侧孢短芽胞杆菌JYC 688鉴定及抑菌促生特性研究

马铃薯疮痂病是由多种致病链霉菌侵染引起薯块表面结痂导致薯块外观品质下降的病害,防控困难,生物防治是其主要防治措施之一。筛选具有良好拮抗作用的生防菌,可为马铃薯病害的生物防治提供优良的微生物资源。本研究从健康马铃薯植株根际土壤中分离获得JYC 688菌株,采用平板对峙和纸碟法筛选验证菌株拮抗性,结合形态学观察、生理生化特性测定及分子生物学手段确定其种属地位,并检测其产生水解酶和嗜铁载体的能力。结果表明,菌株JYC 688是侧孢短芽胞杆菌Brevibacillus laterosporus,其菌株培养液能够显著抑制引起马铃薯疮痂病的酸性疮痂链霉菌Streptomyces acidiscabies和肿胀疮痂链霉菌S.turgidiscabies的生长,抑菌圈直径分别为27.86 mm和31.90 mm;其对马铃薯晚疫病菌Phytophthora infestans、早疫病菌Alternaria alternata、黑痣病菌Rhizoctonia solani也具有良好的抑制活性;菌株JYC 688能够分泌β-1,3葡聚糖酶、纤维素酶、蛋白酶、嗜铁素等多种抗菌相关物质,可溶解大豆卵磷脂、碳酸钙、碳酸镁3种难溶性化合物,有利于磷、钙、镁3种元素的释放,是一株具有开发潜力的生防菌。     

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。     

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。     

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.     

黑龙江省部分地区马铃薯疮痂病菌种类及致病性鉴定

为明确黑龙江省马铃薯疮痂病病原菌的种类及其特征,2012-2013年从黑龙江省克山县、绥化市、哈尔滨市、杜尔伯特蒙古族自治县采集具有疮痂病病斑的马铃薯块茎,从中分离纯化病原菌,根据16SrRNA基因的差异采用分子手段对所分离的菌株进行种类和致病性鉴定,并对txtAB阳性菌株采用萝卜幼苗法或马铃薯致病性试验测定其致病性。共分离出74株菌株,鉴定出致病性菌株26株,其中Streptomyces scabies或S.europaeiscabiei 21株,S.turgidiscabies 3株和S.acidiscabies 2株。所有的致病性菌株中共有4种致病岛基因型,即nec1+/tomA+、nec1-/tomA+、nec1+/tomA-和nec1-/tomA。     

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。     

生物有机肥GSJ-1对马铃薯土壤疮痂病病原菌分布影响及生防效果研究

为了确定生物有机肥 GSJ －1对连作马铃薯土壤疮痂病病原菌分布影响及对马铃薯疮痂病的防治效果,本试验进行了土壤疮痂病病原菌含量测定、马铃薯疮痂病病情田间调查。结果表明,连作土壤疮痂病病原菌在盛花期之前向根际富集,盛花期之后向匍匐茎和薯块富集。施用生物有机肥 GSJ －1显著降低了土壤疮痂病病原菌含量,比对照下降了19．6％～74．9％,且改变了土壤中疮痂病病原菌原有的分布规律：从连作处理的根际多于非根际改变为生物有机肥 GSJ －1处理的根际少于非根际,且对疮痂病的防效达到47．2％。     

香蕉枯萎病拮抗菌筛选及其抑菌活性

为获得对香蕉枯萎病菌具有稳定拮抗作用的微生物菌株,分别从海南省临高县南宝镇新营农场、皇桐村及美台镇美梅村采集感病土壤和健康土壤进行微生物分离,并以香蕉枯萎病菌4号小种(Fusarium oxysporum f.sp.cubense Race 4)等9种病原菌为靶标菌株,采用平板对峙法和含药介质法对拮抗菌的抑菌活性进行评价,同时根据形态特征、培养特征及生理生化特征和16S rDNA序列分析对其进行鉴定。结果表明:6个土壤样品中共分离得到具有拮抗作用的细菌93株和放线菌133株,其中T3-G-59菌株对9种病原菌具有广谱抗性,菌丝生长抑制率为43.58%~86.43%,孢子萌发抑制率为42.22%~81.11%,其中对香蕉枯萎病菌4号小种的菌丝生长和孢子萌发抑制率分别为86.43%和81.00%,经鉴定该菌株为多产色链霉菌Streptomyces polychromogenes,表明该多产色链霉菌T3-G-59菌株具有应用于香蕉枯萎病生物防治的潜力。     

糜子细菌性条斑病病原菌鉴定及其对13种杀菌剂的敏感性

为有效防治糜子细菌性条斑病,采用平板划线分离法对2016年自黑龙江省齐齐哈尔市采集的罹细菌性条斑病的糜子植株进行病原菌分离与纯化,依据柯赫氏法则对其致病性进行测定;采用Biolog自动微生物鉴定系统、16S rDNA和16S-23S rDNA间隔区序列分析及病原菌的生理生化反应对病原菌进行鉴定;并测定该病原菌对13种常用杀菌剂的敏感性。结果显示,从糜子叶片和叶鞘病斑中共分离纯化获得10株培养性状一致的菌株,经柯赫氏法则验证均为细菌性条斑病病原菌。经Biolog鉴定,3株代表菌株与标准菌株燕麦噬酸菌Acidovorax avenae的相似值分别为0.716、0.705、0.723,序列分析显示,这3株代表菌株与燕麦噬酸菌燕麦亚种A. avenae subsp. avenae的一致性为99%以上;该病原菌的革兰氏染色反应及碳源利用等生理生化反应与对照菌株燕麦噬酸菌ICMP3183表现一致。因此,确定糜子细菌性条斑病病原菌为燕麦噬酸菌。糜子细菌性条斑病病原菌代表菌株MZ121101对80%乙蒜素乳油、30%乙蒜素乳油及0.3%四霉素水剂的敏感性较高,抑制中浓度EC50介于389.31～939.94μg/mL之间,这2种药剂可作为防治糜子细菌性条斑病的首选药剂。     

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...     

Phylogenetic relationships of Ralstonia solanacearum species complex strains from Asia and other continents based on 16S rDNA, endoglucanase, and hrpB gene sequences

The 16S rDNA, endoglucanase, and hrpB genes were partially sequenced for Asian strains of Ralstonia solanacearum spp. complex, including 31 strains of R. solanacearum and two strains each of the blood disease bacterium (BDB) and Pseudomonas syzygii. Additional sequences homologous to these DNA regions, deposited at DDBJ/EMBL/GenBank databases were included in the analysis. Various levels of polymorphisms were observed in each of these DNA regions. The highest polymorphism (approximately 25%) was found in the endoglucanase gene sequence. The hrpB sequence had about 22% poly-morphism. The phylogenetic analysis consistently divided the strains into four clusters, as distinctly shown on the phylogenetic trees of 16S rDNA, hrpB gene, and endo-glucanase gene sequences. Cluster 1 contained all strains from Asia, which belong to biovars 3, 4, 5, and N2. Cluster 2 comprised the Asian strains of R. solanacearum (as biovars N2 and 1) isolated from potato and clove, as well as BDB and P. syzygii. Cluster 3 contained race 3 biovar 2 strains from potato, race 2 biovar 1 strains from banana, and race 1 biovar 1 strains isolated from America, Asia, and other parts of the world. Cluster 4 was exclusively composed of African strains. The results of the study showed the distribution and diversity of the Asian strains, which are present in three of the four clusters. The similarity of Asian strains to those in the other regions was also observed.The nucleotide sequence data reported are available in the DDBJ/EMBL/GenBank databases under accession numbers AY464950 to AY465050     

Characterization of <Emphasis Type="Italic">Dickeya</Emphasis> strains isolated from potato and river water samples in Finland

Plant pathogenic enterobacteria in the genera Pectobacterium and Dickeya (formerly classified as Erwinia) were isolated from diseased potato stems and tubers. The isolated bacteria were identified as P. atrosepticum, P. carotovorum and pathogens in the genus Dickeya with PCR tests. Furthermore, Dickeya strains were isolated from river water samples throughout the country. Phylogenetic analysis with 16S-23S rDNA intergenic spacer sequences suggested that the Dickeya strains could be divided into three groups, two of which were isolated from potato samples. Phylogenetic analysis with 16S rDNA sequences and growth at 39°C suggested that one of the groups corresponds to D. dianthicola, a quarantine pathogen in greenhouse cultivation of ornamentals, while two of the groups did not clearly resemble any of the previously characterised Dickeya species. Field trials with the strains indicated that D. dianthicola-like strains isolated from river samples caused the highest incidence of rotting and necrosis of potato stems, but some of the Dickeya strains isolated from potato samples also caused symptoms. The results showed that although P. atrosepticum is still the major cause of blackleg in Finland, virulent Dickeya strains were commonly present in potato stocks and rivers. This is the first report suggesting that Dickeya, originally known as a pathogen in tropical and warm climates, may cause diseases in potato in northern Europe.     

GA1-2菌株的分离鉴定及其对香蕉尖孢镰刀菌的抑菌效果

为了从土壤中分离筛选对香蕉尖孢镰刀菌具有良好拮抗作用的放线菌,采用平板稀释涂布法从四川省会理县干热河谷小麦根际土壤中进行放线菌分离,并采用平板对峙法和孢子萌发法进行筛选,通过形态特征、培养特征、生理生化特征以及16S r DNA序列分析对筛选菌株进行鉴定。结果表明,从四川省会理县干热河谷小麦根际土壤筛选获得一株对香蕉尖孢镰刀菌4号生理小种Fusarium oxysporum f.sp.cubense race 4(FOC4)菌丝和孢子萌发都有很强抑制作用的菌株GA1-2,对FOC4菌丝和孢子萌发抑制率分别为36.34%和94.81%。菌株GA1-2与薰衣草灰链轮丝菌Streptoverticillium lavenduligriseum的亲缘关系最近,相似率达99.85%,且其形态特征、培养特征、生理生化特征也与薰衣草灰链轮丝菌基本相符,因此将菌株GA1-2初步鉴定为薰衣草灰链轮丝菌。     

新疆加工型辣椒一种新的细菌性叶斑病病原的鉴定

2014—2015年在新疆沙湾县加工型辣椒Capsicum annuumL.上发现一种细菌性叶斑病。为明确其病原种类,采用组织分离法分离病原菌,依据柯赫氏法则进行致病性测定,并对菌株进行16S rDNA和持家基因rpoD及gry B的扩增、序列测定和系统发育分析,对其进行鉴定。结果表明,获得的8个细菌菌株间致病力无明显差异。致病细菌与已报道的黄褐假单胞菌Pseudomonas fulva菌株的16S rDNA、rpoD基因、gry B基因的同源性分别达到了99.1%~99.9%、99.4%~99.5%和97.6%~100.0%。结合革兰氏染色反应、菌体形态、培养性状、生理生化反应、寄主范围等特征,将病原菌鉴定为黄褐假单胞菌。该病原菌通过人工接种还能侵染番茄、茄子、马铃薯、黄瓜、西瓜、甜瓜、四季豆、豆角、豇豆、白菜、萝卜、胡萝卜及芹菜等多种植物。     

人参病原菌拮抗细菌的分离筛选与鉴定

为明确土壤拮抗细菌对人参常见病原菌的生防价值,从吉林省抚松县人参根际土壤中分离、纯化获得113株细菌,采用平板对峙法测定菌株的拮抗能力,通过温室盆栽试验研究其在土壤及人参根部的定殖规律,并通过形态、培养特征及16S r DNA序列分析进行分类鉴定。对峙试验结果表明,2株细菌对供试人参病原菌具有良好的拮抗广谱性,且多次验证其抑菌效果稳定。定殖研究结果表明,活性菌株SZ-56与SZ-60的定殖能力均达到显著水平,在土壤中的最大定殖菌量分别为每克土7.51×106CFU和7.28×106CFU,30 d后定殖菌量仍保持在105CFU;21 d后在人参根部的定殖菌量仍保持在每克根105CFU水平。根据形态、培养特征、生理生化指标和16S r DNA序列分析,确定SZ-56为内生芽胞杆菌Bacillus endophyticus,SZ-60为解淀粉芽胞杆菌B.amyloliquefaciens。     

Isolation of antagonistic <Emphasis Type="Italic">Streptomyces</Emphasis> sp. against a potato scab pathogen from a field cultivated with wild oat

Fungi, actinomycetes, and bacteria isolated from soil and plant samples from a potato field in which wild oat (Avena strigosa) had been pre-cultivated were screened for microorganisms that can be used as biocontrol agents for common scab of potato. Of 342 isolates assessed in initial pot trials for their suppressive effect on the severity of potato scab caused by Streptomyces turgidiscabies, 26 isolates were selected as antagonistic candidates based on their ability to reduce disease severity, then tested in a second pot trial. Of the 26, five actinomycetes, isolated from either the rhizosphere soil of wild oat or the soil adhering to potato stolons and tubers, were selected as antagonists. A comparison of partial sequences of 16S rRNA genes from the five isolates indicated that they belong to the genus Streptomyces. Of these five, WoRs-501 most strongly inhibited in vitro mycelial growth of S. turgidiscabies and was also the most effective in suppressing potato scab in a third field pot trial. In that pot trial, a 10% (v/v) mix of WoRs-501 (6.2 × 10⁸ colony-forming units [CFU]/g dry mass) decreased the disease severity by 78–94% in comparison with the untreated control at 5 × 10⁴ to 5 × 10⁶ CFU S. turgidiscabies/g dry soil. WoRs-501 also grew well in vitro at a wide range of pH levels and temperatures. These results suggest that WoRs-501 is a promising candidate for biocontrol of potato scab.     

Biochemical and genetical analysis reveal a new clade of biovar 3 <Emphasis Type="Italic">Dickeya</Emphasis> spp. strains isolated from potato in Europe

Sixty-five potato strains of the soft rot-causing plant pathogenic bacterium Dickeya spp., and two strains from hyacinth, were characterised using biochemical assays, REP-PCR genomic finger printing, 16S rDNA and dnaX sequence analysis. These methods were compared with nineteen strains representing six Dickeya species which included the type strains. A group of twenty-two potato strains isolated between 2005-2007 in the Netherlands, Poland, Finland and Israel were characterised as belonging to biovar 3. They were 100% identical in REP-PCR, dnaX and 16S rDNA sequence analysis. In a polyphasic analysis they formed a new clade different from the six Dickeya species previously described, and may therefore constitute a new species. The strains were very similar to a Dutch strain from hyacinth. On the basis of dnaX sequences and biochemical assays, all other potato strains isolated in Europe between 1979 and 1994 were identified as D. dianthicola (biovar 1 and 7), with the exception of two German strains classified as D. dieffenbachia (biovar 2) and D. dadantii (biovar 3), respectively. Potato strains from Peru were classified as D. dadantii, from Australia as D. zeae and from Taiwan as D. chrysanthemi bv. parthenii, indicating that different Dickeya species are found in association with potato.     

Phytotoxin produced by Streptomyces sp. causing potato russet scab in Japan

A phytotoxin isolated from the Stretomyces sp. causing potato russet scab in Japan (Kamenoko-byo in Japanese) was identified as a 16-membered macrolide, FD-891. It induced necrosis in potato tuber slices above 50 µg/disk, which is about 1/100 the activity of thaxtomin A. The phytotoxin was detected in potatoes infected by the pathogen and was produced by other pathogenic strains with different geographic origins.     

Rapid Identification of Clavibacter michiganensis Subspecies sepedonicus Using Two Primers Random Amplified Polymorphic DNA (TP-RAPD) Fingerprints

Twenty strains of Clavibacter michiganensis subsp. sepedonicus from different geographic origins and other reference strains of the same and different species, including other potato pathogens, were analysed with a new procedure named TP-RAPD that originates fingerprints of bacterial species. This procedure uses two primers to amplify the 16S rDNA gene. At 45 °C of annealing, the PCR product electrophoresed in agarose gels produced a band pattern that was different in all bacterial species studied as well as in the subspecies of C. michiganensis. All strains of C. michiganensis subsp. sepedonicus displayed the same TP-RAPD number of pattern. Unlike Gram negative bacteria, Gram positives of high G + C content, such as Clavibacter, produced low bands in TP-RAPD. By using a different set of two primers also based in the 16S rDNA sequence from Escherichia coli a more adequate amplification of Gram positives of high G + C including a greater number of bands was obtained. TP-RAPD patterns using the new set of primers described in this work is a reliable and fast method to identify C. michiganensis subsp. sepedonicus.     

Phylogenetic Analysis of 16S rRNA Genes and PCR Analysis of the nec1 Gene from Streptomyces spp. Causing Common Scab, Pitted Scab, and Netted Scab in Finland

ABSTRACT The sequences of the 16S rRNA genes (nucleotides 29 to 1,521) from various Streptomyces strains pathogenic to potato were compared. These included 10 pathogenic Streptomyces strains isolated from potato scab lesions in Finland, the type strains of S. aureofaciens NRRL 2209(T) and S. lydicus ATCC 25470(T), 'S. griseus subsp. scabies' ATCC 10246, and two S. griseus strains that were originally deposited to the collection as pathogens. The nucleotide sequence (>94.5% sequence identity [SI]) and length (1,469 to 1,481 nucleotides) of the analyzed region varied. Phylogenetic analysis of 16S rRNA genes placed Finnish strains into three species, supported by previously characterized morphological and physiological traits. Six Finnish strains, including two strains that deviated from the others in one trait (no spiral sporophores or D-xylose utilization), had identical 16S rRNA genes and were identified as S. scabies (99.9% SI to S. scabies ATCC 49173). Three Finnish strains were identified as S. turgidiscabies, a species previously described only in Japan (99.9% SI to S. turgidiscabies ATCC 700248). Finnish strain 317 and S. aureofaciens NRRL 2209 (99.8% SI) were placed in a distinct phylogenetic cluster together with Kitosatospora spp., which suggests that S. aureofaciens may belong to the recently revived genus Kitosatospora. In pathogenicity tests, S. scabies caused characteristic symptoms of common scab, S. turgidiscabies caused mainly pitted scab, and S. aureofaciens caused netted scab and necrotic lesions on stolons of potato cultivars Bintje and Matilda in the greenhouse. The nec1 gene and the intergenic region between nec1 and the 5' transposase pseudogene ORFtnp were successfully amplified by polymerase chain reaction from S. scabies ATCC 49173 and the pathogenic Finnish strains of S. scabies, but not from a nonpathogenic strain of S. scabies, three pathogenic and two nonpathogenic strains of S. turgidiscabies, and S. aureofaciens.