应用PCR方法快速检测黄瓜细菌性角斑病菌

黄瓜细菌性角斑病是黄瓜上的一种重要细菌病害,其病原为丁香假单胞菌黄瓜致病变种(Pseudomonas syringae pv.lachrymans),目前未见到该病害特异性PCR检测方法的报道。通过分析丁香假单胞菌(P.syringae)不同致病变种glyceraldehyde-3-phosphate dehydrogenase 1(gap1)基因序列设计得到一对Psl特异性PCR引物。利用该引物对丁香假单胞菌不同致病变种、假单胞菌属其他种及其他属的共46株菌株进行了PCR扩增,结果表明,所有不同来源的12株黄瓜细菌性角斑病菌均得到179bp的目标片段,而所有其他参试菌株均无扩增条带,PCR检测的灵敏度为7.5×103cfu/mL。利用该方法可从接种后发病的黄瓜叶片总DNA中检测到特异条带,而健康叶片无条带。该引物的PCR检测方法可直接用于植株总DNA的检测,无需进行病原菌的分离培养,快速简便,适用于进出境检验检疫及种苗健康检测等。     

Assessment of resistance pathways induced in Arabidopsis thaliana by hypovirulent Rhizoctonia spp. isolates

Certain hypovirulent Rhizoctonia isolates effectively protect plants against well-known important pathogens among Rhizoctonia isolates as well as against other pathogens. The modes of action involved in this protection include resistance induced in plants by colonization with hypovirulent Rhizoctonia isolates. The qualifications of hypovirulent isolates (efficient protection, rapid growth, effective colonization of the plants, and easy application in the field) provide a significant potential for the development of a commercial microbial preparation for application as biological control agents. Understanding of the modes of action involved in protection is important for improving the various aspects of development and application of such preparations. The hypothesis of the present study is that resistance pathways such as systemic acquired resistance (SAR), induced systemic resistance (ISR), and phytoalexins are induced in plants colonized by the protective hypovirulent Rhizoctonia isolates and are involved in the protection of these plants against pathogenic Rhizoctonia. Changes in protection levels of Arabidopsis thaliana mutants defective in defense-related genes (npr1-1, npr1-2, ndr1-1, npr1-2/ndr1-1, cim6, wrky70.1, snc1, and pbs3-1) and colonized with the hypovirulent Rhizoctonia isolates compared with that of the wild type (wt) plants colonized with the same isolates confirmed the involvement of induced resistance in the protection of the plants against pathogenic Rhizoctonia spp., although protection levels of mutants constantly expressing SAR genes (snc1 and cim6) were lower than that of wt plants. Plant colonization by hypovirulent Rhizoctonia isolates induced elevated expression levels of the following genes: PR5 (SAR), PDF1.2, LOX2, LOX1, CORI3 (ISR), and PAD3 (phytoalexin production), which indicated that all of these pathways were induced in the hypovirulent-colonized plants. When SAR or ISR were induced separately in plants after application of the chemical inducers Bion and methyl jasmonate, respectively, only ISR activation resulted in a higher protection level against the pathogen, although the protection was minor. In conclusion, plant colonization with the protective hypovirulent Rhizoctonia isolates significantly induced genes involved in the SAR, ISR, and phytoalexin production pathways. In the studied system, SAR probably did not play a major role in the mode of protection against pathogenic Rhizoctonia spp.; however, it may play a more significant role in protection against other pathogens.     

Induced Systemic Resistance and Promotion of Plant Growth by Bacillus spp

ABSTRACT Elicitation of induced systemic resistance (ISR) by plant-associated bacteria was initially demonstrated using Pseudomonas spp. and other gram-negative bacteria. Several reviews have summarized various aspects of the large volume of literature on Pseudomonas spp. as elicitors of ISR. Fewer published accounts of ISR by Bacillus spp. are available, and we review this literature for the first time. Published results are summarized showing that specific strains of the species B. amyloliquefaciens, B. subtilis, B. pasteurii, B. cereus, B. pumilus, B. mycoides, and B. sphaericus elicit significant reductions in the incidence or severity of various diseases on a diversity of hosts. Elicitation of ISR by these strains has been demonstrated in greenhouse or field trials on tomato, bell pepper, muskmelon, watermelon, sugar beet, tobacco, Arabidopsis sp., cucumber, loblolly pine, and two tropical crops (long cayenne pepper and green kuang futsoi). Protection resulting from ISR elicited by Bacillus spp. has been reported against leaf-spotting fungal and bacterial pathogens, systemic viruses, a crown-rotting fungal pathogen, root-knot nematodes, and a stem-blight fungal pathogen as well as damping-off, blue mold, and late blight diseases. Reductions in populations of three insect vectors have also been noted in the field: striped and spotted cucumber beetles that transmit cucurbit wilt disease and the silver leaf whitefly that transmits Tomato mottle virus. In most cases, Bacillus spp. that elicit ISR also elicit plant growth promotion. Studies on mechanisms indicate that elicitation of ISR by Bacillus spp. is associated with ultrastructural changes in plants during pathogen attack and with cytochemical alterations. Investigations into the signal transduction pathways of elicited plants suggest that Bacillus spp. activate some of the same pathways as Pseudomonas spp. and some additional pathways. For example, ISR elicited by several strains of Bacillus spp. is independent of salicylic acid but dependent on jasmonic acid, ethylene, and the regulatory gene NPR1-results that are in agreement with the model for ISR elicited by Pseudomonas spp. However, in other cases, ISR elicited by Bacillus spp. is dependent on salicylic acid and independent of jasmonic acid and NPR1. In addition, while ISR by Pseudomonas spp. does not lead to accumulation of the defense gene PR1 in plants, in some cases, ISR by Bacillus spp. does. Based on the strains and results summarized in this review, two products for commercial agriculture have been developed, one aimed mainly at plant growth promotion for transplanted vegetables and one, which has received registration from the U.S. Environmental Protection Agency, for disease protection on soybean.     

Mixtures of plant growth-promoting rhizobacteria enhance biological control of multiple cucumber pathogens

ABSTRACT Plant growth-promoting rhizobacteria (PGPR) strains INR7 (Bacillus pumilus), GB03 (Bacillus subtilis), and ME1 (Curtobacterium flaccumfaciens) were tested singly and in combinations for biological control against multiple cucumber pathogens. Investigations under greenhouse conditions were conducted with three cucumber pathogens-Colletotrichum orbiculare (causing anthracnose), Pseudomonas syringae pv. lachrymans (causing angular leaf spot), and Erwinia tracheiphila(causing cucurbit wilt disease)-inoculated singly and in all possible combinations. There was a general trend across all experiments toward greater suppression and enhanced consistency against multiple cucumber pathogens using strain mixtures. The same three PGPR strains were evaluated as seed treatments in two field trials over two seasons, and two strains, IN26 (Burkholderia gladioli) and INR7 also were tested as foliar sprays in one of the trials. In the field trials, the efficacy of induced systemic resistance activity was determined against introduced cucumber pathogens naturally spread within plots through placement of infected plants into the field to provide the pathogen inoculum. PGPR-mediated disease suppression was observed against angular leaf spot in 1996 and against a mixed infection of angular leaf spot and anthracnose in 1997. The three-way mixture of PGPR strains (INR7 plus ME1 plus GB03) as a seed treatment showed intensive plant growth promotion and disease reduction to a level statistically equivalent to the synthetic elicitor Actigard applied as a spray.     

Induced Systemic Resistance by Fluorescent Pseudomonas spp

ABSTRACT Fluorescent Pseudomonas spp. have been studied for decades for their plant growth-promoting effects through effective suppression of soilborne plant diseases. The modes of action that play a role in disease suppression by these bacteria include siderophore-mediated competition for iron, antibiosis, production of lytic enzymes, and induced systemic resistance (ISR). The involvement of ISR is typically studied in systems in which the Pseudomonas bacteria and the pathogen are inoculated and remain spatially separated on the plant, e.g., the bacteria on the root and the pathogen on the leaf, or by use of split root systems. Since no direct interactions are possible between the two populations, suppression of disease development has to be plant-mediated. In this review, bacterial traits involved in Pseudomonas-mediated ISR will be discussed.     

The Molecular Biology of the Interactions Between Trichoderma spp., Phytopathogenic Fungi, and Plants

ABSTRACT Trichoderma-based biofungicides are a reality in agriculture, with more than 50 formulations today available as registered products worldwide. Several strategies have been applied to identify the main genes and compounds involved in this complex, three-way cross-talk between the fungal antagonist, the plant, and microbial pathogens. Proteome and genome analysis have greatly enhanced our ability to conduct holistic and genome-based functional studies. We have identified and determined the role of a variety of novel genes and gene-products, including ABC transporters, enzymes and other proteins that produce or act as novel elicitors of induced resistance, proteins responsible for a gene-for-gene avirulent interaction between Trichoderma spp. and plants, mycoparasitism-related inducers, plant proteins specifically induced by Trichoderma, etc. We have transgenically demonstrated the ability of Trichoderma spp. to transfer heterologous proteins into plant during root colonization, and have used green fluorescent protein and other markers to study the interaction in vivo and in situ between Trichoderma spp. and the fungal pathogen or the plant.     

Genetic diversity of Pseudomonas syringae pv. lachrymans strains isolated from cucumber leaves collected in Poland

Several strains of Pseudomonas syringae pathovar (pv.) lachrymans and related bacterial pathogens were isolated from cucumber ( Cucumis sativus ) leaves collected in central and southern Poland in 2001 and 2002. Twenty five original strains, together with five reference strains of P. syringae pv. lachrymans , pv. syringae and pv. tomato , were genetically characterized by PCR-RFLP (polymerase chain reaction − restriction fragment length polymorphism), ADSRRS (amplification of DNA fragments surrounding rare restriction sites), and PCR-MP (PCR − melting profiles) fingerprinting techniques. Genetic similarity analyses of the PCR-RFLP and ADSRRS fingerprints showed that strains of P. syringae pv. lachrymans form distinct clusters. The results also indicated that the ADSRRS and the PCR-MP fingerprinting techniques may serve as more efficient tools for evaluating genetic similarity among pathovars and strains of P. syringae than PCR-RFLP. The 25 strains showed diverse pathogenicity to cucumber seedlings and biochemical tests were varied. The syrB gene was identified in four cucumber strains, characterized as P. syringae pv. syringae .     

Different Patterns of Host Genes are Induced in Rice by Pseudomonas syringae, a Biological Inducer of Resistance, and the Chemical Inducer Benzothiadiazole (BTH)

Rice seedlings treated with the synthetic compound benzo(1,2,3)thiadiazole-7-carbothioic acid S-methyl ester (BTH) acquired resistance to subsequent attack by the rice blast fungus Magnaporthe grisea (Hebert) Barr. BTH (trade name Bion) has been released to the market as a plant protecting agent for rice. Here, we analysed the pattern of expressed genes in rice plants treated with BTH, and compared this pattern with those induced by the formerly discovered resistance inducer 2,6-dichloroisonicotinic acid (INA) and by Pseudomonas syringae pv. syringae, a non-host pathogen inducing a hypersensitive response. Both INA and BTH induced similar patterns of genes, suggesting that these compounds are functional analogues. In contrast, the patterns induced by the chemical inducers and by P. syringae were clearly dissimilar.     

The Internal Glycine-Rich Motif and Cysteine Suppress Several Effects of the HpaG(Xooc) Protein in Plants

ABSTRACT HpaG(Xooc), produced by Xanthomonas oryzae pv. oryzicola, is a member of harpin group of proteins that stimulate plant growth, hypersensitive cell death (HCD), and pathogen defense. The protein contains two copies of the glycine-rich motif (GRM), a characteristic of harpins, and a cysteine, which is absent in other harpins. Genetic modification generated the pro-tein mutants HpaG(Xooc)MG (MG) by deleting GRMs and HpaG(Xooc)C47T (C47T) by replacing cysteine with threonine. When applied to tobacco plants, C47T and MG were 1.2- and 1.7-fold stronger, respectively, than HpaG(Xooc) in inducing HCD, which occurred consistently with expression of the marker genes hin1 and hsr203. The proteins markedly alleviated infection of tobacco by Tobacco mosaic virus and Arabidopsis and tomato by Pseudomonas syringae. Treating tobacco plants with HpaG(Xooc), C47T, and MG decreased the viral infection by 58, 81, and 92%, respectively. In Arabidopsis and tomato plants treated with HpaG(Xooc), C47T, or MG, P. syringae multiplication was inhibited; bacterial population multiplied in 5 days in these plants were ca. 160-, 1,260-, or 15,860-fold smaller than that in control plants. So pathogen defense was induced in both plants. Defense-related genes Chia5, NPR1, and PR-1a were expressed consistently with resistance. In response to HpaG(Xooc), C47T, and MG, aerial parts and roots of tomato plants increased growth by 15 and 53%, 25 and 77%, and 46 and 106%, relative to controls. The expansin gene, EXP2, involved in the cell expansion and plant growth was expressed coordinately with plant growth promotion. These results suggest that the presence of GRM and cysteine in HpaG(Xooc) represses the effects of the protein in plants.     

Systemic resistance induced in Arabidopsis thaliana by Trichoderma asperellum SKT-1, a microbial pesticide of seedborne diseases of rice

BACKGROUND: Trichoderma asperellum SKT-1 is a microbial pesticide of seedborne diseases of rice. To investigate the mechanisms of disease suppression in SKT-1, the ability to induce systemic resistance by SKT-1, or its cell-free culture filtrate (CF), was tested using Arabidopsis thaliana Col-0 plants. RESULTS: Both SKT-1 and its CF elicit an induced systemic resistance against the bacterial leaf speck pathogen Pseudomonas syringae pv. tomato DC3000 in Col-0 plants. Involvement of plant hormones in the induced resistance by SKT-1 and CF was assessed using Arabidopsis genotypes such as the jasmonic acid (JA)-resistant mutant jar1, the ethylene (ET)-resistant mutant etr1, the plant impaired in salicylic acid (SA) signalling transgenic NahG and the mutant npr1 impaired in NPR1 activity. In soil experiments using SKT-1, no significant disease suppression effect was observed in NahG transgenic plants or npr1 mutant plants. Expression levels of SA-inducible genes such as PR-1, PR-2 and PR-5 increased substantially in the leaves of Col-0 plants. Expression levels of JA/ET-induced genes such as PDF1.2a, PR-3, PR-4 and AtVsp1 were also induced, but the levels were not as high as for SA-inducible genes. In a hydroponic experiment using CF from SKT-1, all Arabidopsis genotypes showed an induced systemic resistance by CF and increased expression levels of JA/ET- and SA-inducible genes in leaves of CF-treated plants. CONCLUSION: The SA signalling pathway is important in inducing systemic resistance to colonisation by SKT-1, and both SA and JA/ET signalling pathways combine in the signalling of induced resistance by CF. These results indicate that the response of A. thaliana is different from that found in root treatments with barley grain inoculum and CF from SKT-1. Copyright © 2011 Society of Chemical Industry     

Induced systemic protection against tomato late blight elicited by plant growth-promoting rhizobacteria

ABSTRACT Two strains of plant growth-promoting rhizobacteria (PGPR), Bacillus pumilus SE34 and Pseudomonas fluorescens 89B61, elicited systemic protection against late blight on tomato and reduced disease severity by a level equivalent to systemic acquired resistance induced by Phytophthora infestans or induced local resistance by chemical inducer beta-amino butyric acid (BABA) in greenhouse assays. Germination of sporangia and zoospores of P. infestans on leaf surfaces of tomato plants treated with the two PGPR strains, pathogen, and chemical BABA was significantly reduced compared with the noninduced control. Induced protection elicited by PGPR, pathogen, and BABA were examined to determine the signal transduction pathways in three tomato lines: salicylic acid (SA)-hydroxylase transgenic tomato (nahG), ethylene insensitive mutants (Nr/Nr), and jasmonic acid insensitive mutants (def1). Results suggest that induced protection elicited by both bacilli and pseudomonad PGPR strains was SA-independent but ethylene- and jasmonic acid-dependent, whereas systemic acquired resistance elicited by the pathogen and induced local resistance by BABA were SA-dependent. The lack of colonization of tomato leaves by strain 89B61 suggests that the observed induced systemic resistance (ISR) was due to systemic protection by strain 89B61 and not attributable to a direct interaction between pathogen and biological control agent. Although strain SE34 was detected on tomato leaves, ISR mainly accounted for the systemic protection with this strain.     

黄瓜细菌性角斑病菌交叉引物恒温扩增检测技术

黄瓜细菌性角斑病是我国黄瓜生产上的重要病害之一,其病原菌为Pseudomonas syringae pv.lachrymans。根据该病原菌甘油醛-3-磷酸脱氢基因保守序列设计引物和探针,建立了交叉引物恒温扩增和核酸试纸条检测技术。菌体DNA检测灵敏度可达0.55 ng,纯菌直接检测灵敏度基本可达到单个细菌。所测试的5株黄瓜细菌性角斑病菌和染病黄瓜叶片均为阳性,其他13株对照菌株均为阴性。该方法灵敏度高,且操作简单,对设备要求低等,适合基层实验室应用。     

The Relationship Between Pathogen-induced Systemic Resistance (ISR) and Multigenic (horizontal) Resistance in Plants

Plants have developed mechanisms to successfully co-exist in the presence of pathogenic organisms. Some interactions between plants and pathogens are based on recognition of specific elicitor molecules from avirulent pathogen races (avr gene products), which is described in the gene-for-gene resistance theory. Another type of resistance, multigenic (horizontal) resistance, is a less well-studied phenomenon that depends upon multiple genes in the plant host. All plants possess resistance mechamisms which can be induced upon pre-treatment of plants with a variety of organisms or compounds. This general phenomenon is known as induced systemic resistance (ISR). At least in some plant species, ISR depends on the timely accumulation of multiple gene products, such as hydrolytic enzymes, peroxidases or other gene products related to plant defences. The pre-treatment of plants with an inducing organism or compound appears to incite the plant to mount an effective defense response upon subsequent encounters with pathogens, converting what would have been a compatible interaction to an incompatible one. Our studies in three plant–pathogen systems clearly document that multigenic-resistant plants constitutively express specific isozymes of hydrolytic enzymes that release cell wall elicitors, which in turn may activate other defense mechanisms. ISR induces constitutive accumulation of these and other gene products prior to challenge. ISR is known to function against multiple organisms, and there is no specificity observed in the accumulation patterns of defense-related gene products when ISR is induced. It is therefore hypothesized that the constitutive accumulation of specific isozymes of hydrolytic enzymes, or other defense related gene products, is an integral part of both multigenic resistance and the phenomenon of ISR. Further, plants in which ISR has been activated appear to move from a latent resistance state to one in which a multigenic, non-specific form of resistance is active.     

Knot Formation Caused by Pseudomonas syringae subsp. savastanoi on Olive Plants Is hrp-Dependent

ABSTRACT The virulence of Pseudomonas syringae subsp. savastanoi, which causes hyperplastic symptoms (knots) on olive plants, is associated with secreted phytohormones. We identified a Tn5-induced mutant of P. syringae subsp. savastanoi that did not cause disease symptoms on olive plants although it was still able to produce phytohormones. In addition, the mutant failed to elicit a hypersensitive response in a nonhost plant. Molecular characterization of the mutant revealed that a single Tn5 insertion occurred within an open reading frame encoding a protein 92% identical to the HrcC protein of P. syringae pv. syringae. Moreover, sequence analysis revealed that the gene encoding the HrcC protein in P. syringae subsp. savastanoi was part of an operon that included five genes arranged as in other phytopathogenic bacteria. These results imply that hrp/hrc genes are functional in P. syringae subsp. savastanoi and that they play a key role in the pathogenicity of this plant pathogen.     

广东南瓜细菌性叶枯病及其病原鉴定

 在广东省雷州市发生一种南瓜(Cucurbita moschata)叶枯病,病株叶片边缘开始出现水渍状病斑,逐步发展成大病斑,后期病斑焦枯;在叶片上也可形成近圆形水渍状病斑,伴有黄色晕圈,后期病斑联合形成不规则大枯斑;叶柄和匍匐茎被侵染后呈水渍状腐烂。从病斑上分离到一种细菌,在KB培养基上,菌落为椭圆形,乳白色,半透明,边缘参差不齐,紫外灯照射下产生荧光反应。致病性测定结果表明,该病原细菌可侵染6个南瓜品种引起与田间症状相同的叶枯病。生理生化试验结果表明,该病原细菌与丁香假单胞丁香致病变种(Pseudomonas syringae pv. syringae)的特性一致。应用假单胞菌属特异引物Ps-for/Ps-rev和丁香假单胞丁香致病变种组群特异性引物Group III-F/Group III-R,可从该病原细菌中扩增出预期大小分别为1 018 bp和750 bp的目的片段。应用丁香致病变种syrB基因特异性引物B1/B2,可从该病原菌中扩增出预期大小为750 bp的丁香霉素基因片段。基于16S rDNA与gyrB基因序列系统进化分析均表明,南瓜叶枯病菌株与已报道的P. syringae pv. syringae菌株HS191(CP006256)亲缘关系最近,二者聚类在一起形成一个小分支。人工接种条件下,该病原细菌还可侵染西葫芦、丝瓜、茄子、番茄、菜豆、扁豆等植物。这些结果表明,引起广东省南瓜叶枯病的病原为丁香假单胞丁香致病变种(Pseudomonas syringae pv. syringae)。这是首次在中国发现丁香假单胞丁香致病变种引起南瓜叶枯病。     

Comparison of Ethylene Production by Pseudomonas syringae and Ralstonia solanacearum

ABSTRACT Strains of Pseudomonas syringae pv. pisi and Ralstonia solanacearum produced ethylene at rates 20- and 200-fold lower, respectively, than strains of P. syringae pvs. cannabina, glycinea, phaseolicola, and sesami. In the current study, we investigated which ethylene biosynthetic pathways were used by P. syringae pv. pisi and R. solanacearum. Neither the activity of an ethylene-forming enzyme nor a corresponding efe gene homolog could be detected in R. solanacearum, suggesting synthesis of ethylene via 2-keto-4-methyl-thiobutyric acid. In contrast, 2-oxoglutarate-dependent ethylene formation was observed with P. syringae pv. pisi, and Southern blot hybridization revealed the presence of an efe homolog in this pathovar. The efe genes from P. syringae pvs. cannabina, glycinea, phaseolicola, pisi, and sesami were sequenced. Nucleotide sequence comparisons indicated that the efe gene in pv. pisi was not as highly conserved as it was in other P. syringae pathovars. The pv. pisi efe homolog showed numerous nucleotide substitutions and a deletion of 13 amino acids at the C-terminus of the predicted gene product. These sequence alterations might account for the lower rate of ethylene production by this pathovar. All ethylene-producing P. syringae pathovars were virulent on bush bean plants. The overlapping host range of these pathovars suggests that horizontal transfer of the efe gene may have occurred among bacteria inhabiting the same host.     

Pseudomonas syringae pv. syringae on pepper seedlings in Italy

Pseudomonas syringae pv. syringae causing leaf spot on pepper seedlings grown in a plant bed is reported in Italy for the first time. The pathogen was identified by means of biochemical, physiological and pathogenicity tests as well as by SDS-polyacrylamide gel electrophoresis of whole-cell proteins. The bacterial isolates showed positive for ice nucleation and biocide production.     

黄瓜细菌性角斑病免疫胶体金检测试纸条的研制

 应用免疫胶体金技术进行黄瓜细菌性角斑病(Pseudomonas syringae pv.lachrymans)的快速检测研究。为了研制黄瓜细菌性角斑病的免疫胶体金检测试纸条,通过柠檬酸三钠还原法制备胶体金,选择25 nm胶体金标记黄瓜细菌性角斑病菌多克隆抗体(CMb)。采用双抗夹心法,将CMb-胶体金复合物包被在胶体金结合垫上,将羊抗兔二抗和CMb包被在硝酸纤维素膜上,分别作为质控线(C)和检测线(T),组装成试纸条。该试纸条检测灵敏度为106 cfu/mL,与唐菖蒲疮痂病菌(Pseudomonas fluorescens biovarⅡ)等26个菌株无交叉反应,特异性强,检测时间为15 min。稳定性试验表明试纸条在37℃条件下放置15 d可保持检测结果的可靠性。用试纸条对田间采集的病叶进行检测,C线和T线清晰可见,缓冲液对照呈阴性反应。本试纸条可应用于生产上黄瓜细菌性角斑病早期快速检测,进而指导病害防治。