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Pear Blossom Blast Caused by Pseudomonas syringae pv. syringae in China
Agricultural Sciences in China
September 2008
2008, 7(9): 1091-1096
Pear Blossom Blast Caused by Pseudomonas syringae pv. syringae in China QIU Wen1, HUAI Yan1,2, XU Fu-shou3, XU Li-hui1, XIE Guan-lin1, LI Bin1, YU Shan-hong1 and LIU Jun-ying1 1
Institute of Biotechnology, Zhejiang University, Hangzhou 310029, P.R.China
2
Zhejiang Provincial Agricultural Bureau, Hangzhou 310029, P.R.China Hangzhou Plant Protection Station, Hangzhou 310020, P.R.China
3
Abstract This study was done to determine the causal organism of the pear blossom and bud blast in China. It was identified by a bacteriological test, electro-microscopic observation, Koch’s postulate test, Biolog, fatty acid methyl esters (FAMEs), and a polymerase chain reaction (PCR) test, and compared with the standard reference strains. Six representative strains out of 20 pathogenic bacterial isolates from 16 diseased samples showed characteristics similar to three standard strains of Pseudomonas syringae pv. syringae from Belgium. They were identified as P. syringae pv. syringae with a Biolog similarity of 0.57-0.86 and FAMEs similarity of 0.58-0.81. The bacterium was reisolated from the symptomatic plants and blossoms. Identification as P. syringae pv. syringae was confirmed by using PCR primers and sequence tests, and compared with the above-mentioned results. The data supported the fact that the pear blossom and bud blast in China could be caused by P. syringae pv. syringae. Key words: pear blossom blast, pear bud blast, Pseudomonas syringae pv. syringae, confirmation
INTRODUCTION Fire blight, caused by Erwinia amylovora, is not only the world’s first-reported plant bacterial disease, but also one of the most serious diseases in fruit trees. It has been listed as a plant quarantine disease in China and many other countries, and disease management for this is being undertaken (Stoger et al. 2006; Chen et al. 2007). However, some early symptoms caused by E. amylovora are very similar to those of another bacterial disease on pear, namely, pear blossom blast. Until now only three bacterial diseases of pear have been recorded in China, which are crown gall of pear caused by Agrobacterium tumefaciens, bacterial rust, and fruit rot caused by Erwinia spp. There is no official report
about E. amylovora in China. However, the authors have noted pear blossom and bud blast symptoms and little information is available about the pear blossom blast in China. Barker and Beer (1914) first discovered and reported the pear blossom blast in the United Kingdom followed by Italy, France, Spain, the United States, Canada, Chile, South Africa, New Zealand, and Australia (Clara 1932; Yessad et al. 1992). In addition to pear blossom and bud blast, P. syringae pv. syringae also caused pear bud blight, leaf spot, blister bark, and so on (Wilsom 1934; Spotts and Cervantes 1995). As the symptoms of pear blossom blast were easily confused with those caused by E. amylovora, Britain, the United States, and other countries have conducted a series of identifications for the pathogens, as also diagnosis of the symp-
This paper is translated from its Chinese version in Scientia Agricultura Sinica. Correspondence XIE Guan-lin, Professor, Tel: +86-571-86971412, E-mail: [email protected]
© 2008, CAAS. All rights reserved. Published by Elsevier Ltd.
1092
toms (Wilsom 1934; Billing et al. 1960; Yessad et al. 1992; Spotts and Cervantes 1995). It was necessary to confirm whether the pear blossom and bud blight in China was caused by P. syringae pv. syringae or not. The authors found pear blossom and bud blast in the spring of 2006, in an orchard of Hangzhou, China. It could be confirmed as a bacterial disease due to the bacterial streaming observed under the microscope. The initial symptoms were similar to those caused by E. amylovora, however, the diseased pear branch showed no typical symptoms of fire blight, “shepherd’s-crook”. The present study is carried to determine the causal organism of pear blossom and bud blast in China, and differentiate it from other diseases of the pear, based on bacteriological characteristics, electron microscopy, Kock’s postulate test, and molecular biological methods.
MATERIALS AND METHODS The standard reference strains The standard reference strains of P. syringae pv. syringae LMG5570, LMG2230, and LMG 2231 were provided by professor Jean Swings from the Bacterial Cultural Collection Center, University of Ghent, Belgium. Standard strains of Ralstonia solanacearum Rp-1 were provided by Professor Liu Bo from the Fujian Academy of Agricultural Sciences, China. Other materials were obtained from the bacterial laboratory of the institute.
Sample collection and isolation of the pathogenic bacteria Sixteen samples of pear blossom and bud blast were collected from the diseased field. The samples with bacterial streaming observed under microscope were washed with tap water gently. The streaking method was used for isolation of the pathogen on NA medium after surface disinfection with 70% alcohol and 30 min immersion in sterilized distilled water. The representative single colonies were picked three days after incubation at 30°C, purified on KMB medium, and the fluorescent colonies were observed under 365 nm wave-
QIU Wen et al.
length UV light.
Conventional bacteriological and Koch’s postulate tests The colony morphology, cultural characteristics, and hypersensitive reactions on tobacco were determined according to Klement et al. (1990); physiological and biochemical determination were followed as per Schaad et al. (2001). The pathogenicity test in vitro was done as described by Yessad et al. (1992). Koch’s postulate test of the pathogen was measured on the pear variety Cuiguan. The pricking method was used for inoculation on young twigs of pear with the bacterial suspension of 10 8 cfu mL -1 and the disease incidence was recorded 10 d after inoculation. The flowers and leaves were inoculated by spraying method and the incidence observed 3 d after inoculation. All the inoculated young plants of pear were incubated at 26°C in the phytotron. Ice nucleation activity (INA) was determined by using the small droplets freezing method (Lindow 1982) under -2, -3, -4, and -5°C.
Pathogen observation by electron microscope The pathogenic bacteria cultured for 24 h were made into a bacterial suspension with sterile water and filter filtrated, a few drops of fixative (pH 7.2, 0.15% glutaraldehyde in phosphate buffer) were added, and then centrifugated to collect cells. A few drops of fresh glutaraldehyde were added, and fixed in the refrigerator at 4°C overnight, with centrifugation again to collect cells, and then the bacterium concentration was adjusted to 108 cfu mL-1. The final suspension with the equivalent amount of 2% osmium acid was used as a staining solution. It was sucked with sterile capillary suction and dropped in a copper omentum. The free water was removed with the help of a filter paper 4 min later, and the sample was observed using an electron microscopy (TEM, KYKY-1000B, Japan) after it dried.
Biolog identification of the pathogen Six representative bacterial strains were selected to
© 2008, CAAS. All rights reserved. Published by Elsevier Ltd.
Pear Blossom Blast Caused by Pseudomonas syringae pv. syringae in China
confirm their identity by Biolog GN microplates containing 95 carbon source. Bacterial suspension of 150 µL (OD = 0.3) was added to each well. The reaction results were determined by a Biolog reader 24 h after incubation at 30°C and the Biolog specific identification procedures (version 4.1) and directly entered. The operations, in detail, were referred to Xie (2000).
FAMEs Identification of the pathogen FAMEs test was followed according to the instruction of the MIDI Company with a 6890 Gas Chromatography System from USA (Xie et al. 2003). All of the purified strains tested were cultured at NA medium at 30°C for 24 h, and transferred to TSBA solid medium to culture for 24 h again. One loop of the culture was harvested from each strain to extract fatty acids. The identification results were obtained through the microbial identification system software (MIS4.5) and library generation software (LGS4.5) and compared with fatty acid information of standard strains in the database.
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RESULTS Morphology and cultural characteristics of the pathogen All the six pear isolates tested were gram-negative, aerobic rods measuring 0.8-1.5 µm × 1.5-4.0 µm with 2-4 polar flagella (Fig.1-A, B). Fluorescent green diffusible pigment was produced on King’s medium B, which showed typical characteristics of the genus Pseudomonas. They produced levan on sucrose nutrient agar, liquefied gelatin, and produced cloveadriamycin, and cytochrome oxidase. They could not grow at 41°C. Starch hydrolysis, nitrate reduction, and E-glucosidase enzyme reaction were negative (Table 1), indicating that the bacteria had typical characteristics of P. syringae. Three of the six isolates tested displayed INA. A
B
1 µm
0.5 µm
PCR analysis and ITS sequencing of the pathogen PCR amplification of the isolated pathogenic bacteria was done based on the published 16S rDNA universal primer (Edward et al. 1989; Li et al. 2006) of plant pathogenic bacteria in the ITS region. The forward and reverse primers were P1 (5´-AGA GTT TGA TCA TGG CTC AG-3´) and P2 (5´-ACG GTT ACC TTG TTA CGA CTT-3´), respectively. PCR reaction system with PTC-200 thermocycler: DNA 1 µL, P1 (10 µM) 0.5 µL, P2 (10 µM) 0.5 µL, 2 × Taq PCR StarMix 10 µL, and double-distilled water, total volume 20 µL; 94°C pre-denature 5 min, 94°C 25 s, 65°C 25 s, 72°C 1 min, 35 cycles, 72°C 5 min; 1.0% agarose gel electrophoresis, which finally produced a 1.5 kb band. The PCR product was cloned and sequenced by Shanghai Zeheng Biotechnological Company, China. The sequenced results were compared with those in the GenBank (http://www.ncbi. nlm.nih.gov).
Fig. 1 Morphology and flagellum type of the bacterial pathogen of pear blossom blast (transition electromicroscope images. A = 200 000 ×; B = 40 000 ×). Table 1 The comparison of major bacteriological characteristics of the causal organism of pear blossom blast with standard strains Bacteriological test Gram staining Gelatin liquefaction Growth at 41°C Cytochrome oxidase Starch hydrolysis Nitrate reduction Arginine dihydrolase E-Glucosidase Levan formation from sucrose Fluorescent pigment Ice nucleation activity Syringomycin production
Standard strains 1 2 3 4
1
Strains tested 2 3 4 5
6
+ + + + + + +
+ + + + + + +
+ + + + + + +
+ + + + + +
+ + + + + + +
+ + + + + +
+ w + -
+ + + + + +
+ + + + + +
+ + + + + + +
+, -, and w mean the positive, negative, and weak, respectively. Standard strains (1-4) are LMG 5570, LMG2230, LMG 2231, and R. solanacearum Rp-1. Strains tested are L1-L6.
© 2008, CAAS. All rights reserved. Published by Elsevier Ltd.
Pear Blossom Blast Caused by Pseudomonas syringae pv. syringae in China
index is less than 0.2, the results cannot be used; if the index is greater than or equal to 0.5, they can be identified to the species level. The FAMEs identification results of the four standard strains and six blossom blast strains were identical to those of Biolog (Table 2). The six strains of blossom blast were identified as P. syringae pv. syringae with FAMEs similarity index of 0.5840.701 and 0.582-0.671 of the three standard strains, confirming the reliability of the FAMEs identification.
PCR identification of the pathogen The sequences compared with the database of GenBank showed that six strains of blossom blast shared 99% homology with those of P. syringae pv. syringae, P. syringae pv. glycinea (causing soybean bacterial blight) and P. syringae pv. phaseolicola (causing bean bacterial halo). However, the latter two pathogens could be excluded as the possible pear pathogenic bacteria due to the bacteriological characteristics mentioned earlier based on FAMEs, Biolog identification, and the pathogenicity test on the pear trees.
DISCUSSION This study confirmed that pear blossom and bud blast in Hangzhou, Zhejiang Province, China, was caused by P. syringae pv. syringae, which could induce blossom and bud blast, leaf spot, fruit rot, and blister bark by inoculation. The symptoms at the initial stage of blossom and bud blast were very similar to fire blight caused by E. amylovora (Aldwinckle and Beer 1979), which had bacterial streaming when seen under a microscope, in the diseased samples of both. However, fire blight of pear had a typical “shepherd’scrook” symptoms with bacterial oozes on the lesions at higher humidity (Jones and Aldwinckle 1990), whereas, pear blossom and bud blast did not. E. amylovora caused blister bark frequently, whereas, P. syringae pv. syringae did so occasionally (Doidge 1917). The authors found that the appearance of pear blister bark was affected by many factors when they inoculated P. syringae pv. syringae on the pear branches (Whitesides and Spootts 1991). The concentration of 108 cfu mL-1 by wound inoculation supported the best
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result, and those below 106 cfu mL-1 by spraying inoculation did not produce pear blister bark. The inoculation site was also important. It was easy to induce the symptom into young buds near the pear branches. However, the occurrence of pear blister bark in a natural orchard was closely related to pear varieties and spring coldness (Wilsom 1934; Spotts and Cervantes 1995), and wound infection also played an important role. Studies of pear blossom blast in other countries showed P. syringae pv. syringae also caused bacterial blister of apple in addition to producing several pear symptoms (Panagopoulos and Crosse 1963). However, apple blister bark had only been reported in South Africa and differed in the pathogenicity from the pathogen of pear blossom blast (Mansvelt and Hatting 1986). It was necessary to determine whether or not an isolated pathogen of pear blossom and bud blast could also infect apple and cause apple blister bark in China. Fang et al. (1992) reported that in China, P. syringae pv. syringae was the pathogen of rice bacterial sheath rot, with the lesions on the leaves, and sheaths, stems and grains. The disease occurred mainly in the northeast of China. However, there is no pathogen preserved at present, as the study of the disease was done in the 1960s. As a result, the authors can not compare it with the pathogen of pear blossom and bud blast and there is no report on this aspect to date. It has been confirmed that P. syringae pv. syringae has wide ecological diversity, the pathogen may be attached to other fruit trees and some strains have INA, which can cause serious damage in orchards, once the early spring frost occurs (Gross et al. 1983). It can survive in adverse environments in the soil with non-hosts such as other pathogenic bacteria (Wang et al. 2008). Infection cycle of P. syringae pv. syringae causing pear blossom and bud blast and the sources of the inocula in China need to be further studied.
CONCLUSION 1) A pathogenic bacterium causing blossom and bud blast was obtained by isolation from the diseased samples and Kock’s postulate test. 2) It has been confirmed that P. syringae pv. syringae is the causal agent of pear blossom and bud blast in
© 2008, CAAS. All rights reserved. Published by Elsevier Ltd.
1096
China, by phenotypic and molecular identification. 3) This new discovery provides useful diagnostic information for differentiation of pear fire blight from pear blossom blast in management of plant quarantine diseases in China.
Acknowledgements We thank Li Junying from Zhejiang University for electron microscopy assistance; Wang Xiao, Fang Yuan, Lou Miaomiao, and Zhu Bo from Zhejiang University for their assistance; Jean Swings from Ghent University in Belgium and Liu Bo from Fujian Academy of Agricultural Sciences in China for providing standard strains. This work was supported by the National High Technology Research and Development Program of China (2006AA10A211), National Natural Science Foundation of China (30671397), and Hangzhou Agricultural Development Foundation, China (2007-2008).
References Aldwinckle H S, Beer S V. 1979. Fire blight and its control. Horticulture Review, 1, 423-474. Barker B P, Grovo O. 1914. A bacterial disease of fruit blossom. Annals of Applied Biology, 1, 85-97. Billing E, Crosse J E, Garett C M E. 1960. Laboratory diagnosis of fire blight and bacterial blossom blight of pear. Plant Pathology, 9, 19-25. Buyer J S, Roberts D P, Russek C E. 2002. Soil and plant effects on microbial community structure. Canadian Journal of Microbiology, 48, 955-964. Chen C, Chen J, Hu B S, Bai Y Z, Jiang Y H, Liu F Q. 2007. Potential distribution of Erwinia amylovora in China and invasion risk assessment. Scientia Agricultura Sinica, 40, 940947. (in Chinese) Clara F M. 1932. A new bacterial disease of pears. Sciences, 75, 111. Doidge E M. 1917. A bacterial blight of pear blossom occurring in South Africa. Annals of Applied Biology, 4, 50-74. Edward U, Rogall T, Blocker H, Ernde M, Bottger E C. 1989. Isolation and direct complete nucleotide determination of entire genes. Characterization of a gene coding for 16S ribosomal RNA. Nucleic Acids Research, 17, 7843-7853. Fang Z D, Ren X Z. 1992. Notes on list of bacterial plant pathogens in China. Journal of Nanjing Agricultural University, 15, 16. (in Chinese) Gross D C, Cody Y S, Proebsting E L, Radamaker G K, Spotts R A. 1983. Distribution, population dynamics, and
QIU Wen et al.
characteristics of ice nucleation-active bacteria in deciduous fruit tree orchards. Applied and Environmental Microbiotogy, 46, 1370-1379. Jones A L, Aldwinckle H S. 1990. Compendium of Apple and Pear Diseases. APS Press, St Paul, MN, USA. Klement Z, Rudolph K, Sands D C. 1990. Methods in Phytobacteriology. Akademiai Kiado, Budabest. Li B, Xie G L, Zhang J Z, Janssens D, Swings J. 2006. Identification of the bacterial leaf spot pathogen of poinsettia in China. Journal of Phytopathology, 154, 711-715. Lindow S E. 1982. Bacterial ice nucleation: a factor in frost injury to plants. Plants Physiology, 70, 1084-l089. Mansvelt E L, Hatting M J. 1986. Bacterial blister bark and blight of fruit spurs of apple in South Africa. Plant Disease, 70, 403-405. Panagopoulos C G, Crosse J E. 1963. Blossom blight and related symptoms caused by Pseudomonas syringae van Hall on pear trees. Annals Reporter East Mailling Research Statement Kent, 47, 119-122. Schaad N W, Jones J B, Chun W. 2001. Laboratory Guide for Identification of Plant Pathogenic Bacteria. 3rd ed. APS Press, Minnesota, USA. Spotts R A, Cervantes L A. 1995. Factors affecting the severity of bacterial canker of pear caused by Pseudomonas syringae pv. syringae. Plant Pathology, 44, 325-331. Stoger A, Schaffer J, Ruppitsch W. 2006. A rapid and sensitive method for direct detection of Erwinia amylovora in symptomatic and asymptomatic plant tissues by polymerase chain reaction. Journal of Phytopathology, 154, 469-473. Wang G F, Praphat K, Xie G L, Li Bin, Zhu B, Liu B, Zhou Q. 2008. Bacterial wilt of mulberry (Morus alba) caused by Enterobacter cloacae in China. Plant Disease, 92, 483. Whitesides, S K, Spotts R A. 1991. Induction of pear blossom blast caused by Pseudomonas syringae pv. syringae. Plant Pathology, 40, 118-127. Wilsom E E. 1934. A bacterial canker of pear trees new to California. Phytopathology, 24, 534-537. Xie G L, Soad A, Swings J, Mew T W. 2003. Diversity of gram negative bacteria antagonistic against major pathogens of rice seed in the tropic environment. Journal of Zhejiang University (Science), 4, 463-468. Xie G L. 2000. Comparison of the methods for identification of pathogenic bacteria of rice. Journal of Zhejiang University (Agricultural & Life Science), 26, 253-258. Yessad S, Manceau C, Luisetti J. 1992. A detached leaf assay to evaluate virulence and pathogenicity of strains of Pseudomonas syringae pv. syringae on pear. Plant Disease, 76, 370-373. (Edited by ZHANG Juan)
© 2008, CAAS. All rights reserved. Published by Elsevier Ltd.
September 2008
2008, 7(9): 1091-1096
Pear Blossom Blast Caused by Pseudomonas syringae pv. syringae in China QIU Wen1, HUAI Yan1,2, XU Fu-shou3, XU Li-hui1, XIE Guan-lin1, LI Bin1, YU Shan-hong1 and LIU Jun-ying1 1
Institute of Biotechnology, Zhejiang University, Hangzhou 310029, P.R.China
2
Zhejiang Provincial Agricultural Bureau, Hangzhou 310029, P.R.China Hangzhou Plant Protection Station, Hangzhou 310020, P.R.China
3
Abstract This study was done to determine the causal organism of the pear blossom and bud blast in China. It was identified by a bacteriological test, electro-microscopic observation, Koch’s postulate test, Biolog, fatty acid methyl esters (FAMEs), and a polymerase chain reaction (PCR) test, and compared with the standard reference strains. Six representative strains out of 20 pathogenic bacterial isolates from 16 diseased samples showed characteristics similar to three standard strains of Pseudomonas syringae pv. syringae from Belgium. They were identified as P. syringae pv. syringae with a Biolog similarity of 0.57-0.86 and FAMEs similarity of 0.58-0.81. The bacterium was reisolated from the symptomatic plants and blossoms. Identification as P. syringae pv. syringae was confirmed by using PCR primers and sequence tests, and compared with the above-mentioned results. The data supported the fact that the pear blossom and bud blast in China could be caused by P. syringae pv. syringae. Key words: pear blossom blast, pear bud blast, Pseudomonas syringae pv. syringae, confirmation
INTRODUCTION Fire blight, caused by Erwinia amylovora, is not only the world’s first-reported plant bacterial disease, but also one of the most serious diseases in fruit trees. It has been listed as a plant quarantine disease in China and many other countries, and disease management for this is being undertaken (Stoger et al. 2006; Chen et al. 2007). However, some early symptoms caused by E. amylovora are very similar to those of another bacterial disease on pear, namely, pear blossom blast. Until now only three bacterial diseases of pear have been recorded in China, which are crown gall of pear caused by Agrobacterium tumefaciens, bacterial rust, and fruit rot caused by Erwinia spp. There is no official report
about E. amylovora in China. However, the authors have noted pear blossom and bud blast symptoms and little information is available about the pear blossom blast in China. Barker and Beer (1914) first discovered and reported the pear blossom blast in the United Kingdom followed by Italy, France, Spain, the United States, Canada, Chile, South Africa, New Zealand, and Australia (Clara 1932; Yessad et al. 1992). In addition to pear blossom and bud blast, P. syringae pv. syringae also caused pear bud blight, leaf spot, blister bark, and so on (Wilsom 1934; Spotts and Cervantes 1995). As the symptoms of pear blossom blast were easily confused with those caused by E. amylovora, Britain, the United States, and other countries have conducted a series of identifications for the pathogens, as also diagnosis of the symp-
This paper is translated from its Chinese version in Scientia Agricultura Sinica. Correspondence XIE Guan-lin, Professor, Tel: +86-571-86971412, E-mail: [email protected]
© 2008, CAAS. All rights reserved. Published by Elsevier Ltd.
1092
toms (Wilsom 1934; Billing et al. 1960; Yessad et al. 1992; Spotts and Cervantes 1995). It was necessary to confirm whether the pear blossom and bud blight in China was caused by P. syringae pv. syringae or not. The authors found pear blossom and bud blast in the spring of 2006, in an orchard of Hangzhou, China. It could be confirmed as a bacterial disease due to the bacterial streaming observed under the microscope. The initial symptoms were similar to those caused by E. amylovora, however, the diseased pear branch showed no typical symptoms of fire blight, “shepherd’s-crook”. The present study is carried to determine the causal organism of pear blossom and bud blast in China, and differentiate it from other diseases of the pear, based on bacteriological characteristics, electron microscopy, Kock’s postulate test, and molecular biological methods.
MATERIALS AND METHODS The standard reference strains The standard reference strains of P. syringae pv. syringae LMG5570, LMG2230, and LMG 2231 were provided by professor Jean Swings from the Bacterial Cultural Collection Center, University of Ghent, Belgium. Standard strains of Ralstonia solanacearum Rp-1 were provided by Professor Liu Bo from the Fujian Academy of Agricultural Sciences, China. Other materials were obtained from the bacterial laboratory of the institute.
Sample collection and isolation of the pathogenic bacteria Sixteen samples of pear blossom and bud blast were collected from the diseased field. The samples with bacterial streaming observed under microscope were washed with tap water gently. The streaking method was used for isolation of the pathogen on NA medium after surface disinfection with 70% alcohol and 30 min immersion in sterilized distilled water. The representative single colonies were picked three days after incubation at 30°C, purified on KMB medium, and the fluorescent colonies were observed under 365 nm wave-
QIU Wen et al.
length UV light.
Conventional bacteriological and Koch’s postulate tests The colony morphology, cultural characteristics, and hypersensitive reactions on tobacco were determined according to Klement et al. (1990); physiological and biochemical determination were followed as per Schaad et al. (2001). The pathogenicity test in vitro was done as described by Yessad et al. (1992). Koch’s postulate test of the pathogen was measured on the pear variety Cuiguan. The pricking method was used for inoculation on young twigs of pear with the bacterial suspension of 10 8 cfu mL -1 and the disease incidence was recorded 10 d after inoculation. The flowers and leaves were inoculated by spraying method and the incidence observed 3 d after inoculation. All the inoculated young plants of pear were incubated at 26°C in the phytotron. Ice nucleation activity (INA) was determined by using the small droplets freezing method (Lindow 1982) under -2, -3, -4, and -5°C.
Pathogen observation by electron microscope The pathogenic bacteria cultured for 24 h were made into a bacterial suspension with sterile water and filter filtrated, a few drops of fixative (pH 7.2, 0.15% glutaraldehyde in phosphate buffer) were added, and then centrifugated to collect cells. A few drops of fresh glutaraldehyde were added, and fixed in the refrigerator at 4°C overnight, with centrifugation again to collect cells, and then the bacterium concentration was adjusted to 108 cfu mL-1. The final suspension with the equivalent amount of 2% osmium acid was used as a staining solution. It was sucked with sterile capillary suction and dropped in a copper omentum. The free water was removed with the help of a filter paper 4 min later, and the sample was observed using an electron microscopy (TEM, KYKY-1000B, Japan) after it dried.
Biolog identification of the pathogen Six representative bacterial strains were selected to
© 2008, CAAS. All rights reserved. Published by Elsevier Ltd.
Pear Blossom Blast Caused by Pseudomonas syringae pv. syringae in China
confirm their identity by Biolog GN microplates containing 95 carbon source. Bacterial suspension of 150 µL (OD = 0.3) was added to each well. The reaction results were determined by a Biolog reader 24 h after incubation at 30°C and the Biolog specific identification procedures (version 4.1) and directly entered. The operations, in detail, were referred to Xie (2000).
FAMEs Identification of the pathogen FAMEs test was followed according to the instruction of the MIDI Company with a 6890 Gas Chromatography System from USA (Xie et al. 2003). All of the purified strains tested were cultured at NA medium at 30°C for 24 h, and transferred to TSBA solid medium to culture for 24 h again. One loop of the culture was harvested from each strain to extract fatty acids. The identification results were obtained through the microbial identification system software (MIS4.5) and library generation software (LGS4.5) and compared with fatty acid information of standard strains in the database.
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RESULTS Morphology and cultural characteristics of the pathogen All the six pear isolates tested were gram-negative, aerobic rods measuring 0.8-1.5 µm × 1.5-4.0 µm with 2-4 polar flagella (Fig.1-A, B). Fluorescent green diffusible pigment was produced on King’s medium B, which showed typical characteristics of the genus Pseudomonas. They produced levan on sucrose nutrient agar, liquefied gelatin, and produced cloveadriamycin, and cytochrome oxidase. They could not grow at 41°C. Starch hydrolysis, nitrate reduction, and E-glucosidase enzyme reaction were negative (Table 1), indicating that the bacteria had typical characteristics of P. syringae. Three of the six isolates tested displayed INA. A
B
1 µm
0.5 µm
PCR analysis and ITS sequencing of the pathogen PCR amplification of the isolated pathogenic bacteria was done based on the published 16S rDNA universal primer (Edward et al. 1989; Li et al. 2006) of plant pathogenic bacteria in the ITS region. The forward and reverse primers were P1 (5´-AGA GTT TGA TCA TGG CTC AG-3´) and P2 (5´-ACG GTT ACC TTG TTA CGA CTT-3´), respectively. PCR reaction system with PTC-200 thermocycler: DNA 1 µL, P1 (10 µM) 0.5 µL, P2 (10 µM) 0.5 µL, 2 × Taq PCR StarMix 10 µL, and double-distilled water, total volume 20 µL; 94°C pre-denature 5 min, 94°C 25 s, 65°C 25 s, 72°C 1 min, 35 cycles, 72°C 5 min; 1.0% agarose gel electrophoresis, which finally produced a 1.5 kb band. The PCR product was cloned and sequenced by Shanghai Zeheng Biotechnological Company, China. The sequenced results were compared with those in the GenBank (http://www.ncbi. nlm.nih.gov).
Fig. 1 Morphology and flagellum type of the bacterial pathogen of pear blossom blast (transition electromicroscope images. A = 200 000 ×; B = 40 000 ×). Table 1 The comparison of major bacteriological characteristics of the causal organism of pear blossom blast with standard strains Bacteriological test Gram staining Gelatin liquefaction Growth at 41°C Cytochrome oxidase Starch hydrolysis Nitrate reduction Arginine dihydrolase E-Glucosidase Levan formation from sucrose Fluorescent pigment Ice nucleation activity Syringomycin production
Standard strains 1 2 3 4
1
Strains tested 2 3 4 5
6
+ + + + + + +
+ + + + + + +
+ + + + + + +
+ + + + + +
+ + + + + + +
+ + + + + +
+ w + -
+ + + + + +
+ + + + + +
+ + + + + + +
+, -, and w mean the positive, negative, and weak, respectively. Standard strains (1-4) are LMG 5570, LMG2230, LMG 2231, and R. solanacearum Rp-1. Strains tested are L1-L6.
© 2008, CAAS. All rights reserved. Published by Elsevier Ltd.
Pear Blossom Blast Caused by Pseudomonas syringae pv. syringae in China
index is less than 0.2, the results cannot be used; if the index is greater than or equal to 0.5, they can be identified to the species level. The FAMEs identification results of the four standard strains and six blossom blast strains were identical to those of Biolog (Table 2). The six strains of blossom blast were identified as P. syringae pv. syringae with FAMEs similarity index of 0.5840.701 and 0.582-0.671 of the three standard strains, confirming the reliability of the FAMEs identification.
PCR identification of the pathogen The sequences compared with the database of GenBank showed that six strains of blossom blast shared 99% homology with those of P. syringae pv. syringae, P. syringae pv. glycinea (causing soybean bacterial blight) and P. syringae pv. phaseolicola (causing bean bacterial halo). However, the latter two pathogens could be excluded as the possible pear pathogenic bacteria due to the bacteriological characteristics mentioned earlier based on FAMEs, Biolog identification, and the pathogenicity test on the pear trees.
DISCUSSION This study confirmed that pear blossom and bud blast in Hangzhou, Zhejiang Province, China, was caused by P. syringae pv. syringae, which could induce blossom and bud blast, leaf spot, fruit rot, and blister bark by inoculation. The symptoms at the initial stage of blossom and bud blast were very similar to fire blight caused by E. amylovora (Aldwinckle and Beer 1979), which had bacterial streaming when seen under a microscope, in the diseased samples of both. However, fire blight of pear had a typical “shepherd’scrook” symptoms with bacterial oozes on the lesions at higher humidity (Jones and Aldwinckle 1990), whereas, pear blossom and bud blast did not. E. amylovora caused blister bark frequently, whereas, P. syringae pv. syringae did so occasionally (Doidge 1917). The authors found that the appearance of pear blister bark was affected by many factors when they inoculated P. syringae pv. syringae on the pear branches (Whitesides and Spootts 1991). The concentration of 108 cfu mL-1 by wound inoculation supported the best
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result, and those below 106 cfu mL-1 by spraying inoculation did not produce pear blister bark. The inoculation site was also important. It was easy to induce the symptom into young buds near the pear branches. However, the occurrence of pear blister bark in a natural orchard was closely related to pear varieties and spring coldness (Wilsom 1934; Spotts and Cervantes 1995), and wound infection also played an important role. Studies of pear blossom blast in other countries showed P. syringae pv. syringae also caused bacterial blister of apple in addition to producing several pear symptoms (Panagopoulos and Crosse 1963). However, apple blister bark had only been reported in South Africa and differed in the pathogenicity from the pathogen of pear blossom blast (Mansvelt and Hatting 1986). It was necessary to determine whether or not an isolated pathogen of pear blossom and bud blast could also infect apple and cause apple blister bark in China. Fang et al. (1992) reported that in China, P. syringae pv. syringae was the pathogen of rice bacterial sheath rot, with the lesions on the leaves, and sheaths, stems and grains. The disease occurred mainly in the northeast of China. However, there is no pathogen preserved at present, as the study of the disease was done in the 1960s. As a result, the authors can not compare it with the pathogen of pear blossom and bud blast and there is no report on this aspect to date. It has been confirmed that P. syringae pv. syringae has wide ecological diversity, the pathogen may be attached to other fruit trees and some strains have INA, which can cause serious damage in orchards, once the early spring frost occurs (Gross et al. 1983). It can survive in adverse environments in the soil with non-hosts such as other pathogenic bacteria (Wang et al. 2008). Infection cycle of P. syringae pv. syringae causing pear blossom and bud blast and the sources of the inocula in China need to be further studied.
CONCLUSION 1) A pathogenic bacterium causing blossom and bud blast was obtained by isolation from the diseased samples and Kock’s postulate test. 2) It has been confirmed that P. syringae pv. syringae is the causal agent of pear blossom and bud blast in
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China, by phenotypic and molecular identification. 3) This new discovery provides useful diagnostic information for differentiation of pear fire blight from pear blossom blast in management of plant quarantine diseases in China.
Acknowledgements We thank Li Junying from Zhejiang University for electron microscopy assistance; Wang Xiao, Fang Yuan, Lou Miaomiao, and Zhu Bo from Zhejiang University for their assistance; Jean Swings from Ghent University in Belgium and Liu Bo from Fujian Academy of Agricultural Sciences in China for providing standard strains. This work was supported by the National High Technology Research and Development Program of China (2006AA10A211), National Natural Science Foundation of China (30671397), and Hangzhou Agricultural Development Foundation, China (2007-2008).
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