Pathogenicity Groups in Fusarium oxysporum f. sp. lactucae on Horticultural Types of Lettuce Cultivars

Isolates of Fusarium oxysporum f. sp. lactucae obtained from six localities in Japan were divided into three patho-genicity groups. Group 1 was highly pathogenic to lettuce cultivars of crisphead and red leaf types and was less pathogenic to butterhead and green leaf type cultivars. Group 2 was highly pathogenic to butterhead type and less pathogenic to crisphead and leaf types. Group 3 was less pathogenic to all lettuce types than groups 1 and 2. These results indicated pathogenic differentiation in F. oxysporum f. sp. lactucae, roughly relating to horticultural types of host lettuce cultivars. Received 21 February 2001/ Accepted in revised form 28 May 2001     

Physiological Specialization of Fusarium oxysporum f. sp. lactucae, a Causal Organism of Fusarium Root Rot of Crisp Head Lettuce in Japan

In 1995, Fusarium root rot of crisp head lettuce, caused by Fusarium oxysporum f. sp. lactucae, was simultaneously found in the Shiojiri and Kawakami areas of Nagano Prefecture, Japan. The Shiojiri and Kawakami isolates differed in pathogenicity to lettuce cultivars. Because of this distinct physiological specialization, these Shiojiri and Kawakami isolates should be designated as race 1 and race 2, respectively, using lines VP1010 (highly resistant to race 1), VP1013 (highly resistant to race 2) and variety Patriot (highly susceptible to both races) as differential varieties. This is the first report of races of Fusarium oxysporum f. sp. lactucae, Received 21 September 2000/ Accepted in revised form 21 March 2001     

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。     

Occurrence of Fusarium oxysporum f. sp. melonis Race 1 in Japan

In 1994, Fusarium wilt of melon cultivars which are resistant to races 0 and 2 of Fusarium oxysporum f. sp. melonis was observed in southern area of the Lake Biwa region, Shiga prefecture. In commercial fields, mature plants of cv. Amus which were grafted onto cv. Enken Daigi 2, and of cv. FR Amus showed yellowing, wilting and finally death before harvesting of fruits. Diseased plants had vascular and root discolorations, and their stem sections yielded typical colonies of F. oxysporum. When the Shiga strains were tested for their pathogenicity to 12 species of cucurbits, they caused wilts only on melon. Using race differential cultivars of melon, the Shiga strains were classified as race 1 of F. oxysporum f. sp. melonis, which has not been reported in Japan. To further characterize their pathogenicity, the strains were used to inoculate 46 additional cultivars of melon, oriental melon and oriental pickling melon. All the race 1 strains were pathogenic to the cultivars tested, and their host range was apparently different from those of strains belonging to other races (races 0, 2 and 1,2y). DNA fingerprinting with a repetitive DNA sequence, FOLR3, differentiated race 1 strains from strains of races 0 and 2, but not from race 1,2y strains. Received 2 July 1999/ Accepted in revised form 30 September 1999     

Physiological races and vegetative compatibility groups of butterhead lettuce isolates of Fusarium oxysporum f. sp. lactucae in Japan

Races were identified among butterhead lettuce isolates of Fusarium oxysporum f. sp. lactucae collected from three geographical areas of Hokkaido, Shizuoka, and Fukuoka in Japan by inoculation tests using Fujinagas race differential cultivars of lettuce (i.e., Patriot, Costa Rica No. 4, and Banchu Red Fire). Eighteen isolates from Shizuoka and Fukuoka were designated race 3, with two unknown vegetative compatibility groups (VCGs) that differed from Ogisos VCG 1 and 2. These two new VCGs were obtained from both Shizuoka and Fukuoka. On the other hand, three isolates from Hokkaido were classified as race 1 and identified as VCG 1, which represents a VCG of crisphead isolates from Nagano.     

Pathogenic, Genetic and Molecular Characterisation of Fusarium oxysporum f.sp. lilii

Isolates of Fusarium oxysporum from lily were screened for pathogenicity, vegetative compatibility and DNA restriction fragment length polymorphisms, and compared to reference isolates of F. oxysporum f.sp. gladioli and F. oxysporum f.sp. tulipae to justify the distinction of F. oxysporum f.sp. lilii. Twenty-four isolates from different locations in The Netherlands (18 isolates), Italy (4 isolates), Poland and the United States (1 isolate each) shared unique RFLP patterns with probes D4 and pFOM7, while hybridization did not occur with a third probe (F9). Except for a self-incompatible isolate, these 24 isolates all belonged to a single vegetative compatibility group (VCG 0190). Isolates belonging to VCG 0190 were highly pathogenic to lily, but not to gladiolus or tulip, except for a single nonpathogenic isolate. Six saprophytic isolates of F. oxysporum from lily were nonpathogenic or only slightly aggressive to lily, gladiolus and tulip, belonged to unique VCGs and had distinct RFLP patterns. Three pathogenic isolates previously considered to belong to F. oxysporum f.sp. lilii were identified as F. proliferatum var. minus; all three belonged to the same VCG and shared unique RFLP patterns. These three isolates were moderately pathogenic to lily and nonpathogenic to gladiolus and tulip. The reference isolates of F. oxysporum f.sp. tulipae were pathogenic to tulip, but not to lily and gladiolus; they shared a distinct RFLP pattern, different from those encountered among pathogenic and saprophytic isolates from lily, and formed a separate new VCG (VCG 0230). Reference isolates of F. oxysporum f.sp. gladioli belonging to VCG 0340 proved pathogenic to both gladiolus and lily, but not to tulip. These isolates, as well as isolates belonging to VCGs 0341, 0342 and 0343 of F. oxysporum f.sp. gladioli, had RFLP patterns different from those encountered among the isolates from lily or tulip. These findings identify F. oxysporum f.sp. lilii as a single clonal lineage, distinct from F. oxysporum f.sp. gladioli and f.sp. tulipae.     

黄瓜枯萎病菌毒力、营养体亲和性及ISSR分析

 本研究对来自哈尔滨、长春、沈阳、北京、西宁5个城市的70个尖孢镰刀菌黄瓜专化型菌株进行了毒力、营养体亲和性及ISSR分析。毒力测定结果显示黄瓜枯萎病菌在东农803品种上存在明显的毒力分化。在营养体亲和群的测定中有8个菌株没有产生nit突变体,2个菌株经测定为异核体自身不亲和性菌株,不能进行营养体亲和群的测定;其余60个菌株可分为5个营养体亲和群。利用筛选的7个引物对70个菌株进行了ISSR分子标记,聚类分析可将70个菌株分为3个类群,其中IGⅠ的41个菌株均来自东北三省,IGⅡ的21个菌株均来自北京,IGⅢ的8个菌株全部来自西宁。VCGs和ISSRs与菌株的地理来源及毒力存在一定的相关性。     

福建省香蕉枯萎病菌硝酸盐营养突变体的营养体亲和性测定

 Fourteen strains of Fusarium oxysporum f. sp. cubense were induced to produce 146 nitrate-nonutilizing(nit) mutants on a chlorate-containing medium. Among them, there were 117 nit1 mutants(80.14%), 17 nit3 mutants(11.64%) and 12 nitM mutants(8.22%). These strains were divided into two vegetative compatibility groups(VCGs) by the vegetative compatibility tests. Twelve strains of F. oxysporum f. sp. cubense from Musa AAA belonged to VCG1, two trains from Musa ABB belonged to VCG2.     

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。     

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。     

新疆棉花枯萎病菌群体结构的研究

 采自新疆24个不同植棉县(市或团场)的37株棉花枯萎病菌代表菌株,经人工接种于国际通用鉴别寄主,致病性反应均表现为典型的7号生理小种特征。RAPD分析结果也显示出这37个供试菌株与7号小种各对照菌株间基因组DNA的指纹图谱高度相似,属同一遗传相似组,而与3号和8号小种的对照菌株间遗传差异较大,亲缘关系较远,即7号生理小种是组成目前新疆棉花枯萎病菌群体的优势小种,而原分布于新疆吐鲁番等地的3号小种在本研究中未被发现。结合部分自选辅助鉴别寄主对其中18个菌株进行的致病力分化研究表明,在7号小种内部还存在着侵染力的分化,显示出棉花枯萎病菌较强的变异性和适应性。     

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。     

Characterization of Fusarium oxysporum f. sp. radicis-cucumerinum attacking melon under natural conditions in Greece

A severe root and stem rot disease of melon was observed during the 2001 growing season on four glasshouse crops in Heraklio, Greece. A total of 43 isolates of F. oxysporum , obtained in Crete from glasshouse-grown melon and showing fusarium wilt or root and stem rot symptoms, were characterized by pathogenicity and vegetative compatibility. The majority of these isolates was also fingerprinted via amplified fragment length polymorphic (AFLP) analysis. Of the total number of isolates, 22 were identified by pathogenicity tests as F. oxysporum f. sp. melonis , 20 as F. oxysporum f. sp. radicis-cucumerinum , while one isolate was nonpathogenic on cucumber, melon, sponge gourd and pumpkin. All 22 isolates of F. oxysporum f. sp. melonis were assigned to vegetative compatibility group (VCG) 0134, and all 20 isolates of F. oxysporum f. sp. radicis-cucumerinum to VCG 0260. Isolates of F. oxysporum f. sp. radicis-cucumerinum were incompatible with isolates of F. oxysporum f. sp. melonis. AFLP fingerprinting allowed for the clustering of the isolates of the two formae speciales of F. oxysporum along two separate phenetic groups: f. sp. melonis to AFLP major haplotype I, and f. sp. radicis-cucumerinum to AFLP major haplotype II. Overall, pathogenicity, vegetative compatibility grouping and AFLP analysis were correlated and effectively distinguished isolates of F. oxysporum from melon. This appears to be the first report of natural infection of melon by F. oxysporum f. sp. radicis-cucumerinum worldwide.     

甘蓝枯萎病菌REMI转化体系的建立

建立高效、稳定的甘蓝枯萎病菌REMI转化体系,为进一步获得特定表型突变体及基因功能研究建立技术储备.利用REMI(restriction enzyme mediate intergration)转化方法,将线性化的含有潮霉素抗性基因的pUCAT-PH质粒转化甘蓝枯萎病菌A6菌株的原生质体,摸索获得转化子最适的潮霉素筛选浓度以及不同限制性内切酶和酶量对转化效率的影响;利用PCR(polymerase chain reaction)技术对潮霉素抗性转化子进行验证.结果表明转化子的最适潮霉素筛选浓度为50 μg/mL;转化效率较高的限制性内切酶为HindⅢ,并且转化效率最高时的酶量为20 U.利用该转化体系构建了含1 050个转化子的甘蓝枯萎病菌转化子库,对转化子进行Southern验证,证明该转化体系是可行的.     

Vegetative Compatibility Grouping of Fusarium oxysporum f. sp. gladioli from Saffron

Fusarium corm rot of saffron (Crocus sativus L.), incited by Fusarium oxysporum f. sp. gladioli, causes severe yield losses in Italy. Major symptoms during flowering (October–November) include yellowing and wilting of shoots, basal stem rot and corm rot. Sixty-four isolates of F. oxysporum f. sp. gladioli, obtained from infected saffron crops located in Italy (Abruzzi, Tuscany and Umbria) and in Spain, were characterized by pathogenicity and vegetative compatibility. Chlorate-resistant, nitrate-nonutilizing (nit) mutants were used to determine vegetative compatibility among the isolates of the pathogen with the aim of examining the genetic relatedness among populations from different locations. All the isolates belonged to vegetative compatibility group 0340. Since saffron shares susceptibility to F. oxysporum f. sp. gladioli with other ornamental plants of the Iridaceae (Crocus, Gladiolus, Iris and Ixia), it is likely that a clone of the pathogen (VCG 0340) was introduced with other hosts and is responsible for the disease outbreak observed on saffron in Italy. Alternatively, or additionally, the clone of F. oxysporum f. sp. gladioli causing disease on saffron in other countries may have spread to the saffron fields in Italy through the import and dispersal of infested propagation material.     

Isolation of Pathogenicity Mutants of Fusarium oxysporum f. sp. melonis by Insertional Mutagenesis

Restriction enzyme-mediated integration (REMI) mutagenesis was used to isolate mutants of Fusarium oxysporum f. sp. melonis impaired in pathogenicity. The race 2 strain Mel02010 was transformed with linearized pSH75, conferring resistance to hygromycin B, with or without the enzyme used to linearize the plasmid. Addition of restriction enzymes did not affect the transformation frequency. A total of 2929 REMI transformants were tested for pathogenicity to three melon cultivars, Amus, Ogon 9 and Ohi. The race 2 strains are pathogenic to Amus and Ogon 9, but not to Ohi. Of 43 transformants with reduced pathogenicity on susceptible melon cultivars, 12 mutants were examined in detail for pathogenicity, vegetative growth and integrative mode of pSH75. The levels of pathogenicity varied among these mutants. Two mutants (B48 and B137) almost completely lost pathogenicity to both susceptible cultivars, and the others had reduced pathogenicity. Mutants B48, B241, B886 and X36 were also impaired in vegetative growth. Mutant B809 was a biotin auxotroph. By DNA gel blot analysis, nine mutants were found to contain a single copy of the transformation vector. These mutants may thus be useful in isolating genes involved in pathogenicity. Received 22 December 2000/ Accepted in revised form 16 April 2001     

Vegetative compatibility grouping in Fusarium oxysporum f.sp. radicis-lycopersici from the UK, the Netherlands, Belgium and France

The population structure of Fusarium oxysporum f.sp. radicis-lycopersici ( F.o.r.l .), the causal agent of crown and root rot disease in tomato, was studied using the vegetative compatibility grouping approach. Four vegetative compatibility groups (VCGs) were identified among 37 isolates from the UK, the Netherlands, Belgium and France. Three of these VCGs (0090, 0091, 0094) had already been described, whereas VCG 0097 was new. VCG 0094 was dominant in the UK, the Netherlands and Belgium, but not in France. The opposite was true for the cosmopolitan VCG 0091, while the cosmopolitan VCG 0090 was only found in France. Based on hyphal interactions, VCG 0094 was divided into three subgroups, each comprising isolates from at least two countries. One isolate of VCG 0094 did not belong to any of these subgroups, suggesting further variability in this VCG. Isolate FORL-19R from France, previously assigned to VCG 0090 I, was reassigned to VCG 0090 III, a new subgroup of VCG 0090 found in Israel. FORL-19R and additional members of its subgroup manifest cross-VCG compatibility between VCG 0090 and VCG 0092. Along with previous studies, the multiple VCGs and subgroups found among F.o.r.l . in western Europe demonstrate a high level of genetic diversity in this pathogen.     

Phylogenetic position and nucleotide diversity of Fusarium oxysporum f. sp. vanillae worldwide based on translation elongation factor 1α sequences

Fusarium oxysporum f. sp. vanillae is considered the most important fungus affecting vanilla crops around the world, causing rot on vanilla roots and stems. Previous studies showed that the ability to infect vanilla plants is a polyphyletic trait among strains of the Fusarium oxysporum species complex (FOSC). The same studies proposed a single origin for F. oxysporum f. sp. vanillae isolates sampled from Mexico, the centre of origin and distribution of vanilla. The aim of this work was to test the hypothesis of the monophyletic origin of a wider sample of isolates of F. oxysporum f. sp. vanillae infecting Mexican vanilla and estimate nucleotide diversity of pathogen isolates from the main vanilla‐producing countries. Sequence data for the TEF1α gene from 106 isolates was assembled. The phylogenetic analyses suggest that some Mexican isolates of F. oxysporum f. sp. vanillae belong in two well‐supported clades, mixed with isolates from Madagascar, Indonesia, Réunion and Comoros. The phylogenetic position of other Indonesian and Mexican isolates is unresolved. Estimations of nucleotide diversity showed that the population from Mexico is genetically more diverse than the other three populations from Madagascar, Indonesia and Réunion. The results support a polyphyletic origin of vanilla‐infecting isolates of F. oxysporum worldwide, and also reject the proposition that Mexican isolates have a single origin. The phylogenetic optimizations over the strict consensus tree of the ability to infect vanilla plants suggest that pathogenic strains around the world are the product of multiple shifts of pathogenesis and dispersion events.     

农杆菌介导的西瓜枯萎病菌遗传转化

为获得带GFP标记的西瓜枯萎病菌转化株,用于后期观察病原菌侵染过程,采用农杆菌介导的方法,对西瓜枯萎病菌1号生理小种进行了遗传转化。结果表明:共培养时间为36h,枯萎病菌孢子和农杆菌AGL1比例为1∶1时该菌株的遗传转化效率最高,可以达到117.33个转化子/107个孢子。转化株的孢子、菌丝体及萌发的孢子均能发出稳定而强的绿色荧光。转化株的致病力检测显示其致病力与转化前的野生菌株致病力无明显差异。结果表明本研究获得的带GFP标记的西瓜枯萎病菌转化株可用于观察病菌在西瓜根系的侵染过程。