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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

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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.

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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). 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

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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.

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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.

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.

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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 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

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pv. syringae (Pss) but P. syringae pv. 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

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.

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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

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.

Weingart and Volksch (1997) investigated polymerase chain reaction (PCR)

fingerprinting using primers corresponding to repetitive (ERIC and REP) and

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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

IS50-primers 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 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

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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 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 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.

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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.

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 indirect-enzyme-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 non-pathogenic isolates of P. syringae on the basis of

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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:

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 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

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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 (May-Jul.) 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 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

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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, 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 1982-85, 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 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

15

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 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%.

16

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 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 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

17

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.

18

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 Petri-dishs (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).

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.

19

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.

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.

20

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.

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). 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

21

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.

(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:

22

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:

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).

- Peptone yeast extract agar (PYEA).

- Pectate degradation.

- Indole production.

23

- 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

Nucleotide sequence Primer Name 5'-GGTCCCTGAC-3' OPERON A-06 5'-CAATCGCCGT-3' OPERON A-11 5'-CACCGTATCC-3' OPERON D-12 5'-CATACCGTGG-3' OPERON J-08

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) 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

24

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) =100

g/ml 50factor x dilution x OD 260 µ

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 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 isolated Pseudomonas syringe bacteria (Pb-6,

25

Standard curve

0

0.5

1

1.5

2

2.5

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

log of dilution

OD

.

020406080100120140160180200

CFU

OD. CFU

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 40 ْ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 ± 2 ْ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.

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

26

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 incubated at 28 ْ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 Petri-dishes 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,

Cefoperazone, Penicillin, Tetracycline, Cevoran and Chloramphenicol as clear in

Table (3) were tested for their effect on growth of tested pathogenic bacteria and

27

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. 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.

Trade name Active

ingredient Chemical name

Champion (WP) Copper

hydroxide Cu (OH)2 77%

Copper

oxychloride

(WP)

Copper

oxychloride 3Cu (OH)2.CuCl2 50%

kocide 2000 Copper Cu (OH)2 53.8 %

28

(WP) hydroxide

Starner (WP) Oxalinic Acid

5-ethyl-5,8-dihydro-8-oxo-1,3-

dioxolo(4,5) aminoline-7-arboxylic

acid.

Anti-shot (WP) Trial bactericide

WP: Wettable powder

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

Trade name &

molecular weight Active ingredient (%) Chemical formula Producing company

Erythromycin

(M.W. 733.94)

Erythromycin

(80%) C37H67NO13 Pharco

Tetracycline

(M.W. 480.90)

Tetracycline

(83.3%) C22H24N2O8.XH2O Edco

Penicillin

(M.W. 356.37)

Penicillin

(83.3%) C16H17N2O4SNa Cid

Ofloxacin

(M.W. 361.368)

Ofloxine

(83.3%) C18H20FN3O4 Sedico

Chloramphenicol

(M.W. 323.132)

Chloramphenicol

(83.3%) C11H12N2CL2O5

Arabic Co.

Pharmaceuticals

Ceforan

(M.W. 424.386)

Cefuroxime

(83.3%) C16H16N4O8S Aventis

Cefoperazone

(M.W. 667.65)

Cefoperazone

(80%) 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

29

in sodium hypochlorite solution (0.5%) for 2 min., and then rinsed several times in

sterile distilled 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 Disease reduction = Dd C

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) 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.

30

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 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:

31

The number of bacterial spots was estimated as mean number of spots per leaf,

where five leaves were randomly selected from each 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).

32

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 El-Nobaria, 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 Mit-Ghamer, Kafer-El-Gamaal, Toukh and El-Nobaria localities. As well

as, stem samples were collected from Mit-Ghamer, Toukh and Kafer-El-Amar localities

while, the flower samples were collected from El-Nobaria 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 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 Al-8 was isolated from leaf of apricot in the same governorate.

33

Table (4): Source of bacterial isolates collected from different hosts and localities at different

governorates during 2004 growing season.

Code of isolate Locality Governorate Source of

Sample Host

Pb-1 Mit-Ghamr Daqahliya bud Ps-2 Mit-Ghamr Daqahliya stem Pf-4 Mit- Ghamr Daqahliya fruit Pb-5 Kafr-El-Gamal Qalyubiya bud Pb-6 Toukh Qalyubiya bud Ps-13 Toukh Qalyubiya stem

Pl-15 Moshtohor Qalyubiya leaf Pw-9 El-Nobaria Behera flower Pb-14 El-Nobaria Behera bud

Peach

Al-7 El-Amar Qalyubiya leaf

As-12 Kafr-El-Amar Qalyubiya stem Al-8 El-Nobaria Behera leaf

Apricot

Rs-3 Mit-Ghamr Daqahliya stem Moshtohor Qalyubiya flower

Pear

Lb-11 Mit-Ghamr Daqahliya bud Apple

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 Pb-14)

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 yellow-brown discoloration on inoculated cotyledons. (3) on detached fruits of

34

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.

Table (5): Pathogenicity test and virulence of isolates on some differential hosts.

Reaction Code of isolate Tobacco Beans Apricot* Peach* Pear*

Pb-1 - - - - - Ps-2 - - - - - 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 - - - - -

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

35

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). 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.

36

Table (6): Identification of the isolated cankered bacteria which reacted positively on different hosts using the morphological and cultural characters.

Bacterial isolates No. Test

Rs-3 Pb-6 Al-7 Al-8 Lb-11

As-12

Pb-14

Growth and colour on

common media

+ white

+ translucence

+ yellow

+ translucence

+ white

+ white

+ translucence

Gram reaction - - - - - + -

Pigments (K.B) - +

fluorescent -

+ fluorescent

- - +

fluorescent

Spore production

- - - - - + -

Bacterial genera * *

[E] [P] [X] [P] [E] [B] [P]

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

37

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. Table (7): Identification of isolated bacteria from infected hosts, to determine species

using physiological and biochemical tests.

Reaction

Pb-14 As-12 Lb-11 Al-8 Al-7 Pb-6 Rs-3 Test

- + - - + - - Starch hydrolysis

- + + - - - + 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 f o o o f o/f test

Utilization from :

+ - - + d + - Lactose

+ + - + d + - Dextrose

+ d d + - + d Sorbitol

+ - - + + + - Mannose

+ - + + + + + Sucsenic

Ps. Bp. Ea Ps. Xc. Ps. Ea. Bacterial isolate identified

**Ea = E. amylovora , Ps = Pseudomonas syringae

Xc = Xanthomonas campestris , Bp = Bacillus polymyxa *F = fermentative metabolism ; O =oxidative metabolism d = different reaction

38

2. Verification the identification using PCR (Polymerase Chain Reaction)

techniques:

From the above results, the three isolates i.e. Pb-6, Al-8 and Pb-14 were

identified as P. syringae and used only in this test. The traditional identification of

these isolates was confirmed using PCR-RAPD 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, OP-A-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.

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:

39

00.20.40.60.8

11.21.4

15 20 25 30 35 40

Temperature

O.D

.

Pb-6 Al-8 Pb-14

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.

Table (8): Effect of different temperature degrees on growth of Pseudomonas

syringae, in vitro.

CFU*

solid media

Optical density (O.D.) Liquid media

Isolates

Temperature (ºC)

Pb-6 Al-8 Pb-14 Mean Pb-6 Al-8 Pb-14 Mean 15 10 8 4 7.3 0.25 0.20 0.15 0.20 20 32 30 18 26.6 0.59 0.47 0.35 0.40 25 52 40 41 44.3 1.31 1.22 1.19 1.24 30 34 32 29 31.6 0.89 0.78 0.77 0.81

35 25 27 23 25.0 0.72 0.74 0.68 0.71 40 0.0 0.0 0.0 0.0 0.00 0.00 0.00 0.00

Mean 25.5 22.8 19.1 22.4 0.62 0.56 0.52 0.56

• Colony forming unit/plate LSD at 5 % for Isolates 0.015 Temperature 0.021 Interaction 0.036

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

40

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 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.

CFU*

solid media Optical density (O.D.)

Liquid media Isolates

pH values

Pb-6 Al-8 Pb-14 Mean Pb-6 Al-8 Pb-14 Mean 6 1 2 3 2.0 0.11 0.10 0.11 0.10

6.5 16 21 18 18.3 1.03 0.99 1.01 1.01 7 13 23 15 17.0 0.52 0.45 0.50 0.49

7.5 2 2 3 2.3 0.12 0.10 0.11 0.11 8 1 1 2 1.3 0.05 0.05 0.07 0.05

Mean 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 0.012 pH 0.015 Interaction 0.026

41

0

0.2

0.4

0.6

0.8

1

1.2

6 6.5 7 7.5 8

pH

O.D

.

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.

42

01

23

45

67

89

10

50 65 73 80 84 95 100

RH %

No. b

acte

ria ce

lls/p

late

Pb-6 Al-8 Pb-14

Table (10): Effect of relative humidity (R.H) on growth of Pseudomonas syringae

isolates, in vitro.

CFU* Isolates RH %

Pb-6 Al-8 Pb-14 Mean 50 21 29 30 26.6 65 29 30 35 31.3 73 31 34 40 35.0 80 58 54 61 57.6 84 59 55 63 59.0 95 80 86 81 82.3 100 83 92 86 87.0

Mean 51.7 54.2 56.5 54.1

*Colony forming unit/ plate

LSD at 5 % for Isolates 0.171 RH 0.261 Interaction 0.453

Fig.(9):Relationship between relative humidity (R.H) and growth of Pseudomonas

syringae isolates, in vitro.

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

43

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.

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 Isolates

Chemical compound

Rate (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 13.3 800 9 10 9 9.3

Starner

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 10.0 800 9 8 8 8.3

Champion

Mean 10.3 10.3 10.3 10.3 200 20 18 19 19.0 400 18 17 18 17.6 600 18 16 16 16.3 800 16 15 14 15.0

Kocide 2000

Mean 18.0 16.5 16.8 14.4 200 11 11 11 11.0 400 10 10 10 10.0 600 9 9 9 9.0 800 8 8 8 8.0

Copper oxychloride

Mean 9.5 9.5 9.5 9.5 200 10 12 11 11.0 400 9 8 9 8.6 600 7 6 6 6.3 800 5 4 5 4.6

Anti - shot

Mean 7.8 7.5 7.8 7.7 Control 0.0 30 28 25 27.6

LSD at 5 % for Chemical 0.037 Rate 0.037

44

Isolates 0.030 Interaction N.S.

Fig. (10): Relationship between growth of Pseudomonas syringae isolates as count of

bacterial cells and different rates of chemical compounds, in vitro.

Starner

0

0.5

1

1.5

2

0 200 400 600 800

Rate

Bct

eria

l cel

ls c

ou

nt

PB6 AL8 PB14

Champion

00.20.40.60.8

11.21.4

0 200 400 600 800

Rate

Bac

teri

al c

ells

co

un

t

PB6 AL8 PB14

Kocide 2000

0

0.5

1

1.5

2

2.5

0 200 400 600 800

Rate

Bac

teri

al c

ells

co

un

t

PB6 AL8 PB14

Copper oxychloride

0

0.2

0.4

0.6

0.8

1

1.2

0 200 400 600 800

Rate

Bac

teri

al c

ells

co

un

t

PB6 AL8 PB14

Anti - shot

0

0.5

1

1.5

0 200 400 600 800

Rate

Bac

teri

al c

ells

co

un

t

PB6 AL8 PB14

45

Table (12): Effect of different chemical compounds at different rates on growth (inhibition zone (mm)) of Pseudomonas syringae isolates, in vitro.

Inhibition zone (mm) Isolates

Chemical compound

Rate (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 15.6 800 18 16 21 18.3

Starner

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 20.0 800 22 24 25 23.6

Champion

Mean 17.7 17.8 18.7 18.0 200 5 11 4 6.6 400 8 14 11 11 600 10 19 13 14 800 14 22 16 17.3

Kocide 2000

Mean 9.2 16.5 11.0 12.2 200 18 15 17 16.6 400 23 21 20 21.3 600 26 23 28 25.6 800 28 26 31 28.3

Copper oxychloride

Mean 23.7 21.2 24.0 22.9 200 25 24 30 26.3 400 32 32 31 31.6 600 35 34 35 34.6 800 40 40 40 40

Anti - shot

Mean 33.0 32.5 34.0 33.1 Control 0.0 0.0 0.0 0.0 0.0

LSD at 5 % for Chemical 0.043 Rate 0.043 Isolates 0.033 Interaction 0.165

46

Fig. (11): Relationship between growth of Pseudomonas syringae isolates as

inhibition zone (mm) and different rates of chemical compounds, in

vitro. 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

Champion

0

0.5

1

1.5

2

2.5

3

0 200 400 600 800

Rate

Inhi

bitio

n zo

ne (m

m)

Pb-6 Al-8 Pb-14

Kocide 2000

0

0.5

1

1.5

2

2.5

0 200 400 600 800

Rate

Inhi

bitio

n zo

ne (m

m)

Pb-6 Al-8 Pb-14

Anti - shot

0

2

4

6

0 200 400 600 800

Rate

Inh

ibit

ion

zo

ne

(mm

)

Pb-6 Al-8 Pb-14

Starner

0

0.5

1

1.5

2

2.5

0 200 400 600 800

Rate

Inhi

bitio

n zo

ne (m

m)

Pb-6 Al-8 Pb-14

Copper oxychloride

0

1

2

3

4

0 200 400 600 800

Rate

Inh

ibit

ion

zo

ne

(mm

)

Pb-6 Al-8 Pb-14

47

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.

48

Table (13): Effect of different antibiotics, at different rates on growth (count of bacterial cells) of Pseudomonas syringae isolates , in vitro.

Bacterial cells count Isolates Antibiotic Rate

(ppm) Pb-6 Al-8 Pb-14 Mean

50 23 23 23 23.0 100 21 21 20 20.6 150 20 20 19 19.6 200 18 16 17 17.0

Erythromycin

Mean 20.5 20.0 19.8 20.1 50 18 17 17 17.3 100 12 13 12 12.3 150 11 10 10 10.3 200 7 6 7 6.6

Ofloxine

Mean 12.0 11.5 11.5 11.6 50 19 18 18 18.3 100 16 14 16 14.6 150 11 10 12 11.0 200 7 6 7 6.6

Cefoperazone

Mean 13.3 12.0 13.3 12.8 50 25 23 24 24.0 100 24 22 23 23.0 150 21 19 21 20.0 200 17 16 16 16.3

Penicillin

Mean 21.8 20.0 21.0 20.9 50 22 22 21 21.6 100 20 19 18 19.0 150 19 18 17 18.0 200 15 16 15 15.3

Tetracycline

Mean 19.0 18.8 17.8 18.5 50 20 21 19 20.0 100 18 17 17 17.3 150 15 16 15 15.3 200 14 14 13 13.6

Cevoran

Mean 16.8 17.0 16.0 16.6 50 21 21 19 20.3 100 20 19 16 18.3 150 16 15 15 15.3 200 14 13 12 13.0

Chloramphenicol

Mean 17.8 17.0 15.5 16.7 Control 0.0 30 28 25 27.6

LSD at 5 % for Isolates 0.024 Antibiotics 0.037 Rate 0.032 Interaction N.S.

49

Erythromycin

0

0.5

11.5

2

2.5

0 50 100 150 200

Rate

Bac

terial

cel

ls c

ount

PB6 AL8 PB14

Ofloxine

0

0.5

1

1.5

2

0 50 100 150 200

RateB

acte

rial

cel

ls c

ou

nt

PB6 AL8 PB14

Cefoperazone

0

0.5

1

1.5

2

0 50 100 150 200

Rate

Bac

terial

cel

ls

cou

nt

PB6 AL8 PB14

penicillin

00.5

11.5

22.5

3

0 50 100 150 200

Rate

Bac

terial

cel

ls c

ount

PB6 AL8 PB14

Tetracycline

0

0.5

1

1.5

2

2.5

0 50 100 150 200

Rate

Bac

terial

cel

ls c

ou

nt

PB6 AL8 PB14

Cevoran

0

0.5

1

1.5

2

2.5

0 50 100 150 200

Rate

Bac

terial

cel

ls c

ount

PB6 AL8 PB14

Chloramphenicol

0

0.5

1

1.5

2

2.5

0 50 100 150 200

Rate

Bac

teri

al c

ells

coun

t

PB6 AL8 PB14

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

50

Table (14): Effect of different antibiotics, at different rates on growth (inhibition zone (mm)) of Pseudomonas syringae isolates, in vitro.

Inhibition zone (mm) Isolates Antibiotic Rate

(ppm) Pb-6 Al-8 Pb-14 Mean

50 4 5 4 4.3 100 7 7 6 6.6 150 8 10 8 9.0 200 11 13 11 11.6

Erythromycin

Mean 7.5 8.8 7.3 7.8 50 8 13 10 10.3 100 16 18 17 17.0 150 22 23 22 22.3 200 28 27 27 27.3

Ofloxine

Mean 18.5 20.3 19.0 19.2 50 7 7 8 7.3 100 9 10 10 9.6 150 14 16 14 14.6 200 18 18 17 17.6

Cefoperazone

Mean 12.0 12.8 12.3 12.3 50 2 2 2 2.0 100 3 3 3 3.0 150 4 4 4 4.0 200 6 6 6 6.0

Penicillin

Mean 3.8 3.8 3.8 3.8 50 8 8 9 8.3 100 9 9 10 9.3 150 10 10 11 10.3 200 11 11 12 11.3

Tetracycline

Mean 9.5 9.5 10.5 9.8 50 11 13 12 12.0 100 13 15 15 14.3 150 18 17 18 17.6 200 19 19 19 19.0

Cevoran

Mean 15.3 16.0 16.0 15.7 50 11 10 10 10.3 100 15 17 17 16.3 150 17 19 19 18.3 200 20 20 21 20.3

Chloramphenicol

Mean 15.8 16.5 16.7 16.3 Control 0.0 0.0 0.0 0.0 0.0

LSD at 5 % for Isolates 0.247 Antibiotics 0.378 Rate 0.314 Interaction 1.400

51

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

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

Ofloxine

0

10

20

30

0 50 100 150 200

Rate

Inh

ibit

ion

zo

ne

(mm

)

Pb-6 Al-8 Pb-14

Erythromycin

0

5

10

15

0 50 100 150 200

Rate

Inhib

itio

n z

one

(mm

)

Pb-6 Al-8 Pb-14

penicillin

0

5

10

0 50 100 150 200

Rate

Inh

ibit

ion

zo

ne

(mm

)

Pb-6 Al-8 Pb-14

Cefoperazone

0

10

20

0 50 100 150 200

RateIn

hib

itio

n z

on

e (m

m)

Pb-6 Al-8 Pb-14

Cevoran

05

1015

20

0 50 100 150 200

Rate

Inh

ibit

ion

zo

ne

(mm

)

Pb-6 Al-8 Pb-14

Tetracycline

0

5

10

15

0 50 100 150 200

Rate

Inhib

itio

n z

one

(mm

)

Pb-6 Al-8 Pb-14

Chloramphenicol

0

50

0 50 100 150 200

Rate

Inh

ibit

ion

zo

ne

(mm

)

Pb-6 Al-8 Pb-14

52

0

5

10

15

20

25

Pb-6 Al-8 Pb-14

Inhib

itio

n zone

(mm

)

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

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 Bioagent

Pb-6 Al-8 Pb-14

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

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

53

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 – 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.

LSD at 5 % for 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

Chemical compounds

Rate ( mg/l )

A Disease

reduction (%)

B Disease

reduction (%)

C Disease

Reduction (%)

Copper oxychloride

400 600 800

6.0 5.6 4.5

25.0 30.0 43.7

7.2 7.0 5.7

10.0 12.0 28.7

7.0 6.6 5.5

12.0 17.5 31.3

Champion 400 600 800

6.3 5.3 4.3

21.3 33.7 46.2

7.1 6.6 5.3

11.2 17.2 33.7

6.5 5.8 5.2

18.8 27.5 35.0

Starner 400 600 800

5.6 5.3 4.4

30.0 33.7 45.0

7.0 6.4 5.6

12.0 20.0 30.0

6.9 6.2 5.4

13.8 22.5 32.5

Check 0.0 8.0 0.0 8.0 0.0 8.0 0.0

54

Copper oxychloride

0

10

20

30

40

50

0 400 600 800

Dis

ease

red

uct

ion

(%

)

A c B

Starner

0

10

20

30

40

50

0 400 600 800

Dis

ease

red

uctio

n %

A B c

Champion

0

10

20

30

40

50

0 400 600 800

Dis

ease

red

uct

ion

(%

)

A B 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.

55

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 0.622 Rate 0.718 Antibiotics 0.622 Interaction 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

56

ofloxine

0

10

20

30

40

50

60

70

0 500 750 1000

Rate

Dis

ease

red

uct

ion

(%

)

A B c

Chloramphenicol

0102030

40506070

0 500 750 1000

Rate

Dis

ease

red

uct

ion

(%

)

A B C

cefoperazone

0

10

20

30

40

50

60

70

0 500 750 1000

Rate

Dis

ease

red

ucti

on (%

)

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.

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

57

0

5

10

15

20

25

30

35

40

45

50

(Bs3) (Pf1) (Sm)

Bioagent

Dis

ease

sev

erity

%

A B C

effective, where the percentages of disease reduction ranged from 20.0 – 42.5 and

from18.8 – 32.5%, respectively.

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

Bioagents A Disease

reduction (%)

B Disease

reduction (%)

C Disease

reduction(%)

Bacillus subtilis (Bs-3) 6.0 25.0 4.6 42.5 6.4 20.0

Pseudomonas fluorescens (Pf -5) 6.0 25.0 5.4 32.5 6.5 18.8

Serratia marcescns (Sm-1) 5.6 30.0 4.4 45.0 6.2 22.5

Check 8.0 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

Fig. (17): Relationship between different bioagents on reducing the percentage of

bacterial canker disease, using artificial inoculation on peach fruits, in

vitro.

6. Effect of bactericides on bacterial canker disease, under artificial inoculation conditions:

58

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.

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.

Disease reduction

(%)

Infection (%)

Mean No. of spots/leaf

Rate

Treatment

13.8 50.0 10.7 800 mg/l Starner

31.0 40.0 8.8 800 mg/l Copper oxychloride

36.2 37.0 8.0 800 mg/l Champion

22.4 45.0 9.9 200 ppm Cefoperazone

27.0 42.0 9.4 200 ppm chloramphenicol

0.0 58.0 15.0 0.0 check

2.40 3.24 1.62 LSD at 5 %

59

0

0.2

0.4

0.6

0.8

1

1.2

Starne

r

Copp

er oxy

chlorid

e

Cham

pion

Cefope

razo

ne

Chloramph

enicol

chec

k

mea

n of

spo

ts

0

10

20

30

40

50

60

70

Redu

ctio

n of

dis

ease

inci

denc

e

Mean No. of spots/leafDisease reduction (%) Infection (%)

0

10

20

30

40

50

60

Starne

r

Cop

per o

xych

lorid

e

Cha

mpion

Cefop

erazon

e

Chloram

phen

icol

check

Red

uctio

n of

disea

se in

cide

nce

Infection (%) Disease reduction (%)

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. 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.

Disease reduction

(%)

Infection (%)

Rate

Treatment

12.5 44.3 800 mg/l Starner 27.7 36.6 800 mg/l Copper oxychloride 30.8 35.0 800 mg/l Champion 20.8 40.1 200 ppm Cefoperazone 24.9 38.0 200 ppm Chloramphenicol 0.0 50.6 0.0 Check (water)

2.054 2.054 LSD at 5 %

60

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

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 (one-year-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.

Disease parameters on Shoots Leaves

Disease reduction

(%)

Infection (%)

Disease reduction

(%)

Infection (%)

Mean No. of

spots/leaf

Treatment

7.5 46.8 10.3 52 11.7 Bacillus subtilis (Bs-3)

11.1 45.0 12.1 51 10.9 Pseudomonas fluorescens

(Pf-5)

12.6 44.2 13.8 50 10.6 Serratia

marcescens (Sm-1)

0.0 50.6 0.0 58.0 15.0 Check (water)

1.412 3.208 1.633 2.824 1.631 LSD at 5 %

61

(B)

010203040506070

(Bs3) (Pf5) (Sm1) check

%

Infection (%) Disease reduction (%)

(A)

0

10

20

30

40

5060

70

(Bs3

)

(Pf5)

(Sm

1)

chec

k

Mea

n o

f sp

ots

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

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.

62

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 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 Al-8 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

63

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 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 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,

64

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 fifty-four

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 -30Cْ, 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 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.

65

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 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.

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

66

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 bio-

agents 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) 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.

67

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.

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,

68

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, 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, Al-8 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.

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

69

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 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.

70

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العربيالملخصزادت المساحة المنزرعة من أشجار الفاكهة تحت الظروف المصرية بسبب التوسع في زراعتها في

اآلونة األخيرة لإلصابة بالعديد من األمراض في ه األشجار هذت ولقد تعرض.األراضي المستصلحة حديثاً من البكتيريع مصادرها ، و يعتبر مرض التقرح المنزرعة وتنواألصناف فيالبكتيرية وذلك بسبب التباين

ل تحت الظروف المصرية مما سبب العديد من المشاكليصا المحلتلكنتشارا وضررا إأكثر األمراض ري لتلك األمراض من خالل إجراء يالبكت وقد أجرى هذا البحث بغرض التعرف على المسبب. نللمزارعي

المختلفة و كذلك المواسم مناطق الزراعة و في فاكهة مختلفة أجزاء مختلفة من أشجار العزل من اتعملي المزرعية و المورفولوجية و الحيوية للعزالت المختلفة و تأكيد عملية االختبارات بإجراء البكتيرياتعريف تلك

خان الد لهذه العزالت على نباتات اإلمراضية القدرة إختبار وكذلك إجراء PCRالـ باستخدام إختبارالتعريف فاعلية بعض المركبات الكيماوية و المضادات الحيوية و إختبارو . والفاصوليا والمشمش والخوخ والكمثري

علي ثمار بصورة منفردة على شدة اإلصابةإختبارهاالكائنات المضادة لتثبيط نمو البكتريا الممرضة و أيضاً .الخوخ وشتالت المشمش تحت ظروف العدوى الصناعية

- :يليخيص النتائج المتحصل عليها فيما و يمكن تلتثقب حيث سجل وجود بعض أشجار الفاكهة على البكتيري التقرح لوحظت األعراض المميزة لمرض - 1

بنية مستديرة كما لوحظ تقرحات ذبول علي الفروع فلوحظالفروعأما على . لفحة البراعماألوراق وعلي هذا باإلضافة إلي اإلفرازات الصمغية . وذات ملمس خشنوالساقكل من الجذع سطح لي قليالً عومنخفضة

. اللزجة المميزة لعرض التقرح البكتيريوذلك من براعم . المختلفة من ثالث محافظات هي القليوبية والبحيرة والدقهلية المصابةتم جمع العينات - 2

وأجريت عملية العزل مستخدما .ثريوأزهار وثمار وأوراق وسيقان كل من المشمش والخوخ والتفاح والكم .في ذلك بيئات متخصصة وتقليدية

الدخان و الفاصوليا و ( عوائل نباتية مختلفة هي القدرة المرضية لخمسة عشر عزله علي نفذ إختبار- 3موجبة ) Pb-14 و Al-8 وPb-6 ( أظهرت النتائج أن ثالث عزالت ). المشمش و الخوخ و الكمثري

) Lb-11 و Rs-3 (كما وجد أن عزلتي . ة علي إحداث اإلصابة لكل النباتات محل الدراسةلإلختبار أي قادركانت ) As-12وAl-7 (كما وجد إن عزلتي . كانت قادرة علي إحداث إصابة في نباتات الدخان والكمثري

غير قادرة قادرة علي إحداث اإلصابة للدخان فقط في حين أن باقي العزالت كانت سالبة لهذا اإلختبار أي . علي إحداث اإلصابة

نبات الدخان بقع مائية يعلي العوائل المختلفة حيث ظهر عل اإلمراضية سجلت أعراض إختبار القدرة - 4كما أظهرت علي ، ثم تصبح جافة ذات لون بني فاتح وذلك بعد ثالثة أيام . ساعة48في األنسجة المعداة بعد

هذا باإلضافة إلي ظهور أعراض . ون بني مصفر علي الفلقات المعداةبادرات الفاصوليا بقع موضعية ذات لعلي ثمار الخوخ المفصولة في صورة بقع سوداء مع إفرازات صمغية ذات رائحة كريهة علي شتالت كل

عندما أجريت العدوى ، كما أظهرت بقع بنية موضعية علي األوراق المعداة. من الخوخ والمشمش والكمثري .هرت منطقة ميتة بنية سوداء علي األفرع وحدوث موت من أعلي ألسفلبالرش ولكن أظ

المورفولوجية أن جميع العزالت البكتيرية المتحصل عليها كانت عصوية دراسة الصفات أظهرت-5 تفاعل موجب مع أعطت(As-12) ماعدا عزلة واحدة هي . مع صبغة جرام وغير متجرثمةةقصيرة سالب . طويلة ومتجرثمةوكانت عصويةصبغة جرام

عند تنمية مرتفعة نوعاً ذات حافة كاملة و ملساء– مستديرة – العزالت مستعمرات بيضاء كل ت أظهر- 6 أعطت ثالث . (YDC) على بيئة آجار مستخلص الخميرة و الدكستروز و كربونات الكالسيوم العزالت

كانت وعة ذات حافة كاملة ملساء مستعمرات بيضاء مستديرة مرتفAs-12)وLb-11 و(Rs-3 عزالت هي كما كانت مستعمره واحده ذات لون نصف شفافة ) Pb-14 وAl-8 وPb-6(المستعمرات في ثالث عزالت

وجد أن بعض المستعمرات تفرز صبغة ( KB )دراسة الخواص المزرعية علي بيئة وب) Al-7( أصفر ).Pb-14و Al-8 وPb-6(فلورسنتية وهذه العزالت هي

أن العزالت التي تم عزلها وتعريفها طبقا للصفات المورفولوجية والمزرعية أنها تضم أربعة أجناس وجد - 7 .Erwinia sp. :-Bacillus sp. - Xanthomonas sp. -. Pseudomonas sp هي

ختبار إ مع ةنتيجة موجب Lb-1) وRs-3 (عزالتت عند دراسة الخواص الفسيولوجية والحيوية أعط- 8 السكروز ختزالإ ،إختبار فرط الحساسية للدخان،سالة الجيالتين، تكوين الليفان إ، % 3د البوتاسيوم هيدروكسي

NaCl 5%. والنمو علي

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،إختبار تحليل األحماض الدهنية،تحليل البكتات، نتيجة سالبة مع تحليل النشا السابقة العزالتت أظهر - 9نتاج ،إنتاج اليورييز، إختزال النتراتإ ،نتاج االندول ، إ كبريتيد الهيدروجينإنتاج أحمر الميثيل،

،أختبار تحليل األرجنين،العفن الطري في البطاطس ،ختبار األكسيديزم، إ° 50،القدرة علي النمو علي صبغاتo/f. عند دراسة الخواص الفسيولوجية وهذه العزالت أعطت رد فعل مختلف في تحليل مصادر الكربون ،

.والحيوية صبغاتإنتاج تحليل البكتات،ختبارإ مع ة نتيجة موجبPb-14 ) و Al-8 وPb-6 ( عزالتت ط أع- 10

السكروز ، والنمو علي ختزالإ ،إختبار فرط الحساسية للدخان،، تكوين الليفان % 3هيدروكسيد البوتاسيوم ، NaCl 5% عند دراسة الخواص الفسيولوجية والحيويةوذلك .

،تحليل البكتات، نتيجة سالبة مع تحليل النشا Pb-14 ) و Al-8 وPb-6( السابقة ت العزالت أظهركما - 11، ختزال النتراتإ ،نتاج االندول، إ كبريتيد الهيدروجينإنتاج ، إختبار أحمر الميثيل،تحليل األحماض الدهنية

بار القدرة علي إحداث ، إختختبار األكسيديزم، إ° 50، القدرة علي النمو علي نتاج صبغاتإنتاج اليورييز، إ، عند دراسة والدكستروززالالكتوستفادة من واإل.o/f، أختبار تحليل األرجنينالعفن الطري في البطاطس،

.الخواص الفسيولوجية والحيوية Rs-3( أن عزلتين من العزالت البكتيرية الممرضة ختبارات الفسيولوجية والكيموحيويةأوضحت اإل - 12 Pseudomonasهم ) Pb-14و Al-8 وPb-6 ( ، وعزالت Erwinia amylovoraهما ) Lb-11و

syringae والعزلة (Al-7) كانت Xanthomonas campestris والعزلة (As-12) كانت Bacillus polymyxa.

-PCRباستخدام ) Pb-6 وAl-8 وPb-14 ( للعزالت تم تأكيد اختبارات التعريف التقليدية - 13RAPD بواسطة أربعة بريمر والذي أوضحت درجة تشابه بين الثالث عزالت وذلك .

أجريت دراسة تأثير بعض العوامل البيئية مثل درجات الحرارة ومستويات مختلفة من الحموضة - 14 تحت ظروف المعمل وأظهرت النتائج أن Pseudomonas syringaeوالرطوبة النسبية علي نمو بكتيريا

. م° 35م بينما كانت درجة الحرارة العظمي لنفس الميكروب هي °25ي للنمو كانت درجة الحرارة المثليمكنها النمو في مدي واسع من درجات Pseudomonas syringae وقد أظهرت النتائج أن بكتيريا- 15

.6.5 عند pH وكان رقم الحموضة األمثل 7.5 - 6الحموضة يتراوح بين هي الدرجة المثلي والتي حدث عندها أفضل نمو للبكتيريا % 100-80ة وتعتبر درجة الرطوبة النسب- 16

في حين لم يحدث نمو للبكتيريا عند درجات % 50الممرضة بينما كانت أدني درجة رطوبة نسبية للنمو عند .رطوبة نسبية أقل من ذلك

تريا الممرضة مقارنة البكضد نموتأثير مثبط )المبيدات والمضادات الحيوية (وقد أظهرت جميعها - 17 .ستخدام تلك المبيدات البكتيريةإرتفعت كفاءة التثبيط بارتفاع معدالت إوقد . وذلك في المعملبمعاملة المقارنة

-Anti أظهر كل منكما. التأثير المثبط للمضادات الحيوية كان أكثر تأثيرا من المبيدات الكيماوية- 18shot و كمبيد Ofloxineأعلي كفاءة لتثبيط نمو بكتريا كمضاد حيوي .

Erythromycin Penicillin و Starner 20% وKocide 2000بينما كانت المبيدات البكتيرية - 19 Champion 77% ان البكتيرينذات تأثير تثبيطي ضعيف ، في حين كانت المبيدا Tetracyclineو وCevoran و Cefoperazone والمضادات الحيوية Copper oxychloride 77%و

Chloramphenicolذات تأثير تثبيطي متوسط. كفاءة بلبكتريا الممرضة ي ل تأثير تثبيطSerratia marcescens (Sm-1) أظهرت بعض عزالت بكتريا- 20

Bacillus subtilis (Bs-3) وP. fluorescens (Pf-1, Pf-5) مثل في حين أن بعض العزالت.أعلي Pseudomonas putida (Pt-3) في حين أن تأثير. متوسط ضد البكتيريا الممرضةكانت ذات تأثير

.بكترياال في تثبيط نمو اكان ضعيفإلي خفض المرض مقارنة مع ) مبيدات كيماوية و مضادات حيوية ( أدي إستخدام المبيدات البكتيرية - 21

المرض بدرجة كما اختزل. إستخدامهاة تلك المبيدات مع زيادة معدالت ءوزادت كفا. معاملة المقارنةم تلك المبيدات قبل العدوى بالمسبب المرضي في حين أن المضادات الحيوية كانت أكثر املحوظة عند استخد

.فعالية من المبيدات الكيماوية في خفض نسبة المرض وكذلك. بالمرض اإلصابة كفاءة أعلي في خفض Champion و Chloromphincol ي أظهر مركب- 22

كفاءة Starner و Copper oxychloride وOfloxine وCefoperazone ات مركبتأظهر .تحت ظروف العدوى الصناعية علي ثمار الخوخ معمليا بالمرض اإلصابةفي خفض متوسطة

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B. subtilis (Bs-3) وP. fluorescens (Pf-5) مثل اختبرت بعض األنواع البكتيرية المضادة- 23 S. marcescns (Sm-1) لدراسة كفاءتها في مكافحة مرض التقرح البكتيري تحت ةحيوية مضادكعوامل

، وقد أظهرت جميع العوامل الحيوية المختبرة تأثير علي ثمار الخوخ في المعمل الصناعيةالعدوىظروف .بمعاملة المقارنة مقارنة بالمرض اإلصابةواضح في خفض

في . يريا المضادة قبل العدوى بالميكروب الممرض أكثر تأثيرا في تقليل المرضالمعاملة بالبكت كانت - 24 B. subtilis و P. fluorescens (Pf-5) كانت أكثر تأثيرا منS. marcescns (Sm-1)حين أن عزلة

(Bs-3) . ضد المرض

جربة اختزال اإلصابة المضادة التي ذكرت سابقا في تالمبيدات البكتيرية والعوامل الحيوية أمكن تقيم - 25 الصناعيةالعدوى تحت ظروف )عمر سنة( مرة ثانية علي شتالت المشمش صنف كانينو خ ثمار الخوعلي

كمعاملة رش أو حقن األفرع ( بالبكتريا الممرضةالعدوىكمعاملة رش للمجموع الخضري قبل في الحقل . ساعة24بـ ) الصغيرة

والعوامل الحيوية المضادة كفاءة عالية في خفض المرض بالمقارنة مع أظهر استخدام المبيدات البكتيرية - 26. اإلصابةفي خفض المضادة العوامل الحيوية البكتيرية أفضل منتوقد وجد أن المبيدا. معاملة المقارنة

سبة في تقليل اإلصابة بالنواضحا ) المضادة المبيدات البكتيرية والعوامل الحيوية( اإلثنين كالوكان تأثير .لألوراق عن الفروع المصابة

أكثر تأثيرا في تقليل اإلصابة المرضية في Copper oxychlorideو Champion كل من وجد أن- 27باإلضافة . كان متوسط التأثير في تقليل المرضCefoperazone و Chloramphenicolحين أن كال من

. أقل تأثيراكانStarner المبيد إلي أن ذات تأثير متوسط في تقليل P. fluorescens و بكتيريا S. marcescnsن كال من بكتيريا وجد أ- 28

.لكذفي قل تأثيرا كانت األ B. subtilis المرض في حين أن بكتيريا