Determination of Ralstonia (Pseudomonas) solanacearum rDNA subgroups by PCR tests

Rapid and sensitive polymerase chain reaction (PCR) methods are described for determination of the two 16 S rDNA subgroups of Ralstonia solanacearum, the causal agent of bacterial wilt. A third subgroup consisting of Indonesian R. solanacearum isolates belonging to Division II, the blood disease bacterium and Pseudomonas syzygii can also be identified. Primers were designed to sequences within R. solanacearum 16 S rDNA (equivalent to Escherichia coli 16 S rDNA positions 74–97, 455–475, 1454–1474), and the internal transcribed spacer region between the 16 S and 23 S rDNA genes. Different combinations of forward and reverse primers allowed selective PCR amplification of (a) R. solanacearum Division I (biovars 3, 4 and 5), (b) Division II (biovars 1, N2, and 2) including the blood disease bacterium and P. syzygii , or (c) amplification of Division II only except for five biovar 1, 2 or N2 isolates of R. solanacearum from Indonesia, P. syzygii and the BDB. A total of 104 R. solanacearum , 14 blood disease bacterium and 10 P. syzygii isolates were tested. Simultaneous detection of species and subdivision was achieved by designing a multiplex PCR test in which a 288-base pair (bp) band is produced by all R. solanacearum isolates, and an additional 409-bp band in Division I strains.     

烟草感染青枯菌后的组织病理变化

病症观察表明,烟草感染青枯菌后168h叶片全部萎蔫下垂、褐变。茎部切片表明,青枯菌处理72h,个别导管内出现了染色较深的物质,髓部和皮层的部分薄壁细胞出现破损。120h后导管内染色较深的物质增多,导管堵塞程度加大。168h后局部区域的木质部和韧皮部分离。对感病植株的叶进行青枯病菌分离,结果表明:从叶中可分离出致病青枯菌,并可以导致复感植株得病继而死亡。     

Comparison of ethylene-producing Pseudomonas syringae strains isolated from kudzu (Pueraria lobata) with Pseudomonas syringae pv. phaseolicola and Pseudomonas syringae pv. glycinea

The relationships among strains of Pseudomonas syringae pv. glycinea (Psg) and Pseudomonas syringae pv. phaseolicola (Psp) isolated from kudzu ( Pueraria lobata) and bean ( Phaseolus vulgaris) were investigated. All strains tested showed a close phenotypic similarity, with the exception of the utilization of inositol and mannitol as well as the production of toxins. On this basis the strains could be divided into three groups. Group 1 consists of all strains of pathovar glycinea, group 2 includes all Psp strains isolated from kudzu, and all Psp strains isolated from bean belong to group 3. This grouping was also reflected in the genetic fingerprints using the polymerase chain reaction (PCR) with primers that anneal to dispersed repetitive bacterial sequences (rep-PCR). The rep-PCR generated fingerprints were unique for each of the three groups. The strains of group 2, Psp strains isolated from kudzu, possess certain characteristics of group 1 (ethylene production) and group 2 (phaseolotoxin production). The Psp strains from kudzu can be clearly differentiated from Psp strains isolated from bean. They utilize mannitol, produce ethylene, and are strongly pathogenic to kudzu, bean, and soybean. The results obtained show that the Psp strains from kudzu should be separated from the pathovar phaseolicola and should represent their own pathovar.     

Bacterial wilt of bellflower caused by Ralstonia solanacearum in Japan

A sudden wilt of bellflower (Campanula lactiflora) was observed in Japan in 1997. A bacterium that formed white fluidal and mucoid colonies resembling those of Ralstonia solanacearum was isolated from the infected plants. The bacterium was bacteriologically identified as biovar 3 of R. solanacearum. This is the first report of R. solanacearum affecting a plant species of the Campanulaceae family.     

Chlorosis-inducing Phytotoxins Produced by Pseudomonas syringae

Pseudomonas syringae causes a wide range of symptoms on plants including blights, leaf spots, and galls. Phytotoxins generally enhance the virulence of phytopathogenic P. syringae, and their synthesis can substantially increase disease severity. Although several P. syringae phytotoxins cause chlorosis (coronatine, phaseolotoxin, and tabtoxin), they are synthesized by unrelated biosynthetic pathways and have completely different modes of action. Phaseolotoxin and tabtoxin inhibit ornithine carbamoyltransferase and glutamine synthetase, respectively, whereas coronatine functions as a mimic of methyl jasmonate in some plant species. This review focusses on the mode of action, genetics, biosynthesis and regulation of coronatine, tabtoxin, and phaseolotoxin. Current techniques used to detect these toxins and phytotoxin-producing P. syringae pathovars are discussed. The utilization of toxin resistance genes in the development of transgenic plants with phytotoxin tolerance is also reviewed.     

Genetic Diversity of Japanese Strains of Ralstonia solanacearum

ABSTRACT The genetic diversity of 74 Japanese strains of Ralstonia solanacearum was assessed by pathogenicity tests and the repetitive sequencebased polymerase chain reaction (rep-PCR) fingerprint method. Based on their genomic fingerprints, biovar N2 strains were divided into two distinct groups, one consisting of potato isolates belonging to race 3, and the other consisting of tomato, eggplant, pepper, and tobacco isolates belonging to race 1. Biovar 3 strains had low average similarity and were divided into five groups that differed in original host or pathogenicity. Biovar 4 strains consisted of only one group at the 80% similarity level. Comparative analysis of the rep-PCR fingerprints of 78 strains, including six biovars from Japan and various countries, revealed two main clusters. Cluster 1 comprised all biovar 3, 4, and 5 strains, biovar 1 strains from Reunion, and some biovar N2 strains from Japan. Cluster 2 included most of the biovar 1, 2, and N2 strains. The fingerprints showed low average similarity with biovar N2 strains from Japan and Brazil.     

Specific Detection of Biovars of Ralstonia solanacearum in Plant Tissues by Nested-PCR-RFLP

A sensitive and specific assay, based on a Nested-PCR-RFLP protocol, was developed for the detection of biovars of Ralstonia solanacearum, the causal agent of bacterial wilt. Oligonucleotide primer pairs were selected within the hrp gene region. Specific amplification of the hrp fragments was obtained for all R. solanacearum strains and also for two closely related species, Pseudomonas syzygii and the blood disease bacterium. No amplification was observed for a wide range of other bacterial species, including R. pickettii and Burkholderia cepacia. Digestion with HindII provided four distinct restriction profiles specific to biovars or groups of biovars of R. solanacearum: one for biovar 1 strains originating from the Southern part of Africa, one for American biovar 1 and biovars 2 and N2 strains, one for biovars 3 and 4 strains, and one for biovar 5 strains. When applied to either pure culture or infected plant tissues, Nested-PCR allowed detection as low as 10³cfu ml^–1, which corresponds to 1cfu per reaction. Amplification was partially or completely inhibited by compounds contained in plant extracts (potato plant and potato tuber, tomato, tobacco, eggplant, pepper and Pelargonium asperum). A combined PVPP/BSA treatment prior to amplification permitted reliable Nested-PCR detection of R. solanacearum strains in plant samples. Nested-PCR-RFLP, assessed with isolates from Reunion Island but also applicable to any R. solanacearum strain, provides a wide range of possible uses for identification, detection and epidemiological investigations.     

Suppression of Ralstonia solanacearum bacterial wilt disease by an organic hydroponic system

An organic hydroponic system that we developed has potential to control root diseases including bacterial wilt of hydroponically grown tomato. In inoculation tests with Ralstonia solanacearum during tomato plant cultivation in conventional inorganic hydroponics and in our organic system, many of the tomato seedlings in the conventional system wilted and died, but none of the seedlings in the organic hydroponics wilted or developed any symptoms, suggesting that the organic system can suppress this bacterial wilt disease. Interestingly, a rhizosphere biofilm, formed only on roots in the organic hydroponic system, may be responsible for the suppression of the bacterial wilt.     

Induction of pear blossom blast caused by Pseudomonas syringae pv. syringae

Conditions were established for inducing pear blossom blast caused by Pseudomonas syringae pv. syringae on both attached and detached shoots. The incidence of blossom blast was proportional to the logarithm of the P.s. pv. syringae population under optimal temperature, moisture, and bloom developmental stage. Highest incidence of blossom infection followed occurrence of a major exotherm (an increase in temperature caused by the heat of fusion from ice formation within blossom tissue) in the presence of P. s. pv. syringae. The exotherm was detected inside ovary tissue at temperatures ranging from –1.8 to –3.5 C. Wetness duration following the thawing process was less important than wetness during and immediately after the freeze event. Blossoms inoculated, then air-dried or removed from low-temperature treatment prior to occurrence of an exotherm, had a low incidence of infection, The full bloom stage of blossom development was more susceptible to blossom blast than either the open cluster or tight cluster stages of development.     

Pathogenic characters of Japanese potato strains of Ralstonia solanacearum

Ralstonia solanacearum (Rs) strains in phylotypes I and IV isolated from potato in Japan were investigated for pathogenicity on potato, tomato, eggplant, Solanum integrifolium, tobacco, groundnut, and pumpkin. The strains were divided into 17 types based on differences in their pathogenicity on the tested plants. Particularly, the pathogenicity of most phylotype I strains on eggplant was distinctly different from that of the phylotype IV strains. When nine potato varieties (included two breeding lines) were inoculated with several Rs strains, phylotype IV strains were highly virulent on the breeding lines that are regarded as resistant to phylotype I strains.     

Bacterial inflorescence rot of grapevine caused by Pseudomonas syringae pv. syringae

Molecular sequencing (rpoB) and standard pathological and microbiological methods identified Pseudomonas syringae pv. syringae (Pss) as the causal agent of bacterial inflorescence rot of grapevines (Vitis vinifera) in three vineyards in Tumbarumba, NSW, Australia in 2006 and 2007. Pss strains from shrivelled berries and necrotic inflorescences of diseased grapevines were used to inoculate leaves and inflorescences of potted cv. Semillon grapevines. Pss caused disease symptoms similar to those experienced in the field, including angular leaf lesions, longitudinal lesions in shoot tissues and rotting of inflorescences from before flowering until shortly after fruit set. High humidity promoted symptom severity. The necrotic bunch stem and leaf lesions were susceptible to the development of Botrytis cinerea infections. Cryo‐scanning electron microscopy (cryoSEM) indicated that Pss entered leaves and inflorescence tissues via distorted, open, raised stomata surrounded by folds of tissue that appeared as ‘star‐shaped’ callose‐rich complexes when viewed by UV light microscopy. In necrotic tissues, cryoSEM revealed Pss within petiole parenchyma cells and air‐filled rachis xylem vessels. This is the first report of inflorescence and hence fruit loss caused by Pss in grapevines. The disease is described as ‘bacterial inflorescence rot’ and regarded as one that expands the previously reported pathology of grapevines caused by P. syringae. This study also indicated that infection by Pss might promote destructive B. cinerea infections when the fungus is already present but latent, although further experimentation is needed to prove such an interaction.     

Molecular Diversity of Ralstonia solanacearum Isolated from Ginger in Hawaii

ABSTRACT The genetic diversity of Ralstonia solanacearum strains isolated from ginger (Zingiber officinale) growing on the island of Hawaii was determined by analysis of amplified fragment length polymorphisms (AFLPs). Initially 28 strains of R. solanacearum collected from five host plant species worldwide were analyzed by AFLP. A second analysis was conducted on 55 R. solanacearum strains collected from three ginger farms along the Hamakua Coast of Hawaii, the principle area of ginger cultivation in the state. From the initial analysis, R. solanacearum strains from ginger in Hawaii showed a high degree of similarity at 0.853. In contrast, the average genetic similarity between R. solanacearum strains from heliconia and ginger was only 0.165, and strains from ginger showed little similarity with strains from all other hosts. The second analysis of 55 strains from ginger on different Hawaiian farms confirmed that they were distinct from race 1 strains from tomato. Strains from ginger also showed greater diversity among themselves in the second analysis, and the greatest diversity occurred among strains from a farm where ginger is frequently imported and maintained. Our results provide evidence that R. solanacearum strains from ginger in Hawaii are genetically distinct from local strains from tomato (race 1) and heliconia (race 2).     

温度和病原接种浓度对番茄青枯病菌侵染的影响

本试验以不同浓度的Pseudomovas solanacearum悬浮液接种番茄品种湘引79-1号(抗)和粤农2号(感),置于不同温度的光照培养箱内,观察发病情况.当菌液浓度为3×108CFU/mL、3×106CFU/mL在温度25℃以上时9天或更短时间就表现青枯病症状,发病的最低温度是22℃,但潜育期较长,需20天,温度30℃与25℃的病情指数没有显著差别,但30℃潜育期短,只需5天.25℃潜育期稍长,则要9天.病情的发展随着菌液浓度的增高而严重,3×108CFU/mL浓度的病情指数最高达90%,3×102CFU/mL浓度的病情指数最低为40%.     

中生菌素对马铃薯青枯病菌的溶菌作用

马铃薯青枯病菌［Ｐｓｅｕｄｏｍｏｎａｓｓｏｌａｎａｃｅａｒｕｍ（Ｓｍｉｔｈ）Ｄｏｗｓｏｎ］是一种寄主范围非常广泛的植物病原细菌,可以侵染所有茄科和茄科以外一百多种植物。中生菌素（农抗７５１）是中国农科院生防所研制成功的一种新型农用抗生素,属于Ｎ－糖苷...     

β-氨基丁酸和茄青枯菌hrp-突变体协同防治辣椒疫病研究

将具有不同作用机理的植物化学诱抗剂β-氨基丁酸(BABA)与无致病力的茄青枯菌hrp-突变体菌株联合使用,研究不同配比对辣椒疫病的协同防治效果和使用技术。结果表明,BABA与hrp-菌株按一定配比混合后于接种前使用一次,与单剂相比对辣椒疫病均有不同程度的增效作用。其中0.08%BABA+hrp-菌株1×10~7 cfu/m L、0.1%BABA+hrp-菌株1×10~6 cfu/m L和0.1%BABA+hrp-菌株1×10~5 cfu/m L等3种配比组合控制辣椒疫病的效果均在80%以上;与BABA单独使用相比,增效作用超过23%;以0.1%BABA+hrp-菌株1×10~6 cfu/m L配比效果最佳,其增效达到43%。适当增加使用次数后,防治效果及持效期均得到进一步提高。本研究为探索多种生防措施的协调和配合应用开辟了一条新途径。     

Pathogenicity and virulence factors of Pseudomonas syringae

In 1994, Oku reported that plant pathogens, mainly fungal pathogens, require three essential abilities to infect plants: to enter plants, to overcome host resistance, and to evoke disease. Because the infectious process of phytopathogenic bacteria differs from that of fungal pathogens, we have attempted to characterize pathogenicity, the ability of a pathogen to cause disease, using the phytopathogenic bacterium Pseudomonas syringae as a representative pathogen. To establish infection and incite disease development, bacteria first have to enter a plant. This process requires flagella- and type IV pili-mediated motility, and active taxis is probably necessary for effective infection. After bacteria enter a plant’s apoplastic spaces, they need to overcome host plant resistance. To do this, they secrete a wide variety of hypersensitive response and pathogenicity (Hrp) effector proteins into the plant cytoplasm to interfere with pathogen/microbe-associated molecular pattern- and effector-triggered immunity, produce phytohormones and/or phytotoxins to suppress plant defense responses and extracellular polysaccharides to prevent access by antibiotics and to chelate Ca²⁺, and activate the multidrug resistance efflux pump to extrude antimicrobial compounds for successful colonization. Furthermore, to evoke disease, bacteria produce toxins and Hrp effectors that compromise a plant’s homeostasis and injure plant cells. The expression of these virulence factors depends on the infection processes and environmental conditions. Thus, the expression and function of virulence factors interact with each other, creating complex networks in the regulation of bacterial virulence-related genes.