马铃薯疮痂病菌致病相关基因的克隆及表达

 A pathogenic-related gene nec1 was cloned in Streptomyces scabies CPS-1, potato scab strain. Analysis results showed that the length of open reading frame(ORF) for nec1 gene was 666 bp, and the GC content was 54.2%. Sequence alignment indicated that a 650 bp up-stream sequence shared 91% similarity with IS 256 family transposase nucleotide sequences by BLASTn searches against GenBank. The segments obtained by PCR amplification were digested by enzymes SphⅠand SacⅠ, and linked to the expression vector pIJ702. The recombinants were transformed into nonpathogenicity strain Streptomyces lividans 66 TK24. Bioas-say results suggested that the transformants possessed the same symptoms as pathogenic strain on potato tuber slices and radish seedlings, which implied that nec1 gene was associated with the pathogenicity of S. scabies CPS-1.     

马铃薯疮痂病菌毒素及其致病性的研究

 本文对分离自我国不同地区的马铃薯疮痂病菌产生的毒素进行了研究。通过对不同菌株进行毒素提取、纯化和产毒分析发现各致病菌株均能产生同一类毒素,而非致病菌株均未见毒素产生。毒素的活性检测结果表明,毒素能使马铃薯薯片和萝卜幼苗产生与病菌作用相同的症状,说明毒素是疮痂病菌侵染过程中的主要致病因子。     

黑龙江省马铃薯干腐病菌种类鉴定及致病性

本研究将采自黑龙江省不同地区的马铃薯干腐病病样进行分离和病原菌纯化,得到27个镰刀菌菌株,通过致病性鉴定,其中的18个菌株具有致病性。运用培养性状和形态特征综合分析的方法,对上述18个菌株进行鉴定,结果显示为6种镰刀菌,分别为拟枝孢镰孢(Fusarium sporotrioides)、茄镰孢(F.solani)、接骨木镰孢(F.sam-bucinum)、拟丝孢镰孢(F.trichothecioides)、燕麦镰孢(F.avenaceum)和茄病镰孢蓝色变种(F.solanivar.coerule-um)。同时对上述6种镰刀菌进行致病性测定,结果表明不同种类镰刀菌致病性不同,以接骨木镰孢、燕麦镰孢和拟丝孢镰孢致病力最强,拟枝孢镰孢致病力最弱。     

我国马铃薯疮痂病及其防治研究进展

马铃薯疮痂病(potato common scab)是由放线菌目链霉菌属的链霉菌Streptomyces spp.引起的土传兼种传病害,广泛分布于世界各马铃薯种植区,不仅影响马铃薯的外观品质和销售价格,严重时还会导致马铃薯出苗延迟甚至引起幼苗死亡,造成产量下降,给马铃薯产业带来巨大的经济损失,已经成为全球危害马铃薯生产的第四大病害。2015年我国确立马铃薯主粮化战略,推动了马铃薯产业的发展。近年来,随着种植区域和规模不断扩大,马铃薯疮痂病在我国很多省(自治区)有不同程度的发生,并有逐年扩大和加重的趋势,严重影响商品薯、加工原料薯和种薯的生产,成为制约我国马铃薯生产的主要病害。本文对马铃薯疮痂病症状、发病因素、传播规律、致病机理、分类方法以及我国马铃薯疮痂病发生情况、种类及分布进行归纳,并对马铃薯疮痂病防治措施进行总结,以期为我国马铃薯疮痂病的研究和防治奠定理论基础。     

云南省马铃薯疮痂病致病链霉菌种类组成研究

 为明确云南省马铃薯疮痂病病原菌(Streptomyces spp.)的种类及其生物学特征,自2013年从云南省13个马铃薯主产区采集疮痂病病样,共分离到200株链霉菌,通过温室盆栽致病性试验筛选出67株致病菌。通过形态学、生理生化指标、致病性测定及16S rDNA序列分析对获得的菌株进行鉴定。结果显示,引起云南地区马铃薯疮痂病的病原为10种链霉菌,分别为S. caviscabies、S. anulatus、S. scabies、S. turgidiscabies、S. acidiscabies、S. europaeiscabiei、S. luridiscabiei、S. enissocaesilis、S. griseus和S. aureofaciens。其中S. enissocaesilis和 S. anulatus为优势种群,S. caviscabies、S. anulatus和S. luridiscabiei为国内首次报道的病原菌。因此,认为云南省马铃薯疮痂病菌种类复杂多样。     

西北地区马铃薯疮痂病病原菌鉴定及其生物学特性

为明确西北地区马铃薯疮痂病病原菌的种类和生物学特性,分别采用常规组织分离法和土壤混悬液分离法从宁夏、陕西和甘肃3个省区采集的29份疮痂病发病薯块和8份发病地块土壤中进行病原菌分离,并利用形态特征、生理生化特性和16S rDNA序列分析对病原菌进行鉴定。结果表明,从发病薯块和发病土壤中共分离到50株链霉菌Streptomyces spp.,通过回接法验证获得6株马铃薯疮痂病致病菌株。6株致病菌株的培养特性和形态特征差别较大;其中菌株G4-1、G9和SYN13不能以果糖和木糖为单一碳源,菌株SYNT3不能以棉子糖为单一碳源;除菌株NLG4-1外,其余5株菌株均能在络氨酸琼脂培养基上产生黑色素。经16S rDNA序列分析,菌株G4-1、G9与疮痂病链霉菌S. scabiei的相似率分别达99.47%和99.34%,菌株NLG4-1、SYNT3与S. enissocaesilis的相似率分别达97.90%和98.18%,菌株GBH2与加利利链霉菌S. galilaeus的相似率达99.93%,菌株SYN13与S. turgidiscabies的相似率达97.56%,表明西北地区马铃薯疮痂病病原菌至少存在4个种。     

甘肃马铃薯疮痂病病原初步鉴定

对甘肃不同地区马铃薯疮痂病病原进行了分离鉴定.采用盆栽方法进行致病性测定、形态特征、生理生化特性测定和16S rDNA序列分析.结果表明:共有6株菌能使马铃薯块茎出现疮痂症,分别为GP-1、GP-2、GH-1、GH-2、JB-1和JB-2.经鉴定菌株GP-1、JB-2的形态特征、生理生化特性及16S rDNA序列与Streptomyces scabies 87 22菌株均一致;GP-2、JB-1的16S rDNA序列与S.scabies菌株相似性为99％.菌株GH-2和GH-1与S.griseus的16S rDNA序列相似性分别为100％和99％,形态特征一致,但不能以棉籽糖和肌醇为单一碳源,暂定为S griseus.     

义乌市马铃薯疮痂病发生原因及防控措施

马铃薯疮痂病已成为威胁义乌市马铃薯生产的最主要病害。当田间有大量病原菌存在时,该病的发生程度与土壤酸碱度、土壤湿度、品种抗性等因素密切相关。选用抗病品种、水旱轮作、施用酸性肥料、调节土壤酸碱度、利用太阳能杀灭土壤病菌,块茎生长期间保持土壤湿度、选用无病种薯并进行消毒、防治好地下害虫等防控措施,可有效地抑制疮痂病的发生。     

马铃薯疮痂病菌在植株和田间的分布与动态分析

为探明马铃薯疮痂病菌在植株和土壤中的分布情况及种群动态变化特点,利用常规PCR和定量PCR(qPCR)技术对不同环境的马铃薯疮痂病株和田间植株不同生育期的土壤样品进行病原菌的定性定量检测.结果 表明,病田、温室盆栽和微型薯苗床中马铃薯疮痂病重度发病植株的根、匍匐茎、块茎、地上茎、叶片等组织样品均可检测到184 bp的疮...     

甘肃省定西市马铃薯疮痂病新病原Streptomyces galilaeus的分离、鉴定及生物学特性研究

对甘肃省定西市安定区的马铃薯疮痂病病原进行了分离、鉴定和生物学特性研究。结果表明,菌株5T-1具有较强致病性,菌落表面呈灰色,有金属光泽,平均直径为4.68mm,可产生黄褐色素,孢子圆形或圆柱形,孢子丝松散,革兰氏染色呈阳性。5T-1的16SrDNA序列与加利利链霉菌Streptomyces galilaeus菌株的相似度为99%,结合形态特征将菌株5T-1鉴定为Streptomyces galilaeus,为甘肃省新报道病原菌。菌株5T-1生长最适温度为30℃,最适光照条件为全黑暗,最适pH为8.5,最佳碳源和氮源分别为肌醇和天冬氨酸。该研究结果为甘肃省马铃薯疮痂病诊断和综合防治提供了依据。     

Distribution and characterization of Streptomyces species causing potato common scab in Germany

Field‐grown potatoes showing scab infections were sampled in two successive years and analysed for prevailing Streptomyces strains. In 2008 and 2009, 293 Streptomyces isolates were collected in Germany and analysed for morphology, pathogenicity and strain type. Isolates varied in mycelium colour, sporulation and pigmentation. Based on their morphology, no clear differentiation of species was possible. At the genetic level, sampled isolates, as well as a number of type strains from culture collections, were characterized by PCR using 16S rRNA‐specific primers and PCR‐RFLP of the 16S–23S internal transcribed spacer (ITS) region with Hpy99I. Using this fingerprinting approach, Streptomyces species could be differentiated genotypically. The data from this study show that diversity among scab‐causing species in Germany is much higher than previously thought. Isolates belonged to various Streptomyces spp. previously associated with common scab. This is apparently the first report of pathogenic strains of S. europaeiscabiei, S. stelliscabiei, S. acidiscabiei, S. turgidiscabiei and S. bottropensis within Germany. Streptomyces europaeiscabiei was the predominant species found. Other scab‐causing species were identified, but their local distribution was uneven. For most of the isolates, the presence of the txtAB gene was demonstrated, indicating pathogenicity. This analysis is one of the first reports to examine the distribution of common scab‐causing species in Germany.     

Detection and characterization of Streptomyces causing potato common scab in Western Europe

A PCR-based diagnostic method was developed for direct detection from tuber lesions of pathogenic Streptomyces causing common scab of potato. Primers were designed to amplify a fragment of the txtAB ( txtA and txtB ) genes, which are pathogenicity determinants in the main pathogenic Streptomyces species. The method was evaluated on 84 naturally infected potato samples, comprising 19 potato cultivars, harvested in the years from 2000 to 2004 in the Netherlands, the UK, France, Germany and Spain. Pathogenic Streptomyces in tuber lesions were detected by PCR in 70 samples and were also successfully isolated from these 70 samples. All pathogenic isolates showed the basic general phenotypic traits of the S. scabiei phenetic cluster. RFLP analysis of amplified rRNA sequences, together with carbon source utilization and repetitive BOX profiles, allowed most isolates to be assigned to S. europaeiscabiei , which emerged as the main cause of potato common scab in Western Europe.     

Differences in host range, pathogenicity to potato cultivars and response to soil temperature among Streptomyces species causing common and netted scab in France

The pathogenicity and ecology of some isolates representative of the four main Streptomyces species ( S. scabies , S. europaeiscabiei , S. stelliscabiei and S. reticuliscabiei ) identified as pathogenic to potato tubers were investigated. Three pathogenicity groups could be distinguished. Group 1 included all isolates of S. scabies , S. europaeiscabiei and S. stelliscabiei from common scab lesions of potato and other susceptible root crops. All these produced similar symptoms and were pathogenic to potato, carrot and radish. Group 2 included all isolates from S. reticuliscabiei netted scab lesions; they were pathogenic to both tubers and roots of only a few potato cultivars, and did not infect carrot or radish. Group 3 included three isolates of S. europaeiscabiei from netted scab lesions on cv. Bintje, which produced either common or netted scab symptoms depending on the potato cultivar or plant species. In an experiment on a few isolates from each of the three groups, held at various soil temperature regimes, the three from group 1 were most pathogenic at higher temperatures (20°C or 20/30°C), the two from group 2 were most pathogenic at a lower temperature (17°C). The group 3 isolate caused netted scab symptoms on susceptible cultivars at low temperatures (≤ 20°C) and deep-pitted lesions at higher temperatures. Since the groups identified differ in ecological requirements, it is important to adapt the control methods to the pathogenic species present in the soil.     

Isolation and characterization of Streptomyces species from potato common scab lesions in Norway

The present survey was conducted to isolate and characterize Streptomyces species from common scab lesions of potato in Norway. Bacteria were isolated from scab lesions on tubers sampled in two consecutive years at different locations in Norway spanning ～1400 km from south to north. In total, 957 independent isolations from individual tubers were performed, with 223 putative pathogenic isolates obtained from 29 different potato cultivars and 130 different fields. Streptomyces europaeiscabiei was the most abundant species isolated from common scab lesions (69%), while 31% of the isolates obtained were S. turgidiscabies. Streptomyces scabies was not found. Pathogenicity of selected Streptomyces isolates was tested on potato. The ability of the bacterial isolates to infect potato was consistent with the presence of the txtAB operon. The results revealed no pattern in geographical distribution of S. europaeiscabiei and S. turgidiscabies; both could be found in the same field and even the same lesion. Four different pathogenicity island (PAI) genotypes were detected amongst the txtAB positive isolates: nec1+/tomA+, nec1–/tomA+, nec1+/tomA? and nec1?/tomA?. The current findings demonstrate that there is genetic variability within species and that the species are not spread solely by clonal expansion. This is thought to be the most comprehensive survey of Streptomyces species that cause common scab of potato in a European country.     

Occurrence and survival of potato scab pathogens (Streptomyces species) on tuber lesions: quick diagnosis based on a PCR-based assay

A time-saving and cost-effective polymerase chain reaction (PCR)-based method was developed for species-specific detection of the scab pathogens ( Streptomyces scabies and S. turgidiscabies ) prevalent in potato ( Solanum tuberosum ) in northern Scandinavia. Species specificity of primers was verified using a collection of previously characterized Streptomyces strains isolated from potato scab lesions in Finland and Sweden. A total of 1245 scab lesions was tested from potato cvs Matilda and Sabina grown in the field in two geographic regions of Finland in 2000 and 2001. Freshly harvested or stored potato tubers were incubated at room temperature (18–21°C) under humid conditions for a few days. Bacterial growth was collected from scab lesions for DNA isolation and PCR. The two scab pathogens were detected in the same potato fields, tubers and scab lesions. The relative incidence of S. scabies was high in freshly harvested tubers but was much lower than that of S. turgidiscabies following storage. Both pathogens were seed-transmitted in Matilda and Sabina after 24 weeks of storage at 4°C.     

Quantification of potato common scab pathogens in soil by quantitative competitive PCR with fluorescent quenching‐based probes

Common scab of potato tubers caused by pathogenic Streptomyces spp. is a cause of serious economic loss worldwide. For the rapid and accurate quantification of pathogenic Streptomyces spp. residing in soil, a new competitive real‐time PCR method using fluorescent quenching‐based probes (quantitative competitive quenching probe PCR: QCQP‐PCR) was developed. The virulence gene of pathogenic Streptomyces spp., nec1, was selected as the target for QCQP‐PCR. A specific primer set to amplify the nec1 gene, and a fluorescently labelled probe that specifically hybridizes with the nec1 amplicon were designed. For QCQP‐PCR, an internal standard DNA (IS DNA) that is identical to the nec1 amplicon but has a 4‐base mismatch in the probe‐hybridizing region, and a fluorescently labelled probe IS, which specifically hybridizes with IS DNA at the mutagenized region, were PCR‐synthesized. The target nec1 gene was co‐amplified with the known copy number of IS DNA by PCR using the same primer set in the presence of the specific probes. The PCR products were monitored in real‐time by measuring the fluorescence intensity (quenching) of each probe. The initial amount of the nec1 gene was quantified based on the ratio of the PCR products of the same PCR cycle. The results revealed that QCQP‐PCR could be used to precisely quantify the nec1 gene, even in the presence of PCR inhibitors in the soil samples examined. The lower limit of quantification was 20 copies per tube, which corresponded to 1500 copies per g dry soil. The quantification achieved by this method was completed within 5 h, i.e. the duration of the entire analysis. These results demonstrate the usefulness of the present method for monitoring pathogenic Streptomyces species in soil.     

Genotypic and phenotypic characterization of Streptomyces species associated with potato crops in the central part of Colombia

In Colombia, Streptomyces scabiei (syn. S. scabies) is commonly believed to be the causal organism of scab disease in local potato crops. However, very little is known about this organism and about the diversity and pathogenicity of the Streptomyces species associated with potato crops in Colombia. This study, therefore, aimed to elucidate aspects regarding the diversity of these bacteria associated with potato crops in a particular region of Colombia and evaluate their pathogenicity. We obtained 33 isolates of Streptomyces from netted, superficial and deep-pitted potato scab lesions from two main potato-producing regions in Colombia. Of these, 17 were pathogenic based on in vitro and in planta assays. None of these isolates carried the txtA, txtB, or nec1 genes, commonly associated with pathogenicity in Streptomyces, and characteristic of the pathogenicity island (PAI). We also characterized all isolates based on phenotypic characteristics and analysed their phylogenetic relationships using the 16S rRNA, atpD, recA, rpoB, and trpB genes. The isolates were highly diverse, placed in nine clades with 15 different phenotypes. The 17 pathogenic isolates were placed into three clades, namely S. pratensis, S. xiamenensis, and unknown species. This study is a preliminary investigation towards understanding scab disease in Colombia through the study of both pathogenic and nonpathogenic species present in scab disease lesions in potatoes. Also, this is the first report of Streptomyces species associated with potato tubers in Colombia.