Phylogenetic Relationships Among Global Populations of Pseudomonas syringae pv. actinidiae

ABSTRACT Pseudomonas syringae pv. actinidiae, the causal agent of canker in kiwifruit (Actinidia spp.) vines, was first detected in Japan in 1984, followed by detections in Korea and Italy in the early 1990s. Isolates causing more severe disease symptoms have recently been detected in several countries with a wide global distribution, including Italy, New Zealand, and China. In order to characterize P. syringae pv. actinidiae populations globally, a representative set of 40 isolates from New Zealand, Italy, Japan, South Korea, Australia, and Chile were selected for extensive genetic analysis. Multilocus sequence analysis (MLSA) of housekeeping, type III effector and phytotoxin genes was used to elucidate the phylogenetic relationships between P. syringae pv. actinidiae isolates worldwide. Four additional isolates, including one from China, for which shotgun sequence of the whole genome was available, were included in phylogenetic analyses. It is shown that at least four P. syringae pv. actinidiae MLSA groups are present globally, and that marker sets with differing evolutionary trajectories (conserved housekeeping and rapidly evolving effector genes) readily differentiate all four groups. The MLSA group designated here as Psa3 is the strain causing secondary symptoms such as formation of cankers, production of exudates, and cane and shoot dieback on some kiwifruit orchards in Italy and New Zealand. It is shown that isolates from Chile also belong to this MLSA group. MLSA group Psa4, detected in isolates collected in New Zealand and Australia, has not been previously described. P. syringae pv. actinidiae has an extensive global distribution yet the isolates causing widespread losses to the kiwifruit industry can all be traced to a single MLSA group, Psa3.     

Occurrence of Pseudomonas syringae pv. actinidiae on kiwifruit in Italy

Pseudomonas syringae pv. actinidiae has been isolated from kiwifruit plants for the first time in Italy. Biochemical tests were consistent with those characterizing the type-strain; pathogenicity tests yielded severe blights in the inoculated kiwifruit plants and no symptoms on lilac, pear and peach. Nutritional tests as well as whole-cell protein profiles revealed slight differences between the strains isolated in Japan and those of the present study. The main symptoms observed in the field are a red-rusty exudation covering the bark of twigs and trunks, blight of young canes and plants, angular leaf spots surrounded by chlorotic haloes and tiny cankers along the twigs.     

Genomic and phenotypic characterization of the bacterium causing blight of kiwifruit in New Zealand

Strains of the pathogen causing bacterial blight of kiwifruit in New Zealand, previously identified as Pseudomonas viridiflava, were examined using phenotypic and genotypic methods. Percentage DNA–DNA reassociation values for strains of the pathogen, with the type strains representing P. viridiflava and P. savastanoi, and representative strains within P. syringae , were obtained using the S1 endonuclease method. Strains of the pathogen were most similar to the type strain of P. savastanoi. This similarity was supported by examination of the Δ T _m between representative strains. It is concluded that the pathogen can be considered as a member of the P. savastanoi genomic species. The pathogen from kiwifruit in New Zealand was also differentiated in genomic terms from P. syringae pv. actinidiae . Strains of the kiwifruit pathogen compared using the Biomerieux API Biotype 100 system exhibited consistent determinative tests which distinguished the pathogen from P. viridiflava and P. syringae pv. actinidiae . The origins of the pathogen in New Zealand are discussed.     

猕猴桃溃疡病菌在中国的适生性分析

通过分析猕猴桃溃疡病菌在中国的适生性,为科学制定有效的检疫监管措施,防范其入侵和扩散,确保猕猴桃产业健康发展提供理论依据。本研究根据前人研究结果,采用模糊数学综合评判的原理和方法,定量分析猕猴桃细菌性溃疡病菌(Pseudomonas syringae pv.actinidiae)在我国各个地区的适生性。猕猴桃溃疡病菌在我国最适宜的省份主要分布在四川、云南、贵州、福建、安徽、湖南、湖北、河南、江西、陕西、浙江、重庆、西藏。鉴于该病具有发生发展迅速,危害性强,防治难度大等特点,应当加强猕猴桃种苗等繁殖材料的检疫,加强对果园的管理和病害监测,积极采取有效的防治措施并加强抗病育种方面的研究。     

猕猴桃溃疡病菌的分子检测技术研究

 猕猴桃溃疡病是猕猴桃生产上的主要病害,为建立该病的快速诊断技术,本实验通过RAPD分析获得一条1 300 bp左右的致病菌的特异片段,对该片段进行克隆测序,在测序的基础上设计并合成一对特异引物F7/R7,优化特异引物扩增条件,并验证引物的特异性和灵敏性。利用该特异引物对包括猕猴桃溃疡病菌在内的14个菌株基因组DNA进行PCR扩增表明,只有猕猴桃溃疡病菌能扩增出1条约为950 bp的特异条带,其他菌株及对照均未扩增出特异条带。对采自果园的染病枝干组织和接种致病菌的枝干组织的检测表明,该特异引物能特异性地检测到猕猴桃溃疡病菌的存在,其在组织中的检测灵敏度为100 fg/μL。因此,利用设计合成的特异引物F7/R7,参考优化的体系和程序,结合简单的试剂盒法提取猕猴桃溃疡病菌或植物组织DNA,可以在短时间内完成对该病原菌的分子检测。     

Infection of horse chestnut (Aesculus hippocastanum) by Pseudomonas syringae pv. aesculi and its detection by quantitative real-time PCR

Pseudomonas syringae pv. aesculi is a pathogenic bacterium causing bleeding canker disease of horse chestnut ( Aesculus hippocastanum ). This is a serious disease which has been affecting horse chestnut in several European countries over the last five years; however, very little is known about the biology of the causal agent. One of the obstacles to studying this pathogen is the lengthy procedure associated with confirming its presence on the host. In this study, P. syringae pv. aesculi was isolated from lesions on different parts of horse chestnut and its pathogenicity confirmed on horse chestnut saplings using two inoculation techniques. Real-time PCR primers were developed based on gyrase B gene sequence data for the specific detection of P. syringae pv. aesculi . Primer specificity was tested on isolates of the target pathogen as well as on a broad range of related non-target bacteria and other bacterial spp. which inhabit horse chestnut. The real-time primers reliably amplified P. syringae pv. aesculi down to 1 pg of extracted DNA, with and without the presence of host DNA, and also amplified unextracted DNA in whole cells of the bacterium down to at least 160 colony forming units. Detection and quantification of the target pathogen in phloem and xylem of both naturally infected and inoculated horse chestnut tissues was also demonstrated. This quantitative real-time PCR assay provides the facility to study several important aspects of the biology of P. syringae pv. aesculi on horse chestnut including its potential for dissemination in different substrates.     

猕猴桃品种酚类物质及可溶性蛋白含量与抗溃疡病的关系

以安徽省猕猴桃主栽品种金魁、早鲜、魁蜜、华美2号、秦美、金丰为研究对象,于展叶孕蕾期分别取发病的枝条、叶片,以未发病健株的相应组织为对照,分析枝条、叶片中酚类物质和可溶性蛋白的含量变化。结果表明：抗病品种健株枝条、叶片中可溶性蛋白含量显著高于易感病品种,说明枝条中可溶性蛋白含量与品种抗性成正相关。自然发病后,感病品种枝条中可溶性蛋白含量增加,抗病品种可溶性蛋白含量降低。抗病品种健枝条、叶片中酚类物质含量高于易感病品种的健枝、叶,发病后抗感品种酚类物质含量都增加。     

豆薯细菌性角斑病的病原鉴定

 在安徽滁州的豆薯叶片上发现一种由细菌侵染引起角斑症状的病害,从角斑上分离到具有致病性的非荧光的杆状细菌,菌株的表型特征、细菌学特征、LOPAT试验和生理生化试验表明该细菌与丁香假单胞菌(Pseudomonas syringae van Hall)相似,BIOLOG系统鉴定结果与丁香假单胞菌豌豆致病变种(P.syringae pv.pisi)相近,接种试验表明豆薯菌株能侵染大豆、菜豆和眉豆,但对豌豆的致病性差;在豇豆、绿豆和蚕豆上不表现症状。结果表明豆薯细菌性角斑病是一种新病害,病原菌属于丁香假单胞菌群的一个新的致病变种,命名为P.syringae pv.pachyrhizus nov.     

Analysis of the natural infection of European horse chestnut (Aesculus hippocastanum) by Pseudomonas syringae pv. aesculi

Pseudomonas syringae pv. aesculi (Psa) is an emerging bacterial pathogen responsible for a recent epidemic of bleeding canker of European horse chestnut (Aesculus hippocastanum) in northwest Europe. Very little is known about the infection biology of this pathogen, which can cause lethal cankers in the branches and stem of its host. In this study, branches and whole trees of European horse chestnut naturally infected with Psa were subjected to detailed morphological and histological examination to identify the primary infection sites, the time of infection, and the patterns of subsequent lesion expansion within the host. Lesions developed during the host dormant season on the 2003–2009 extension growth increments and were centred mainly on lenticels, leaf scars and nodes. The oldest lesion developed in the 2004/2005 dormant season and the number of new lesions increased in each subsequent year. The lesions developed in the cortex and phloem and extended into the cambium to cause cankers, but there was no evidence of necrosis in the xylem. All lesions on the branches were discrete and apparently contained by a necrophylactic periderm, although there was evidence that Psa could survive within such periderms and subsequently breach them. Examination of two whole 30‐year‐old trees revealed extensive, continuous cankers in the phloem and cambium which had formed within a single growing season. Thus, the success of Psa as a tree pathogen and the causal agent of a large‐scale epidemic may in part reflect an ability to infect the aerial woody parts of its host directly.     

Transgenic production of cytokinin suppresses bacterially induced hypersensitive response symptoms and increases antioxidative enzyme levels in Nicotiana spp

Responses of cytokinin overproducing transgenic Nicotiana plants to infections with compatible and incompatible Pseudomonas syringae pathovars were compared. Plants used were transformed with the ipt(isopentenyl transferase) gene that catalyzes the synthesis of cytokinin. In cytokinin overproducing lines that carry the ipt gene fused to the CaMV 35S (Nt+ipt), the wound-inducible proteinase inhibitor II (Ntx+ipt), or the light-inducible Rubisco small subunit protein (Npl+ipt) promoter, development of the hypersensitive response (HR) after infection with incompatible bacteria (P. syringae pv. tomato) was significantly inhibited as compared to the untransformed (Nt) controls. Over a 12 h period following inoculation, P. syrinage pv. tomato populations were slightly reduced in leaves of the cytokinin-overproducing Nt-ipt line compared with the Nt control. When the compatible P. syringae. pv. tabaci was used to infect the ipt transformed lines, slight or no significant differences in necrosis development were observed. Following infection, the titer of P. syringae pv. tabaci increased rapidly in both the transgenic and control lines but was higher in Nt+ipt plants. Leaf superoxide dismutase and catalase enzyme activities were about 60% higher in ipt leaf extracts than in the controls. This augmented antioxidant capacity likely decreased the amount of H(2)O(2) that may be associated with the higher tolerance of plants to pathogen-induced necrosis. In addition, the Nt+ipt lines had a significantly lower molar ratio of free sterols to phospholipids. The more stable membrane lipid composition and the higher antioxidant capacity likely contributed to the suppressed HR symptoms in the cytokinin overproducing Nt+ipt plants. In conclusion, the overproduction of cytokinins in tobacco appears to suppress the HR symptoms induced by incompatible bacteria.     

Phloem and xylem modifications of Vitis vinifera stems in response to flavescence dorée phytoplasma infection

Anatomical modifications of xylem and phloem tissues of grapevine (Vitis vinifera) stems of shoots infected by the flavescence dorée phytoplasma (FDp) were first observed and described in the 1960s, but never quantified in detail. In this paper, we describe and quantify the impact of FDp on grapevine stem tissues, and relate it to the level of expression of symptoms and to cultivar-specific FDp susceptibility. For this purpose, we measured and quantified the anatomical parameters of xylem and phloem tissues of a tolerant (Merlot) and a susceptible (Chardonnay) cultivar. For each cultivar, thin sections of eight shoots with symptoms from FDp-infected grapevines, eight symptomless shoots from the same FDp-infected grapevines, and eight symptomless shoots from symptomless grapevines (control) were compared. Results showed general inhibition of xylem growth and proliferation of phloem tissues (hyperplasia) with lack or irregular arrangement of the fibre-sclereids in the axial phloem of the stems from shoots with symptoms, irrespective of the cultivar. Xylem vessels of infected Merlot shoots were partly occluded by tyloses and a higher number of smaller vessels were produced than in control plants. Thus, the anatomical responses confirmed the detrimental effect of FDp on stems of infected grapevine shoots, including impaired stem development and lack of periderm formation. Statistically significant differences were found between the two cultivars with different levels of susceptibility to FDp infection.     

Pseudomonas syringae pv. coryli, the Causal Agent of Bacterial Twig Dieback of Corylus avellana

ABSTRACT Thirty-eight bacterial strains isolated from hazelnut (Corylus avellana) cv. Tonda Gentile delle Langhe showing a twig dieback in Piedmont and Sardinia, Italy, were studied by a polyphasic approach. All strains were assessed by fatty acids analysis and repetitive sequence-based polymerase chain reaction (PCR) fingerprinting using BOX and ERIC primer sets. Representative strains also were assessed by sequencing the 16S rDNA and hrpL genes, determining the presence of the syrB gene, testing their biochemical and nutritional characteristics, and determining their pathogenicity to hazelnut and other plants species or plant organs. Moreover, they were compared with reference strains of other phytopathogenic pseudomonads. The strains from hazelnut belong to Pseudomonas syringae (sensu latu), LOPAT group Ia. Both fatty acids and repetitive-sequence-based PCR clearly discriminate such strains from other Pseudomonas spp., including P. avellanae and other P. syringae pathovars as well as P. syringae pv. syringae strains from hazelnut. Also, the sequencing of 16S rDNA and hrpL genes differentiated them from P. avellanae and from P. syringae pv. syringae. They did not possess the syrB gene. Some nutritional tests also differentiated them from related P. syringae pathovars. Upon artificial inoculation, these strains incited severe twig diebacks only on hazelnut. Our results justify the creation of a new pathovar because the strains from hazelnut constitute a homogeneous group and a discrete phenon. The name of P. syringae pv. coryli is proposed and criteria for routine identification are presented.     

Leaf necrosis and twig dieback of sweet persimmon (Dyospiros kaki L.) caused by Pseudomonas syringae pv. syringae in Italy

In spring 1996, extensive leaf necrosis and twig dieback were observed on young sweet persimmon (Dyospiros kaki L.) trees, cultivars O'Gosho, Hachija, Mercatelli and Kaki-tipo planted in the Abruzzo region (central Italy). Many trees were killed. When the dieback reached the trunk, in many cases, new vegetation was noticed above the graft point. The cultivar Jiro-C was not affected by the disease. During 1997, no symptoms were observed on any plant. The orchard was planted in a clay soil with a very low content of organic matter. Biochemical, nutritional and pathogenicity tests indicated Pseudomonas syringae pv. syringae van Hall as the causal agent of the disease. This is the first report of this bacterium as a pathogen of sweet persimmon in Europe.     

Pseudomonas syringae pv. solidagae pv. nov., the Causal Agent of Bacterial Leaf Spot of Tall Goldenrod Solidago altissima L.

A new bacterial disease of tall goldenrod (Solidago altissima L., “Seitaka-awadachiso” in Japanese), one of the most serious weeds in non-agricultural land, was discovered in Ibaraki Prefecture, Japan. Characterized by angular or round, dark brown necrotic spots on leaves, this disease resulted in defoliation and terminal dieback of the plants in severe cases. The disease was named “bacterial leaf spot”. The causal bacterium was identified as Pseudomonas syringae based on its bacteriological properties including those determined by LOPAT tests. The present bacterium was pathogenic to tall goldenrod alone but not to many other tested plants including weeds, flowers, trees and crops. In addition, P. syringae pv. syringae and other pathovars did not show any pathogenicity to tall goldenrod. Because no pathovars of P. syringae pathogenic to tall goldenrod have been reported, the present bacterium was concluded to be a new pathovar of P. syringae. We propose the name P. syringae pv. solidagae pv. nov. , and strain Sei 1 (MAFF 810063) is designated as the pathotype strain and has been deposited in the MAFF collection with two reference strains (MAFF 810064 and MAFF81066). Received 9 May 2001/ Accepted in revised form 18 June 2001     

中国猕猴桃细菌性花腐病菌的鉴定

 从福建、湖南和湖北猕猴桃病花上分离到能引起花腐病的32个细菌菌株,经细菌学和BiologGN测试板测定,可以看出中国的猕猴桃细菌性花腐病菌与新西兰的猕猴桃花腐病菌、丁香假单胞菌丁香致病变种Pseudomonas syringae pv.syringae和绿黄假单胞菌P.viridiflava相似,与萨氏假单胞菌P.savastanoi和猕猴桃溃疡病菌P.syringae pv.actinidiae有更多的不同,但是DNA/DNA同源性测定结果却显示出中国的菌株可分为2个类型:第1个类型与新西兰猕猴桃花腐病菌和萨氏假单胞菌有很高的同源性,第2类型与绿黄假单胞菌有很高的同源性,说明中国菌株分别属于这2个种。第1类型来自于福建和湖北,第2类型来自于湖南。     

Presence of Brome mosaic virus in Barley Guttation Fluid and Its Association with Localized Cell Death Response

ABSTRACT Water exits from inside the leaf through transpiration or guttation. Under conditions to promote guttation, surface fluid (guttation fluid) from Brome mosaic virus (BMV)-infected barley, wheat, and maize plants was analyzed for the presence of the virus by biological and serological assays. We also investigated the route by which BMV exited infected cells to the intercellular space of the barley leaf. BMV was detected in guttation fluid from systemically infected barley leaves when the initial viral symptoms were observed on these leaves. The virus was also detected in guttation fluid from systemically infected wheat leaves, but not in maize leaves showing either systemic necrosis or chlorotic streaks. Interestingly, in BMV-infected barley leaves, but not in maize leaves showing chlorotic streaks, cell death occurred within and adjacent to veins. Staining of xylem and phloem networks in infected barley leaves with fluorescent dyes showed that xylem, and to a lesser extent phloem, were severely damaged and thus became leaky for dye transport. No such damage was observed in BMV-infected maize leaves showing chlorotic streaks. We propose that in infected barley leaves, BMV exits from damaged vein cells (especially the xylem elements), accumulates in intercellular spaces, and then reaches the surface of the leaves through stomata during guttation or transpiration. In nature, BMV may be carried to adjacent plants and cause infection by movement of vertebrate and invertebrate vectors among infected plants exuding guttation fluid.     

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.     

First report of bacterial canker of kiwifruit in Iran

Symptoms of bacterial canker of kiwifruit consisting of darkening and shrivelling along the bark were observed in kiwifruit orchards in Tonkabon, Iran. Longidutinal sections showed darkening of cortical and woody tissues. A fluorescent pseudomonad was consistently isolated from canker tissues. According to biochemical, physiological and pathogenicity tests the causal agent was identified as Pseudomonas syringae pv. syringae. This is the first report of the disease in Iran.     

Expression analysis of defense-related genes in wild kiwifruit (Actinidia rufa) tolerant to bacterial canker

Journal of General Plant Pathology - Kiwifruit bacterial canker, which is caused by Pseudomonas syringae pv. actinidiae biovar 3 (Psa3), is found throughout kiwifruit-growing countries. Here, we...     

猕猴桃品种中糖分及木质素含量 与抗溃疡病的关系

以安徽省猕猴桃主栽品种金魁、早鲜、魁蜜和金丰为研究对象，于展叶孕蕾期分别取发病的枝条、叶片，以未发病的健株相应组织为对照，采用生理生化的方法，分析枝条、叶片中木质素和可溶性糖的含量变化以及与抗溃疡病的关系。结果表明：抗病品种金魁健株枝条、叶片中可溶性糖及木质素含量显著高于感病品种金丰。自然发病后，抗感病品种枝条、叶片中可溶性糖含量都降低，感病品种降低更多，金魁叶片可溶性糖含量下降4．20％，金丰叶片可溶性糖含量下降55.35％；木质素含量都升高，且抗病品种金魁叶片中的木质素含量比感病品种金丰高得多，其变化率分别为7．17％、3．01％，枝条中的木质素含量变化率分别为110．39％、68．98％，其差异达到显著水平。相关分析表明，枝条中木质素、可溶性糖含量与品种发病率也呈负相关，γ分别为-0．9583和-0．9282；叶片中木质素、可溶性糖含量与品种发病率呈负相关，γ分别为-0．8099和-0、8266。从而说明枝条、叶片中可溶性糖及木质素含量与品种抗性呈正相关。抗感品种淀粉含量无明显规律性变化，与品种抗性关系不大。