 12 10.5 12 15 18 19 16.0 10 17 19 21 16.7 0.0

0.247 0.378 0.314 1.400

64

4.3 6.6 9.0 11.6 7.8 10.3 17.0 22.3 27.3 19.2 7.3 9.6 14.6 17.6 12.3 2.0 3.0 4.0 6.0 3.8 8.3 9.3 10.3 11.3 9.8 12.0 14.3 17.6 19.0 15.7 10.3 16.3 18.3 20.3 16.3 0.0

Erythrom ycin

30 20 10 0 0

50

15 10

(mm)

Inhibition zone

Inhibition zone (mm)

Ofloxine

5 0 0

100 150 200

50

Pb-6

Al-8

Pb-6

Pb-14

10 5 0 50

Al-8

100 150 200

Al-8

0 0

50

Pb-6

Pb-14

100 150

Al-8

Pb-14

Te tracycline 15 10

(mm)

Inhibition zone

Inhibition zone (mm)

Al-8

5 0 0

200

50

100

150

200

Rate

Rate Pb-6

100 150 200 Rate

20 15 10 5 0 50

Pb-14

10

Cevoran

0

200

20

Rate Pb-6

150

Cefoperazone Inhibition zone (mm)

Inhibition zone (mm)

penicillin

0

100 Rate

Rate

Pb-6

Pb-14

Al-8

Pb-14

Inhibition zone (mm)

Chloramphenicol

50 0 0

50

100

150

200

Rate Pb-6

Al-8

Pb-14

Fig.(13): Relationship between growth of Pseudomonas syringae isolates as inhibition zone (mm) and different rates of antibiotics, in vitro.

65

Experimental Results

3. Effect of bioagents on growth of plant pathogenic bacteria: Five isolates of antagonistic bacteria were tested for their ability to inhibit the growth of P. syringae isolates, using nutrient agar (NA) medium, in vitro. Data in Table (15) and Fig. (14) indicate that

Serratia marcescens (Sm.) isolate was the best antagonistic bacterium in reducing the growth of P. syringae isolates, where the inhibition zone ranged from 18 – 21 mm. Meantime, P. fluorescens isolates (i.e. Pf-1, Pf-2) and Bacillus subtilis (Bs-3) showed moderately effect in reducing the growth of P. syringae. Meanwhile, P. putida was the lesser effective one in reducing the growth of tested pathogenic bacteria, where the obtained inhibition zone ranged between 7-9 mm.

Table (15): Effect of different bioagents on growth of Pseudomonas syringae isolates as inhibition zone (mm), in vitro Inhibition zone (mm) Isolates Pb-6 Al-8 Pb-14

Bioagent Pseudomonas fluorescens (PF-1)

10

10

11

Pseudomonas fluorescens (PF-5)

11

12

11

Pseudomonas putida (Pt-13)

9

7

7

Bacillus subtilis (Bs-3)

11

12

12

Serratia marcescns (Sm-1.)

18

21

19

Experimental Results

66

Inhibition zone (mm)

25 20 15 10 5 0 Pb-6

Al-8

Pb-14

Pseudomonas fluorescens (PF-1) Pseudomonas fluorescens (PF-2) Pseudomonas putida (Pt-13) Bacillus subtilis (Bs-3) serratia marcescns (Sm )

Fig.(14): Relationship between growth of Pseudomonas syringae isolates and different bioagents as inhibition zone (mm), in vitro. 4. Effect of bactericides in controlling canker disease on immature peach fruits: Application of chemical compounds and antibiotics (as bactericides) reduced the infection with canker bacteria comparing with the control treatment as clear in Tables (16 & 17) and Figs. (15 & 16). Efficiency of bactericides was increased with increasing their concentrations. Treatment with bactericides before inoculation with the pathogen reduced the disease incidence to values ranged between 12.0 – 35.0 % for chemical compounds and 18.8 – 46.2 % for antibiotics. Meanwhile, the treatment with bactericides and the pathogen at the same time reduced the disease of tested bacteria to values ranged between 10.0 – 33.7 % for chemical compounds and between 13.8 – 42.5% for antibiotics. Whereas, the treatment with bactericides after inoculation with the pathogen reduced the disease incidence to values ranged between 21.3 – 46.2 % for chemical compounds and between 24.0 – 58.7 % for antibiotics. Application of antibiotics was more effective than chemical compounds in reducing the disease, where, the percentages of disease reduction were 13.8 –

67

Experimental Results

58.7% and 10.0- 46.2%, respectively. Meanwhile, Champion (compound) and Chloramphenicol (antibiotic) were the most effective in reducing the disease incidence, where the percentages of disease reduction were ranged from 11.2 – 46.2 and 21.3 – 58.7%, respectively. Starner, copper oxychloride, cefoperazone and ofloxine exhibited moderate effect, where the percentages of disease reduction were 12.0 – 45.0, 10.0 – 43.7, 17.5 – 55.0 and 13.8 – 50.0%, respectively.

Table (16): Effect of different chemical compounds, on percentage of bacterial canker disease caused by P. syringea, using artificial inoculation on peach fruits, in vitro. Chemical Rate compounds ( mg/l ) Copper oxychloride Champion Starner Check

400 600 800 400 600 800 400 600 800 0.0

A 6.0 5.6 4.5 6.3 5.3 4.3 5.6 5.3 4.4 8.0

Disease reduction (%) 25.0 30.0 43.7 21.3 33.7 46.2 30.0 33.7 45.0 0.0

LSD at 5 % for

B 7.2 7.0 5.7 7.1 6.6 5.3 7.0 6.4 5.6 8.0

Disease reduction (%) 10.0 12.0 28.7 11.2 17.2 33.7 12.0 20.0 30.0 0.0

C 7.0 6.6 5.5 6.5 5.8 5.2 6.9 6.2 5.4 8.0

Time 0.502 Rate 0.058 Chemical 0.502 Interaction 1.737 A,B and C = diameter mean of canker on infected fruits A = Appling the pathogen after chemical compounds by 24 - h B = Appling the pathogen and chemical compounds at the same time C = Appling the pathogen before chemical compounds by 24 - h % = percentage of disease reduction

Experimental Results

68

Disease Reduction (%) 12.0 17.5 31.3 18.8 27.5 35.0 13.8 22.5 32.5 0.0

Copper oxychloride D is e a s e re d u c t io n ( % )

D isease red u ctio n (% )

Champion

50

50 40 30 20 10

40 30 20 10 0

0 0

400

600 A

c

0

800

400 A

B

600 B

800 c

Starner Disease reduction %

50 40 30 20 10 0 0

400

600

A

B

800 c

A = Appling the pathogen after chemical compounds by 24 - h B = Appling the pathogen and chemical compounds at the same time C = Appling the pathogen before chemical compounds by 24 – h

Fig. (15): Relationship between different rates of chemical compounds and percentage of bacterial canker disease caused by P. syringea, using artificial inoculation on peach fruits, in vitro.

69

Experimental Results

Table (17): Effect of different antibiotics, on percentage of bacterial canker disease, using artificial inoculation on peach fruits, in vitro caused by P. syringae.

Antibiotic

Rate (ppm)

A

Disease reduction (%)

B

Disease reduction (%)

C

Disease reduction (%)

Ofloxine

100 150 200

6.0 5.3 4.0

25.0 33.7 50.0

6.9 6.3 5.5

13.8 21.3 31.2

6.5 6.0 5.3

18.8 25.0 33.7

Chloramphenicol

100 150 200

5.3 5.0 3.3

33.7 24.0 58.7

6.3 5.0 4.6

21.3 24.0 42.5

5.6 5.0 4.3

30.0 37.5 46.2

Cefoperazone

100 150 200

5.6 4.6 3.6

30.0 42.5 55.0

6.6 5.9 5.0

17.5 42.5 37.5

6.3 5.7 4.8

21.3 28.8 40.0

check

0.0

8.0

0.0

8.0

0.0

8.0

0.0

LSD at 5 % for

Time Rate Antibiotics Interaction

0.622 0.718 0.622 2.154

A,B and C = diameter mean of canker on infected fruits A = Appling the pathogen after antibiotics by 24 - h B = Appling the pathogen and antibiotics at the same time C = Appling the pathogen before antibiotics by 24 - h % = percentage of disease reduction

Experimental Results

70

Chloramphenicol

ofloxine 70 60 50 40 30 20 10 0

D i s e a s e r e d u c ti o n (% )

D i s e a s e r e d u c ti o n (% )

70 60 50 40 30 20 10

0

0 0

500

750

A RateB

500

1000

750

1000

Rate A

c

B

C

cefoperazone Disease reduction (%)

70 60 50 40 30 20 10 0 0

500

750

1000

Rate

A

B

C

A = Appling the pathogen after antibiotics by 24 - h B = Appling the pathogen and antibiotics at the same time C = Appling the pathogen before antibiotics by 24 - h

Fig. (16): Relationship between different rates of antibiotics and percentage of bacterial canker disease, caused by P. syringea using artificial inoculation on peach fruits, In vitro.

71

Experimental Results

5. Effect of some bioagents in controlling canker disease on immature peach fruits: Application

of

bioagents

i.e.

Bacillus

subtilis

(Bs-3),

Pseudomonas fluorescens (Pf-5) and Serratia marcescns (Sm-1) (the most effective bioagents in reducing the growth of P. syringae under laboratory conditions) reduced the infection of canker bacteria on peach fruits compared with the control as clear in Table (18) and Fig.

(17). The treatment with bioagents before pathogen inoculation was the most effective, where, the percentage of disease reduction ranged from 32.5 – 45.0%. Meanwhile, the treatment with bioagents and the pathogen at the same time or the treatment with bioagents after the pathogen exhibited moderate effect, where the percentages of disease reduction ranged from 25.0 – 30.0% and from 18.8 – 22.5%, respectively. However, Serratia marcescens was the most effective treatment in reducing the disease, where percentages of disease reduction ranged from 22.5 – 45.0 %. Application of Bacillus subtilis and Pseudomonas fluorescens were moderately effective, where the percentages of disease reduction ranged from 20.0 – 42.5 and from18.8 – 32.5%, respectively.

Experimental Results

72

Table (18): Effect of different bioagents on reducing the percentage of bacterial canker disease (Pseudomonas syringae), using artificial inoculation on peach fruits, in vitro. Disease Disease A reduction B reduction (%) (%)

Bioagents

Bacillus subtilis 6.0 (Bs-3) Pseudomonas fluorescens 6.0 (Pf-5) Serratia marcescns 5.6 (Sm-1) Check 8.0

Disease C reduction (%)

25.0

4.6

42.5

6.4

20.0

25.0

5.4

32.5

6.5

18.8

30.0

4.4

45.0

6.2

22.5

0.0

8.0

0.0

8.0

0.0

LSD at 5 % for

Time 0.730 Bioagents 0.843 Interaction 1.460 A,B and C = diameter mean of canker (infection zone) on infected fruits A = Inoculation of the pathogen and bioagents at the same time B = Inoculation of the pathogen after bioagents by 24 – hrs C = Inoculation of the pathogen before bioagents by 24 - hrs % = percentage of disease reduction 50 45 Disease severity %

40 35 30 25 20 15 10 5 0 (Bs3)

(Pf 1)

(Sm) Bioagent

A

B

C

Fig. (17): Relationship between different bioagents on reducing the percentage of bacterial canker disease, using artificial inoculation on peach fruits, in vitro.

73

Experimental Results

6. Effect of bactericides on bacterial canker disease, under artificial inoculation conditions: Starner, Copper oxychloride, Champion, Cefoperazone and Ofloxine were applied as bactericides against bacterial canker disease of apricot. Apricot seedlings (one-year-old) was used in this experiment. These seedlings were inoculated with the pathogen as spray on the foliage treatment and as injection treatment in the branches. The bactericides were applied as spray treatment before inoculation by the pathogen by 24 hrs. Data in Tables (19 & 20) and

Figs. (18 & 19) indicate that all tested bactericides reduced the disease incidence compared with the control treatment. The tested bactericides were more effective in reducing the disease on leaves than on branches of apricot seedlings, where the percentages of disease reduction were 13.8 - 36.2% and 12.5 – 30.8%, respectively. Champion and Copper oxychloride were the most effective bactericides in reducing the disease, where the percentages of disease reduction were 36.2 and 31.0% on leaves and was 30.8 and 27.7% on branches,

respectively.

Meanwhile,

Chloramphenicol

and

Cefoperazone were moderately effective, where the percentages of disease reduction were 27.0 and 22.4% on leaves and were 24.9 and 20.8% on branches, respectively. Starner was the lesser effective one, where the percentage of disease reduction was 13.8% on leaves and was 12.5% in branches.

Experimental Results

74

Table (19): Effect of different chemical compounds & antibiotics on percentage of bacterial canker disease incidence on leaves of apricot, under artificial inoculation conditions, in vivo. Rate

Starner

800 mg/l

10.7

50.0

Disease reduction (%) 13.8

Copper oxychloride Champion

800 mg/l 800 mg/l

8.8 8.0

40.0 37.0

31.0 36.2

Cefoperazone

200 ppm

9.9

45.0

22.4

chloramphenicol

200 ppm

9.4

42.0

27.0

check

0.0

15.0

58.0

0.0

3.24

2.40

1.62

70

1.2

60

1

50

0.8

40 0.6

30

0.4

20

0.2

10

Disease reduction (%)

S ta rn er

0 C C ha op m pe pi ro on xy ch lo rid e

0 C ch hl or e am ck ph en ic C ol ef op er az on e

Reductionof disease incidence

LSD at 5 %

Infection (%)

meanof spots

Treatment

Mean No. of spots/leaf

Mean No. of spots/leaf Infection (%)

Fig. (18): Efficiency of some chemical compounds and antibiotics on reducing percentage of bacterial canker disease of apricot on leaves, under artificial inoculation conditions, in vivo.

75

Experimental Results

Table (20): Effect of different chemical compounds & antibiotics on percentage of bacterial canker disease incidence on branches of apricot, under artificial inoculation conditions, in vivo. Treatment

Rate

Infection (%)

Starner Copper oxychloride Champion Cefoperazone Chloramphenicol Check (water)

800 mg/l 800 mg/l 800 mg/l 200 ppm 200 ppm 0.0

44.3 36.6 35.0 40.1 38.0 50.6

Disease reduction (%) 12.5 27.7 30.8 20.8 24.9 0.0

2.054

2.054

60 50 40 30 20 10

C op pe r

Infection (%)

ch ec k

C ha m pi on C ef op er az C on hl e or am ph en ic ol

0 S ta rn er ox yc hl or id e

Reductionof diseaseincidence

LSD at 5 %

Dis eas e reduction (%)

Fig.(19): Efficiency of some chemical compounds and antibiotics on bacterial canker disease incidence of apricot under artificial inoculation conditions, in vivo.

Experimental Results

76

7. Effect of bioagents on bacterial canker disease, under artificial inoculation conditions: Isolates of Bacillus subtilis (Bs-3), Pseudomonas fluorescens (Pf-5) and Serratia marcescens (Sm-1) were applied as bacterial bioagents against bacterial canker disease of apricot cv. Canino (oneyear-old seedling). Results in Table (21) and Fig. (20) indicate that all tested bioagents reduced the disease incidence compared with the control. The tested bacterial bioagents were more effective on leaves than in branches of apricot seedlings in reducing the canker disease, where, the percentages of disease reduction ranged between 10.3 – 13.8 and between 7.5 – 12.6%, respectively. Isolates of S. marcense (Sm-1) and P. fluorescens (Pf-5) were highly effective, where the percentages of disease reduction were 13.8 and 12.1% on leaves and were 12.6 and 11.1% on branches, respectively.

Table (21): Effect of different bioagents on percentage of bacterial canker disease of apricot (leaves and branches), under artificial inoculation conditions, in vivo. Treatment

Bacillus subtilis (Bs-3) Pseudomonas fluorescens (Pf-5) Serratia marcescens (Sm1)

Mean No. of spots/leaf

Disease parameters on Leaves Shoots Disease Disease Infection Infection reduction reduction (%) (%) (%) (%)

11.7

52

10.3

46.8

7.5

10.9

51

12.1

45.0

11.1

10.6

50

13.8

44.2

12.6

Check (water)

15.0

58.0

0.0

50.6

0.0

LSD at 5 %

1.631

2.824

1.633

3.208

1.412

77

Experimental Results

(A)

(B) 70 60 50 40 30 20 10 0

60 50 40 30

%

Mean of spots

70

20 10 1)

ch ec k

(S m

(P f5 )

(B s3 )

0

(Bs3)

Infection (%) Disease reduction (%) Mean No. of spots/leaf

(Pf5)

(Sm1)

check

Infection (%) Disease reduction (%)

Fig. (20): Efficiency of different bioagents on bacterial canker disease of apricot on leaves (A) and branches (B), under artificial inoculation conditions, in vivo.

Meanwhile, B. subtilis (Bs-3) was the lesser effective one in reducing the disease, where the percentage of disease reduction was 10.3% on leaves and was 7.5% on branches. Also, all tested bacterial bio-agents clearly reduced the number of spots of bacterial canker disease on leaves of apricot comparing to control treatment.

Experimental Results

78

DISCUSSION Bacterial canker of stone fruits caused by P. syringae has become a serious problem in many parts of the world (Cameron, 1962 and Mohammadi et al., 2001). Pseudomonas syringae causes many important and common diseases including bacterial canker of stone fruit, blossom blight or blast of pear, brown spot of bean, citrus blast and black pit, as well as blights and leaf spots of pea, cowpea and lilac (Elliott 1951, Stapp 1961 and Hayward and Waterston,1969). The disease occurs on the aboveground parts of the trees, and may results in localized canker or death of entire limbs of trees. In mature trees, under the right climatic conditions, infection can spread quickly, killing large branches in a matter of weeks. Symptoms vary widely between hosts and different climatic conditions, but are more commonly initiated on green foliage. As for sampling and isolation of the canker bacteria, the bacterial canker disease occurs on branches, flowers, twigs, buds, leaves, and fruits. The most conspicuous symptoms are the cankers that exude gum during late spring and summer on apricot, peach and pear trees. Gumming is common on stone fruit trees, whether on trunks, limbs, twigs or fruits when injuries occur. Cankers on the twigs are darkened areas often at the base of buds. On limbs or trunks, they are often darker than the normal bark, sunken in their centers and they may extend for a considerable distance. Moreover, the grown leaves and shoots may be cankered, wilted and died during the growing season. In contrast, leaves and flowers from the other infected buds may remain symptomless. Leaf infections appear as water-soaked spots then become brown and dry. Also, fifteen bacterial isolates were isolated from different parts of peach, apricot, pear and apple which collected from different localities of Egypt. In this respect, the bacterial isolates coded as Pb-1, Ps-2 and Pf-4 were

79

Discussion

isolated from buds, stems and fruits respectively of peach in Daqahlyia (Mit-Ghamr). Meanwhile, the isolates coded as Rs-3 and Lb-11 were isolated from stem and bud of pear and apple respectively in the same governorate (Mit-Ghamr). On the other hand, the bacterial isolates coded as Pb-5, Pb-6, Ps-13 and Pl-15 were isolated from peach in Qualubia governorate while, Al-7 and As-12 were isolated from apricot in the same governorate. Meanwhile, the isolate coded as Rf-10 was isolated from pear in Qualubia governorate (Moshtohor), while, the isolates coded as Pf-9 and Pb-14 were isolated from flower and buds of peach respectively in Beheira governorate. The isolate Al8 was isolated from leaf of apricot in the same governorate. Isolation from such tree parts are in harmony with those obtained by Crosse (1959) and English and Davis (1960) who revealed that Pseudomonas syringae pv. syringae and P. syringae pv. morsprunorum can be readily isolated from leaf surfaces of peach and apricot during the growing season and Crosse (1966) who isolated P. syringae pv. morsprunorum from cherry and plum and P. syringae pv. syringae from leaf scars of different hosts in autumn. Also, Vock (1978) reported that the bacterial canker of stone fruit is a serious disease of cherry trees, but may also causes early death in apricot, peaches, nectarines, plums and prunes in Australia. Moreover, Guevara et al.(2000) mentioned that symptoms of dieback disease on branches of peach (Prunus persica) in Trujillo, Aragua and Miranda, Venezuela appeared as cankers with gum exudates between healthy and diseased areas and red spots with yellow halos on leaves. The causal agent was identified using biochemical and physiological tests as Pseudomonas syringae pv. syringae. Also, Hetherington, (2005) verified our obtained results on symptoms of bacterial canker disease and isolation. Concerning identification of isolated bacteria using the traditional techniques according to their inspected morphological and

Discussion

80

cultural characteristics, these traditional tests revealed that these isolates may be belong to four genera i.e., Erwinia, Bacillus, Xanthomonas and Pseudomonas. Also, the other testes based on the biochemical and physiological characteristics of isolated bacteria revealed finally that two isolates i.e. Rs-3 and Lb-11 could be identified as Erwinia amylovora, while, the isolates i.e. Pb-6, Al-8 and Pb-14 could be identified as Pseudomonas syringae. Meanwhile, the isolate Al-7 could be identified as Xanthomonas campestris but the isolate As-12 is Bacillus polymyxa. In this respect, the identification of isolated bacteria was achieved and agreed with the findings of Schaad, (1980); Fahy and Persley (1983); Krieg and Holt (1984), Leliott & Stead (1987) and Little et al, (1998). On the other hand, PCR-RAPD amplification by using the primer- OP-A-11 (5'CAATCGCCGT-3') for the three bacterial isolates i.e. Pb-6, Al-8 and Pb-14 which identified as P. syringae by the traditional identification verified that these three isolates are P. syringae as well as the three other random primers verified that they also different isolates. Also, similar results were obtained by Abu-Ashraf et al., (2000) who used the polymerase chain reaction (PCR) technique for differentiating the pathovars of Pseudomonas syringae and considered this technique is rapid, simple and reproductive to identify and classify phytopathogenic P. syringae at pathovar level, and it may be a useful diagnostic tool for these important plant pathogens. While, Mohammadi et al.(2001) isolated 27 bacterial strains from cankerous tissues of apricot, nectarine, peach, plum, sour cherry and sweet cherry trees in Tehran province and identified them as Pseudomonas syringae pv. syringae, the causal agent of the bacterial canker disease, based on the levan production, oxidase test, potato rot, arginine dihydrolase and tobacco hypersensitive reaction (LOPAT), and gelatin liquefaction, aesculin hydrolysis, tyrosinase activity and Na-tartrate utilization (GATTa's) group tests. Pss strains showed slight differences in morphology, phenotypic (biochemical and

81

Discussion

physiological) characteristics. On the other hand, Kotan and Sahin (2002) isolated and identified Pseudomonas syringae pv. syringae from typical bacterial canker symptoms on apricot trees in Turkey and confirmed its pathogenicity. Also, Vasinauskiene and Baranauskaite (2003) used morphological, biochemical and serological analysis to identify P. syringae pv. syringae the causal organism of blossom infection, shoot dieback and blight similar to fire blight on pear trees in Lithuania. On the other hand, Vicente et al. (2004) isolated fiftyfour Pseudomonas syringae isolates from cherry and 13 isolates from pear and lilac and characterized them by physiological, biochemical, serological and pathogenicity tests. As for factors affecting the growth of P. syringae in vitro, the optimum growth of P. syringae was recorded at 25 -30ْC, pH ranged between 6.5-7.5 and 80-100% relative humidity. In this respect, similar results on the bacterial canker pathogen (Pseudomonas syringae) under natural conditions were obtained by Schmidle and Zeller (1976) who revealed that leaves can be successfully infected between -0.5 and -2ºC and the optimal temperature range for symptom development is 15-25ºC. Also, Wimalajeewa and Flett (1985) found that the epiphytic populations of the bacterial canker pathogen (Pseudomonas syringae pv. syringae) on leaves, buds and shoots of apricot and cherry were lowest during mid- to late summer. While, the high proportions of tree contamination and high populations coincided with periods when max. temps. were 19–25ºC and when rainfall was moderately high. In addition, Süle and Seemüller (1987) indicated that P. syringae pv. syringae on sour cherry orchards under cool and wet weather conditions may cause considerable crop damage and serious economic losses. While, Cao et al. (1999) reported that freezing at -5°C for 12 to 24h produced significantly larger lesion on infected peach with bacterial canker disease than did inoculations performed before freezing. Latorre et al. (2002) mentioned that

Discussion

82

freezing temperatures may predispose pears to the infection with P. syringae pv. syringae.. Regarding disease control trials of the bacterial canker pathogen (Pseudomonas syringae) using chemical compounds and antibiotics under in vitro conditions, all tested chemical compounds and antibiotics (as bactericides) effectively reduced the growth of P. syringae compared with the control. Meanwhile, increasing the rates of chemical compounds and antibiotics increased the growth reduction %. In this respect, Anti–shot was the most effective compound in reducing the growth of P. syringae, followed by Copper oxychloride and Champion while, Kocide 2000 was the lesser effective one in reducing the growth of the tested pathogenic bacteria. On the other hand, Ofloxine and Cefoperazone were the most effective in reducing the growth of P. syringae while, Cevoran and Chloramphenicol were moderately effective whereas, Penicillin was the lesser effective one. On the other hand, application of chemical compounds and antibiotics (as bactericides) on immature peach fruits reduced the infection with canker bacteria comparing with the control treatment. In this respect, the treatment with chemical compounds before inoculation with the pathogen was better than the that at the same time and/or after inoculation with the pathogen although all of them effectively reduced the disease incidence. Also, application of antibiotics was more effective than chemical compounds in reducing the disease. Champion and Chloramphenicol were the most effective compounds in reducing the disease incidence. Meanwhile, Starner, Copper oxychloride, Cefoperazone and Ofloxine were moderately effective. As for the effect of bactericides on bacterial canker disease under artificial inoculation conditions, all tested bactericides reduced the disease incidence compared with the control treatment. The tested bactericides were more effective in reducing the disease on leaves than on branches of apricot seedlings. Champion and Copper oxychloride

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were the most effective bactericides in reducing the disease on leaves and branches respectively. Meanwhile, Chloramphenicol and Cefoperazone were moderately effective, on leaves and branches respectively. While, Starner was the lesser effective one on leaves and branches. These results could be interpret in light the findings of Gaignard et al. (1976) who found that Oxytetracyclin controlled greatly peach bacterial canker with minimum infected leaf scars and numbers of bacteria/leaf in the spring. While, Bordeaux mixture and Copper oxychloride reduced the disease severity to the half, but were somewhat phytotoxic. Also, Menkissoglu and lindow (1991) suggested that the bactericidal effects of copper compounds in growth media due to the concentration of free copper ions as well as in field, small quantities of copper salts are solubilized when leaves are wetted by rain or dew but the copper ions are probably complexed with organic compounds leached from leaf surface. While, free copper ions are considered more toxic to microorganisms than complexed form of this element. While, Lye (1997) interpreted the effect of Cupprous oxide and Copper oxychloride mainly as surface protectants and as enzyme inhibitors on several of phytopathogens. On the other hand, many investigators like Stall et al. (1986); Sundin et al. (1989); Bender, et al. (1990) and Cooksey (1990) mentioned that copper resistance (Cur) have been localized to plasmid DNA in all phytopathogenic bacteria. While, Andersen, et al., (1991) attributed the inadequate control of P. syringae pv. syringae attributed to the occurrence of strains resistant to antibiotics and resistant to copper in almond and citrus orchards. Moreover, Sundin and Blender, (1993) isolated resistant strains of P. syringae to both copper and streptomycin from ornamental pear trees. They attributed these resistance to strA,strB genes of the broad-host-range entobacteria plasmid Rslolo. Also, Penrose, (1998) reduced blossom blast (P. syringae pv. syringae) significantly on pears when antibiotics treatments applied after each wearing. Discussion

84

Concerning the effect of bio-agents in controlling canker bacteria in vitro, the tested isolate of Serratia marcescens (Sm.) was the best antagonistic bacterium in reducing the growth of P. syringae isolates, followed by P. fluorescens isolates (i.e. Pf-1, Pf-2) while, Bacillus subtilis (Bs-3) showed moderately effect in reducing the growth of P. syringae. Meanwhile, P. putida was the lesser effective one in this respect. Also, application of bio-agents i.e. Bacillus subtilis (Bs-3), Pseudomonas fluorescens (Pf-5) and Serratia marcescens (Sm-1) on immature peach fruits reduced the infection of canker bacteria compared with the control. The treatment with bioagents before pathogen inoculation was the most effective. However, Serratia marcescens was the best effective treatment in reducing the disease. Application of Bacillus subtilis and Pseudomonas fluorescens were moderately effective. On the other hand, all tested bio-agents i.e. Bacillus subtilis (Bs-3), Pseudomonas fluorescens (Pf-5) and Serratia marcescens (Sm-1) reduced the disease incidence compared with the control when tested under artificial inoculation conditions. The tested bacterial bio-agents were more effective on leaves than branches of apricot seedlings in reducing the canker disease. Isolates of Sm-1 and Pf-5 were moderately effective on leaves and branches, respectively. Meanwhile, Bs-3 was the lesser effective one in reducing the disease on leaves and branches. Also, all tested bacterial bio-agents clearly reduced the number of spots of bacterial canker disease on leaves of apricot comparing to control treatment. These results could be interpret in light the findings of Kloepper et al., (1980) who reported that fluorescent Pseudomonas strains on King's B and potato dextrose agar media produced adverse array of inhibitory compounds (Siderophores), which inhibited growth of phytopathogens. While, Suslow and Schroth (1982) mentioned that production of both antibiotics and siderophores has been cited as a factor relation to the abitty of PGPR to displace or excluded soil borne pathogens and deleterious rhizosphere microorganisms. Whereas, Palleroni (1984)

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observed that fluorescent Pseudomonas had great potentiality to produce a broad spectrum of secondary metabolites that may be toxic to other microorganisms. Moreover, Toyota and Kimura (2000) found that colonization of the rhizosphere with fluorescent Pseudomonas has been successfully employed to reduce the amount of pathogen inoculum reaching the roots and they promote plant growth. Deboer et al. (2003) revealed that the production of antimicrobial compounds by some strains of Pseudomonas spp. has been recognized as a major factors in suppression of many root pathogens. Schoofs et al. (2002) found that the use of Serratine-P, a phage tail-like bacteriocin, produced by Serratia plymiticum, shows an interesting antibacterial activity against E. amylovora. Its mode of action consists in the perforation of the cytoplasmic membrane of the target cell, inducing perturbations in cellular exchanges and a final lysis of the bacterial cell.

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SUMMARY The present investigation was planned to study bacterial canker problem of some fruit trees in Egypt, Pseudomonas canker (Pseudomonas syringae Van Hall, 1902).This disease resulted great loss in yield and limited the production of stone fruit in Qalyubiya and Behera governorates through the late years. The results obtained can be summarized as follows: 1. Syndrome typical for bacterial canker disease on some fruit trees caused by Pseudomonas syringae observed as shot hall on leaves, blast blossom, wilt shoot and canker in trunk and stem. The most conspicuous symptoms are the cankers that exude gum. 2. All collected samples of different hosts which included (leaves, branches, flowers, buds and fruits ) were positive to isolate bacteria, using common and selective media. 3. Fifteen bacterial isolates were examined for their reaction on differential hosts (tobacco, beans, apricot, peach and pear). Three isolates (Pb-6, Al-8 and Pb-14) were positive on all tested plants and two isolates (Rs-3 and Lb-11) were positive on tobacco and pear plants. Meanwhile, two isolates (Al-7 and As-12) were positive on only tobacco plants and other isolates were negative on all tested plants. 4. On tobacco plants symptoms were appeared as water-soaking of inoculated tissue within 48 hrs followed by dry, light-brown localized necrosis with 3 days, and on bean seedlings appeared as yellow-brown discoloration on inoculated cotyledons. Also, on detached fruits of peach symptoms was appeared as black localized necrosis with bacterial ooze in inoculated area and on peach, apricot and pear seedlings showed brown spots on inoculated leaves and small dark-green lesion at entry and exit points on inoculated branches, die-back symptoms were observed.

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Summary and Conclusions

5. All isolates were, Gram negative, short rods and non spore formers except one isolate (As-12) was Gram positive, long rods and formed spores. 6. All isolates on (YDC) medium were grown with different colour, white in three isolates (Rs-3, Lb-11 and As-12) , translucence in three isolates (Pb-6, Al-8 and Pb-14) and yellow in one isolate (Al-7). Meanwhile on king's B medium colonies produced florescent pigments in three isolates (Pb-6, Al-8 and Pb-14). 7. The aforementioned tested appeared that these isolates belong to four genera i.e. Erwinia sp., Bacillus sp., Xanthomonas sp. and Pseudomonas sp. according to morphological and cultural characteristics. 8. Two isolates (Rs-3 and Lb-11) were positive with gelatin liquefaction, KOH 3 %, Levan test, tobacco hypersensitivity and reducing substance from sucrose and growth on 5% NaCl. 9. These isolates gave negative reaction with starch hydrolysis, pectate degradation, fats hydrolysis, methyl red test (M.R.), production of H2S, production of indole, urease production, nitrate reduction, production of pigments, growth at 50°C, oxidase reaction, potato rot, arginine dihydrolase and fermentative metabolism in o/f test. This isolate gave different reaction in utilizing carbon source. 10. Three isolates i.e. (Pb-6, Al-8 and Pb-14) gave positive reaction with pectate degradation, production of pigments, KOH 3 %, Levan test, tobacco hypersensitivity and reducing substance from sucrose and growth on 5% NaCl. 11. These isolates i.e. (Pb-6, Al-8 and Pb-14) gave negative reaction with starch hydrolysis, gelatin liquefaction, fats hydrolysis, methyl red test (M.R.), H2S production , production of indole, urease production, nitrate reduction, growth at 50°C, oxidase reaction,

Summary and Conclusions

88

potato rot, arginine dihydrolase and oxidative metabolism in o/f test. These isolates were able to utilize L (+) Lactose, Dextrose. 12. Biochemical and physiological properties revealed that the pathogenic bacterial isolates were as follows: two isolates (Rs-3 and Lb-11) belong to Erwinia amylovora, three isolates (Pb-6, Al8 and Pb-14) belong to Pseudomonas syringae, one isolate (Al-7) belong to Xanthomonas campestris and one isolate (As-12) belong to Bacillus polymyxa. 13. The three isolates i.e. Pb-6, Al-8 and Pb-14 were identified as P. syringae and used only in this study. The traditional identification of these isolates was confirmed using PCR-RAPD amplification of DNA with four random primers. In this respect, the four tested primers revealed the similarity and diversity between the three tested isolates with superiority of the primer OPERON A-11 in revealing high similarity between the three tested P. syringe isolates. The developed bands of the three isolates showed very close similarity with this primer and confirmed that this primer could be useful in differentiating the P. syringe isolates 14. The optimum temperature was 25º C for growth of Pseudomonas syringae, while, the maximum temperature was 35° C . 15. Isolates of Pseudomonas syringae were able to grow at pH values 6 -7.5 and the optimum pH level for their growth was 6.5. 16. Isolates of Pseudomonas syringae were able to grow at wide range of relative humidity (RH).The maximum growth for them was observed at 80-100 RH, while the minimum growth was at 50%RH. 17. All tested chemical compounds and antibiotics as bactericides have inhibitory effect against growth of Pseudomonas syringae bacteria, compared with the control, in vitro. Efficiency of inhibition was increased with increasing the rates of tested bactericides.

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18. Inhibitory effect of antibiotics was more effective than chemical compounds on growth of P. syringae Anti-shot as chemical compound and Ofloxin as antibiotic were the most effective against growth of the pathogen . 19. Kocide 2000 or Starner as chemical compounds and Erythromycin or Tetracycline or Penicillin as antibiotics were less effective. Copper oxychloride or Champion as chemical compound and Cefoperazone or Cevoran or Chloramphenicol as antibiotics were moderately effective. 20. Isolates of Serratia marcescns were the most effective against growth of P. syringae bacterium. Meanwhile, P. fluorescens (Pf-1, Pf-5.) and Bacillus subtilis (Bs-3) isolates showed moderately effective against growth of P. syringae, and P. putida isolate showed the lowest effect. 21. Three chemical compounds (Copper oxychloride, Champion and Starner) and three antibiotics (Ofloxine, Cefoperazone and Chloromphincol) as bactericides were tested against bacterial canker disease, using artificial inoculation on peach fruits, in vitro. 22. Chloramphenicol and Champion as bactericides were the most effective to decrease the disease reduction. Meanwhile, Cefoperazone, Ofloxine, Copper oxychloride and Starner were moderately effective against the disease. 23. Application of B. subtilis (Bs-3), P. fluorescens (Pf-5)and S. marcescns (Sm-1) isolates as bio-agents were tested against the disease, using artificial inoculation on peach fruits, in vitro. All of them led to reduce disease reduction compared with the control. 24. Treatment with bio-agents before inoculation by the pathogen was the most effective to decrease the disease. Meanwhile, isolate of S. marcescns (Sm-1) was more effective than isolates P. fluorescens (Pf-5) and B. subtilis (Bs-3) against the disease. 25. Also, all bactericides and bio-agents which previously mentioned in experiment disease reduction on peach fruits were assessed on Summary and Conclusions

90

apricot seedling cv. Canino (one-year-old), under artificial inoculation condition, in vivo. Bactericides and bioagents were applied as spray treatment before inoculation with the pathogen by 24-hrs. 26. Application of bactericides or bioagents were effective to control bacterial canker disease compared with the control. Bactericides were more effective than bio-agents to reduce the disease. Efficiency of bactericides or bio-agents were more effective to decrease the disease on leaves than branches. 27. Champion and Copper oxychloride were the most effective to reduce the disease. Meanwhile, Chloramphenicol and Cefoperazone were moderately effective against the disease and Starner exhibited the lowest effect. 28. Isolates of S. marcescns and P. fluorescens were moderately effective against the disease and B. subtilis isolate showed the lowest effect.

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REFERENCES Abd El-ghafar, N.Y. (1988): Studies on the bacterial blight of pears in Egypt M.Sc. Thesis, Faculty of Agricultural, Ain-shams university, Cairo, Egypt.151pp. Abu-Ashraf-Khan; Furuya, N; Matsumoto, M; Matsuyama, N. (2000): Differentiation of phytopathogenic Pseudomonas and Xanthomonas pathovars and strains by PCR analysis for DNA topoisomerase genes. Journal of the Faculty of Agriculture, Kyushu University,45(1): 1-6. Allen, W.R. and Dirks, V.A. (1978): Bacterial canker of sweet cherry in the Niagara Peninsula of Ontario Pseudomonas species involved and cultivar susceptibilities. Canadian Journal of Plant Science. 58(2): 363-369. Andersen, G.L.; Menkissoglou, O. and Lindow, S.E. (1991): Occurrence and properties of copper tolerant strains of Pseudomonas syringae isolated from fruit trees in California. Phytopathology, 81:648-656. Bender, C.L., Malvick, D.K., Conway, K.E., George, S., and Pratt, P. (1990): Characterization of pXV10A,a copper resistance plasmid in Xanthomonas campestris pv. vesicatoria. Appl. Environ. Microbial. 56:170-175. Berger, H.J. (2004): Epidemiology of Pseudomonas syringae Pathovars Associated with Decline of Plum Trees in the Southwest of Germany. Phytopathology 94. 153- 160. Bordjiba, O. and Prunier, J.P. (1991): Establishment of an epiphytic phase by three species of Pseudomonas on apricot trees.ActaHorticulturae.(293): 487-494. Burki, T. (1968): Studies on Pseudomonas species pathogenic to fruit trees in Switzerland. Schweizerische Landwirtschaftliche Forschung. 7(3/4):125-265.

References

92

Burkowicz, A.; Rudolph, K. and Cinar, O. (1978): Pseudomonas syringae Van Hall as incident of bacterial canker of apricot (Prunus armeniaca L.) in the Malatya-Gurun region of Turkey. Phytopathologia-Mediterranea.17(1): 45-51. Cameron, H.R. (1962): Disease of deciduous fruit trees incited by Pseudomonas syringae Van Hall. Oregen Agric. Exp.st. Tech. Bull.66.64p. Cancino, L.; Latorrc, B. and Larach, W. ( 1974): Pear blast in Chile. Plant Disease Reporter.58 (6):568-570. Cao, T.; Sayler, R.J.; Dejong, T.M.; Kirkpatrick, B.C.; Bostock, R.M. and Shackel, K.A. (1999): In flounce of stem diameter, water content and freezing-thawing on bacterial canker development in excised stems of dormant stone fruit. Phytopathology 89:962-966. Claflin, L. (2003): Control of Pseudomonas syringae pathovars. Proceedings of the 1st International Symposium on Biological Control of Bacterial Plant Diseases Germany, 23rd - 26th October 2005. Cooksey, D.A. (1990): Genetics of bactericide resistance in plant pathogenic bacteria. Annu. Rev. Phytopathology, 28:201-219. Crosse, J.E. (1959): Bacterial canker of stone-fruits .IV. Investigation of a method for measuring the inoculum potential of cherry trees. Annals of Applied Biology 47:306-317. Crosse, J.E. (1966): Epidemiological relations of Pseudomonad pathogens of deciduous fruit trees. Annual Review of Phytopathology 4:291-310. Daniell, J.W. and Chandler, W.A. (1974): Effect of temperature on bacterial canker in peach seedlings grown in old and new peach soil. Phytopathology, 64(10): 1284-1286. Deboer, M.; Bom, P.; Kind, T.; Keurentijes, J.B.; Vander Sulis, I.; Van Leon, L.C. and Bakker, P.A.H.M. (2003): Control of Fusarium wilt of radish by combining Pseudomonas putida

93

References

strains that have different disease suppressive mechanisms. Phytopathology, 93:626-632. Dorozhkin, N.A. and Grigortsevich, L.N. (1976): Harmfulness of bacterial canker to fruit trees.Zashchita-Rastenii. 12:39.(Abstract). Dowler W.M. and Weaver, D.J. (1974): Isolation and Characterization of Fluorescent Pseudomonads from Apparently Healthy Peach trees. Phytopathology, 64:233-236. Dowler, W.M., and Petersen D.H. (1966): Induction of bacterial canker of peach in the field. Phytopathology, 56:989-990. Edgecomb, D.W. and Manker, D. (2006): Bacillus subtilis strain QST 713, bacterial disease control in fruit, vegetable and ornamental production. Biocontrol of Bacterial Plant Diseases, 1st Symposium 2005. Mitt. Biol. Bundesanst. LandForstwirtsch. 408, 2006; 167. Elliott, C. (1951): "Manual of bacterial plant pathogens".2nd edition.Waltham,Massachusetts:Chronica,Botanica.186pp. Endert, E. and Ritchie, D.F. (1984): Overwintering and survival of Pseudomonas syringae pv. syringae and symptom development in peach trees. Plant Disease 68:468-470. English, H. and Davis, J.E. (1960): The source of inoculum for bacterial canker and blast of stone fruit trees. Phytopathology, 50:634. .(Abstract.) English, H. and Davis, J.E. (1964): Influence of soil fumigation on growth and canker resistance of young fruit trees in California. Down Earth 20 (3):6-8. English, H.; Davis, J.K.; Devay, J.E. and Lownsbery, B.F. (1980): Bacterial canker ,on important decline disease of apricot and other fruits in California. Acta Hortic. 85:235-242.

References

94

Ercolani, G.L. and Ghaffer, A. (1985): Outbreaks and new records. Afghanistan. Bacterial canker and gummosis of stone fruit. FAO-Plant-Protection-Bulletin.33(1): 37-39. Fahy, P.C. and Persley, G.J. (1983): Plant bacterial disease, a diagnostic guide. Academic press, London, handbook, 377 pp. FAO-Stat Database (2005): Food and Agriculture Organization, United Nations. C.f. Economic Research Service, USDA, Vegetables and Melons Outlook/VGS-297/June, 2005. Fiori, M.; Cicconi, L. and Scortichini, M. (2003): Bacterial canker of hazelnut (Corylus avellana L.) in Sardinia (Italy).Occurrence of Pseudomonas syringae strains Presentations from the 6th International Conference on Pseudomonas syringae pathovars and related pathogens, Maratea, Italy, September 15-19, 2003; 617-625. French, W.J. and Miller, J.W. (1974): Bacterial canker of peach in Florida.Proceedings-of-the-Florida-State-HorticulturalSociety. 86: 310-311. Gaignard, J.L.; Gardan, L.; Luisetti, J.; Prunier, J.P. and Minodier, R. (1976): Chemical control of Pseudomonas persicae agent of peach bacterial canker. Results of experiments in 1973-74.. Arboriculture-Fruitier, (271): 21-26. Gomez, K.A., and Gomez, A.A. (1984): Statistical Procedures for Agricultural Research, 2nd ed. John Wiley and Sons Ltd., New York,680 pp. Guevara, Y.; Rondon, A. and Maselli, A. (2000): Bacterial canker of peach in Venezuela.Agronomia-Tropical-Maracay.50(2): 229-239. Hattingh, M.J.; Roos, I.M.M. and Mansvelt, E.L. (1989): Infection and systemic invasion of deciduous fruit trees by Pseudomonas syringae in South Africa. Plant-Disease, 73 (10): 784-789.

95

References

Hayward, A.C. and Waterston, J.M. (1969): Pseudomonas syringae. C.M.I. Descriptions of pathogenic fungi and bacteria No.45.Kews Common- wealth Mycological Institute. Heimann, M. (1973): Bacterial canker of apricot trees. Mitteilungen Rebeund Wein, Obstbau und Fruchteverwertung.23(5/6): 391402. Hetherington, S. (2005): Bacterial canker of stone fruit. Research Horticulturist Health Sciences, Science Alliance and Evaluation, Orange Agricultural Institute. Hickey, K.D. and Zwet, T.V. (1995): Efficacy of antagonistic bacteria for control of fire blight on apple. Acta Horticulturae 410: 299-302. Isabel, M., Roos, M. and Hattingh, M. J. (1988): Systemic invasion of immature Sweet Cherry Fruit by Pseudomonas syringae pv. morsprunorum through blossoms. Phytopathology, 78: 26-32. Jacobs, M.B. and Gerstein, M.J. (1960): Handbook of microbiology. Princeton: D. Van. Nostrand Co. p 3-150. Jindal, K.K. and Rana, H.S. (1992): Studies on germplasm resistance and chemical control of bacterial canker of apricot. Plant Disease Research.7(1): 7-10. Ju-Luric (1978): Hand Book of Analytical Chemistry. (Translated from Russian), MIR Publishers, Moscow, USSR, 129-820. Kearns, A.M.; Freeman, R.; Steward, M. and Magee, J.G. (1998): A rapid polymerase chain reaction technique for detecting M tuberculosis in a variety of clinical specimens., Clinical Pathology, 51: 922-924 King, E.O.; Ward, M.K. and Raney, D.E. (1954): Two simple media for the demonstration of pyocyanin and fluorescein. Journal of Laboratory and Clinical Medicine 44: 301-307. Klement, Z. (1977): Bacterial canker and dieback disease of apricots (Pseudomonas syringae van Hall). EPPO-Bulletin.7(1): 57-68.

References

96

Kloepper, T.W.; Leog, J.;Teintze, M. and Schroth, M.N. (1980): Enhanced plant growth by siderophores produced by plant growth-promoting rhizobacteria. Nature 286:885-886. Kotan, R. and Sahin, F. (2002): First record of bacterial canker caused by Pseudomonas syringae pv. syringae, on apricot trees in Turkey. Plant-Pathology. 51(6):798.(Abstract). Krieg, N.R. and Holt, J.R. (1984): Bergey’s manual of systematic bacteriology. Baltimore, Williams and Wilkins, 38-46 pp. Latorre, B.A.; Lillo, C. and Rioja, M.E. (2002): Effects of temperature, free moisture duration and inoculum concentration on infection of sweet cherry by Pseudomonas syringae pv. syringae. Phytoparasitica 30:410-419. Lelliott, R.A. and Stead, D.E. (1987): Method for the diagnosis of bacterial diseases of plants. Methods in plant pathology. Blackwell Scientific Publications, Oxford, London, 216pp. Lindow, S.E.; McGourty, G. and Elkins, R. (1996): Interactions of antibiotics with Pseudomonas fluorescens strain A506 in the control of fire blight and frost injury to pear. Phytopathology, 86: 841-848. Little, E.L.; Bostock, R.M. and Kirkpatrick, B.C. (1998): Genetic characterization of Pseudomonas syringae pv. syringae strains from stone fruits in California. Applied and Environmental Microbiology, 64(10): 3818-3823. Lownsbery, B.F.; English, H.; Noel, G.R. and Schick, F.J. (1977): Influence of nemaguard and lovell rootstocks and Macroposthonia xenoplax on bacterial canker of peach .J.Nematol.9:221-224. Lye, H. (1997): Mechanism of action fungicides in plant disease :An advanced treatise. Vol.(I):How Disease is Managed, Academic Press, New York, pp.239-261.

97

References

Menkissoglu, O. and Lindow, S.E. (1991): Chemical forms of copper on leaves in relation to the bactericidal activity of cupric hydroxide deposits on plants. Phytopathology, 81:12631270. Mohammadi, M.; Ghasemi, A. and Rahimian, H. (2001): Phenotypic characterization of Iranian strains of Pseudomonas syringae pv. syringae Van Hall, the causal agent of bacterial canker disease of stone fruit trees. Journal of Agricultural Science and Technology, 3(1): 51-65. Pabitra-Kalita; Bora, L.C. and Bhagabati, K.N. ( 1996): Phylloplane microflora of citrus and their role in management of citrus canker. Indian-Phytopathology, 49(3): 234-237. Pajk, P. (2004): Possibilities of application of biotic methods for supression of bacteria Erwinia amylovora (Burr.) Winsl. et al. in SloveniaZbornik referatov 1 Slovenskega sadjarskega kongresa z mednarodno udelezbo, Krsko, Slovenia, 24-26marec-2004-Del-2. 2004; 449-453.(c.f. CABI Publishing, UK, 370 pp.) Palleroni, N.J. (1984): Family I. Pseudomonacea,. Wilson, Broadhurst, Buchanan, Krumwide, Rogers and Smith 1917.pp.143-213 In: Bergey's manual of systematic Bacteriology, vol. I ( Krieg, N.R. and Holt,K.G.,eds), Wiliams and Wilkins, Baltimore, M.D. Penrose, L.J. (1998): Disease control in stone fruit in Australia. Pesticide outlook 9 (2) 13-17. (C.F. Review of Plant Pathology 77: No.11). Prunier, J.P.; Gaignard, J.L.; Gardan, L.; Luisetti, J. and Vigouroux, A. (1976): Peach bacterial canker in south-east Franc a disquieting progression. Arboriculture-Fruitiere.(274): 21-24.

References

98

Raaijmakers, J.M.; Vlami, M. and De Souza, J.T. (2002): Antibiotic production by bacterial biocontrol agents. Antonie Van Leeuwenhoek 81: 537-547. Ronald, M.A. (1946): Handbook of media for environmental microbiology. CRC press, Inc.london, p 349. Roos, I.M.M. and Hattingh, M.J. (1983a): Bacterial canker of stone fruit in South Africa. Deciduous-Fruit-Grower.33(11): 405409. Roos, I.M.M. and Hattingh, M.J. (1983b): Fluorescent Pseudomonads associated with bacterial canker of stone fruit in South Africa Plant-Disease.67(11): 1267-1269. Roos, I.M.M. and Hattingh, M.J. (1986a): Weeds in orchards as potential source of inoculum for bacterial canker of stone fruit. Phytophylactica, 18(1): 5-6. Roos, I.M.M. and Hattingh, M.J. (1986b): Pathogenic Pseudomonas spp. In stone fruit buds. Phytophylactica, 18(1): 7-9. Sambrook, J.; Fritsch, E.F. and Maniatis, T. (1989): Molecular Cloning - A Laboratory Manual, 2nd Edition. Cold Spring Habour Laboratory Press, New York. Sands, D.C. and Kolias, D.A. (1974): Pear Blast in Connecticut. Plant Disease Reporter Vol. 58, No.1. Schaad, N.W. (1980): Laboratory guide for identification of plant pathogenic bacteria. American Phytopathological Society, 3340 Pilot Knob Road, St. Paul, Minnesota, 55121. Schmidle, A. and Zeller, W. (1976): The influence of temperature and relative humidity on the infection of Cherry (Prunus cerasus). Phytopathology, 66:274-283. Schoofs, H.; Vandebroek, K.; Pierrard, A.; Thonart, P.; Lepoivre, P.; Beaudry, T. and Deckers, T. (2002): Bacteriocin Serratine-P as a biological tool in the control of fire blight Erwinia amylovora. Mededelingen Faculteit

99

References

Landbouwkundige en Toegepaste Biologische Wetenschappen, Universiteit Gent. 2002; 67(2): 361-368. Scortichini, M.; Marchesi, U.; Dettori, M.T. and Rossi, M.P. (2003): Genetic diversity presence of the syrB gene, host preference and virulence of Pseudomonas syringae pv. syringae strains from woody and herbaceous host plants. Plant Pathology, 52(3): 277-286. Scortichini, M; Pellegrino, S. and Berra, L. (1999): Susceptibility of apricot germplasm to natural infection by Pseudomonas syringae pv. syringae in Piedmont. Rivista di Frutticoltura edi Ortofloricoltura. 61(9): 83-86.(in Italian) .(c.f. CABI Publishing, UK, 230 pp.) Severin, V.; Stancescu, C. and Foad, I. (1986): Bacterial canker of peach in Romania.Analele-Institutului-de-Cercetari-pentruProtectia-Plantelor 19:55-58. (in Romanian).(c.f. CABI Publishing, UK, 378 pp.) Shams-Bakhsh, M. and Rahimian, H. (1997): Comparative study on agents of citrus blast and bacterial canker of stone fruits in Mazandaran. Iranian-Journal-of-Plant-Pathology, 33: (3-4), 44-47. Shane, W.W. and Baumer, J.S. (1987): Population dynamics of Pseudomonas syringae pv. syringae on spring wheat. Phytopathology, 77: 1399-1405. Shurtlett, M.C. and Averre C.W. (1997): The plant disease clinic and field diagnosis of a biotic disease. 223-224 pp. Aps press, Minnesota, USA. Sobiczewski, P. (2001): The present state and perspectives of the protection of orchards and fruit nurseries against bacterial diseases. Progress-in-Plant-Protection. 41(1): 291-298. Sobtczewski, P. and Jones, A.L. (1992): Effect of exposure of freezing temperature on necrosis in sweet cherry shoots References

100

inoculated with Pseudomonas syringae pv. syringae or P. s. pv. Morsprunorum. Plant Dis.,76:447-451. Spotts, R.A. and Cervantes, L.A. (1995a): Factors affecting the severity of bacterial canker of pear caused by Pseudomonas syringae pv. syringae. Plant-Pathology.,44(2): 325-331. Spotts, R.A. and Cervantes, L.A. (1995b): Copper, oxytetracycline, and streptomycin resistance of Pseudomonas syringae pv. syringae strains from pear orchards in Oregon and Washington. Plant Dis., 79:1132-1135. Stall, R.E.; Loscke, D.C. and Jones, J.B. (1986): Linkage of copper resistance and a virulence loci on a self-transmissible plasmid in Xanthomonas campestris pv. vesicatoria. Phytopathology, 76:240-243. Stapp, C. (1961): Bacterial plant pathogens. Oxford University Press.292pp. Süle, S. and Seemüller, E. (1987): The role of ice formation in the infection of sour cherry leaves by Pseudomonas syringae pv. syringae Phytopathology, 77: 173-177. Sundin, G. W., and Bender C. L. (1993): Ecological and genetic analysis of copper and streptomycin resistance in Pseudomonas syringae pv. syringae. Appl. Environ. Microbiol. 59:1018–1024. Sundin, G.W.; Jones, A.L. and Fulbright, D.W. (1989): Copper resistance in Pseudomonas syringae pv. syringae from cherry orchards and its associated transfer in vitro and in planta with a plasmid. Phytopathology, 79:861-865. Suslow, T.V. and Schroth, M.N. (1982): Role of deleterious rhizobacteria as minor pathogens in reducing crop growth.Phytopathol.72:111-115.

101

References

Takanashi, K. (1988): Bacterial canker of Japanese plum caused by Pseudomonas syringae pv. morsprunorum. Bulletin-of-theFruit-Tree-Research-Station,-A-Yatabe,-Japan, 15, 117-125. Takikawa, Y.; Serizawa, S.; Ichikawa, T.; Tsuyumu, S. and Goto, M. (1989): Pseudomonas syringae pv. actinidiae: the causal bacterium of canker of kiwi fruit in Japan.Annals of the Phytopathological Society of Japan. 55(4): 437-444. Tawfik, A.E.; El-Shall, S.A.; Hanna, A.I.; Gomah, A.A.; ElGhareeb, L.A. and Mahmoud, S.M. (2002): Efficacy of bactericides and dormancy-breaking agents on the incidence of fire blight and fruit production of pear in Egypt. Annals Agriculture Science, Faculty of Agriculture, Ain-Shams University, Cairo, Egypt. 47 (1): 389-404. Thornberry, H.H. (1950): A paper disc method for the quantitative evaluation of fungicides and bactericides. Phytopathology 40: 419-429. Tominaga, T.; Takanashi, K.; Nishiyama, K. and Kishi, K. (1983): Identification of the organism causing bacterial canker of Japanese apricot (Prunus mume Sieb. et Zucc.). Annals-of-thePhytopathological-Society-of-Japan. 49(5): 627-632. Toyota, K. and Kimura, M. (2000): Suppression of Ralstonia solanacearum in soil following colonization by other strains of R. solanacearum .Soil Science and plant nutrition 46:449459.[Rev.Plant Pathology 80:105(753)]. Vanneste, J.L. and Yu, J. (1996): Biological control of fire blight using Erwinia herbicola EH252 and Pseudomonas fluorescens A506 separately or in combination. Acta- Horticulturae., 411: 351-354. Vasinauskiene, M. and Baranauskaite, L. (2003): Pseudomonas syringae pv. syringae the causal agent of bacterial canker on pear trees in Lithuania Sodininkyste-ir-Darzininkyste. 22(3):217-22. References

102

Vicente, J.G.; Alves, J.P.; Russell, K. and Roberts, S.J. (2004): Identification and discrimination of Pseudomonas syringae isolates from wild cherry in England European Journal of Plant Pathology.110(4): 337-351. Vock, N.T. (1978): "Handbook of plant diseases" vol.1 and 2 Brisbane: Queensland Department of Primary Industries. Weaver, D.J.; Wehunt, E.J. and Dowler, W.M. (1974): Association of tree site, Pseudomonas syringae , Criconemoides xenoplax, and pruning date with short life of peach trees in Georgia, Plant Dis. Rept., 58:76-79. Weingart, H. and Volksch, B. (1997): Genetic fingerprinting of Pseudomonas syringae pathovars using ERIC., REP., and IS50-PCR. Phytopathology, 87(8/9): 339-345. Wimalajeewa, D.L.S. (1987): Seasonal variation in susceptibility of apricot to Pseudomonas syringae pv. syringae (bacterial canker), and site of infection in apricot and cherry. AustralianJournal-of-Experimental-Agriculture. 27(3): 475-479. Wimalajeewa, D.L.S. and Flett, J.D. (1985): A study of populations of Pseudomonas syringae pv. syringae on stone fruits in Victoria. Plant Pathology, 34(2): 248-254. Wimalajeewa, D.L.S.; Cahill, R.; Hepworth, G.; Schneider, H.G. and Washbourne, J.W. (1991): Chemical control of bacterial canker (Pseudomonas syringae pv. syringae) of apricot and cherry in Victoria. Australian Journal of Experimental Agriculture., 31(5):705-708.

103

References

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