Genetic variation and population structure of Fusarium oxysporum f.sp. vasinfectum in Australia

Genetic variation among 348 isolates of Fusarium oxysporum f.sp. vasinfectum (Fov) collected from diseased cotton plants in 31 fields in six cotton-growing regions in New South Wales and Queensland in 2002 and 2004 was analysed using amplified fragment length polymorphisms (AFLPs). Twenty-eight haplotypes were identified based on 146 polymorphic bands generated with four Eco RI and Mse I and four Hind III and Mse I primer combinations. The haplotypes separated into two distinct groups (37% similarity), with 21 in group I and seven in group II. The two unique vegetative compatibility groups of Fov known to occur in Australia (VCG 01111 and VCG 01112) were correlated to the two AFLP groups, with both VCG 01111 reference isolates being included in group I and both VCG 01112 reference isolates in group II. Group I was widespread, occurring in all regions sampled and all but one of the fields, while group II was limited to three fields in the Boggabilla region. Group I was further divided into two subgroups. The two haplotypes in subgroup I-B (I-20 and I-21) may represent the emergence of a new form of Fov based on their marked genetic discrimination from the subgroup I-A haplotypes. No spatial population differentiation was discernible at the national level, as only 3·9% of total genetic variation was attributed to differences among regions ( P =  0·4868). When each region was analysed separately, clear differentiation was found in the Boggabilla region, with 86·3% of total genetic variation resulting from differences among fields ( P <  0·0001).     

香蕉枯萎病菌生理分化研究摘要本研究

本研究对香蕉枯萎病菌菌株FOCAAA9(来自香蕉)和FOCABB1(来自粉蕉)进行培养试验和接种试验;在含粉蕉和香蕉组织浸提液的培养基上2个菌株的培养性状、菌丝生长速度、孢子形态、大小型孢子比率和产孢量显示出差异;接种结果FOCAAA9能侵染香蕉(MusaAAA)品种巴西蕉、红香蕉和台蕉引起枯萎病,而FOCABB1对3个香蕉品种无致病性。研究结果表明侵染香蕉和粉蕉的古巴尖镰孢[Fusariumoxysporumf.sp.cubense(E.F.Smith)Snyder]存在生理分化现象。     

香蕉枯萎病菌生理分化研究

本研究对香蕉枯萎病菌菌株FOCAAA9（来自香蕉）和FOCABB1（来自粉蕉）进行培养试验和接种试验；在含粉蕉和香蕉组织漫提液的培养基上2个菌株的培养性状、菌丝生长速度、孢子形态、大小型孢子比率和产孢量显示出差异；接种结果FOCAAA9能侵染香蕉（Musa AAA）品种巴西蕉、红香蕉和台蕉引起枯萎病，而FOCABB1对3个香蕉品种无致病性。研究结果表明侵染香蕉和粉蕉的古巴尖镰孢[Fusarium oxysporum f．sp．cubeilse（E．F．Smith）Snyder]存在生理分化现象。     

Nonspecific toxins as components of a host‐specific culture filtrate from Fusarium oxysporum f. sp. cubense race 1

Bananas and plantains (Musa spp.) are among the most important crops in the world providing staple food for hundreds of millions of people. However, banana production has been devastated by fungal infestations caused by Fusarium oxysporum f. sp. cubense (Foc). Despite the fact that there is very little known on the role of microbial metabolites in the molecular mechanism of Foc infections, it has been proposed that the toxins fusaric acid and beauvericin produced by Foc play an important role during pathogenesis. The aim of this contribution was to study the toxic components of culture filtrates (CF) of Foc and to isolate the extracellular microbial metabolites involved in the plant response. An in vitro bioassay was used to evaluate the production of phytotoxic metabolites as well as the specificity of culture from a strain of Foc belonging to VCG 01210 (race 1). A host‐specific CF was obtained and the phytotoxic compounds characterized as fusaric acid, beauvericin and fumonisin B1. Despite the presence of these nonspecific toxins, a water‐soluble extract from the CF induced protection to the main phytotoxic fraction, measured by lesion area. This hydrophilic fraction induced a fast and strong response of just jasmonic acid (JA)‐dependent defence genes rather than salicylic acid (SA)‐ and ethylene (ET)‐response genes in resistant cultivars. Extracellular proteins isolated from CF of Foc provide an important source for further investigations on the molecular basis of the interaction between Foc and banana.     

香蕉枯萎病菌生理小种鉴定及其SCAR标记

 通过室内人工接种蕉类鉴别寄主,对采集于广东蕉区的18个蕉类枯萎病菌菌株进行鉴定,KP021、KP022、GZ981和JL021 4个菌株属Racel,其余14个菌株属Race4,说明广东蕉区同时存在尖孢镰刀菌古巴专化型Race1和Race4。用RAPD技术对上述18个菌株进行分析,从200条随机引物中筛选出8条引物可产生生理小种RAPD标记12个,其中标记Racel的8个,标记Race4的4个。对这些RAPD标记带分别进行回收、克隆、测序,根据这些特异片段序列分别设计相应的SCAR引物,通过对18个菌株的PCR扩增检验,有4个RAPD标记成功地转化为SCAR标记,其中Race1-SCAR标记1个、Race4-SCAR标记2个、同时能鉴定出2个小种的SCAR标记1个。应用这4个SCAR标记同时对采自田间的9个病菌分离物进行检测,能够准确地鉴定出广东蕉区的尖孢镰刀菌古巴专化型Racel和Race4,这为下一步开展香蕉枯萎病菌生理小种的分子鉴定及各生理小种田间流行动态监测奠定了基础。     

香蕉枯萎病菌群体多样性研究进展

香蕉枯萎病严重威胁世界香蕉产业,而目前尚无有效防治药剂。开发快速诊断技术以加强检疫,控制其传播速度,同时加快选育抗病品种是有效控制该病的根本策略。然而,无论是研发快速诊断的分子技术还是进行抗病品种的选育,都需要深入了解病原菌的群体结构及其基因多样性背景。香蕉枯萎病菌经过一个多世纪的变异与进化,已分化出4个生理小种,23个营养亲合群和多个基因多样性类群。本文从香蕉枯萎病的起源及其病原菌培养性状、生理小种、致病性、营养亲合群及基因多样性等方面研究进展进行梳理和分析,以期为下一步的研究提供思路和启发。     

Genetic diversity in Fusarium oxysporum f.sp. dianthi and Fusarium redolens f.sp. dianthi

Pathogenic isolates were selected representing all known vegetative compatibility groups (VCGs) and races of Fusarium oxysporum sensu lato from Dianthus spp. On basis of differences in the internal transcribed spacer region of the ribosomal DNA, six VCGs were classified as F. oxysporum f.sp. dianthi and four as F. redolens f.sp. dianthi. All VCGs of F. oxysporum f.sp. dianthi were characterized by unique restriction fragment length polymorphisms (RFLPs), unique overall esterase profiles, and unique virulence spectra, supporting a clonal lineage concept. Two VCGs of F. oxysporum f.sp. dianthi nevertheless comprised more than one race, but races within the same VCG shared the same distinct overall virulence spectrum. VCGs belonging to F. redolens f.sp. dianthi also had unique RFLPs and unique virulence spectra, but had grossly identical esterase profiles. Three new races (9, 10 and 11) are described for F. oxysporum f.sp. dianthi, and four for F. redolens f.sp. dianthi. Two races previously considered lost were recovered; race 7 was identified as a member of VCG 0021 of F. oxysporum f.sp. dianthi while race 3 was identified as a distinct VCG and race of F. redolens f.sp. dianthi. A summary of races and VCGs in F. oxysporum f.sp. dianthi and F. redolens f.sp. dianthi is presented.     

Sensitivity of Fusarium oxysporum f. sp. cubense to phosphonate

Phosphonate (0.1 mM) significantly reduced growth of Fusarium oxysporum f. sp. cubense (Foc) race 4 grown at an optimal phosphate concentration of 0.3 mM in vitro. At higher phosphate concentrations, closer to physiological conditions within the plant, the sensitivity of Foc race 4 to phosphonate was greatly reduced, with 25 mM phosphonate required to reduce growth by 50% at 1 mM phosphate. Two isolates of Fusarium oxysporum f. sp. dianthi and another race of Foc, race 1, were shown to be similar to Foc race 4 in their sensitivity to phosphonate, while another species of Fusarium, F. avenaceum , was more sensitive to phosphonate in vitro.     

Genetic diversity and population structure of Lasiodiplodia theobromae from different hosts in northeastern Brazil and Mexico

Lasiodiplodia theobromae is one of the most frequent fungal pathogens associated with dieback, gummosis, leaf spot, stem-end rot and fruit rot symptoms in cashew, mango, papaya and grapevine. In this study, the variation in the genetic diversity of 117 L. theobromae isolates from northeastern Brazil (n = 100) and Mexico (n = 17), which were collected from these four crops, was analysed using microsatellite markers. The results revealed low genetic diversity among L. theobromae populations and the existence of two genetic groups. All Mexican isolates were grouped with Brazilian isolates, suggesting a low level of differentiation between these populations. Furthermore, no evident host or climate-based population differentiation was observed for L. theobromae in Brazil. The populations studied were mostly clonal, but additional studies are needed to better understand the mode of reproduction of the pathogen. The low genetic diversity of L. theobromae populations in northeastern Brazil suggests that resistant cultivars could be used as a durable management strategy to reduce the impact of the diseases caused by this pathogen.     

假单胞菌对香蕉枯萎病菌的抑制作用

从已感染香蕉枯萎病的果园中分离到1株对香蕉枯萎病菌（Fusarium oxysporum f.sp.cubense）抑制作用很好的菌种,命名为G1。采用固体和液体培养法从不同角度证实了G1对香蕉枯萎病菌生长的抑制作用。结果表明,G1的发酵液、无菌滤液、挥发性物质、非挥发性代谢产物的平均抑菌率分别为92.5%、27.4%、73.8%、57.7%,可见抑制作用与活菌体有直接关系,其中产生挥发性物质尤其重要。液体培养的病原菌浓度为1.0×107cfu/mL经G1作用10d后,取样镜检发现G1通过抑制病原菌菌丝正常生长以至不能产孢,从而导致菌丝消融;通过吸附在孢子的周围,融解细胞壁,造成原生质泄露致使孢子死亡。     

根癌农杆菌介导的香蕉枯萎病菌4号生理小种的转化

 本文针对香蕉枯萎病菌4号生理小种这一对我国香蕉生产构成了极大威胁的检疫性有害生物,建立了根癌农杆菌介导的该生理小种的转化体系,确定了影响转化效率主要因子的最佳条件分别是:共培养乙酰丁香酮(AS)浓度为200μmol/L、共培养时间为48 h、培养温度为25℃、诱导培养基pH值为5.5。此条件下,转化效率达到21~24个转化子/10⁴香蕉枯萎病菌孢子。PCR和Southern杂交分析表明外源的T-DNA已经成功随机地整合到该病原菌基因组中,且多为单拷贝。应用该转化体系已获得近25 000个转化子,为研究该生理小种致病机制奠定了基础。     

Resistance sources to Fusarium oxysporum f. sp. cubense tropical race 4 in banana wild relatives

Target trait evaluation in crop wild relatives is an important prerequisite for efficiently using the potential useful genes located in this valuable germplasm. Over recent decades, Fusarium oxysporum f. sp. cubense tropical race 4 (Foc‐TR4) has seriously threatened worldwide banana plantations. Breeding new resistant cultivars from wild banana species is expected to provide invaluable additional resources. However, knowledge on resistance to Foc‐TR4 in wild Musa species is very limited. In this study, eight genotypes of wild banana relatives (Musa acuminata subsp. burmannica, M. balbisiana, M. basjoo, M. itinerans, M. nagensium, M. ruiliensis, M. velutina and M. yunnanensis) were characterized for resistance to Foc‐TR4 in both greenhouse and field conditions. Most wild bananas showed higher resistance levels to Foc‐TR4 than the reference cultivars ‘Brazilian’ (AAA, susceptible) and ‘Goldfinger’ (AAAB, moderate resistance). Among the wild species, M. balbisiana showed the highest levels of disease intensity followed by M. acuminata subsp. burmannica. Some individuals of M. yunnanensis, M. nagensium, M. ruiliensis and M. velutina showed low levels of rhizome discolouration in greenhouse conditions, but were resistant in the field. No symptoms were observed on M. basjoo and M. itinerans, suggesting higher levels of resistance to Foc‐TR4. The results revealed different sources of resistance to Foc‐TR4 in banana wild relatives, which constitute a valuable genetic resource for banana breeding programmes aiming to produce cultivars resistant to fusarium wilt.     

香蕉枯萎病菌侵染香蕉根系的组织学过程

 为探明香蕉枯萎病菌侵染香蕉根系的过程,利用绿色荧光蛋白标记的香蕉枯萎病菌4号生理小种（Fusarium oxysporum f. sp. cubense race 4 tagged with green fluorescent protein,GFP-FOC4）,接种香蕉根系以观察病原菌侵染香蕉根系的组织学过程。结果表明,接种1 d后病原菌以菌丝体、大型分生孢子和小型分生孢子的形式附着于根系表皮细胞,优先沿细胞胞间层生长。接种7 d后,观察到病原菌以菌丝体、大型分生孢子和小型分生孢子的形式直接侵染维管束,在维管束内以两种方式扩展繁殖,一种在维管束内横向扩展,菌丝体随机分支,逐步形成网状分布;另一种是菌丝体在维管束内纵向生长,倾向于呈束状沿维管束单侧生长繁殖,形成大量菌丝体。本研究首次从组织病理学的角度观察并分析了GFP-FOC4侵染香蕉根系的过程,为研究香蕉枯萎病菌的致病过程机理提供参考。     

香蕉枯萎病颉颃放线菌的分离和筛选

从采自内蒙古、海南的土样中经分离、纯化得到64株优势放线菌,采用平板对峙培养法对香蕉枯萎病菌(Fusarium oxysporum f.sp.cubense)进行颉颃放线菌的筛选.通过皿内颉颃试验,获得4株抑菌效果较好的颉颃放线菌,以HN3菌株的抑菌效果最为明显,发酵液抑茵试验表明,其抑菌物质存在于菌丝内.     

Genetic Variation Among Vegetative Compatibility Groups of Fusarium oxysporum f. sp. cubense Analyzed by DNA Fingerprinting

ABSTRACT Genetic variation within a worldwide collection of 208 isolates of Fu-sarium oxysporum f. sp. cubense, representing physiological races 1, 2, 3, and 4 and the 20 reported vegetative compatibility groups (VCGs), was analyzed using modified DNA amplification fingerprinting. Also characterized were 133 isolates that did not belong to any of the reported VCGs of F. oxysporum f. sp. cubense including race 3 isolates from a Heliconia species and isolates from a symptomatic wild banana species growing in the jungle in peninsular Malaysia. The DNA fingerprint patterns were generally VCG specific, irrespective of geographic or host origin. A total of 33 different genotypes were identified within F. oxysporum f. sp. cu-bense; 19 genotypes were distinguished among the isolates that belonged to the 20 reported VCGs, and 14 new genotypes were identified among the isolates that did not belong to any of the existing VCGs. DNA fingerprinting analysis also allowed differentiation of nine clonal lineages within F. oxysporum f. sp. cubense. Five of these lineages each contained numerous closely related VCGs and genotypes, and the remaining four lineages each contained a single genotype. The genetic diversity and geographic distribution of several of these lineages of F. oxysporum f. sp. cubense suggests that they have coevolved with edible bananas and their wild diploid progenitors in Asia. DNA fingerprinting analysis of isolates from the wild pathosystem provides further evidence for the coevolution hypothesis. The genetic isolation and limited geographic distribution of four of the lineages of F. oxysporum f. sp. cubense suggests that the pathogen has also arisen independently, both within and outside of the center of origin of the host.     

Genetic diversity in Fusarium oxysporum f. sp. melonis*

Fusarium oxysporum f. sp. melonis is an important vascular wilt pathogen of melon. Races 1, 2 and 1–2 of this fungus have been identified in Portugal by pathogenicity tests with appropriate hosts. The aim of this research was to examine the relationships between different races of F. o. melonis of Portuguese and French origin through analysis of random amplified polymorphic DNAs (RAPDs). DNA fingerprint profiles were developed for all the accessions. Each isolate showed 5–10 DNA bands with each of the 16 primers employed. A total of 126 bands was obtained. The size of amplified DNA fragments generated with these primers ranged from 0.5 to 3.2 kb. A phenogram based on the Jaccard coefficient of similarity was computed by the unweighed pair group method using arithmetic averages (UPGMA). It was found that Portuguese race 2 is very similar to French race 1, while French race 2 is the most dissimilar being clearly separated from all other races. The genetic diversity of these isolates is also being studied for vegetative compatibility by using the nit mutant system.     

西瓜枯萎病菌遗传多样性的相关序列扩增多态性分析

对30个西瓜枯萎病菌Fusarium oxysporum f.sp.niveum菌株基因组DNA进行相关序列扩增多态性(SRAP)分子标记分析,以探究其遗传多样性与地理来源的关系。采用尖孢镰刀菌西瓜专化型Fusarium oxysporumf.sp.niveum0、1、2号生理小种的基因组DNA为模板,对225对SRAP引物进行筛选,筛选出20对多态性、重复性较好且条带清晰的引物,对30个菌株进行PCR扩增,共扩增出386条带,其中多态性条带有371条,多态性比率为96.11%,平均每对引物扩增出19.3个位点和18.55个多态性位点。UPGMA法聚类分析结果显示,供试菌株两两之间的遗传相似系数范围为0.69~0.90,平均为0.79,说明尖孢镰刀菌西瓜专化型的遗传多样性较为丰富。基于SRAP标记聚类分析表明,30个菌株在遗传相似系数为0.70处被划分为3个类群,I类群包含24个菌株,其中18个来自湖南省,Ⅱ类群只包含1个来自黑龙江省哈尔滨市的菌株,它和另一个来自黑龙江地区的菌株被划分到不同的类群,且遗传距离相对较远;Ⅲ类群包含了5个菌株,其中3个来自海南三亚,其余两个来自湖南省。根据菌株的分布情况来看,菌株的聚集与地理来源没有明显的相关性。     

香蕉枯萎病生防菌对杀虫剂的适应性研究

将4株香蕉枯萎病生防细菌bio ZK11、bio HN2、bio HN10和bio F4分别与杀虫剂噻唑磷、毒死蜱、辛硫磷混合,测定其混合液对香蕉枯萎病病菌孢子萌发的抑制作用以及杀虫剂对生防菌生长繁殖的影响。结果显示,噻唑磷对生防菌bio ZK11的抑菌作用没有影响,而毒死蜱和辛硫磷增强bio ZK11的抑菌作用;噻唑磷和毒死蜱增强bio HN2的抑菌作用,辛硫磷则对bio HN2的抑菌作用没有影响;噻唑磷、毒死蜱和辛硫磷增强了生防菌bio HN10的抑菌作用;噻唑磷、毒死蜱和辛硫磷对生防菌bio F4的抑菌作用没有影响。辛硫磷对生防菌bio HN2的菌落生长有一定的影响,对另外3种生防菌没有影响;噻唑磷对4种生防菌菌落大小都没有影响;毒死蜱对香蕉枯萎病生防菌bio HN10的菌落大小没有影响,但使生防菌bio ZK11、bio HN2和bio F4的菌落变小。3种杀虫剂除了对生防菌bio F4的生长量没有显著影响,对其他3种拮抗菌的生长都有一定的影响。     

香蕉枯萎病菌SIX2基因序列及特异性分析

依据侵染的香蕉品种范围不同,引起香蕉枯萎病的尖孢镰刀菌古巴专化型(Fusarium oxysporum f.sp.cubense,Foc)分为4个生理小种。Foc在寄主植物木质部分泌的SIX(secreted in xylem)蛋白可能与不同生理小种侵染的香蕉品种范围不同存在密切关系,找到Foc 4号生理小种(Foc4)特有的SIX蛋白编码基因将有利于进一步分析Foc4寄主范围更广的原因,从而开展抗病育种工作。采用PCR方法比较分析了国内不同地理区域及来源于澳大利亚与南非的Foc1、Foc2、Foc3、Foc4共56株菌株中的SIX2基因,并以8个以上其他专化型或其他种或属的热带作物病原菌共39株菌株分析了Foc4 SIX2基因序列的特异性,分析了SIX2基因的灵敏度及利用其检测感病植株。仅从供试的Foc4菌株基因组DNA中扩增出SIX2基因序列,检测的DNA灵敏度达5pg/25μL,并可用于检测感病的球茎组织。Foc4中特有的SIX2基因序列为特异性鉴定感病植株病原菌种类提供了快速分子检测技术,为明确该基因是否决定性影响Foc4对寄主差异性的选择研究提供了基础。     

香蕉枯萎病菌RAPD分析及4号生理小种的快速检测

 用随机扩增多态性DNA(RAPD)技术,对采自广东、广西的香蕉和粉蕉上的30个香蕉枯萎病菌(Fusarium oxysporum f.sp.cubense)菌株和3个其它尖孢镰刀菌专化型的菌株进行比较及聚类分析。在遗传相似系数0.67时,可将供试菌株划分为3个RAPD群(RGs),其中香蕉枯萎病菌4号生理小种(FOC4)共15个菌株属于RGⅠ,1号生理小种(FOC1)共15个菌株属于RGⅡ,供试的其它尖孢镰刀菌专化型的3个菌株则属于RGⅢ。这说明香蕉枯萎病菌和供试3个其它专化型菌株与致病性间存在明显的相关性。1号生理小种内菌株间的遗传分化大于4号生理小种内菌株间的遗传分化。从90条RAPD随机引物中筛选出2条引物可产生4号生理小种的RAPD标记2个。将这2个RAPD标记电泳切胶回收、克隆及测序,并根据这2个特异片段序列设计SCAR上下游特异引物,通过对30个菌株的PCR扩增检验,其中一个RAPD标记成功地转化为SCAR标记,初步建立了以此为基础的4号生理小种快速检测技术,其检测灵敏度为2 ng新鲜菌丝。对采自不同地区的显症样品、吸芽、室内接种未显症的香蕉苗以及发病的香蕉植株不同部位进行检测,能够准确灵敏地鉴定出4号生理小种,从而为香蕉枯萎病菌的快速检测及防治奠定了基础。同时,快速检测结果发现,田间发病植株果柄的各部位及果实内并没有枯萎病菌的存在。