香蕉上的镰孢菌种类及其系统发育关系(英文)

镰孢菌属真菌是香蕉上的重要病原菌,主要引起香蕉枯萎病以及香蕉冠腐病,在我国已明确引起香蕉枯萎病的病原为尖孢镰孢古巴专化型 Fusarium oxysporum f. sp. cubense(FOC)1号和4号生理小种,但是引起香蕉冠腐病的镰孢菌种类还未明确。为了解香蕉上镰孢菌在种间及种内水平上的多样性,2008–2011 年间作者从华南地区不同的水果市场及香蕉果园采集香蕉样品90份,分离得到143株镰孢菌。通过形态学观察及基于 EF-1α基因的系统进化分析鉴定出10种镰孢菌,即F. oxysporum、F. solani、F. camptoceras、F. pallidoroseum、F. stiloides、F. chlamydosporum、F.verticillioides、F. proliferatum、F. concentricum、F. sacchari,以及藤仓赤霉复合种(Gibberella fujikuroi species complex,GFC)中 3 个未定名的类群。轮纹镰孢 F. concentricum 及甘蔗镰孢 F.sacchari 是香蕉果实中最常见种,前菌为我国首次报道,后菌是首次报道与香蕉有关。对从香蕉上分离的藤仓赤霉复合种(GFC)及尖孢镰孢复合种(FOSC)的EF-1α序列进行了系统发育分析,其GFC中的27个菌株组成的单系群可分为7个不同的亚群,分别为 F.verticillioides、F. proliferatum、F. concentricum、F. sacchari 以及3个没有描述过的菌系 Fusarium sp. 1、Fusarium sp.2和 Fusarium sp.3;FOSC中的50个菌株形成2大分枝共12个谱系,分离自我国华南地区的21株尖孢镰孢形成7个谱系,其中 13株已知的香蕉枯萎病病原菌分布在3个谱系中,我国大陆的香蕉枯萎病病原菌菌株与来源于台湾地区及东南亚的菌株亲缘关系较近,FOC1号生理小种的遗传分化大于4号生理小种,FOC 1号生理小种与分离自香蕉果实上的尖孢镰孢菌的亲缘关系比与FOC 4号生理小种的亲缘关系更近。研究结果表明,我国香蕉上存在着丰富的镰孢菌种类,而且种内遗传多样性丰富。     

海南省香蕉枯萎菌生理小种的RAPD分析

利用随机扩增多态性DNA(RAPD)分子标记方法对海南省香蕉枯萎病菌2个生理小种(小种1和小种4)进行遗传多样性分析,以筛选出的15个随机引物对采自海南省各市县发病蕉区的分别属于1号生理小种和4号生理小种的16个代表菌株及广东省2个1号和4号生理小种对照菌株进行RAPD-PCR扩增,结果产生97个RAPD分子标记,其中多态性的条带有76条,通过聚类分析探讨了供试小种间的亲缘关系,并寻找到了1、4号生理小种的特异性条带,为在分子水平上进行香蕉枯萎病菌生理小种鉴定提供更为便利的手段。     

pH值对香蕉枯萎病菌4号生理小种生长的影响

【背景】香蕉枯萎病菌4号生理小种(镰刀菌)是香蕉产业的致命威胁。已有研究表明土壤pH值越高,香蕉枯萎病发病率越低,但是现有pH值对镰刀菌影响的研究大都是用强酸强碱调节pH值,pH值没有缓冲体系保护,而且尚未检测试验终点时介质的pH值。此外,关于pH值对香蕉枯萎病菌4号生理小种(Foc4)影响的研究尚不系统,难以用于指导生产实践。【目的】为系统地了解土壤酸碱度对Foc4生长的影响。【方法】在pH 3.0-11.0之间设定9个pH值梯度,模拟酸性到碱性土壤pH值条件,于室内培养条件下系统研究pH值对Foc4生长、产孢、孢子萌发的影响及其生长过程对环境pH值的影响。【结果】弱酸性至中性环境(pH 5.0-7.0)最适宜于香蕉枯萎病菌的生长、产孢和孢子萌发。弱碱性处理(pH8.0和pH9.0)孢子平均萌发率较弱酸性环境处理(pH5.0和pH6.0)下降了73.1%。与pH 6.0酸性处理相比,pH 8.0和pH 9.0处理的产孢量分别下降了52.3%和68.1%。【结论】香蕉枯萎病菌Foc4生长和萌发过程会产酸,但是在缓冲体系液体培养基中,除了pH 9.0和pH10.0处理终点培养液pH值分别下降了0.34和0.27个单位外,其它处理起始和终点的pH值无差异。说明在缓冲体系液体培养基中的研究结果可以反映环境pH值对Foc4生长和萌发的影响。在作物可以生长的pH值范围内(pH5.0-9.0),碱性和微碱性条件(pH8.0-9.0)能明显抑制Foc4生长、产孢和孢子萌发。     

氨基酸影响尖孢镰孢菌古巴专化型厚垣孢子的形成

香蕉枯萎病是由尖孢镰孢菌古巴专化型Fusarium oxysporum f. sp. cubense（Foc）侵染引起的一种土传真菌病害,已严重威胁香蕉产业的健康发展。该病菌产生的厚垣孢子可在土壤中存活多年,是香蕉枯萎病的初侵染源。本研究通过氨基酸添加试验,证明添加甘氨酸可抑制厚垣孢子的形成;通过对该病菌厚垣孢子形成前期、初期、中期和后期的转录组分析,发现氨基酸合成通路中有93个基因的表达水平在厚垣孢子形成过程中发生了显著变化;In silico 分析表明其中10个基因参与调控真菌的氨基酸合成,11个基因参与调控真菌种的生长发育和产孢,19个基因参与调控真菌种的致病性和毒素产生。由此推测,氨基酸合成通路不仅与尖孢镰孢菌古巴专化型厚垣孢子的形成相关,其有可能参与调控该病菌的致病性。     

几种尖孢镰刀菌专化型中SIX1、SIX4、SIX6、SIX8同源基因分析

尖孢镰刀菌在与寄主的相互作用中分泌几个特定的富含半胱氨酸的小分子量蛋白进入木质部中启动致病力,被称为SIX(secreted in xylem)蛋白,为明确其在不同寄主中的作用,本研究比较分析了几种尖孢镰刀菌专化型中SIX1、SIX4、SIX6、SIX8同源基因序列。根据已完成的尖孢镰刀菌古巴专化型1号(Foc1)与4号生理小种(Foc4)全基因组测序序列信息及相关SIX基因序列设计引物,应用PCR方法扩增分析56株尖孢镰刀菌古巴专化型与18株其它专化型及非致病型尖孢镰刀菌菌与其它种或属共21株菌株中的SIX1、SIX4、SIX6、SIX8基因。结果表明:设计的SIX1、SIX4、SIX6、SIX8基因引物均不能从非致病性尖孢镰刀菌与其它镰刀属种或其它属的菌株DNA中扩增出目的条带;SIX1基因的2个引物均能从供试的Foc菌株DNA中扩增出目的条带,同时可从部分其它专化型菌株DNA中扩增出目的条带;SIX4基因引物仅能从供试的尖孢镰刀菌番茄专化型与部分甘蓝专化型菌株DNA中扩增出目的条带;SIX6引物仅能从供试Foc1、Foc2、Foc4菌株DNA中扩增出目的条带;SIX8基因引物能从所有供试的致病尖孢镰刀菌中DNA扩增出目的条带。研究发现的SIX6基因序列提供了快速鉴定尖孢镰刀菌古巴专化型的检测方法,同时为深入研究尖孢镰刀菌各个专化型中SIX基因的功能奠定基础。     

尖镰孢古巴专化型Focr4-1701突变体的生物学表型研究

由尖镰孢古巴专化型(Fusarium oxysporum f.sp.cubense,Foc)引起的香蕉枯萎病是香蕉生产中的毁灭性病害之一。Foc有4个小种,其中4号小种(Focr4)因其寄主范围广且无高抗病性的香蕉品种,对香蕉产业的危害最大,其致病机制至今尚不清楚。为研究其致病机制,本文对Focr4野生型菌株(Focr4-193-6)、因其T-DNA插入导致致病性严重减弱的突变体Focr4-1701、T-DNA插入标签基因敲除子△Focr4-1701及敲除基因互补子△Focr4-1701-cp-1为材料,从致病性测定、玻璃纸穿透试验、孢子形态观察、产孢量与孢子萌发率测定、粗毒素测定等方面对其与致病性相关的生物学表型进行了测定。结果表明:T-DNA插入突变体Focr4-1701和基因敲除突变体△Focr4-1701接种后的香蕉植株叶片没有表现发病症状,敲除基因互补菌株与野生型菌株一样表现出发病症状;T-DNA插入突变体和基因敲除突变体不能穿透玻璃纸生长,T-DNA插入突变体及基因敲除突变体在产孢量、孢子萌发率和产毒素能力上均极显著低于野生型菌株及互补菌株。这些表型性状差异说明Focr4-1701致病性严重减弱可能是T-DNA插入位点标签基因失活后导致了菌丝体侵染能力下降及产毒素能力降低造成的。     

解淀粉芽孢杆菌SC1150 的抑菌活性及其液体发酵条件的优化

通过比较解淀粉芽孢杆菌SC1150 菌株发酵液对香蕉枯萎病菌4 号生理小种的抑菌活性, 对解淀粉芽孢杆菌SC1150 菌株液体发酵条件进行了优化研究。结果显示: 在固体培养条件下解淀粉芽孢杆菌SC1150 菌株对测试的8 种作物病原菌均有抑制作用, 其中对香蕉枯萎病菌4 号生理小种的抑制作用特别强烈, 其抑菌圈直径达到23.31 mm。以香蕉枯萎病菌4 号生理小种为指示菌, 对该活性SC1150 菌株的液体发酵条件进行优化, 液体发酵培养基各组分的最佳配比为0.5%可溶性淀粉(碳源)、1.5%蛋白胨+酵母粉(1︰1)的混合氮源、0.1%NaCl; 最佳培养条件: pH 6.5、30 ℃、摇床转速确180 rmin–1。优化后SC1150 菌株对香蕉枯萎病菌4 号生理小种的抑菌圈直径达到 28.33 mm。     

海南省香蕉枯萎病病原菌的鉴定

香蕉枯萎病在海南省为首次报道。在Komada改良培养基鉴定的基础上,用温室人工接种法对采自海南省各市县香蕉种植区的18个香蕉和粉蕉假茎分离物进行鉴定。结果表明香蕉枯萎病菌的两种分离物在培养特性和致病性上存在明显区别,分离自粉蕉的12个菌株为1号生理小种,而分离自香蕉的6个菌株为4号生理小种。     

淡紫紫孢菌PLF-1对感染尖刀镰孢菌百合种球的促生作用及其相关防御酶活性变化

为了探究淡紫紫孢菌(Purpureocillium lilacinum)PLF-1对百合种球的促生作用及对百合尖刀镰孢菌的防治效果,采用平板对峙法评估淡紫紫孢菌对尖孢镰刀菌的拮抗效果,以及淡紫紫孢菌对百合抗尖孢镰刀菌的抗性作用。同时监测百合种球中超氧化物歧化酶（SOD）、过氧化物酶（POD）和过氧化氢酶（CAT）的活性变化情况。研究结果表明：浓度为4.34×10⁴ CFU/mL和4.34×10⁵ CFU/mL的淡紫紫孢菌孢子悬浮液对百合种球表现为促进作用,浓度为4.34×10⁴ CFU/mL时最高茎长达11 cm。平板拮抗实验中该淡紫紫孢菌菌株能有效抑制尖孢镰刀菌生长,抑制率高达72%。接种淡紫紫孢菌和病原菌的百合种球茎长会增长37.6%,根长会增长33%。该菌株能提高感染尖刀镰孢菌百合种球中的超氧化物歧化酶（SOD）、过氧化物酶（POD）和过氧化氢酶（CAT）活性,有效抑制尖刀镰孢菌的毒害作用,促进植株健康生长。     

香蕉枯萎病菌粗毒素对地衣芽胞杆菌生长和培养液上清蛋白组成的影响

本文研究了香蕉枯萎病菌4号生理小种湛江菌株(Foc 4-zj)产生的粗毒素对地衣芽胞杆菌R21菌株生长及其培养液中蛋白组成变化的影响。实验结果表明, Foc 4-zj的粗毒素能够抑制R21菌株的生长, 缩短其生长周期; 减少培养液上清蛋白含量以及改变蛋白质的种类; 低剂量的粗毒素有利于拮抗蛋白的积累, 而高剂量的粗毒素则相反。     

香蕉枯萎病菌Fow1基因的克隆及序列分析

为了解Fow1基因在尖镰刀菌古巴专化型侵染香蕉过程中的作用,及其与尖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Fow1基因,并对扩增产物进行了克隆测序及相似序列搜索和比对,还对基因编码的蛋白进行了结构预测和功能分析。研究结果表明2个生理小种Fow1基因开放阅读框均为957bp,编码318个氨基酸,基因序列和氨基酸序列差异小,而且两个生理小种Fow1基因所编码的蛋白均具有酵母线粒体载体蛋白典型的结构特征,推测Fow1基因可能为香蕉枯萎病菌在香蕉组织中定殖所必需。从Fow1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Fow1基因并无明显对应关系,这为进一步研究Fow1基因功能奠定了基础。     

Genetic diversity of Fusarium oxysporum strains from common bean fields in Spain.

Fusarium wilt is an endemic disease in El Barco de Avila (Castilla y León, west-central Spain), where high-quality common bean cultivars have been cultured for the last century. We used intergenic spacer (IGS) region polymorphism of ribosomal DNA, electrophoretic karyotype patterns, and vegetative compatibility and pathogenicity analyses to assess the genetic diversity within Fusarium oxysporum isolates recovered from common bean plants growing in fields around El Barco de Avila. Ninety-six vegetative compatibility groups (VCGs) were found among 128 isolates analyzed; most of these VCGs contained only a single isolate. The strains belonging to pathogenic VCGs and the most abundant nonpathogenic VCGs were further examined for polymorphisms in the IGS region and electrophoretic karyotype patterns. Isolates belonging to the same VCG exhibited the same IGS haplotype and very similar electrophoretic karyotype patterns. These findings are consistent with the hypothesis that VCGs represent clonal lineages that rarely, if ever, reproduce sexually. The F. oxysporum f. sp. phaseoli strains recovered had the same IGS haplotype and similar electrophoretic karyotype patterns, different from those found for F. oxysporum f. sp. phaseoli from the Americas, and were assigned to three new VCGs (VCGs 0166, 0167, and 0168). Based on our results, we do not consider the strains belonging to F. oxysporum f. sp. phaseoli to be a monophyletic group within F. oxysporum, as there is no correlation between pathogenicity and VCG, IGS restriction fragment length polymorphism, or electrophoretic karyotype.     

A differential medium for the identification of races 1 and 4 of Fusarium oxysporum f.sp. cubense

Three distinctive colony types were produced when Fusarium oxysporum f.sp. cubense (Foc ) races 1 and 4 were cultured on a defined basal medium containing an appropriate carbon source and bromothymol blue as a pH indicator. These distinctive cultural characteristics have been used as a specific and reliable method for the differentiation of races 1 and 4 of Foc from other species of Fusarium.     

香蕉枯萎病菌Argonaute基因功能分析及其调控小RNA发生

尖孢镰刀菌古巴专化型Fusarium oxysporum f.sp.cubense是威胁香蕉生产的重要土传病原真菌,其中4号生理小种(Foc4)能感染几乎所有的栽培品系.Argonaute蛋白(AGO)介导的RISC复合体在RNAi干扰中起到重要作用.Foc4含有两个进化上高度保守的AGO蛋白,本研究利用同源重组技术获...     

Production of monoclonal antibodies to Fusarium oxysporum f.sp. cubense race 4

The production of monoclonal antibodies (mab) to Fusarium oxysporum f.sp. cubense (Foc ) race 4 is described. Heat-killed conidia of this fungus were toxic to female Balb/c mice, but this toxic reaction was not found with fractionated hyphal walls. A simple and reproducible enzyme immunoassay using a standard 9 cm polystyrene Petri dish as a solid phase was devised for screening culture supernatant fluids. Sixteen stable hybridoma clones secreting mabs of the IgM class were isolated by fusing splenic lymphocytes from immunized female Balb/c mice with P3-NSl-Ag4-l mouse myeloma cells. Monoclonal antibodies produced by eight of the 16 hybridoma clones were selected and the specificity of the mabs was determined by an indirect immunofluorescence test. Of the eight mabs, only one displayed an exceptionally high degree of specificity to the thick-walled chlamydospores of Foc race 4. This specific reactivity allowed differentiation of Foc race 4 from other races.     

Genetic Diversity of Fusarium oxysporum f.sp. cubense Isolates (Foc) of India by Inter Simple Sequence Repeats (ISSR) Analysis

To find out the genetic diversity of Indian Foc isolates of banana, a total of 107 isolates of Fusarium which includes 98 Foc isolates obtained from different banana growing regions of India and seven Foc isolates belong to all known VCGs obtained from Australia and two non-pathogenic Fusarium oxysporum (npFo) isolates were subjected to ISSR analysis. In the initial screening of ISSR primers, out of 34, 10 primers which generated more polymorphic bands were selected for further analysis. The Phylogenetic analysis carried out based on the fingerprints obtained through ISSR analysis indicated the presence of wide genetic diversity among the Foc isolates of India and also its polyphyletic nature. Totally, seven different clusters were obtained and these clusters differentiated the Foc isolates of India based on the races/VCGs. Besides, the cluster analysis clearly distinguished the freshly emerged Foc strain obtained from cv. Grand Naine (Cavendish-AAA) and Poovan (Mysore-AAB) from the other Foc isolates. The non-pathogenic F. oxysporum isolates which have been included for comparison purpose also clustered separately. All these above said findings indicates for the first time the discriminatory power of ISSR to clearly distinguish and separate the Foc isolates according to its race/VCGs and also its virulence. This study would be useful not only to design and develop effective management strategies but also useful for quarantine purposes.     

Development of an in vitro hydroponic system for studying the interaction between banana plantlet and Fusarium oxysporum f. sp. cubense

Plant Cell, Tissue and Organ Culture (PCTOC) - Fusarium wilt of banana, caused by Fusarium oxysporum f. sp. cubense (Foc), is one of the most destructive diseases of banana. Several experimental...