猕猴桃果实中Neofabraea actinidiae的快速分子检测

通过对已报道的猕猴桃果腐病菌(Neofabraea actinidiae)及明孢盘属其他种真菌负责编码RNA聚合酶II的第2大亚基(the second largest RNA polymerase subunit,RPB2)的基因序列进行同源性比对,设计合成了一对用于猕猴桃果实中N. actinidiae的检测引物NACF1/NACR1。利用该引物对包括N. actinidiae在内的8种9株Neofabraea属真菌的基因组DNA进行扩增,建立了猕猴桃果实中N. actinidiae的快速分子检测方法,并进行了灵敏度试验、病菌接种与病果中N. actinidiae的检测。结果表明:该引物特异性强,稳定扩增出片段大小为582 bp单一条带,检测灵敏度为0.2 ng/μL,可以特异性地检测到接种发病果实组织中N. actinidiae。该方法能够实现猕猴桃果实中N. actinidiae的快速检测,为口岸提供了特异、高效、可靠的检测方法。     

葡萄霜霉病菌PCR检测方法的建立与应用

通过对已测序和已报道的葡萄霜霉病菌［Plasmopara viticola (Berk et Curtis) Ber.et de Toni］细胞色素c氧化酶亚型2（cytochrome c oxidase Ⅱ,cox2）的基因序列进行同源性比对分析,设计合成了一对用于P.viticola的检测引物F Pv/R Pv。利用该引物对包括P.viticola在内的30种常见植物病原真菌（包括15种Plasmopara属真菌、8种葡萄常见致病菌）的基因组DNA进行了PCR扩增,验证引物F Pv/R Pv的特异性,结果表明：该引物特异性强,仅P.viticola基因组DNA作为模板的PCR扩增产物呈现一条600 bp左右的特异性条带,其他参照菌株及阴性对照均无任何条带;灵敏度验证结果表明,该检测法可以检测出3.3 pg/μL 水平的P.viticola基因组DNA;应用该方法对来自全国不同葡萄产区的不同品种的78份葡萄霜霉病菌进行了检测,结果表明,该检测法适用范围广,且检测准确率达100%。     

Fusarium boothii特异性PCR检测方法的建立与应用

根据报道的Fusariumspp.的reductase-like基因序列,设计合成了1对用于Fusarium boothii的特异性检测引物F-Fg/R-Fg。利用该对引物对包括F.boothii在内的35株镰刀菌的基因组DNA进行了扩增。结果表明:该引物特异性强,仅从F.boothii基因组DNA中扩增出300bp左右的特异性条带,其他参照菌株及阴性对照均无任何条带;灵敏度验证结果表明,该检测法可以检测出50pg F.boothii基因组DNA。     

双重PCR检测马铃薯晚疫病和环腐病方法的建立

通过克隆马铃薯环腐病菌和晚疫病菌转录间隔区(ITS)序列,并对测序结果进行同源性比较,选取差异位点分别设计了两对引物P.IN1/P.IN2和C.IN1/C.IN2,并检测了引物的特异性及方法的灵敏度。引物P.IN1/P.IN2可扩增出1条363bp马铃薯晚疫病菌的特异性条带,在DNA水平上其灵敏度达18fg/μL;引物C.IN1/C.IN2可扩增出1条218bp马铃薯环腐病菌的特异性条带,在细菌数上检测灵敏度为104 cfu/mL。混合这两对引物构建双重PCR反应体系,能从马铃薯环腐病菌和晚疫病菌的混合DNA及感染这两种菌的马铃薯植株中同时扩增到363bp和218bp的特异片段。实现了同时对马铃薯晚疫病菌和环腐病菌的快速可靠检测。     

猕猴桃果腐病菌PCR和实时荧光PCR检测

为了快速、准确地鉴定猕猴桃果腐病菌(Neofabraea actinidiae),根据GenBank中N. actinidiae的β-tubulin序列设计特异引物NAC-F/R和探针NAC-P,建立了常规PCR和实时荧光PCR检测方法。利用引物NAC-F/R扩增供试的4株N. actinidiae能得到389 bp的预期目标条带,但扩增其他20个非N. actinidiae供试菌株不能得到预期产物,检测灵敏度为140 pg菌丝体DNA;探针NAC-P对供试4株N. actinidiae表现为阳性扩增,而对其他菌株和空白对照均表现为阴性扩增,检测灵敏度可达14 pg菌丝体DNA,比常规PCR高10倍。样品检测试验结果表明两种PCR方法可用于口岸植物检疫中快速、准确地检测猕猴桃果腐病菌。     

稻曲病菌的SCAR标记及其PCR检测

为建立稻曲病菌Ustilaginoidea virens快速、灵敏的PCR早期检测技术,以S380为引物,对稻曲病菌和其它参试病原菌的DNA进行RAPD-PCR扩增。稻曲病菌RAPD特异片段经回收、克隆和测序后,根据序列用Primer 5.0软件设计了特异性SCAR引物US-SF（5’-TCGCCTCAGACCTCAATC-3’）/US-SR（5’-GAGCCTCAAATGCCTTCC-3’）和巢式PCR引物US-NF（5’-AGCGTCTCCTGCAACC-AC-3’）/US-NR（5’-GAGCCTCAAATGCCTTCC-3’）。利用引物US-SF/US-SR通过常规PCR对供试稻曲病菌均可扩增出1条大小约257 bp的清晰条带,对其它植物病原菌DNA的PCR产物均无扩增条带,最低可检测到1 pg/μL 的病菌基因组DNA;而利用引物US-SF/US-SR和US-NF/US-NR通过巢式PCR可从10 fg/μL的病菌基因组DNA中扩增出1条大小约210 bp的特异性条带。试验表明,巢式PCR检测灵敏度比常规PCR提高了100倍,且巢式PCR可从接菌的水稻幼颖和自然感染的谷粒中检测出稻曲病菌。     

香蕉炭疽菌rDNA ITS区的分子鉴定与检测

 香蕉炭疽病菌(Colletordchum muscat)是一种引起香蕉采后病害的最重要病原,本研究用真菌18S~28S间的内转录间隔区(internal transcribed spacer,ITS)通用引物18SF和28SR扩增香蕉炭疽菌和其它外群真菌的基因组DNA,扩增出约510bp的片段;通过克隆测序香蕉炭疽菌的ITS全序列并与GenBank中炭疽菌属其它种的ITS序列比对,设计出香蕉炭疽菌的特异性引物ColM1和ColM2。用此特异引物可以从香蕉炭疽菌株中扩增出382bp的特异性片段,而其余20个参试菌株和香蕉组织的PCR反应结果为阴性,灵敏度实验证明可以检测到目标DNA的浓度为0.1Pg。该方法可用于快速、准确和灵敏地检测香蕉炭疽菌,为快速监测组织中有无香蕉炭疽病菌潜伏侵染与及早采取防治措施提供积极的指导意义。     

冬生疫霉(Phytophthora hibernalis)的快速分子检测

 由冬生疫霉(Phytophthora hibernalis)引起的疫病是一类植物检疫性病害。为建立该病原菌的快速检测技术,本文比较分析了冬生疫霉和其它疫霉的ITS序列,在此基础上设计了一对检测冬生疫霉的特异性引物751F/752R,该对引物从冬生疫霉中扩增得到一条616bp的条带,而其它19种疫霉和其它真菌菌株均无扩增条带,表明该对引物对冬生疫霉具有特异性。在25μL PCR反应体系中,引物751F/752R检测灵敏度为10龟基因组DNA;而以卵菌ITS区通用引物ITS1/ITS4和751F/752R进行套式PCR扩增,能够检测到10ag的基因组DNA,使检测灵敏度提高了1000倍。该检测体系对灭菌水中游动孢子的检测灵敏度可达0.5个游动孢子。结合快速碱裂解法提取发病组织的DNA,采用该PCR检测技术,在1个工作日内即可从人工接种发病的植物组织中特异性的检测到该病原菌。表明本研究建立的检测方法可用于冬生疫霉的快速分子检测。     

利用多重PCR诊断水稻白叶枯病和细菌性条斑病的复合发生

为准确检测水稻白叶枯病菌、细菌性条斑病菌及这两种病菌的复合发生,利用软件DNAStar分析比较这两种菌的部分核酸序列,设计了检测这两种病菌的特异性引物。引物Xoo F-Xoo R能特异性扩增出水稻白叶枯病菌中一条大小162 bp的条带;引物Xooc F1-Xooc R1和Xooc F2-Xooc R2能够分别特异性扩增出水稻细菌性条斑病菌中690 bp和945 bp的条带。通过优化PCR反应条件,成功建立了多重PCR技术,可以对不同国家的水稻白叶枯病菌和细菌性条斑病菌进行准确检测,对由这两种病菌引起的复合侵染实现了准确诊断。     

双重PCR检测马铃薯晚疫病菌和青枯病菌方法的建立及应用

 利用真菌通用引物ITS1和ITS4扩增马铃薯晚疫病菌转录间隔区并进行序列测定,通过序列比较,设计了1对马铃薯晚疫病菌的特异引物INF1/INF2,并对15种不同真菌、细菌和7种疫霉属和腐霉属卵菌基因组DNA进行PCR扩增,结果只有不同来源的马铃薯晚疫病菌株可获得324 bp的特异带。将引物INF1/INF2与卵菌通用引物进行巢式PCR扩增后,其检测灵敏度在DNA水平上可达30 fg。运用设计的引物与马铃薯青枯病菌特异引物结合建立了双重PCR体系,能从马铃薯晚疫病菌和马铃薯青枯病菌总基因组DNA以及人工接种和自然发病的马铃薯植株中分别或同时扩增到324 bp和281 bp的特异片段。实现了同时对马铃薯晚疫病菌和马铃薯青枯病菌的快速可靠检测。     

Bacterial canker on kiwifruit in Italy: anatomical changes in the wood and in the primary infection sites

The bacterial canker of kiwifruit caused by Pseudomonas syringae pv. actinidiae is a severe threat to kiwifruit production worldwide. Many aspects of P. syringae pv. actinidiae biology and epidemiology still require in-depth investigation. The infection by and spread of P. syringae pv. actinidiae in xylem and phloem was investigated by carrying out artificial inoculation experiments with histological and dendrochronological analyses of naturally diseased plants in Italy. We found that the bacterium can infect host plants by entering natural openings and lesions. In naturally infected kiwifruit plants, P. syringae pv. actinidiae is present in the lenticels as well as in the dead phloem tissue beneath the lenticels, surrounded by a lesion in the periderm which appears to indicate the importance of lenticels to kiwifruit infection. Biofilm formation was observed outside and inside plants. In cases of advanced stages of P. syringae pv. actinidiae infection, neuroses of the phloem occur, which are followed by cambial dieback and most likely by infection of the xylem. Anatomical changes in wood such as reduced ring width, a drastic reduction in vessel size, and the presence of tyloses were observed within several infected sites. In the field, these changes occur only a year after the first leaf symptoms are observed suggesting a significant time lapse between primary and secondary symptoms. It was possible to study the temporal development of P. syringae pv. actinidiae-induced cambial dieback by applying dendrochronology methods which revealed that cambial dieback occurs only during the growing season.     

Rapid Diagnosis of Pseudomonas syringae pv. papulans, the Causal Agent of Blister Spot of Apple, by Polymerase Chain Reaction Using Specifically Designed hrpL Gene Primers

ABSTRACT The identification and detection of Pseudomonas syringae pv. papulans, the causal agent of blister spot of apple, on apple leaves and fruit was achieved by polymerase chain reaction amplification of a specific DNA fragment of the hrpL sequence. The consensus primers hrpL(1) and hrpL(2) were designed based on the alignment of pseudomonad hrpL gene sequences available in nucleic acid data banks. This primer set produced a 631-bp amplicon from 37 of the 57 pseudomonads strains tested. These strains belonged to genomospecies 1 and 2, as described by Gardan et al. (8). The amplicon obtained from 30 of these strains was digested with eight restriction enzymes. Three different restriction patterns were produced from strains belonging to genomospecies 1, resulting in A1 and A2 patterns, while strains belonging to genomospecies 2 were characterized by a B pattern. Patterns A1 and A2 differed at only two sites, a Bsp 143I site located at nucleotide 360 and a MseI site located at nucleotides 22-24. Group A2 consisted solely of P. syringae pv. papulans strains. The hrpL gene in P. syringae pv. papulans strain CFBP3323 was sequenced. Two primer sets, Pap1/Pap2 and Pap1/Pap3, were designed and tested for specificity to P. syringae pv. papulans. These primers amplified expected fragments of 242 and 303 bp, respectively. Pap1/Pap2 amplified a fragment only with P. syringae pv. papulans DNA, while Pap1/Pap3 amplified all tested strains belonging to genomospecies 1. A diagnostic procedure using the Pap1/Pap2 primer set was successful for the detection of P. syringae pv. papulans in diseased fruit and artificially inoculated leaves.     

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.     

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。     

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.     

Detection of Pseudomonas syringae pv. actinidiae using polymerase chain reaction (PCR) primers based on the 16S–23S rDNA intertranscribed spacer region and comparison with PCR primers based on other gene regions

Several published polymerase chain reaction (PCR) primers to identify Pseudomonas syringae pv. actinidiae, the causal organism of bacterial canker of kiwifruit, were found not to be specific. Two new sets of PCR primers, PsaF1/R2 and PsaF3/R4, were designed to be complementary to a portion of the 16S–23S rDNA intertranscribed spacer (ITS) regions. These primers amplified a DNA fragment from strains of P. syringae pv. actinidiae, but not from 56 strains of bacteria from six genera and 17 species, except for a strain of the tea pathogen, P. syringae pv. theae. When tested against DNA extracted from a further 20 strains from Japan, Korea, Italy and the USA deposited in culture collections as P. syringae pv. actinidiae, all except six cultures produced the expected product of 280 bp with PsaF1/R2 and 175 bp with PsaF3/R4. Results of multilocus sequence analysis using five housekeeping genes (gyrB, acnB, rpoD, pgi and cts) showed that none of these six strains was phylogenetically similar to P. syringae pv. actinidiae. In contrast to the P. syringae pv. actinidiae type strain, these strains were positive in the determinative tests for ice nucleation and syringomycin production. It is suggested that these six strains were incorrectly identified as P. syringae pv. actinidiae. It was not possible to distinguish P. syringae pv. actinidiae from the phylogenetically similar P. syringae pv. theae using the ITS, gyrB, acnB, rpoD, pgi or cts gene regions to design PCR primers. Because P. syringae pv. theae is unlikely to be found on kiwifruit, primers PsaF1/R2 and PsaF3/R4 are recommended for screening bacteria isolated from kiwifruit tissue.     

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。     

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.     

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...     

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.