Occurrence of bacterial rot of onion bulbs caused by Burkholderia cepacia in Japan

In 2001, a bacterial rot of onion (Allium cepa L.) bulbs was observed in Japan. The causal agent was identified as Bukholderia cepacia (Palleroni & Holmes 1981 ex Burhkolder 1950) Yabuuchi, Kosako, Oyaizu, Yano, Hotta, Ezaki, and Arakawa 1993. The identified bacteria were divided into two groups (Y and W) based on colony colors, and several phenotypic and genetic characteristics. Based on recA polymerase chain reaction assays, the strains of the Y and W groups belong to genomovar I (B. cepacia sensu stricto) and genomovar III (B. cenocepacia), respectively.The nucleotide sequence data reported are available in the DDBJ/EMBL/GenBank databases under the accession numbers AB162427 and AB162428     

Stem and root rot of scarlet runner bean (Phaseolus coccineus) caused by Pythium myriotylum

A severe rot was found on the stems and roots of scarlet runner bean (Phaseolus coccineus) in Ibaraki Prefecture (Japan) in August 2004. The causal fungus was identified as Pythium myriotylum. We propose the name of stem and root rot of scarlet runner bean (“Kuki-negusare-byo” in Japanese) for this new disease.     

青海省青稞根腐病调查及病原菌鉴定

为明确青海省青稞根腐病发生情况及其病原菌,于2016年6—9月对青海省海北藏族自治州海晏县、刚察县,海东市互助土族自治县苗期及成株期青稞上根腐病发生情况进行调查,采集19份根腐病样品,采用常规组织分离法对样品进行分离,并对分离菌株进行致病性测定,通过形态学特征及分子生物学方法对其进行鉴定。结果显示,青海省青稞根腐病分布广泛,发病率较高,为10%～20%;发病植株长势弱、发黄,茎基部缢缩腐烂,穗白粒瘪,茎秆发褐或黑红;19份样品中共分离纯化得到93株病原菌菌株,其中73株为燕麦镰孢Fusarium avenaceum,9株为木贼镰孢F. equiseti,11株为锐顶镰孢F. acuminatum;木贼镰孢的绝对病情指数最强,为79.43,燕麦镰孢次之,为50.90,锐顶镰孢最弱,为23.70,各病原种间及种内致病性差异显著。根据分离率及各病原菌致病性综合确定燕麦镰孢为青海省青稞根腐病的优势病原菌。     

Phylogenetic relationships between the lettuce root rot pathogen Fusarium oxysporum f. sp. lactucae races 1, 2, and 3 based on the sequence of the intergenic spacer region of its ribosomal DNA

The genetic relationship between the vegetative compatibility groups (VCGs) and between physiological races of Fusarium oxysporum f. sp. lactucae (FOL), the causal pathogen of lettuce root rot, was determined by analyzing the intergenic spacer (IGS) region of its ribosomal DNA. A total of 29 isolates containing a type strain were tested: 24 Japanese isolates, 2 Californian isolates, and 3 Italian isolates. Three races (races 1, 2, and 3) were found in Japan, and race 1 was also distributed in California and Italy. Races 1, 2, and 3 each belonged to a distinct VCG: VCG-1, VCG-2, and VCG-3 (VCG-3-1, VCG-3-3), respectively. Phylogenetic (neighbor-joining) analysis of the IGS sequences revealed that races 1, 2, and 3 coincided with three phylogenetic groups (PG): PG-1, PG-2, and PG-3, respectively. These results indicate that the three races are genetically quite different and have a strong correlation with VCGs and phylogenetic groupings. The nucleotide sequence data reported are available in the DDBJ/EMBL/GenBank databases under the accession no. AB195218     

口红吊兰菌核病病原鉴定及其生物学特性分析

为鉴定1株分离自口红吊兰叶部病斑上的疑似核盘菌菌株,利用柯赫氏法则验证其致病性并通过形态观察和ITS序列分析对该病原菌进行分类鉴定,结合温度、酸碱度等生理指标研究其生物学特性,并利用菌丝生长速率法测定了扑海因、多菌灵、苯醚甲环唑、腐霉利4种药剂对该病原菌的抑制作用。结果显示:在PDA平板上该病原菌菌丝为白色,均匀生长;约7 d后开始产生菌核,直径3~5 mm;菌核萌发后可产生1~3个子囊盘,内含8个大小为8.0~12.0μm×4.0~5.5μm的子囊孢子。该菌株ITS序列系统进化分析结果显示,其与核盘菌的同源性高达99%。综上所述,初步确定该菌株为核盘菌Sclerotinia sclerotiorum。该病原菌在PDA培养基上的最适生长温度为20~25℃、最适生长p H为5~7。室内毒力测定发现供试4种药剂中扑海因对该病原菌菌丝生长有较好的抑制效果,且其EC_(50)最小,仅为0.62 mg/L,证明扑海因对核盘菌毒力最强。     

枸杞根腐病病原菌鉴定及其拮抗菌的筛选

为了针对根腐病实施有效的生物防治,分离鉴定枸杞根腐病病原菌及其拮抗菌,采用组织分离法获得腐根上的病原菌,结合形态学与分子生物学特征鉴定病原菌;利用稀释涂布、滤纸片法和共培养法从健康根际土壤筛选拮抗细菌,通过形态学观察、生理生化鉴定和16S rDNA序列分析对拮抗效果较好的菌株进行鉴定。结果表明：从枸杞根腐病分离得到腐皮镰刀菌（Fusarium solani）和尖孢镰刀菌（Fusarium oxysporum）两种主要致病菌,从健康枸杞根际土壤中分离获得12株对两种镰刀菌具有拮抗作用的细菌,其中J7-3、J7-8、J7-9和J10-8对腐皮镰刀菌的抑菌率均在80%以上,J7-3和J10-8对尖孢镰刀菌的抑菌率均在70%以上。菌株J7-3和J10-8对根腐病病原菌的抑菌作用较好,分别鉴定为解淀粉芽孢杆菌（Bacillus amyloliquefaciens）和枯草芽孢杆菌（Bacillus subtilis）,其中J10-8的拮抗作用和溶菌效果最好,可作为研发微生物菌剂的菌种资源。     

安徽省大豆疫霉根腐病菌的鉴定及rDNA-ITS序列分析

为明确安徽省夏大豆疫霉根腐病的病原菌种类,对采集自涡阳、怀远、固镇3个县的夏大豆病株及土样分离纯化后获得28株菌株,选取6株代表性菌株,通过形态学观察及核糖体DNA-ITS序列分析对其进行鉴定,并测定了其致病型。结果表明,6株菌株在利马豆培养基上菌落白色,质地均匀;菌丝无隔,致密,具近直角分枝;在10%V8C培养液中,游动孢子囊顶生,不脱落,卵形至椭圆形,无明显乳突,有内层出现象,长宽比大于1.6∶1;同宗配合,在利马豆培养基上单株培养产生大量卵孢子,藏卵器球形,雄器大多侧生;接种合丰35大豆品种后出现典型的大豆疫霉根腐病症状。r DNA-ITS序列分析表明,6株菌株与Gen Bank中大豆疫霉Phytophthora sojae的ITS序列同源性高达100%;菌株GY4、GY8、HY11、HY16、GZ10、GZ21的毒力公式分别为1b,2,3a,3b,4,5,6,7;1b,1d,3a,3b;1d,3a,3b,3c,4,5,6,7;2,3c,4,5,6,7;1b,3a,3c,5,8;3a,3b,5,6,7,8;属于6个不同的致病型。研究表明,这6株菌株均为大豆疫霉。     

非洲菊根腐病病原的鉴定与ITS序列分析

 从南京花卉苗圃的非洲菊腐烂病植株的根部分离到46株真菌菌株,将所有菌株回接到健康的植株上,结果发现只有11个疫霉菌菌株可以引起非洲菊典型的根腐病症状。对上述疫霉菌的形态特征、致病性及核糖体DNA-ITS序列进行分析,结果为所有菌株在LBA平板上菌落圆形、呈放射状、菌丝致密、气生菌丝较丰富、菌丝无隔,能形成大量菌丝膨大体,水培后产生大量椭圆形孢子囊,孢子囊无乳突,游动孢子在孢子囊内形成。进一步克隆并分析供试菌株核糖体DNA-ITS区域的序列,结果是分离菌株与GenBank中隐地疫霉的ITS序列的同源性均为99.87%,仅有1个碱基的差异,进一步明确了本研究中从非洲菊腐烂病植株根部分离的病原菌为隐地疫霉(Phytophthora cryptogea)。     

117个玉米品种对禾谷镰孢根腐病的抗性鉴定及根腐病对苗势与产量的影响

为评价东北地区玉米主推品种对禾谷镰孢根腐病的抗性水平,探究根腐病发生与苗势、产量损失间的关系,采用人工接种方法鉴定东北地区117个玉米主推品种对禾谷镰孢根腐病的抗性水平,应用室内盆栽试验分析自交系LN810在施用氮、磷、钾及复合肥后对禾谷镰孢根腐病发生和苗势的影响,在田间对先玉335接种禾谷镰孢菌Fusarium graminearum后探究根腐病发生程度对其苗势及产量的影响。结果显示,117个玉米品种对禾谷镰孢根腐病的抗性差异明显,鉴定出高抗品种14个,抗病品种44个,中抗品种47个,感病品种12个,其中表现中抗以上的品种占89.74%,且中早熟材料均表现为抗性。与未施肥相比,施用磷钾肥后玉米禾谷镰孢根腐病发生率最低,为22.03%。按照禾谷镰孢根腐病发生程度从轻到重将先玉335群体划分为一、二、三类苗,级别越高苗势越弱,产量测定发现二、三类苗的平均产量较一类苗的平均产量分别下降了39.97%和76.39%。表明东北地区主推玉米品种大部分对禾谷镰孢根腐病表现出抗性,但仍有部分品种存在较大感病风险,且该病害的发生程度与幼苗长势和产量呈负相关,施用磷钾肥可降低该病害的发生率。     

Root and stem rot of chrysanthemum caused by five <Emphasis Type="Italic">Pythium</Emphasis> species in Japan

Root and stem rot with wilt of above ground parts of cultivated chrysanthemums was first found in Ibaraki, Toyama and Kagawa prefectures, Japan in 2002 and 2003. Pythium species were isolated from the diseased tissues and identified as P. dissotocum, P. oedochilum, P. sylvaticum, P. ultimum var. ultimum and asexual strains of P. helicoides based on their morphologies and sequences of rDNA-ITS region. All the Pythium species were strongly pathogenic to chrysanthemums in pot conditions and were reisolated from the inoculated plants. Because Pythium root and stem rot of chrysanthemum has never been reported in Japan, we propose that this is a new disease that can be caused by the five Pythium species.     

辽宁省大豆根腐病病原菌的分离鉴定及其生物学特性和对常用杀菌剂的敏感性

为明确辽宁省沈阳市大豆根腐病病原菌的分类地位,使用形态学特征和分子生物学技术对分离菌株进行鉴定,于室内对其最适生长温度、碳源及氮源进行测定,采用分生孢子悬浮液蘸根测定其对大豆品种合丰47的致病力,并采用菌丝生长速率法和分生孢子萌发率法测定16种常用杀菌剂对其的毒力。结果表明,从感根腐病大豆样品中共分离到31株镰孢菌菌株,基于培养性状从中选择3株疑似锐顶镰孢菌Fusarium acuminatum菌株进行鉴定和分析。这3株菌株在PDA培养基上形成的菌落呈白色绒毡状,产生黄色色素;大型分生孢子大小为3.3～7.2μm×29.0～62.0μm,小型分生孢子大小为2.5～5.7μm×9.1～16.8μm;结合翻译延伸因子(translation elongation factor,TEF)序列系统发育分析结果将3株菌株最终鉴定为锐顶镰孢菌F.acuminatum。该菌菌丝生长和孢子萌发的最适温度为25℃;最适生长碳源为葡萄糖和蔗糖,最适生长氮源为硝酸钠、硝酸钾和尿素;其对大豆合丰47的致病力较弱,发病植株的株高、根长和鲜重较空白对照显著降低了22.8%、12.2%和26.7%。在16种常用杀菌...     

基于环介导等温扩增技术检测由强雄腐霉引起的玉米茎腐病

为建立强雄腐霉Pythium arrhenomanes的快速分子检测技术,基于环介导等温扩增技术(loop-mediated isothermal amplification,LAMP)以β-tubulin基因为靶标序列,设计强雄腐霉的特异性引物,建立了一种准确快速的LAMP检测方法,并对该检测方法的特异性、灵敏度和实际应用效果进行评估。25μL LAMP最终反应体系为:10×ThermoPol Buffer 2.5μL、10 mmol/L dNTPs 3.5μL、6 mmol/L MgSO_4 2μL、5 mol/L甜菜碱4μL、40μmol/L FIP-1/BIP-1各1μL、5μmol/L F3-1和B3-1各1μL、40μmol/L LB-1 1μL、Bst DNA polymerase 1μL、2.5 mmol/L HNB 1.9μL、模板DNA 2μL、灭菌水3.1μL。LAMP体系在等温64℃条件下反应60 min,HNB显色反应显示天蓝色即为阳性反应;扩增产物经2%琼脂糖凝胶电泳验证为梯形条带,也可判定为阳性反应。特异性检测结果显示,LAMP体系能特异性地检测出强雄腐霉,而其它卵菌近缘种和常见植物病原真菌均未检测出。灵敏度检测结果显示,LAMP体系的最低检测灵敏度为10 pg/μL,是普通PCR反应灵敏度的1 000倍。在实际应用中,LAMP体系能够快速检测出人工接种和实际发病玉米组织中的病原菌。表明本研究建立的快速、准确、可视化的LAMP检测方法能在60 min内检测出强雄腐霉。     

山东省小麦根腐病病原菌的分离鉴定

为明确山东省小麦根腐病的病原菌种类,于2012—2014年从山东省10个地市采集小麦病株,通过组织分离法获得了185株分离物,利用形态学鉴定方法,结合基于5.8S r DNA-ITS序列或TEF-1α基因序列分析的分子鉴定方法对分离物进行了鉴定。结果表明:分离物中共得到135株麦根腐平脐蠕孢Bipolaris sorokiniana,占分离病原菌总数的72.97%,属优势种群;50株镰孢属Fusarium菌株,其中14株尖孢镰孢菌Fusarium oxysporum、19株层出镰孢菌Fusarium proliferatum和17株黄色镰孢菌Fusarium culmorum;按照柯赫氏法则进行致病性测定,证实了4种病原菌对鲁麦21号具有致病性,麦根腐平脐蠕孢的致病力较强,病情指数显著高于镰孢菌属真菌。研究表明,山东小麦根腐病主要是由麦根腐平脐蠕孢和镰孢属真菌侵染引起的,麦根腐平脐蠕孢为优势菌群。     

Grapevine crown gall caused by <Emphasis Type="Italic">Rhizobium radiobacter</Emphasis> (Ti) in Japan

In 2005, characteristic symptoms of crown gall on grapevines (Vitis vinifera L. cv. Muscat of Alexandria, and cv. Seto Giants) were observed in a commercial greenhouse-orchard in Okayama Prefecture, Japan. Isolations from diseased tissues consistently yielded bacterial colonies that were white, glistening, and produced abundant polysaccharide on potato semi-synthetic agar (PSA) medium. Ten representative isolates were chosen for further characterization. A multiplex polymerase chain reaction (PCR) assay showed these strains were not Rhizobium vitis but did possess a Ti plasmid. The bacteriological characteristics of the isolates corresponded well with R. radiobacter. The almost complete 16S ribosomal DNA sequences of isolates AT06-1 and AT06-2, selected from 10 grapevine isolates, were determined and corresponded to sequences of R. radiobacter. The pathogenicity of the isolates was tested on young grapevine and tomato (Lycopersicon esculentum Mill.) plants. Gall symptoms developed on both plant species after inoculation, and bacteria with the same colony morphology as those inoculated were reisolated. Based on these results, the isolates were identified as R. radiobacter (Ti). This report is the first of the occurrence of R. radiobacter (Ti) on grapevine in Japan. Phylogenetic analyses using the partial nucleotide sequences of virC operon located on a Ti plasmid showed that the isolate of R. radiobacter (Ti) isolated from grapevine and some strains of R. vitis (Ti) belonged to the same monophyletic group, which differed from the groups of R. radiobacter (Ti) isolated from plants other than grapevine and of the majority of R. vitis (Ti) strains isolated from grapevine. The nucleotide sequence data reported are available in the DDBJ/EMBL/GenBank databases under accessions AB306890, AB306891, and AB465432–AB465459.     

Internal fruit rot of netted melon caused by <Emphasis Type="Italic">Pantoea ananatis</Emphasis> (=<Emphasis Type="Italic">Erwinia ananas</Emphasis>) in Japan

An internal fruit rot with a malodor was found in netted melons (Cucumis melo L.) in commercial greenhouses in Kochi Prefecture, Japan, in 1998, despite their healthy appearance and lack of water-soaking or brown spots on the surface. A yellow bacterium was consistently isolated from the affected fruits. To confirm the pathogenicity of eight representative isolates of the yellow bacterium, we stub-inoculated ovaries (immature-fruits) 5–7 days after artificial pollination, with a pin smeared with bacteria. After the melon fruits had grown for 60 more days, an internal fruit rot resembling the natural infection appeared, and the inoculated bacterium was reisolated. The melon isolates had properties identical with Pantoea ananatis, such as gram-negative staining, facultative anaerobic growth, indole production, phenylalanine deaminase absence, and acid production from melibiose, sorbitol, glycerol, and inositol. Phylogenetic analysis based on 16S rDNA sequences showed that the melon bacterium positioned closely with known P. ananatis strains. The melon bacterium had indole acetic acid (IAA) biosynthesis genes (iaaM and iaaH) and a cytokinin biosynthesis gene (etz). The bacterium could be distinguished from the other ‘Pantoea’ group strains by rep-PCR genomic fingerprinting. From these results, the causal agent of internal fruit rot was identified as a strain of P.ananatis [Serrano in (Philipp J Sci 36:271–305, 1928); Mergaert et al. in (Int J Syst Bacteriol 43:162–173, 1993)]. The nucleotide sequence data reported are available in the DDBJ database under accessions AB297969, AB373739, AB373740, AB373741, AB373742, AB373743 and AB373744.     

宁夏马铃薯镰刀菌根腐病病原菌的ISSR-PCR标记体系建立及其遗传多样性分析

为明确宁夏马铃薯镰刀菌根腐病病原菌种内的遗传差异与亲缘关系,利用ISSR-PCR技术对影响PCR扩增效果的因素和ISSR引物进行优化和筛选,建立适合马铃薯镰刀菌根腐病病原菌的ISSR-PCR反应体系,并通过ISSR分子标记技术对30株地理来源不同的镰刀菌进行遗传多样性分析。结果显示:马铃薯镰刀菌根腐病病原菌的ISSR-PCR扩增最适引物为807,20μL反应体系中2×Easy Taq PCR Super Mix为8μL、引物浓度为0.6μmol/L、DNA模板浓度为56 ng/μL,循环次数为36次,退火温度为54℃。在选用的18条ISSR引物中有13条对30株镰刀菌扩增出84条条带,大小多分布在250～2 000 bp之间,其中多态性条带为82条,多态性比例平均为98.3%。30株镰刀菌的遗传相似系数在0.349～0.976之间,在0.478水平上可划分为2个类群,其中类群I包括尖孢镰刀菌Fusarium oxysporum、木贼镰刀菌F. equiseti、茄病镰刀菌F. solani和锐顶镰刀菌F. acuminatum;类群II全部为接骨木镰刀菌F. sambucinum;在0...     

Leaf mold of tomato caused by races 4 and 4.11 of Passalora fulva in Japan

Leaf mold of tomato was found on cv. Momotaro-fight in 2003 in Ehime Prefecture. The symptoms were marginal indefinite yellowing on the upper leaf surface, and downy, gray to brown sporulation on the lower surface underneath the spots. The symptoms and morphology were the same as seen with Passalora fulva. The fungal isolates were identified as races 2.4, 2.4.11, 4, and 4.11 by inoculation tests. Races 4 and 4.11 have never before been found in Japan.     

Suppression of Rhizoctonia root-rot of tomato by Glomus mossae BEG12 and Pseudomonas fluorescens A6RI is associated with their effect on the pathogen growth and on the root morphogenesis

Rhizoctonia solani root-rot is a major soilborne disease causing growth and yield depression. The ability of Glomus mosseae BEG12 and Pseudomonas fluorescens A6RI to suppress this soilborne disease in tomato was assessed by comparing the shoot and root growth of plants infested with R. solani 1556 when protected or not by these beneficial strains. The epiphytic and parasitic growth of the pathogenic R. solani 1556 was compared in the presence and absence of the biocontrol agents by microscopical observations allowing the quantification of roots with hyphae appressed to epidermal cells (epiphytic growth) and of roots with intraradical infection (parasitic growth). The root architecture of the tomato plants under the different experimental conditions was further characterized by measuring total root length, mean root diameter, number of root tips and by calculating degree of root branching. G. mosseae BEG12 and P. fluorescens A6RI fully overcame the growth depression caused by R. solani 1556. This disease suppression was associated with a significant decrease of the epiphytic and parasitic growth of the pathogen together with an increase of root length and of the number of root tips of inoculated tomato plants. The combined effects of G. mosseae BEG12 and P. fluorescens A6RI on pathogen growth and on root morphogenesis are suggested to be involved in the efficient disease suppression.     

Pythium rot of chingensai ( Brassica campestris L. chinensis group) caused by Pythium ultimum var. ultimum and Pythium aphanidermatum

Severe rot was found at the base of leaves and stems of chingensai (Brassica campestris L. chinensis group) in Okayama Prefecture in 2000. The causal fungi were morphologically identified as Pythium ultimum Trow var. ultimum and P. aphanidermatum (Edson) Fitzpatrick. This is the first report of rot caused by Pythium species on chingensai. We named this disease Pythium rot of chingensai.     

Trichoderma Biocontrol of Colletotrichum acutatum and Botrytis cinerea and Survival in Strawberry

Trichoderma isolates are known for their ability to control plant pathogens. It has been shown that various isolates of Trichoderma, including T. harzianum isolate T-39 from the commercial biological control product TRICHODEX, were effective in controlling anthracnose (Colletotrichum acutatum) and grey mould (Botrytis cinerea) in strawberry, under controlled and greenhouse conditions. Three selected Trichoderma strains, namely T-39, T-161 and T-166, were evaluated in large-scale experiments using different timing application and dosage rates for reduction of strawberry anthracnose and grey mould. All possible combinations of single, double or triple mixtures of Trichoderma strains, applied at 0.4% and 0.8% concentrations, and at 7 or 10 day intervals, resulted in reduction of anthracnose severity; the higher concentration (0.8%) was superior in control whether used with single isolates or as a result of combined application of two isolates, each at 0.4%. Only a few treatments resulted in significant control of grey mould. Isolates T-39 applied at 0.4% at 2 day intervals, T-166 at 0.4%, or T-161 combined with T-39 at 0.4% were as effective as the chemical fungicide fenhexamide. The survival dynamics of populations of the Trichoderma isolates (T-39, T-105, T-161 and T-166) applied separately was determined by dilution plating and isolates in the mixtures calculated according to the polymerase chain reaction (PCR) using repeat motif primers. The biocontrol isolates were identified to the respective species T. harzianum (T-39), T. hamatum (T-105), T. atroviride (T-161) and T. longibrachiatum (T-166), according to internal transcribed spacer sequence analysis.