Characterization of Pseudomonas syringae pv. actinidiae (Psa) isolated from France and assignment of Psa biovar 4 to a de novo pathovar: Pseudomonas syringae pv. actinidifoliorum pv. nov.

Since 2008, bacterial canker of kiwifruit (Actinidia deliciosa and A. chinensis) caused by Pseudomonas syringae pv. actinidiae (Psa) has resulted in severe economic losses worldwide. Four biovars of Psa can be distinguished based on their biochemical, pathogenicity and molecular characteristics. Using a range of biochemical, molecular and pathogenicity assays, strains collected in France since the beginning of the outbreak in 2010 were found to be genotypically and phenotypically diverse, and to belong to biovar 3 or biovar 4. This is the first time that strains of biovar 4 have been isolated outside New Zealand or Australia. A multilocus sequence analysis based on four housekeeping genes (gapA, gltA, gyrB and rpoD) was performed on 72 strains representative of the French outbreak. All the strains fell into two phylogenetic groups: one clonal corresponding to biovar 3, and the other corresponding to biovar 4. This second phylogenetic group was polymorphic and could be divided into four lineages. A clonal genealogy performed with a coalescent approach did not reveal any common ancestor for the 72 Psa strains. Strains of biovar 4 are substantially different from those of the other biovars: they are less aggressive and cause only leaf spots whereas Psa biovars 1, 2 and 3 also cause canker and shoot die‐back. Because of these pathogenic differences, which were supported by phenotypic, genetic and phylogenetic differences, it is proposed that Psa biovar 4 be renamed Pseudomonas syringae pv. actinidifoliorum pv. nov. Strain CFBP 8039 is designated as the pathotype strain.     

Redefining the global populations of Pseudomonas syringae pv. actinidiae based on pathogenic,molecular and phenotypic characteristics

Knowing the population structure of a pathogen is fundamental for developing reliable phytosanitary legislation, detection techniques, and control strategies based on the actual aggressiveness and distribution of the pathogen. Currently, four populations of Pseudomonas syringae pv. actinidiae (Psa) have been described: Psa 1, Psa 2, Psa 3 and Psa 4. However, diagnostic assays specific for Psa populations do not detect Psa 4, the less virulent (LV) strains isolated in New Zealand. Similarly, multilocus sequence typing (MLST) of housekeeping genes, or broad Psa strain genome comparisons, revealed that Psa 4‐LV strains clustered separately from other Psa populations. In order to examine whether the placement of Psa 4 in the pathovar actinidiae was appropriate, various tests were carried out. It was shown that the Psa 4‐LV strains induced leaf and shoot wilting in Prunus cerasus, extensive necrotic lesions in Capsicum annuum fruits, and no significant symptoms in Actinidia deliciosa. Moreover, repetitive‐sequence PCR fingerprinting, type III secretion system effector protein genes detection and colony morphology clearly indicated the distinctiveness of Psa 4‐LV strains from the other three Psa populations. Rep‐PCR molecular typing revealed a high similarity of the Psa 4‐LV strains with members of Pseudomonas avellanae species. The Psa 4‐LV strains, most probably, belong to a new, still unnamed pathovar. It was concluded that the Psa 4‐LV strains isolated in New Zealand do not belong to the pathovar actinidiae, and, consequently, three Psa populations pathogenic to Actinidia spp. should currently include Psa 1, Psa 2 and Psa 3.     

Real‐time and qualitative PCR for detecting Pseudomonas syringae pv. actinidiae isolates causing recent outbreaks of kiwifruit bacterial canker

Since 2008, Pseudomonas syringae pv. actinidiae virulent strains (Psa‐V) have quickly spread across the main areas of kiwifruit (Actinidia deliciosa and A. chinensis) cultivation causing sudden and re‐emerging outbreaks of bacterial canker to both species. The disease caused by Psa‐V strains is considered worldwide as pandemic. Recently, P. syringae strains (ex Psa‐LV, now called PsD) phylogenetically related to Psa‐V have been isolated from kiwifruit, but cause only minor damage (i.e. leaf spot) to the host. The different biological significance of these bacterial populations affecting kiwifruit highlights the importance of having a diagnostic method able to detect Psa‐V, which is currently solely responsible for the severe damage to the kiwifruit industry. In order to improve the specific molecular detection of Psa‐V, a real‐time PCR assay has been developed based on EvaGreen chemistry, together with a novel qualitative PCR (PCR‐C). Both methods are based on specific primer sets for the hrpW gene of Psa. The real‐time PCR and PCR‐C were highly specific, detecting down to 50 and 200 fg, respectively, and were applied to a range of organs/tissues of kiwifruit with and without symptoms. These methods are important tools for both sanitary and certification programmes, and will help to avoid the spread of Psa‐V and to check possible inoculum sources. In addition to being used as routine tests, they will also enable the study of the biology of Psa‐V and the disease that it causes, whilst avoiding the detection of other populations of related P. syringae present in kiwifruit.     

Highly specific assays to detect isolates of Pseudomonas syringae pv. actinidiae biovar 3 and Pseudomonas syringae pv. actinidifoliorum directly from plant material

Pseudomonas syringae pv. actinidiae (Psa) is responsible for bacterial canker of kiwifruit. Biovar 3 of Psa (Psa3) has been causing widespread damage to yellow‐ and green‐fleshed kiwifruit (Actinidia spp.) cultivars in all the major kiwifruit‐producing countries in the world. In some areas, including New Zealand, P. syringae pv. actinidifoliorum (Pfm), another bacterial pathogen of kiwifruit, was initially classified as a low virulence biovar of Psa. Ability to rapidly distinguish between these pathovars is vital to the management of bacterial canker. Whole genome sequencing (WGS) data were used to develop PCR assays to specifically detect Psa3 and Pfm from field‐collected material without the need to culture bacteria. Genomic data from 36 strains of Psa, Pfm or related isolates enabled identification of areas of genomic variation suitable for primer design. The developed assays were tested on 147 non‐target bacterial species including strains likely to be found in kiwifruit orchards. A number of assays did not proceed because although they were able to discriminate between the different Psa biovars and Pfm, they also produced amplicons from other unrelated bacteria. This could have resulted in false positives from environmental samples, and demonstrates the care that is required when applying assays devised for pure cultures to field‐collected samples. The strategy described here for developing assays for distinguishing strains of closely related pathogens could be applied to other diseases with characteristics similar to Psa.     

软枣猕猴桃种质资源溃疡病抗性鉴定方法的建立与评价

以51份软枣猕猴桃Actinidia arguta种质资源为材料,以中华猕猴桃Actinidia chinensis Planch'红阳'、美味猕猴桃Actinidia chinensis var.deliciosa'徐香'为对照,利用丁香假单胞菌猕猴桃致病变种Pseudomonas syringae pv.actin...     

Current situation and characterization of Pseudomonas syringae pv. actinidiae on kiwifruit in Galicia (northwest Spain)

Bacterial canker of kiwifruit, caused by Pseudomonas syringae pv. actinidiae (Psa), is a disease that is spreading rapidly in several kiwifruit‐producing countries, causing significant economic losses. In 2011, it was detected for the first time in Spain, in the south of Galicia (northwest Spain). Kiwifruit orchards were therefore inspected and sampled in 2011 and 2012 to determine the pathogen distribution, and the isolates obtained were characterized by morphology, fatty acids profile, biochemical tests and molecular techniques. Isolates were obtained from Actinidia deliciosa ‘Hayward’ (from leaves, canes, flower buds, fruits and roots), from A. deliciosa ‘Summer’, from Actinidia chinensis ‘Jin Tao’ (from canes and leaves) and from A. chinensis pollinator ‘Belén’ (from canes). Results of the analysis of the cfl gene (phytotoxin production‐related), the tox–argK gene cluster and phylogenetic analysis of the cts gene demonstrated that all Psa isolates from northwest Spain correspond to the Psa3 population, which includes strains of haplotype 2. This is the first record of Psa3 and haplotype 2 in Spain.     

Evaluation of the wild Actinidia germplasm for resistance to Pseudomonas syringae pv. actinidiae

Bacterial canker of kiwifruit caused by Pseudomonas syringae pv. actinidiae (Psa) is a catastrophic disease that threatens the global kiwifruit industry. As yet, no cure has been developed. Planting resistant cultivars is considered as one of the most effective ways to control Psa. However, most existing cultivars lack Psa-resistance genes. Wild Actinidia resources contain rich genetic diversity and may have powerful disease-resistance genes under long-term natural selection, but lack of knowledge about the resistance to Psa for most Actinidia species results in some excellent wild resistant genotypes being underutilized. In this study, the response to Psa of 104 wild genotypes of 30 Actinidia species (including 37 taxa) was tested with an in vitro bioassay, and a considerable number of individuals from different species with tolerance or high resistance to Psa were identified. The results showed high consistency between years. This is the first large-scale evaluation of diverse Actinidia species with resistance to Psa through an in vitro bioassay. The resistant genotypes of A. chinensis identified could be used in future kiwifruit improvement programmes. The findings should help provide an understanding of the resistance to Psa.     

Similarity between Copper Resistance Genes from Pseudomonas syringae pv. actinidiae and P. syringae pv. tomato

Twenty-eight strains of Pseudomonas syringae pv. actinidiae isolated in 1984, 1987 and 1988 from kiwifruit orchards in Japan were tested for their resistance to copper sulfate. All strains isolated in 1984 were copper sensitive with a minimum inhibitory concentration (MIC) of cupric sulfate of 0.75 mM. However, some strains isolated in 1987 and 1988 were resistant, with the MIC ranging from 2.25 to 3.0 mM. All copper-resistant strains contained at least one of two plasmids, pPaCul (about 70.5 kb) or pPaCu2 (about 280 kb), or both. In a copper-resistant strain Pa429, the location of the copper-resistance gene(s) was examined by insertional inactivation with Tn5. The MIC of copper sulfate in the copper-sensitive mutant obtained by Tn5 tagging decreased from 2.75 to 0.75 mM. The 14.5 kb BamHI fragment, designated pPaCuB14, containing the same locus mutagenized with Tn5 was cloned from pPaCu1. However, pPaCuB14 did not confer copper resistance in the transformant of copper-sensitive strain Pa21R, suggesting that this clone did not contain a full set of copper-resistance gene(s). Then a cosmid library of pPaCu1 was constructed and six cosmid clones hybridized with pPaCuB14 were selected. One of the six cosmids, designated pPaCuC1, conferred a near wild-type level of copper resistance in the transformant of the copper-sensitive strain. pPaCuC1 had a homologous region that hybridized with all of the PCR-amplifled fragments of copA, copB, copR, and copS genes of P. syringae pv. tomato. DNA sequence analysis of the homologous region revealed the existence of four open reading frames (ORF A, B, R and S) oriented in the same direction. The predicted amino acid sequences of ORF A, B, R and S had 80, 70, 97 and 95% identity with CopA, B, R and S of P. syringae pv. tomato, respectively. Received 5 July 2001/ Accepted in revised form 27 September 2001     

Nucleotide Sequence and Organization of Copper Resistance Genes from Pseudomonas syringae pv. actinidiae

Copper-containing bactericides have been used to control bacterial canker of kiwifruit, caused by Pseudomonas syringae pv. actinidiae. However, the efficacy of copper has been reduced by the occurrence of copper-resistant strains. Analysis of the DNA sequence of a cluster region containing the copper-resistance genes from P. syringae pv. actinidiae suggested the presence of three possible different systems for copper resistance: copper-trapping, copper-efflux and copper-transport systems. Transposon insertional inactivation analysis indicated that the copper-trapping system was essential for copper resistance.     

湘西地区猕猴桃细菌性溃疡病抗性资源筛选及其抗性机理研究

由Pseudomonas syringae pv.actinidiae(Psa)引起的溃疡病是猕猴桃生产中威胁最大的细菌性病害。种植抗病品种是防治猕猴桃溃疡病最有效途径,猕猴桃抗源是抗病育种的物质基础。本试验通过离体接种,在室内评价了4个不同种的7个猕猴桃资源及品种对溃疡病的抗性。结果表明:供试材料离体叶片、枝条接种溃疡病菌后,其发病时间、病斑大小、发病率差异明显。按病斑大小排序,其抗性强弱依次为毛花猕猴桃‘M9808’>对萼猕猴桃‘S9801’>美味猕猴桃‘M-06’>美味猕猴桃‘米良1号’>中华猕猴桃‘Z-12’>中华猕猴桃‘东红’>中华猕猴桃‘红阳’(CK)。其中,毛花猕猴桃‘M9808’抗病性最强,表现为发病最晚,离体叶片、枝条分别于接种后10 d、20 d发病,比对照推迟了8 d、10 d;病斑最小,为对照的1/25、1/11;发病率最低,为对照的1/28、1/10。抗病性相关酶活性比对发现,不同材料的PAL、CAT、POD酶活性差异较大,抗性材料均高于感病材料,且峰值出现时间早于感病材料。其中,毛花猕猴桃‘M9808’的PAL峰值最高,接种后5 d出现,比对照早10 d;CAT、POD峰值接种后10 d出现,比对照早5 d。说明毛花猕猴桃‘M9808’对溃疡病抗性最强,可作为今后猕猴桃抗病育种或抗性砧木的理想材料。这为猕猴桃抗病育种提供了理论依据,为猕猴桃溃疡病的防控提供了参考。     

Genetic diversity of Pseudomonas syringae pv. actinidiae,pathogen of kiwifruit bacterial canker

Bacterial canker disease of kiwifruit currently occurs in at least 15 countries, causing serious damage. The causative agent of the disease is Pseudomonas syringae pv. actinidiae (Psa), which is genetically diverse and is currently classified into five biovars, namely, biovars 1, 2, 3, 5 and 6. In Japan, four biovars except biovar 2 have been found so far. These biovars have been confirmed to have differences in the virulence and composition of pathogenicity-related genes, such as toxin biosynthesis and type III effector genes. Biovars 1 and 6 possess the tox island, a genomic island of approximately 38 kb, which contains phaseolotoxin biosynthesis genes (argK-tox cluster) and is confirmed to have been acquired from other bacteria through horizontal transfers. Also, on the megaplasmid possessed by biovar 6, there exist coronatine biosynthesis genes, and biovar 6 has the ability to produce two phytotoxins, phaseolotoxin and coronatine. In 2014, biovar 3, considered to be of foreign origin, was confirmed for the first time in Japan. Biovar 5, whose virulence is relatively weak, is distributed only in a limited area. In addition to the tox island and various plasmids, a large number of mobile genetic elements are confirmed to be present on the Psa genomes, which might have played a major role in helping Psa to acquire new features. In order to understand how Psa acquired the ability to infect kiwifruit systemically, it is important to make polyphasic comparisons with related pathovars, such as P. syringae pv. theae and pv. actinidifoliorum.     

Survival,cold tolerance and seasonality of infection of European horse chestnut (Aesculus hippocastanum) by Pseudomonas syringae pv. aesculi

Bleeding canker of European horse chestnut, caused by the pathogenic bacterium Pseudomonas syringae pv. aesculi (Pae), is now an established disease throughout several countries in northwest Europe after first emerging in 2001–2002. Pae infects the woody branches of horse chestnut directly via natural discontinuities in the bark, such as lenticels, leaf scars and leaf traces, and nodes. However, the timing of infection in relation to seasonality of host development, and the dispersal mechanisms of Pae, in particular its ability to survive and spread in soil and water, remains unknown. In this study, infection of freshly cut horse chestnut shoots by Pae was assessed at monthly intervals over a 12 month period. Pae infected the greatest numbers of lenticels and leaf scars of horse chestnut when inoculated onto actively elongating shoots in spring and early summer, whereas lesion extension from artificial wound sites was greatest after inoculation during early dormancy. Soil survival experiments showed that Pae was still detectable and viable after 50 weeks' incubation in sterile soil and 41 weeks' incubation in nonsterile soil in the absence of host debris. Pae also remained viable and pathogenic after 1 year's storage in King's B broth at ?20 and ?80°C, and was not killed by freeze/thaw treatments. Thus Pae is able to survive independently for extended periods in soil and water, and can tolerate lengthy periods of freezing at very low temperatures. Such information may facilitate a greater understanding of the epidemiology and spread of Pae in northern Europe.     

Molecular and phenotypic features of Pseudomonas syringae pv. actinidiae isolated during recent epidemics of bacterial canker on yellow kiwifruit (Actinidia chinensis) in central Italy

Pseudomonas syringae pv. actinidiae (Psa) was identified as the causal agent of severe epidemics of bacterial canker on Actinidia chinensis (yellow kiwifruit) in central Italy occurring during 2008–9. A total of 101 strains were obtained from infected leaves, twigs, branches and trunks of cvs Hort16A, Jin Tao and CK3. Outbreaks were also found on A. deliciosa cv. Hayward. A representative set of 21 strains were compared with other Psa strains isolated from previous outbreaks in Japan and Italy as well as with P. s. pv. syringae strains obtained from A. chinensis and with strains of genomospecies 8. Repetitive‐sequence PCR (rep‐PCR) typing using BOX and ERIC primer sets revealed that all Psa strains obtained during 2008–9 showed the same fingerprinting profile. This profile, however, was different from those of strains previously isolated in Japan and Italy. Multilocus sequence typing (MLST) of gapA, gltA, gyrB and rpoD revealed a higher genetic variability among the strains than rep‐PCR, with some of them showing the same sequence pattern although isolated from different areas, cultivars and years. None of the recently obtained strains possessed genes coding for phaseolotoxin or coronatine, and all had an effector protein, namely hopA1, differentiating them from the strains causing past outbreaks in Japan and Italy. All isolates were inhibited in vitro by copper‐based compounds, antibiotics, geraniol, citronellol and by a chitin‐based organic compound. The recent epidemics found in central Italy on yellow kiwifruit appear to have been caused by a different Psa population than those previously recorded in Japan, South Korea and Italy.     

Characterization of the pathogenicity of strains of Pseudomonas syringae towards cherry and plum

Bacterial canker is a major disease of Prunus avium (cherry), Prunus domestica (plum) and other stone fruits. It is caused by pathovars within the Pseudomonas syringae species complex including P. syringae pv. morsprunorum (Psm) race 1 (R1), Psm race 2 (R2) and P. syringae pv. syringae (Pss). Psm R1 and Psm R2 were originally designated as the same pathovar; however, phylogenetic analysis revealed them to be distantly related, falling into phylogroups 3 and 1, respectively. This study characterized the pathogenicity of 18 newly genome‐sequenced P. syringae strains on cherry and plum, in the field and laboratory. The field experiment confirmed that the cherry cultivar Merton Glory exhibited a broad resistance to all clades. Psm R1 contained strains with differential specificity on cherry and plum. The ability of tractable laboratory‐based assays to reproduce assessments on whole trees was examined. Good correlations were achieved with assays using cut shoots or leaves, although only the cut shoot assay was able to reliably discriminate cultivar differences seen in the field. Measuring bacterial multiplication in detached leaves differentiated pathogens from nonpathogens and was therefore suitable for routine testing. In cherry leaves, symptom appearance discriminated Psm races from nonpathogens, which triggered a hypersensitive reaction. Pathogenic strains of Pss rapidly induced disease lesions in all tissues and exhibited a more necrotrophic lifestyle than hemibiotrophic Psm. This in‐depth study of pathogenic interactions, identification of host resistance and optimization of laboratory assays provides a framework for future genetic dissection of host–pathogen interactions in the canker disease.     

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。     

中国4省猕猴桃细菌性溃疡病菌的生物型检测和遗传多样性分析

由丁香假单胞菌猕猴桃致病变种Pseudomonas syringae pv. actinidiae (Psa)侵染引起的猕猴桃细菌性溃疡病(kiwifruit bacterial canker)是全球猕猴桃生产上最具毁灭性的细菌病害。为探明福建、安徽、四川和陕西4省Psa菌株的生物型和遗传多样性,用5对PCR特异性引物PsaJ-F/-R、PsaK-F/-R、Tac-F/-R、Con002-F/-R和avrRps4-F1/-R2检测Psa菌株的生物型;用4对PCR引物27F/1492R、PsaF1/PsaR2、gapA-Fps/Rps和rpoD+364s/-1222ps分别扩增16S rRNA、ITS、gapA和rpoD基因,进行多基因联合分析Psa菌株的遗传多样性。结果表明,特异性引物Tac-F/-R从47株Psa菌株中均能扩增出一条545 bp的特异条带,其他4对引物未扩增出任何条带,说明供试Psa菌株的生物型均为biovar 3。多基因联合分析表明,4省Psa存在丰富的遗传多样性,4个群体共检测出27个单倍型,单倍型多样性为0.955。安徽、福建、四川和陕西群体的单倍型数差异较大,分别为1、8、12个和12个。4个群体的多态性位点数、核苷酸多样性和平均核苷酸差异数差异极显著(P<0.01),其中福建群体的多态性最丰富,而安徽群体的多态性最低。AMOVA分析表明,3.6%的遗传变异来源于种群间,而96.4%的遗传变异来源于种群内,说明种群内变异是遗传变异的主要来源。遗传分化分析表明,安徽省Psa群体与其他3个群体间的遗传分化极高(Fst>0.175),福建、四川和陕西群体间的遗传分化水平较低(Fst<0.017)。研究结果有利于了解福建省Psa的来源,为阻断Psa的传播和猕猴桃细菌性溃疡病的长期可持续控制提供了理论参考。     

利用EMAqPCR建立快速检测猕猴桃溃疡病菌活菌的方法

利用叠氮溴乙锭(ethidium monoazide bromide,EMA)与实时荧光定量PCR技术相结合(EMA-qPCR),建立了一种有效快速检测猕猴桃溃疡病菌活菌的方法。以猕猴桃溃疡病菌ITS序列为检测靶标,菌体经EMA渗透处理,再进行qPCR特异性扩增。结果显示,qPCR检测灵敏度为2cfu;当EMA的浓度为2.0μg/mL时,能有效抑制1.0×10~7 cfu/mL经高温灭活的死菌的扩增,对活菌的扩增没有影响。当活菌数在1.0×10~1~1.0×10~5 cfu范围内,每个qPCR反应体系中活菌数与Ct值呈线性相关(R~2=0.988)。不同温度处理活菌菌悬液后用EMA-qPCR检测猕猴桃溃疡病菌的存活情况并与平板计数法进行比较,结果表明待检样品可在4℃和20℃短期保存。对疑似带病猕猴桃材料进行EMA-qPCR检测,结果表明能减少猕猴桃溃疡病菌PCR的假阳性结果。本研究建立的EMAqPCR方法是一种有效检测猕猴桃溃疡病菌活菌的方法,能有效避免PCR检测实际样品可能造成的假阳性结果。