尖孢镰孢霉种下分类鉴定新技术：营养体亲和性鉴定方法

尖孢镰孢是许多重要作物枯萎病的病原真菌。迄今已报道的专化型有８０多个，专化型下又划分为许多生理小种。专化型和生理小种的鉴别，对于抗病育种，植物检疫以及病害防治，具有重要的理论和实际意义，传统的鉴别方法是利用人工接种的办法，但这种方法较费时费工，易受环境条件和寄主因素的影响，营养体亲和性鉴定方法，是国外最近发明的一种新的变通技术，这种方法利用了突变体的互补性以及遗传关系接近的菌株才能形成异核体的原理     

香蕉枯萎病菌1号和4号生理小种菌株侵染和定殖巴西蕉和粉蕉根系的观察

尖孢镰刀菌古巴专化型1号和4号生理小种菌株对巴西蕉和粉蕉的致病性不同,其在巴西蕉和粉蕉根系的侵染和定殖过程是否不同仍未阐明。笔者将绿色荧光蛋白标记的1号（T320）和4号（B2-63）生理小种菌株分别接种巴西蕉和粉蕉,借助激光共聚焦显微镜观察侵染过程。发现这2个生理小种菌株菌丝均可沿巴西蕉和粉蕉根系表层细胞之间的凹槽生长,并进一步侵入维管束;而且B2-63侵入粉蕉根系维管束快于其侵入巴西蕉根系维管束。在接种T320 菌株15 d后,在轻微褐变的巴西蕉球茎组织中未观察到其菌丝体,而在褐变粉蕉球茎中可观察到。推测T320在粉蕉根系维管束中的扩展较其在巴西蕉根系维管束中的扩展容易。结果为进一步研究尖孢镰刀菌古巴专化型生理小种与不同基因型香蕉的互作奠定基础。     

尖孢镰刀菌古巴专化型4号生理小种fpd1基因敲除与表型分析

为了研究尖孢镰刀菌古巴专化型4号生理小种fpd1基因的功能,构建了该基因敲除突变体并对其表型特征进行了分析。结果表明,与野生型菌株相比,fpd1基因敲除突变体生长减慢,产孢量显著降低,对巴西蕉的致病性明显减弱;fpd1基因可能在尖孢镰刀菌古巴专化型的产孢、生长发育和致病性等方面具有重要作用。本研究结果为进一步研究fpd1基因的功能和尖孢镰刀菌的致病机理奠定了基础。     

香蕉枯萎病菌pgx4基因的克隆与序列分析

为了解pgx4基因在尖孢镰刀菌古巴专化型侵染香蕉过程中的作用,及其与尖孢镰刀菌古巴专化型生理小种1号和生理小种4号之间的致病力差异的关系,以尖孢镰刀菌古巴专化型1号生理小种和4号生理小种的基因组DNA和cDNA为材料,采用PCR和RT-PCR方法扩增了2个生理小种的pgx4基因,并对扩增产物进行克隆测序后再用生物信息学软件对序列进行比对分析.结果表明:2个生理小种pgx4基因的开放阅读框均为1 395 bp,编码465个氨基酸,且前17个氨基酸均构成该基因的信号肽.通过DNAStar 7.1比对发现,尖孢镰刀菌古巴专化型2个生理小种pgx4基因的核苷酸序列存在25个碱基的差异,而其编码的465个氨基酸中,也有3个氨基酸不同,分析这些核苷酸序列和氨基酸序列的差异可能与尖孢镰刀菌古巴专化型1号生理小种和4号生理小种在侵染蕉类不同栽培种时的识别专性寄主机制有一定关系.     

大蕉枯萎病病原菌的分离、鉴定和致病性测定

从表现枯萎病症状的大蕉球茎病组织中获得10株分离物,采用形态学分类方法将这些分离物鉴定为尖孢镰刀菌.利用分离物DJ1的rDNA-ITS区序列和IGS序列开展的系统发育分析迸一步确定DJ1为尖孢镰刀菌.对不同香蕉品系的致病性测定结果表明,DJ1对粉蕉(Musa sp.ABB)的致病性最强,其次是特威(Musa sp.AAA)、巴西蕉(Musa sp.AAA)和泰蕉(Musa sp.AAA),对皇帝蕉(Musa sp.AA)的致病性较弱,根据DJ1的寄主范围确定DJ1为尖孢镰刀菌古巴专化型生理4号小种.对IGS序列的进一步比对分析表明DJ1不属于热带4号生理小种菌株.这些结果为大蕉枯萎病的防治提供了重要依据和指导.     

利用ITS1和ITS4通用引物扩增香蕉枯萎病菌核酸片段鉴定其生理小种

利用真菌核糖体rDNA区通用引物ITS1和ITS4,扩增采集自香蕉的13株镰刀菌包括18S rDNA部分序列、ITS1-5.8S-ITS2全部序列和28S rDNA部分序列的片段,通过BLAST序列比对分析,确定13株菌为尖孢镰刀菌[Fusarium oxysporum]。采用最大简约法,以层出镰刀菌[F.proliferatum(EU151490)]和茄病镰刀菌[F.solani(GQ376116)]为外群,将13株菌的序列与BLAST检索获得的尖镰孢古巴专化型(F.oxysporum f.sp.Cubense)相应序列构建了系统发育树。系统发育分析结果显示,13株菌与NCBI登录的4个尖镰孢古巴专化型菌株一起被分成3个亚群,亚群聚类结果与回接鉴定结果及文献报道的生理小种结果完全一致。     

东北三省大豆根腐镰孢菌种类及其致病力分析

于2007~2010年连续4年从东北三省大豆主产区采集大豆根腐病株,分离大豆根腐镰孢菌,并进行了形态学鉴定和致病力测定。结果表明,分离到的1199个镰孢菌菌株属于 8 个种,分别为尖镰孢菌（F. oxysporum）、 腐皮镰孢菌（F. solani）、 禾谷镰孢菌（F. graminearum）、 半裸镰孢菌（F. semitactum）、 轮枝镰孢菌（F. verticillioides）、燕麦镰孢菌（F. avenaceum）、木贼镰孢菌（F. equiseti）和厚垣镰孢菌（F. chlamydosporum）,其出现频率依次为36.45 %、25.35 %、11.43 %、10.76%、6.76%、4.42%、3.84%和1.00 %。以上8种镰孢菌在黑龙江省都存在,且在黑龙江省首次发现了厚垣镰孢菌。在吉林省没有分离到F. avenaceum和F. verticillioides,在辽宁省没有分离到F. avenaceum和F. chlamydosporum。以合丰25号大豆品种为接种材料进行的致病力测定结果显示：8种镰孢菌对大豆均有致病力,其中尖镰孢菌为强致病力,腐皮镰孢菌、禾古镰孢菌、半裸镰孢菌、燕麦镰孢菌、木贼镰孢菌和厚垣镰孢菌均为中等致病力,轮枝镰孢菌为弱致病力;8种镰孢菌引起的大豆根腐病症状不尽相同。     

Red Fluorescent Protein Gene (DsRed) Transformation of Fusarium oxysporum f.sp.cubense race 4

采用PEG-CaCl2介导的原生质体转化体系,成功地将红色荧光蛋白基因DsRed转入到尖孢镰刀菌古巴专化型4号生理小种B2菌株中.在显微镜下观察连续转接6代的转化子,结果都可以观察到很强的红色荧光,表明DsRed基因在转化的B2菌株中能稳定遗传.通过PCR验证,也表明红色荧光蛋白基因DsRed已经转入到尖孢镰刀菌中.对香蕉的致病力测定结果表明,转化子与野生型菌株B2相比没有明显差异.     

尖孢镰刀菌古巴专化型4号生理小种红色荧光蛋白基因转化

采用PEG-CaCl2介导的原生质体转化体系,成功地将红色荧光蛋白基因DsRed转入到尖孢镰刀菌古巴专化型4号生理小种B2菌株中。在显微镜下观察连续转接6代的转化子,结果都可以观察到很强的红色荧光,表明DsRed基因在转化的B2菌株中能稳定遗传。通过PCR验证,也表明红色荧光蛋白基因DsRed已经转入到尖孢镰刀菌中。对香蕉的致病力测定结果表明,转化子与野生型菌株B2相比没有明显差异。     

香蕉枯萎病菌fga1基因的克隆与序列分析

为了解fga1基因在尖孢镰刀菌古巴专化型侵染香蕉过程中的作用,及其与尖孢镰刀菌古巴专化型生理小种1号和生理小种4号之间的致病力差异的关系,采用PCR和RT-PCR方法扩增了2个生理小种的fga1基因,并对扩增产物进行了测序及相似序列搜索和比对,还对基因编码的蛋白进行了氨基酸序列比对和功能分析.研究结果表明2个生理小种fga1基因开放阅读框均为1 062 bp,编码353个氨基酸,基因同源性为99.5%,氨基酸序列相同.推测fga1基因可能与香蕉枯萎病菌自身繁殖和附着胞的形成有关.从fga1基因序列及其编码蛋白的氨基酸序列看,2个生理小种致病力的差异与fga1基因并无明显对应关系,这为进一步研究fga1基因功能奠定了基础.     

2种激发子对西瓜枯萎病的诱抗作用

分别测定了来源于西瓜枯萎病菌和香蕉枯萎病菌的细胞壁来源的水溶性、热稳定激发予对西瓜枯萎病的诱抗作用。结果表明：2种来源的激发子均有明显的诱导西瓜苗细胞壁木质化的作用，但来源于香蕉枯萎病菌的激发子对木质化的诱导活性比来源于西瓜枯萎病菌的要高；西瓜苗分别经2种激发子处理后，体内过氧化物酶、多酚氧化酶和苯丙氨酸解氮酶的活性均有异常的增加，但来源于西瓜枯萎病菌的激发子对这3种防御酶的诱导速度比来源于香蕉枯萎病菌的要快；经激发子诱导处理后瓜苗对枯萎病菌的抗性有了明显的提高，但来源于西瓜枯萎病菌的激发予的诱抗效果相对较好。     

10份香蕉种质对枯萎病的抗性评价(简报)

采用苗期、田间抗性评价方法,对引进的10份香蕉种质(台蕉1号、台蕉3号、台蕉4号、台蕉7号、GCTCV-106、GCTCV-119、GCTCV-247、FHIA-03、FHIA-18、FHIA-25)进行枯萎病4号小种的抗性评价.结果表明:10份香蕉种质中,GCTCV-119、FHIA-18、FHIA-25抗性为高抗;台蕉1号、台蕉4号、GCTCV-247、FHIA-03抗性为抗:台蕉3号、台蕉7号、GCTCV-106抗性为中抗.     

培养基营养成分对香蕉枯萎病尖孢镰刀菌生长的影响

通过对香蕉枯萎病发病株病组织的分离、纯化,得到香蕉枯萎病病原菌--尖镰孢菌古巴专化型[Fusarium oxysporum f.sp.cubense(E.F.Smith)Snyder et Hansen].该病原菌在不同pH值培养基上培养4 d后,发现尖孢镰刀菌菌落在pH值4.0～9.0范围内均可生长,pH值为6.0～7.0时最适,菌落直径平均在3.7～3.9 cm之间.尖孢镰刀菌在不例营养成分的培养基上培养,结果表明,N源对尖孢镰刀菌菌丝的生长影响大于C源.生长到第6天时,全糖型培养基产孢量最大,且小型分生孢子的产孢量也最大,可使香蕉苗发病率达到100%.而大型分生孢子的产生最佳培养基为低氮型培养基.     

Methyl bromide alternatives for weed and soilborne disease management in tomato (Lycopersicon esculentum)

Two field trials were conducted to examine the efficacy of methyl bromide (MBr) alternatives in the control of Cyperus rotundus L. (purple nutsedge) and Fusarium oxysporum Schlecht. f.sp. lycopersici (Sacc.) Snyder & Hansen (Fusarium wilt) in fresh market tomato (Lycopersicon esculentum Mill.). Various treatments compared combinations of soil fumigants and herbicides based on methyl bromide, chloropicrin, 1,3-dichloropropene, metam sodium, dazomet, and pebulate. The data show that consistently 1,3-dichloropropene plus chloropicrin at both doses in combination with the herbicide pebulate can be considered alternatives to methyl bromide in fresh market tomato. These treatments had excellent purple nutsedge and Fusarium wilt control, and marketable tomato yields were similar to that for methyl bromide plus chloropicrin.     

中草药提取物对香蕉镰刀菌枯萎病病原菌的抑制作用

本研究测试了 123 种常见中草药提取物对香蕉镰刀菌枯萎病病原菌的抑菌作用。结果表明,供试提取物 100 倍稀释液对菌株 B1 的菌丝生长和孢子萌发的抑制作用在 50 %以上的分别有 15 种和 14 种;对菌株B2 的菌丝生长和孢子萌发抑制作用在 50 %以上的提取物分别有 7 种和 9 种。进一步测试发现,经过 200 倍 稀释,只有丁香提取物和厚朴提取物对菌株 B1 的菌丝生长和孢子萌发抑制作用均达到 50 %以上。丁香提取物对菌株 B1 和菌株 B2 菌丝生长的抑制中浓度分别为 3 396.31 μg/mL 和 3 375.87 μg/mL。     

抑制尖孢镰刀菌人工肽适体的筛选和鉴定

香蕉枯萎病严重危害香蕉产业的发展。本研究从肽适体文库中筛选获得对香蕉枯萎病致病菌尖孢镰刀菌有抑制作用的人工肽适体SNP-D4。通过构建人工肽适体体外重组表达载体,获得SNP-D4体外重组蛋白。结果表明,通过抑菌活性检测表达人工肽适体SNP-D4的大肠杆菌全蛋白提取液,能够特异性抑制尖孢镰刀菌孢子的萌发,使其萌发率降低至19.60%。本研究筛选得到的人工肽适体SNP-D4可应用于研发环境友好型特异抗真菌生物制剂,为香蕉枯萎病防治提供新的技术手段。     

Control of Fusarium wilt of carnation using organic amendments combined with soil solarization,and report of associated Fusarium species in southern Spain

Fusarium wilt is a disease that restricts carnation (Dianthus caryophyllus L.) yield worldwide. Efficacies in reducing the Fusarium wilt of carnation (FWC), of various types of organic amendments (fresh or pelletized poultry manure, pelletized Brassica carinata and olive residue compost) combined with soil solarization, were compared in two biennial field trials conducted in a greenhouse with a history of carnation monoculture over 8 years. Soil treatments combining organic amendments and soil solarization significantly reduced disease incidence (86–99%) and increased the number of commercial carnation stems by 5–9 times compared to non-treated plots. Twenty-one Fusarium spp. isolates, with different colony morphologies were recovered from soil samples taken in the greenhouse, before the application of treatments in June 2013. Nineteen of them were morphological and molecularly characterized. Additionally, two pathogenicity tests with 17 isolates recovered from greenhouse soils and two isolates recovered from organic amendments were performed. Fusarium species associated with carnation cultivation were identified as Fusarium oxysporum (43%), Fusarium proliferatum (24%), and Fusarium solani (33%). The phylogenetic analysis of the translation elongation factor 1 alpha (EF-1α) region distinguished highly aggressive isolates of F. oxysporum f. sp. dianthi, from low aggressive isolates. The pathogenicity tests showed that FWC has a complex etiology, with several Fusarium spp. identified as causal agents. F. proliferatum and F. solani are associated with carnation wilt for the first time in Spain.     

Chemical management of Fusarium wilt of watermelon

Watermelon yield loss due to Fusarium wilt is increasing in the U.S., due in part to the emergence of the virulent race 2 of Fusarium oxysporum f. sp. niveum, and to the shift in production to triploid cultivars, which generally have less host resistance than previously grown diploid cultivars. One potential management strategy is the use of soil-applied fungicides to reduce Fusarium wilt. The U.S. national program, interregional project 4 (IR-4) supported multistate trials of soil-applied chemicals to manage Fusarium wilt of watermelon. Greenhouse trials were conducted in Maryland, Indiana and Georgia to test the efficacy of 14 chemicals on Fusarium wilt. Based on the performance of these chemicals in the greenhouse, six in Maryland and Delaware and eight in Indiana were selected for subsequent field evaluations. These chemicals were applied once, as a drench at planting, in field trials in Maryland, Indiana, and Delaware in 2008. The fungicides prothioconazole, acibenzolar-S-methyl, and thiophanate-methyl resulted in the greatest reduction in Fusarium wilt, and caused no phytotoxicity. In Maryland and Indiana in 2009, these chemicals were applied through the drip irrigation line alone and in combination, at 0, 2 and 4 weeks after planting. The experiment was repeated in 2010 in Maryland. Prothioconazole alone and in combination with acibenzolar-S-methyl or/and thiophanate-methyl resulted in the greatest decrease in the area under the disease progress curve (AUDPC) of Fusarium wilt of watermelon in Maryland in 2009. The same trend was observed in 2010 in Maryland where three of the prothioconazole treatments ranked the lowest of all treatments and prothioconazole in combination with thiophanate-methyl had significantly lower Fusarium wilt AUDPC compared to the non-treated control. All chemical applications except for acibenzolar-S-methyl in combination with prothioconazole reduced Fusarium wilt AUDPC in Indiana in 2009. Prothioconazole alone and prothioconazole in combination with thiophanate-methyl ranked lowest in Fusarium wilt AUDPC, although not significantly lower than most other treatments. These studies are the first to demonstrate that the soil-applied fungicides prothioconazole and thiophanate-methyl may provide an additional field management option for Fusarium wilt of watermelon.     

不同香蕉品种根系分泌物对香蕉枯萎病致病菌的影响及组成分析

从根系分泌物角度阐释不同香蕉品种对香蕉枯萎病的抗(耐)性机理,通过离位溶液培养法收集对香蕉枯萎病抗(耐)性不同的3个香蕉品种‘GCTCV-119’、‘桂蕉9号’和‘桂蕉1号’的根系分泌物,测定其根系分泌物对香蕉枯萎病致病菌的菌落生长和孢子萌发的影响,并分析其根系分泌物中游离氨基酸、可溶性总糖、有机酸的含量。结果表明,抗(耐)病品种根系分泌物抑制香蕉枯萎病致病菌的菌落生长和孢子萌发,而感病品种根系分泌物则促进香蕉枯萎病致病菌的菌落生长和孢子萌发。抗(耐)病品种根系分泌物中,可溶性总糖及游离氨基酸含量明显低于感病品种,有机酸含量则比感病品种高。3个香蕉品种中,共检测到13种氨基酸。其中,甘氨酸、谷氨酸、胱氨酸、丙氨酸、丝氨酸、酪氨酸仅在感病品种‘桂蕉1号’中检测到;苯丙氨酸和脯氨酸只在抗(耐)病品种‘GCTCV-119’和‘桂蕉9号’中检测到。