出口萝卜种子中芸薹生链格孢的分离与鉴定

本文对酒泉出境萝卜种子上发现的链格孢菌(Alternariasp.)进行了形态学和分子生物学鉴定,根据结果,将这种链格孢鉴定为芸薹生链格孢(Alternariabrassicicola);该种病原菌是危害十字花科蔬菜种子生产的世界性病原真菌,在甘肃酒泉繁种基地过去未见报道。     

引起葡萄叶斑病的链格孢种类的初步鉴定

为明确北京市大兴区、顺义区和山西省襄汾县葡萄树上叶斑病的病原菌,利用组织分离法对病样进行分离纯化,采用多基因系统发育学分析(ITS,Alt a 1和gpd)和形态学对纯化后菌株进行鉴定,并结合柯赫氏法则对其进行验证。结果表明,共获得37株链格孢属真菌菌株,分属链格孢Alternaria alternata、葡萄链格孢A. viniferae和乔木链格孢A. arborescens,柯赫氏法则检测结果显示接种菌株对所接叶片与果穗均具致病性,说明这3种链格孢种均为葡萄叶斑病的病原菌;葡萄链格孢为主要的病原菌,有28株,故将该类病害命名为葡萄链格孢叶斑病;此外,该类菌株在2～40℃之间均能生长,25℃为最适生长温度。     

河南省小麦黑胚病菌的分离鉴定及致病性测定

 为明确我国小麦主产区河南省小麦黑胚病菌的主要类群,为该病的防治工作提供理论依据,对河南省不同地区种植的50个小麦品种(系)的黑胚籽粒进行了分离鉴定和致病型测定。鉴定结果表明：河南省小麦黑胚籽粒病原分离物包括链格孢(Alternaria alternate)、麦根腐蠕孢（Bipolaris sorokiniana）、细极链格孢(A. tenuissima)和小麦链格孢（A. triticina）,平均分离频率分别为63.0%、18.0%、12.2%和6.8%,不同品种小麦黑胚籽粒各种病原菌分离频率有一定差异。扬花后10 d利用孢子悬浮液接种进行分离物的致病性测定,结果表明链格孢(A.alternate)和麦根腐蠕孢（B. sorokiniana）致病力较强。结合分离频率和致病性测定结果,确定链格孢(A. alternate)是河南省小麦黑胚病的优势病原菌。     

北京地区桃黑斑病新症状的发现与诊断

2019年-2020年在北京地区桃园病害调研普查时发现一种桃树病害的新发症状-新叶坏死,主要症状表现为当年新生枝条顶端新叶和生长点腐烂坏死。为明确这种新叶坏死症状是否由病原菌侵染引起,从北京平谷2个乡镇3个桃园采集35份病梢样品,经病样组织分离和菌株纯化,选取12个代表性菌株进行形态学鉴定、系统进化分析以及致病性测定。结果显示,通过形态学鉴定和多基因(ITS,Alt a1,gpd)系统发育分析,以上分离株均鉴定为链格孢Alternaria alternata;3株代表性菌株的致病性测定验证了A.alternata是引起新叶坏死症状的病原菌,并且该病原菌也能导致桃枝条和果实发病,产生黑斑病症状。这是首次报道A.alternata除了引起桃果实黑斑症状之外,还可以引起桃树新叶坏死症状,结果进一步丰富了链格孢侵染桃树的症状多样性,为链格孢引起的病害的整体防控提供了理论基础。     

南疆骏枣黑斑病症状表现及病原菌鉴定

为明确南疆骏枣黑斑病的症状表现及病原菌种类,2013—2014年在新疆建设兵团第一师选取具有代表性的5个骏枣园,对枣叶、花、果等组织上黑斑病的症状进行了系统调查,采用组织分离法和回接试验分离病原菌及测定其致病性,并根据病菌形态特征、ITS序列和β-tubulin序列分析对病原菌进行了鉴定。结果表明:枣叶、花和果均可感染黑斑病菌,室内接种和田间骏枣黑斑病症状相同,叶、花和果中的病原菌可以相互侵染,并在枣果上均表现出黑斑病典型症状;不同发病组织中分离纯化得到357株菌株,形态观察表明,侵染叶、花和果的菌株均为链格孢属链格孢菌Alternaria alternata(Fr.)Keissler;通过ITS序列和β-tubulin序列分析,并结合形态学特征进一步确定引起骏枣不同组织的黑斑病病原菌为链格孢菌A.alternata。     

一种波斯菊叶枯病新病原菌的分离与鉴定

波斯菊是一种重要的观赏植物,叶枯病的发生影响其生长及市场价值。本文从陕西杨凌地区采集波斯菊病叶,通过保湿培养、单孢分离方法获得2株分离物。将分离菌的纯培养物接种健康寄主植物,发病后再分离的分离物与原接种病原菌菌株特征相同。采用形态学及rDNA-ITS、EF-1α和GAPDH基因序列分析相结合的方法对分离菌进行鉴定,结果表明,两株菌株均为侵染向日葵链格孢Alternaria helianthiinficiens。这是国内外波斯菊上A.helianthiinficiens的首次报道。     

福建省柑橘褐斑病菌的分离、鉴定和系统进化分析

为明确在福建省南平市的橘柚和三明市的温州蜜橘上发现的疑似柑橘褐斑病的病原菌种类,采用组织分离法获得纯化菌株,通过回接法验证菌株的致病性,利用形态学特征对病原菌进行初步鉴定,并采用最大似然法以多聚半乳糖醛酸酶基因endoPG为靶标对本研究以及国内外已报道的链格孢菌株构建系统发育树,分析其遗传多样性。结果表明,从病组织中共分离获得26株纯培养菌株,经形态学鉴定均为链格孢菌Alternaria spp.。利用分生孢子液接种橘柚离体嫩叶发现,有22株菌株能侵染橘柚叶片并产生与田间相似的褐斑病症状,确认该病害为链格孢引起的柑橘褐斑病。系统发育树分析结果显示,分离所得的26株菌株均聚在已报道的4个柑橘链格孢进化分支Clade1～Clade4中,其中21株菌株聚在国内特有的分支Clade4中,有3株菌株和1株菌株分别聚在国内外兼有的分支Clade3和Clade1中,1株菌株聚在国外特有的分支Clade2中,表明在福建省采集的这些柑橘褐斑病菌均为链格孢菌,且遗传多样性较丰富。     

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。     

青海省樱桃叶斑病病原菌的分离与鉴定

为明确引起青海省樱桃叶斑病病原菌的种类,本研究从西宁市城北区、海东市乐都区和贵德县的樱桃上采集有叶斑症状的叶片,采用组织分离法分离出病原菌,通过观察病原菌的形态特征,结合rDNA-ITS、EF-1α和Alt a 1基因序列分析对病原菌的种类进行了鉴定,并用柯赫氏法则进行验证。结果表明,共获得279株病原菌菌株,分属链格孢Alternaria alternata、细极链格孢A.tenuissima和刺盘孢属Colletotrichum spp.,分离频率分别为84.95%、5.02%和10.03%。采用柯赫氏法则进行离体叶片致病性测定,接种叶片100%发病,说明引起青海省樱桃叶斑病的病原菌为链格孢、细极链格孢和刺盘孢属真菌,且链格孢为主要病原菌。该研究结果可为青海省樱桃叶斑病的综合防治提供理论依据。     

树莓茎溃疡病病原菌鉴定及其生物学特性研究

在辽宁省沈阳市树莓园发现了一种新的茎溃疡病害,对其进行了组织分离、单孢纯化、致病性测定、形态学和分子生物学鉴定。结果表明,细极链格孢(Alternaria tenuissima)是引起此病害的病原菌。病原菌生物学特性研究表明,该病原菌菌落生长的最适培养基为PDA,最适碳源为麦芽糖,最适氮源为半胱氨酸,最适温度为25℃,光照对其无明显影响。本研究为细极链格孢(A. tenuissima)在我国引起树莓茎溃疡的首次报道。     

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。     

瓠瓜果斑病症状及其病原菌的鉴定

为有效防控瓠瓜果斑病,自浙江省象山县田间采集具有典型果斑病症状的瓠瓜样本,对其进行病原菌分离、形态观察、致病性测定及分子生物学鉴定,并利用特异性引物PL1/PL2 PCR扩增和基质辅助激光解吸电离飞行时间质谱（matrix assisted laser desorption ionization time-of-flight massspectrometry,MALDI-TOF-MS）技术对其病原菌进行亚群鉴定。结果表明：瓠瓜果斑病田间典型症状是发病叶片和果实上病斑由水渍状小斑点逐渐发展为伴有黄色晕圈的褐色不规则病斑,果实腐烂、有臭味。通过菌体形态和培养特性观察、PCR鉴定、16S rDNA序列分析和7个看家基因的系统发育分析将其病原菌鉴定为西瓜噬酸菌Acidovorax citrulli。从瓠瓜上分离的菌株均属于西瓜噬酸菌亚群I,从西瓜上分离的菌株均属于西瓜噬酸菌亚群II。     

Characterisation of fungal pathogens causing basal rot of lettuce in Belgian greenhouses

Basal rot is a common disease in lettuce greenhouses. A 3-year study on the diversity of pathogens associated with basal rot in Belgium was carried out. A total of 150 isolates were collected originating from 56 greenhouses. Four pathogens appeared to be involved. Rhizoctonia solani was found to be the causal agent at 23 locations, Sclerotinia spp. at 14, Botrytis cinerea at 17 and Pythium spp. at seven. The isolates of R. solani were further characterised to anastomosis groups and subgroups using morphological characteristics, pectic zymogram and PCR-RFLP. Five anastomosis groups could be distinguished: AG1-1B, AG4 HGI, AG10, AG2-1, AG2-1 Nt and AG3, with isolates of AG4 HGI and AG1-1B being the most prevalent and the most aggressive. Sclerotinia sclerotiorum was found at 13 locations, while S. minor was found at only one location. Based on ITS-sequencing Pythium isolates were assigned to three different species. At 20°C, isolates of all pathogens were able to cause lesions on detached lettuce leaves, except isolates of R. solani AG3 and AG2-1 Nt. A correlation could be found between the occurrence of the pathogens and the growing season. Botrytis cinerea was the most common pathogen in winter, whereas R. solani was most frequently isolated in summer. Sclerotinia spp. and Pythium spp. were isolated in spring, summer and autumn. The information obtained in this study will be most useful in the development of an alternative control strategy for causal agents of basal rot.     

福建省大豆炭疽病病原菌的分离与鉴定

为明确引起福建省大豆炭疽病的病原菌种类,2018—2019年从福建省福州、三明、莆田、泉州和南平市采集具有大豆炭疽病症状的大豆豆荚,采用组织分离法分离病原菌,结合形态学特征和多基因系统发育分析对病原菌进行鉴定。结果表明,从采集的豆荚样本中共分离获得152株炭疽菌菌株,经形态学初步鉴定分属于3种类型,从3种类型的病原菌中分别选取3株代表菌株进行多基因系统发育分析,这9株代表菌株分别与Colletotrichum plurivorum、平头炭疽菌C.truncatum和胶孢炭疽菌C.gloeosporioides聚在一起。形态学和多基因系统发育分析结果表明,引起福建省大豆炭疽病的病原菌有3种,分别为C.plurivorum、平头炭疽菌和胶孢炭疽菌,其占比分别为10.53%、50.00%、39.47%。致病性测定结果表明,C.plurivorum、平头炭疽菌和胶孢炭疽菌对大豆均有致病作用,但不同的菌株致病力存在差异。表明平头炭疽菌是引起福建省大豆炭疽病的主要病原菌,而C.plurivorum为引起我国大豆炭疽病的新纪录种。     

Gnomonia fragariae, a Cause of Strawberry Root Rot and Petiole Blight

Gnomonia fragariae has been occasionally listed among the fungi associated with diseased strawberry plants. However its pathogenicity has not been established. During the investigation on strawberry decline in Latvia and Sweden, a fungus was repeatedly recovered from discoloured root and crown tissues of severely stunted plants. Attempts to induce sporulation of the isolates grown on several agar media resulted in the formation of mature ascomata only on potato carrot agar and oatmeal agar. On morphological grounds and comparisons with reference herbarium specimens these isolates were identified as Gnomonia fragariae. The pathogenicity of the fungus was evaluated initially in the detached leaf assay and subsequently in three bioassays on strawberry plants. All the bioassays showed that G. fragariae was pathogenic on strawberry and capable of causing severe root rot and petiole blight. The symptoms that developed in the greenhouse experiments closely resembled those observed in the fields. The fungus did not cause rapid plant death but growth and development of inoculated strawberry plants was severely affected. To our knowledge this is the first time when pathogenicity of G. fragariae as a root rot pathogen has been clearly established. Our study shows that G. fragariae is one of the serious pathogens involved in the root rot complex of strawberry in Latvia and Sweden.     

莴笋链格孢叶斑病病原鉴定

正莴笋(Lactuca sativa L.var.asparagina Baily)为菊科舌状花亚科莴苣属,又名茎用莴苣,属一年生或两年生草本植物,原产地中海沿岸,约在七世纪初经西亚传入我国,全国各地普遍栽培。2016年分别在河北省及内蒙古莴笋种植区发现一种叶斑病,病斑呈圆形或近圆形、黑褐色、具同心轮纹,     

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。     

广东省马铃薯块茎软腐病病原菌鉴定

为明确广东省惠州市马铃薯块茎软腐病的病原菌,采用常规病组织分离法获得4株菌株,通过生理生化特征和分子生物学特性对其进行鉴定,并测定了该病原菌对20个马铃薯品种的致病力。结果显示,该病原菌为革兰氏阳性菌,在LB平板上菌落有透明和不透明2种形态;除葡萄糖发酵阳性、对链霉素和青霉素具有抗性等特征不同外,BP-hd-1、BP-hd-2、BP-hd-3和BP-hd-4菌株与短小芽胞杆菌Bacillus pumilus菌株的其它生理生化特征均相同。系统进化分析结果表明,4株菌株的16S rDNA序列分别与短小芽胞杆菌AI-Khrj18(KY123871)、ML270(KC692158)、NCTC10337(LT906438)和ARD21(KX023236)的相似性为100.0%;4株菌株的gyrB基因序列均与短小芽胞杆菌AUEC29菌株(HM585095)的gyrB基因相似性最高,为99.7%～99.8%。生理生化特征及分子生物学鉴定结果表明,引起广东省马铃薯块茎软腐病的病原菌为短小芽胞杆菌。致病力测定结果显示,接种菌株BP-1后20个马铃薯品种的发病率均为100.0%,表明该病原菌对马铃薯有强致病力。     

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。     

Fungi on roots and stem bases of asparagus in the Netherlands: species and pathogenicity

A survey was made to identify the most important soilborne fungal pathogens of asparagus crops in the Netherlands. Ten plants were selected from each of five fields with a young (1–4 y) first planting, five fields with an old (6–13 y) first planting and five fields with a young replanting. The analysis included fungi present in the stem base and the roots of plants with symptoms of foot and root rot or showing growth decline without specific disease symptoms. Isolates of each species were tested for pathogenicity to asparagus on aseptically grown plantlets on Knop's agar. Symptoms were caused byFusarium oxysporum, F. culmorum, Botrytis cinerea, Penicillium verrucosum var.cyclopium, Cylindrocarpon didymum, Phialophora malorum, Phoma terrestris andAcremonium strictum. F. oxysporum was by far the most common species and was isolated from 80% of the plants. Not all of its isolates were pathogenic to asparagus. Symptoms were caused by 67%, 78% and 93% of the isolates obtained from young first plantings, old first plantings and replantings, respectively.F. culmorum was isolated from 31% of the plants. Two other notorious pathogens of asparagus,F. moniliforme andF. proliferatum, did not occur in our samples.Species causing symptoms in the vitro test that were found on more than 5% of the plants were additionally tested for their pathogenicity in pot experiments.F. oxysporum f.sp.asparagi caused severe foot and root rot, significantly reduced root weights and killed most of the plants.F. culmorum caused lesions on the stem base often resulting in death of the plant.P. terrestris, a fungus only once reported as a pathogen of asparagus, caused an extensive root rot, mainly of secondary roots that became reddish. The fungus was isolated in only a few samples and is not to be regarded as an important pathogen in Dutch asparagus crops.P. malorum caused many small brown lesions on the stem base and incidentally also on the upper part of small main roots. This is the first report of its pathogenicity to asparagus. The fungus is one of the organisms inciting spear rust and it reduced crop quality rather than crop yield.P. verrucosum var.cyclopium andC. didymum did not cause symptoms in pot experiments.Because of its predominance on plants with foot and root rot and its high virulence,F. oxysporum f.sp.asparagi was considered to be the main soilborne pathogen of asparagus in the Netherlands.