呼和浩特市苜蓿根腐病病原菌鉴定及致病性测定

为确定引起呼和浩特市苜蓿根腐病的病原菌种类,采用常规组织分离法对采集的疑似苜蓿根腐病病样进行病原菌分离与培养,利用形态学观察结合分子生物学方法对分离物代表菌株进行鉴定,并采用土壤接种法对代表菌株的致病性进行测定。结果表明,共分离获得6类形态学特征不同的分离物,各随机选择1株代表菌株进行鉴定,结合分子生物学鉴定结果确定呼和浩特市苜蓿根腐病病原菌有6种,分别是麦根腐平脐蠕孢菌Bipolaris sorokiniana、立枯丝核菌Rhizoctonia solani、木贼镰刀菌Fusarium equiseti、变红镰刀菌F. incarnatum、锐顶镰刀菌F. acuminatum和织球壳枯萎菌Plectosphaerella cucumerina,分别分离到1、7、14、26、7和14株菌株,占总分离菌株数的1.45%、10.14%、20.29%、37.68%、10.14%和20.29%。其中,立枯丝核菌的致病力最强,接种苜蓿幼苗发病的病情指数达82.67,其次为木贼镰刀菌、变红镰刀菌、锐顶镰刀菌、麦根腐平脐蠕孢菌和织球壳枯萎菌,病情指数分别为72.67、62.67、58.67、52....     

浙江省铁皮石斛根腐病病原真菌的鉴定

为明确铁皮石斛根腐病病原真菌,于其主产地浙江省金华市武义县收集铁皮石斛根腐病病株,采用平板分离方法对病原真菌进行分离,使用镰刀菌种特异性引物并结合ITS和TEF序列分析及形态学鉴定确定该镰刀菌的分类地位。结果表明,共分离纯获得真菌117株,其中有105株镰刀菌;经分子生物学分析及形态学鉴定结果显示,分离出的镰刀菌为层出镰刀菌Fusarium prolife‐mum、茄病镰刀菌F. solani、尖孢镰刀菌F. oxysporum和厚垣镰刀菌F. chlamydosporum四个种,其中层出镰刀菌在数量上具有优势地位,占总镰刀菌数的44.8%;茄病镰刀菌、尖孢镰刀菌、厚垣镰刀菌分别占总镰刀菌数的21.0%、15.2%和19.0%。在致病性测定中发现层出镰刀菌和茄病镰刀菌并不具备致病性,尖孢镰刀菌的致病性明显弱于厚垣镰刀菌,表明厚垣镰刀菌为浙江省金华市武义县铁皮石斛根腐病的主要致病菌。     

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。     

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。     

拮抗丹参根腐病病原菌的丹参根际放线菌筛选研究

为获得高效拮抗丹参根腐病病原菌的生防菌株,该研究通过平板对峙法和滤纸片法对丹参根际土壤中的放线菌进行筛选。结果表明,从丹参根际土壤中分离得到的247株放线菌中,成功筛选得到了10株具有抑菌效果的生防菌株。其中zyzx-001、zyzx-013、zyzx-124抑菌效果较好,其抑菌率超过60%。通过滤纸片法对10株生防菌株进行复筛。结果表明,这10株生防菌的发酵液均可对丹参根腐病病原菌产生抑制作用。说明这10株菌株均可产生抑制丹参根腐病病原菌的活性物质。综合比较,菌株zyzx-001抑制作用最强。经鉴定,菌株zyzx-001属于链霉菌属。     

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。     

黄芪根腐病生防放线菌筛选鉴定及其优化培养

为获得对黄芪根腐病具有强抗菌活性的生防菌株,采用平板对峙法、抑制菌丝生长速率法对分离于河西走廊盐碱土中的172株放线菌进行拮抗菌的筛选,获得优菌株DA4-4-1,根据其形态、培养特征、生理生化特性和16S rDNA基因序列进行系统发育学分析鉴定,并根据单因素试验与正交试验对培养基条件进行优化。结果表明,菌株DA4-4-1对黄芪根腐病的2种主要致病菌尖孢镰刀菌Fusarium oxysporum和茄腐镰刀菌F.solani均有较好的抑制作用,抑菌圈直径最大分别为20.16 mm和19.33 mm,抑菌率最大分别为34.62%和32.44%。结合16S rDNA基因序列的系统发育分析,将菌株DA4-4-1鉴定为链霉菌属蓝紫链霉菌Streptomyces tuirus。培养基最佳碳源为麦芽糖,氮源为(NH_4)_2SO_4,其优化条件为麦芽糖15 g/L、(NH_4)_2SO_4 0.5 g/L、NaCl 10 g/L、pH 7,在此条件下菌株DA4-4-1的抑菌率可达到86.72%。表明菌株DA4-4-1是1株具有较强抗菌活性的放线菌,具有良好的生物防治和开发应用潜力。     

太子参根腐病病原菌的鉴定及防治药剂筛选

为有效防控太子参根腐病,本研究于2017年3月-2019年6月自福建省宁德市柘荣县太子参主产区采集具有典型根腐病症状的植株和块根病样,分离纯化获得病原菌,利用形态学特征、分子生物学特性及致病性测定对其进行鉴定;同时通过测定平板抑制率和扫描电镜观察分析6种杀菌剂对太子参根腐病病原菌菌丝生长的抑制效果。结果表明：太子参根腐病典型症状为苗期和生长期植株萎蔫、根部和茎基部褐变;块根表皮呈现开裂、组织褐变和干腐状。分离纯化后获得22株培养性状一致的菌株,结合菌株的形态学特征、EF-1α基因序列和特异性引物FOF1/FOR1检测,将该病原菌鉴定为尖孢镰刀菌Fusarium oxysporum。致病性测定结果表明其发病症状与田间表现一致,且符合科赫氏法则。经筛选6.6%嘧菌酯·1.1%咯菌腈·3.3%精甲霜灵悬浮种衣剂和25%吡唑醚菌酯乳油对该病原菌有良好的抑制效果,稀释至2 000倍液的抑制率分别为78.52%和75.36%;扫描电镜观察显示这2种杀菌剂处理组的菌丝体发育畸形、断裂、表面皱缩或消解,未见产孢细胞;而无菌水对照处理组的菌丝体生长正常,可见产孢细胞。表明筛选到的2种杀菌剂可用于防治由尖孢镰刀菌引起的太子参根腐病。     

宁夏马铃薯黄萎病病原菌分离鉴定及寄主范围测定

 为明确引起宁夏马铃薯黄萎病病原菌种类,本试验通过常规组织分离法对采自宁夏马铃薯主栽地区固原和西吉的27株马铃薯黄萎病标样进行了分离,共获得11株分离物。采用形态学并结合肌动蛋白(ACT)、延伸因子-1α(EF1α)、3-磷酸甘油醛脱氢酶(GPD)、色氨酸合成酶(TS)的多基因系统发育分析方法,11株分离物均被鉴定为非苜蓿轮枝菌(Verticillium nonalfalfae)。致病性测定结果表明,该轮枝菌能够侵染马铃薯植株幼苗并引致典型的黄萎病症状。非苜蓿轮枝菌对茄科、菊科、豆科、十字花科、葫芦科和锦葵科等6科13种双子叶作物的致病性测定结果表明,其对西葫芦、黄瓜、茄子、烟草、豇豆、棉花和向日葵的致病性强,对番茄、辣椒、生菜和白菜致病力弱且发病缓慢,对西蓝花和苜蓿无致病性。     

京津冀设施大棚根际土壤中筛选出的防治根腐病的生防镰孢菌

 由致病性尖孢镰孢菌(Fusarium oxysporum)引起的根腐病严重危害果蔬生产,但非致病性镰孢菌可作为潜在的生防菌。为筛选防治根腐病的非致病生防镰孢菌,从京津冀设施大棚采集茄科、葫芦科果蔬78份根际土样中分离2 402株真菌,筛选出对致病性尖孢镰孢菌(F. oxysporum)具有拮抗效果的真菌173株。利用镰孢菌通用引物进行PCR扩增,从中筛选出28株候选镰孢菌;通过镰孢菌发酵液泡根进行安全性测试,筛选出对寄主黄瓜幼苗安全无害的镰孢菌菌株4株(1418、1441、1436和1473)。进一步通过镰孢菌测序通用引物TEF1αF/TEF1αR结合菌落和分生孢子的形态学特征,1418菌株和1441菌株被鉴定为尖孢镰孢菌(F. oxysporum)、1436菌株被鉴定为茄病镰孢菌(F. solani)。盆栽测试发现,除1441菌株外,1418菌株、1436菌株和1473菌株对黄瓜根腐病的防效均在50%以上,其中1418菌株的防效为70%,与杀菌剂咪酰胺的防效相当,具有很好的应用潜力。本研究筛选获得的具有生防潜力的镰孢菌不仅为镰孢菌致病力分化的研究提供了实验材料,也为新型生防产品的研制奠定了基础。     

南方镰孢Fusarium meridionale特异性PCR检测方法的建立与应用

为建立快速、稳定的南方镰孢Fusarium meridionale特异性检测方法,对已报道的镰孢属reductase-like基因部分序列进行比对分析,寻找特异性SNP位点,设计出特异性检测引物F-Fm/R-Fm3。利用该引物对包括南方镰孢在内的30株镰孢的基因组DNA进行PCR扩增。结果显示仅在7株南方镰孢中均扩增出400 bp左右的特异性条带。PCR灵敏度试验结果表明该方法的检测灵敏度达到500 pg基因组DNA。     

Diversity of pathogenic Fusarium populations associated with asparagus roots in decline soils in Spain and the UK

Asparagus decline is a disease associated with several species of Fusarium . In order to assess the relative significance of causative species, single-stranded conformational polymorphism (SSCP) analysis of the ITS2 (internal transcribed sequence) region of the ribosomal DNA was used to rapidly and objectively identify the fusarial populations associated with the roots of two intensively sampled asparagus crops, one in the UK and the other in Spain. Over 360 fusarial isolates were obtained from fields showing symptoms of asparagus decline, and most were easily differentiated by SSCP into four principal species, F. oxysporum f. sp. asparagi , F. proliferatum , F. redolens and F. solani . Fusarium oxysporum f. sp. asparagi (Foa) was most frequently isolated from the UK site (69%), whilst Foa and F. proliferatum were found in similar proportions overall (40 and 39%, respectively) from the Spanish site, although individual fields showed considerable intraregional variation. Other minor populations, such as F. culmorum , were also found. Most isolates were highly pathogenic to asparagus in vitro , although F. solani isolates comprised both pathogenic and nonpathogenic populations. Two populations of Foa were distinguished by a single ITS2 base transition, and the dominance of these two populations differed between Europe and the USA. Fusarium proliferatum was more abundant in Spain than in the UK. Phylogenetic analysis using EF1α sequences indicated that isolates of F. oxysporum pathogenic to asparagus are spread across a number of clades within the species complex, supporting the hypothesis that pathogenicity to asparagus in this species is a relatively unspecialized trait.     

Soybean sudden death syndrome: Fungal pathogenesis and plant response

Soybean sudden death syndrome (SDS) is a fungal disease caused by members of clade 2 of the Fusarium solani species complex (FSSC). These fungi are soilborne pathogens that infect soybean plants through the roots and produce toxins that translocate to aerial parts of the plant, inducing foliar chlorosis and necrosis followed by premature defoliation. Here, we first give the current state of knowledge of early pathogen detection and infection establishment for the SDS pathosystem. Subsequently, we discuss the nature and activity of secreted toxins, followed by an overview of changes in plant metabolism and factors that influence fungus–soybean interaction. Finally, we summarize the advances in plant disease resistance, symptom evaluation, and treatment.     

Comparison of genetic diversity and pathogenicity of fusarium head blight pathogens from China and Europe by SSCP and seedling assays on wheat

The genetic diversity and pathogenicity of isolates of Fusarium graminearum and F. asiaticum isolated from wheat heads in China were examined and compared with those of isolates of F. graminearum , F. asiaticum and F. meridionale from Europe, USA and Nepal. Genetic diversity was assessed by SSCP (single strand conformation polymorphism) and AFLP (amplified fragment length polymorphism) analysis and by molecular chemotyping. SSCP analysis of the Fg16F/Fg16R PCR amplicon differentiated F. graminearum , F. asiaticum and F. meridionale and revealed three haplotypes among sequence-characterized amplified region (SCAR) type 1 F. graminearum isolates. AFLP analysis showed a high level of genetic diversity and clustered the majority of Chinese isolates in one group along with other isolates of Asian origin. The second cluster contained F. graminearum isolates from China, Europe and the USA. Of the Chinese isolates, 79% were F. asiaticum and 81% of these were of the 3-AcDON chemotype, with only 9·5% of either chemotype 15-AcDON or NIV. All the Chinese and USA isolates of F. graminearum were 15-AcDON, whereas among the isolates from Europe, 21% were NIV and 8% were 3-AcDON chemotype. No evidence was found for possible differences in aggressiveness between F. graminearum and F. asiaticum . Highly aggressive isolates were present in each region and no evidence was found for any association between aggressiveness and geographical origin or chemotype among the isolates examined. No difference was observed in pathogenicity towards wheat seedlings between Chinese isolates and those from Europe, the USA or Nepal.     

我国小麦茎基腐病菌致病力比较分析

为明确我国不同种、地理来源和毒素化学型小麦茎基腐病菌的致病力分化情况,采用纸塔法对来自全国9个省市80个采样点分离的224株小麦茎基腐病菌进行致病力分析。结果表明,不同种镰刀菌的致病力不同,黄色镰刀菌Fusarium culmorum,禾谷镰刀菌F.graminearum,假禾谷镰刀菌F.pseudograminearum及亚洲镰刀菌F.asiaticum致病力强于其他种。F.culmorum致病力显著高于F.pseudograminearum和F.asiaticum,而F.pseudograminearum,F.graminearum及F.asiaticum三者之间无显著性差异。中华镰刀菌F.sinensis,木贼镰刀菌F.equiseti,锐顶镰刀菌F.acuminatum致病力较弱,三者间苗期致病力无显著性差异;多数省份F.pseudograminearum群体间致病力无显著差异,仅山东F.pseudograminearum群体的致病性显著低于河南群体;此外,产毒类型为3ADON的F.pseudograminearum群体致病力显著高于15ADON群体。     

寄生隐丛赤壳菌ITS分子检测技术研究

寄生隐丛赤壳菌是引起板栗疫病的致病菌。为建立该菌的分子检测技术,本研究首先采用通用引物ITS1/ITS4对分离自四川雅安、泸州及重庆的寄生隐丛赤壳菌及其他参试菌株的ITS区进行PCR扩增和测序比对。根据该片段与GenBank中隐丛赤壳属其他种的ITS序列差异,设计了寄生隐丛赤壳菌的特异性引物ITSP1/ITSP2,片段扩增大小为462bp。利用该引物对菌株基因组DNA进行扩增,可以将寄生隐丛赤壳菌与其他参试菌区分开,检测灵敏度达30pg。而以引物ITS1/ITS4和ITSP1/ITSP2进行的巢氏PCR,可检测到30fg基因组DNA,其灵敏度较常规PCR提高了1 000倍。利用巢氏PCR检测体系对发病程度不同的组织和携菌组织进行检测,均能快速稳定地检测出寄生隐丛赤壳菌。     

美国大豆中镰刀菌的分离鉴定

为加强对美国输华大豆真菌病害的监测力度,降低外来生物入侵风险,本文通过对美国进境大豆病菌分离,共得到32个菌株,并对其中3株镰刀菌进行了形态学和分子生物学鉴定,确认了它们分别是尖孢镰刀菌(Fusarium oxysporum)、木贼镰刀菌(F.equiseti)和拟枝孢镰刀菌(F.sporotrichioides)。本研究从32个分离菌株中得到的3株镰刀菌分属于不同种,不仅证实了美国大豆中镰刀菌的多样性,也可为港口的植物检疫工作提供借鉴。     

陕西省大葱叶部镰刀菌枯萎病病原菌的鉴定

<正>0引言大葱(Allium fistulosum L. var. giganteum Makino)为石蒜科(Amaryllidaceae)葱属(Allium)植物，是常见的调味品，具有杀菌祛痰、强心降压等多种功效。已报道的大葱叶枯病病原菌包括匍柄霉属(Stemphylium)、泛菌属(Pantoea)、疫霉属(Phytophthora)^[1-3]等。调查发现陕西乾县漠西大葱种植基地一种大葱叶部枯萎病发病率较高，成为影响当地大葱生产的主要病害之一。     

Genetic diversity of Fusarium oxysporum and related species pathogenic on tomato in Algeria and other Mediterranean countries

In order to characterize the pathogen(s) responsible for the outbreak of fusarium diseases in Algeria, 48 Fusarium spp. isolates were collected from diseased tomato in Algeria and compared with 58 isolates of Fusarium oxysporum originating from seven other Mediterranean countries and 24 reference strains. Partial sequences of the translation elongation factor EF‐1α gene enabled identification of 27 isolates as F. oxysporum, 18 as F. commune and three as F. redolens among the Algerian isolates. Pathogenicity tests confirmed that all isolates were pathogenic on tomato, with disease incidence greater at 28°C than at 24°C. All isolates were characterized using intergenic spacer (IGS) DNA typing, vegetative compatibility group (VCG) and PCR detection of the SIX1 (secreted in xylem 1) gene specific to F. oxysporum f. sp. lycopersici (FOL). No DNA polymorphisms were detected in the isolates of F. redolens or F. commune. In contrast, the 27 Algerian isolates of F. oxysporum were shown to comprise nine IGS types and 13 VCGs, including several potentially new VCGs. As none of the isolates was scored as SIX1+, the 27 isolates could be assigned to F. oxysporum f. sp. radicis‐lycopersici (FORL). Isolates from Tunisia were also highly diverse but genetically distinct from the Algerian isolates. Several Tunisian isolates were identified as FOL by a PCR that detected the presence of SIX1. The results show that isolates from European countries were less diverse than those from Tunisia. Given the difference between Algerian populations and populations in other Mediterranean countries, newly emergent pathogenic forms could have evolved from local non‐pathogenic populations in Algeria.     

长江中下游地区亚洲镰刀菌NX-2毒素群体的检测

由禾谷镰刀菌复合种(Fusarium graminearum species complex,FGSC)引起的麦类赤霉病,是农业生产上的重要病害。为明确中国长江中下游冬小麦主产区小麦赤霉病菌种的构成及其地理分布,对2008年从江苏、浙江和湖北3省采集的656株小麦赤霉病菌株进行了分类鉴定。结果显示,其中558个菌株为亚洲镰刀菌(Fusarium asiaticum),98个菌株为禾谷镰刀菌(Fusarium graminearum sensu stricto),表明中国长江中下游冬小麦主产区小麦赤霉病的主要致病菌是亚洲镰刀菌。选择亚洲镰刀菌(F.asiaticum)为研究对象,通过PCR-RFLP的方法对其进行产NX-2毒素菌株的检测。结果没有检测到产NX-2毒素菌株,表明中国长江中下游冬小麦主产区并未出现NX-2毒素群体。本研究旨在了解NX-2毒素群体在中国长江中下游地区的地理分布,为进一步研究麦类赤霉病菌群体遗传多样性和演化趋势奠定基础,为麦类赤霉病的防治和毒素污染的控制提供理论依据。