Variation in potential effector genes distinguishing Australian and non‐Australian isolates of the cotton wilt pathogen Fusarium oxysporum f.sp. vasinfectum

This study identified genes that distinguish Australian Fusarium oxysporum f.sp. vasinfectum (Fov) isolates from related co‐localized non‐pathogenic F. oxysporum isolates and from non‐Australian Fov isolates. One gene is a homologue of the F. oxysporum f.sp. lycopersici (Fol) effector gene SIX6, encoding a 215‐residue cysteine‐rich secreted protein. The Six6 proteins from Fol and Fov contained eight conserved cysteine residues, five of which occurred in the highly diverged 48‐amino‐acid region where FovSix6 differs from FolSix6 at 32 residues. Two other potential effector genes, PEP1 and PEP2, were identified in a cDNA library of Fov genes expressed during infection of cotton. The presence of FovSIX6 and other differences in DNA fingerprints clearly distinguished Australian Fov isolates from non‐Australian Fov isolates and these differences further support the hypothesis based on earlier phylogenetic analysis that Australian Fov is different from Fov in other cotton‐growing areas. A specific diagnostic for Fov based on FovSIX6 is described.     

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。     

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     

内生细菌与木霉复合处理诱导甜瓜对枯萎病的抗性

在对甜瓜枯萎病温室防效试验的研究中发现，内生枯草芽孢杆菌B6菌株与绿色木霉T23菌株复合处理的相对防效达82．22％，比B6和T23的单独处理分别提高32．8％和146．7％。分析比较了B6和T23单独和复合处理甜瓜幼苗后，甜瓜根部防御反应相关酶系苯丙氨酸解氨酶、过氧化物酶、多酚氧化酶、β-1，3-葡聚糖酶比活性的变化趋势。结果表明：内生细菌B6和木霉T23复合接种，其苯丙氨酸解氨酶、过氧化物酶、多酚氧化酶和β-1，3-葡聚糖酶比活性比单独接种有不同程度的增强，这种变化在挑战接种甜瓜枯萎病菌之后更加明显。     

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。     

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     

Development of a Fusarium oxysporum f. sp. melonis functional GFP fluorescence tool to assist melon resistance breeding programmes

Fusarium wilt, caused by Fusarium oxysporum f. sp. melonis (Fom), is one of the most widespread and devastating melon diseases. This vascular disease is caused by the colonization of melon xylem vessels by any of the four Fom races reported (r0, r1, r2 and r1,2, subdivided into r1,2w and r1,2y). The macroscopic evaluation of disease symptoms (disease rating, DR) at several days post‐inoculation (dpi) with Fom spores has been the traditional method to determine the resistance of melon accessions to this fungal pathogen. In this study, one isolate from each Fom race was transformed by Agrobacterium tumefaciens to constitutively express the green fluorescent protein (GFP). Fom‐GFP transformants, as virulent as the corresponding wildtype races, were selected to develop an inoculation assay based on the non‐invasive evaluation of the fluorescence emitted by Fom‐GFP. It was determined that melon root neck was the appropriate area to follow Fom‐GFP and a fluorescence signal rating (FSR) was established in parallel to DR determination. This method allowed the evaluation of GFP signal in the root neck of inoculated melon seedlings at 11–15 dpi. The GFP signal was scored in 62 melon accessions/breeding lines inoculated with different Fom‐GFP, followed by evaluation of the macroscopic DR in the aerial part of melon seedlings at 20–28 dpi. Correlation analysis demonstrated a direct and significant relationship between FSR and DR. This method has shown to be an effective and reliable tool that can assist Fom resistance breeding programmes in melon.     

In Vitro Suppression of Mycelial Growth of Fusarium oxysporum by Extracellular Chitosanase of Sphingobacterium multivorum and Cloning of the Chitosanase Gene csnSM1

A chitosan-degrading bacterium, isolated from field soil that had been amended with chitin, was identified as Sphingobacterium multivorum KST-009 on the basis of its bacteriological characteristics. The extracellular chitosanase (SM1) secreted by KST-009 was a 34-kDa protein and could be purified through ammonium sulfate precipitation, gel permeation column chromatography and SDS polyacrylamide gel electrophoresis. A chitosanase gene (csnSM1) was isolated from genomic DNA of the bacteria, and the entire nucleotide sequence of the gene and the partial N-terminal amino acid sequence of the purified SM1 were determined. The csnSM1 gene was found to encode 383 amino acids, 72 N-terminal amino acid residues were processed to produce the mature enzyme during the secretion process. Germinated microconidia of four formae speciales (lycopersici, radicis-lycopersici, melonis, and fragariae ) of Fusarium oxysporum were treated with SM1. Chitosanase treatment caused morphological changes, such as swelling of hyphal cells or indistinctness of hyphal cell tips and cessation or reduction of mycelial elongation. Received 2 May 2001/ Accepted in revised form 21 June 2001     

连作条件下土壤中甘蓝枯萎病菌的致病力变化和种群遗传结构分化

 枯萎病菌致病力的变化可能是连作条件下甘蓝枯萎病严重发生的重要原因之一。本研究在建立适度感染的人工病圃的基础上连续种植甘蓝5茬,每茬收获后随机采集土样。利用驹田氏培养基通过稀释平板法对连作土壤中尖孢镰刀菌种群数量的监测结果表明,连作后尖孢镰刀菌的数量由第二茬后的3.047×10⁴ cfu·g^-1土壤增加到第五茬收获后的1.608×10⁵ cfu·g^-1 土壤。对各茬后所分离30株甘蓝枯萎病菌（Fusarium oxysporum f. sp. conglutinans, Foc）的培养性状的观察结果表明,连作后Foc菌株在菌落形态、菌落扩展速率和产孢量等方面均发生明显的变化。用浸根法进行的致病力测定结果表明,随着连茬次数增加,弱致病力菌株占总供试菌株的比例逐渐变小,到第三茬后由第一茬的6.7%下降为0;而强致病力菌株的比例逐渐上升,由第一茬后的6.7%上升到第四茬后的16.7%。利用11条寡聚核苷酸随机引物对受试菌株进行PCR-ISSR扩增,结果显示从第三茬后Foc群体遗传结构出现分化。UPGMA聚类分析结果表明,第三、四和五茬后的Foc菌株都分为A和B两个类群,每个类群又分为Ⅰ和Ⅱ两个亚类群,但同一类群和亚类群中包含不同致病力的菌株,未发现病菌的致病力变化与遗传结构分化之间的相关。     

Physiological Races and Vegetative Compatibility Groups of Fusarium oxysporum f. sp. lactucae Isolated from Crisphead Lettuce in Japan

One hundred and sixteen isolates of Fusarium oxysporum f. sp. lactucae obtained from 85 fields in three crisphead lettuce-producing areas in Nagano Prefecture, Japan were typed for races using differential cultivars Patriot, Banchu Red Fire and Costa Rica No. 4. They were also grouped into vegetative compatibility groups (VCGs) using complementation tests with nitrate non-utilizing (nit) mutants. Two California strains reported as F. oxysporum f. sp. lactucum, a type culture of F. oxysporum f. sp. lactucae, and 28 avirulent isolates of F. oxysporum obtained from crisphead lettuce were included for comparison. Among Nagano isolates, 66 isolates were identified as race 1, and 50 as race 2. Race 1 strains derived from Shiojiri and Komoro cities and race 2 from Kawakami village and Komoro city. All isolates of race 2 were biotin auxotrophs, and the race could be distinguished based on its requirement for biotin on minimal nitrate agar medium (MM). Pathogenic isolates were classified into two VCGs and three heterokaryon self-incompatible isolates. Strong correlations were found between race and VCG. All the race 1 strains were assigned to VCG 1 except self-incompatible isolates, and all the race 2 strains to VCG 2. The 28 avirulent isolates of F. oxysporum were incompatible with VCG 1 and VCG 2. California strains was vegetatively compatible with VCG 1, and they were assigned to race 1. Based on vegetative compatibility, these two races of F. oxysporum f. sp. lactucae may be genetically distinct, and F. oxysporum f. sp. lactucae race 1 is identical to F. oxysporum f. sp. lactucum. Received 7 May 2002/ Accepted in revised form 6 September 2002     

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     

不同抗性黄瓜品种连作对枯萎病菌群体遗传分化的影响

尖孢镰刀菌黄瓜专化型(Fusarium oxysporum f. sp.cucumerinum,Foc)是引起黄瓜枯萎病的病原真菌,为了明确不同抗性黄瓜品种连作对枯萎病菌遗传分化的影响,本研究采用TEF1-α、H3基因片段对不同抗性黄瓜品种连作及对轮作后分离的枯萎病菌群体进行遗传分化分析。结果表明,感病品种连作及轮作后菌群分化程度较低(0.007ST<0.045);与种植抗病品种一茬后的菌群相比,连作三、五茬后的菌群均发生显著分化(F_ST> 0.050),其中连作三茬后的菌群遗传分化程度最高(F_ST> 0.300)。单倍型分析发现,抗、感病品种连作后部分菌株形成新的单倍型,且分离自抗病品种的病原菌群体分化产生的单倍型数量多于感病品种。主成分分析结果进一步证明,与感病品种连作及轮作相比,抗病品种连作后病原菌菌群呈现出更显著的菌群分化。综上所述,抗病品种连作能够加剧黄瓜枯萎病菌群体的遗传分化。     

sfp基因转化增强了Bacillus subtilis 168的定殖能力和对黄瓜茎内枯萎病菌的抑制作用

枯草芽胞杆菌B006是一株防治黄瓜枯萎病的高效菌株,可产生脂肽类抗生素表面活性素surfactin和芬枯草菌素fengycin。为深入了解surfactin在根际的作用,本研究通过同源重组的方法将生防芽胞杆菌B006菌株中编码4'-磷酸泛酰巯基乙胺转移酶Sfp的sfp基因整合到不产生surfactin的模式菌株B.subtilis168(B168)基因组中,获得了重组菌株B168S。重组菌株B168S在1%淀粉平板上不水解淀粉,在5%血平板上产生透明溶血圈;HPLC-ESI-MS检测表明菌株B168S在牛肉膏蛋白胨培养液中培养48 h后可产生surfactin。平板对峙试验表明菌株B168S在黄瓜根分泌物培养基上对Fusarium oxysporum f. sp.cucumerinum(Foc)菌丝生长有一定的抑制作用,抑制率R2/R1为1.22;平板菌落计数法测定表明,将菌悬液(10⁶ cfu/mL)浸泡90 min的黄瓜种子播种于无菌石英砂3 d后,菌株B168S在黄瓜根基部和根尖的定殖量分别为菌株B168的2~3倍和9~10倍;温室盆栽试验表明,黄瓜苗用浓度为106 cfu/mL的B168S和B168菌悬液蘸根后,移栽到接种Foc孢子(10⁵孢子/g)的病土中,移栽后第3周菌株B168S和B168处理的防效分别为21.1%和17.9%;对不同高度黄瓜茎中Foc的组织分离结果表明,菌株B168S处理后Foc在黄瓜茎内蔓延的相对高度低于菌株B168和Foc处理。上述结果进一步明确了surfactin可明显促进芽胞杆菌在黄瓜根部的定殖,并通过抑制枯萎病菌的侵染和在黄瓜茎中的蔓延增强对黄瓜枯萎病的防治效果。     

Potential role for salicylic acid in induced resistance of asparagus roots to Fusarium oxysporum f.sp. asparagi

Pre-inoculation of asparagus ( Asparagus officinalis ) roots with selected nonpathogenic isolates of Fusarium oxysporum (np Fo ) has previously been shown to induce systemic resistance against infection by F. oxysporum f.sp. asparagi ( Foa ) through activation of plant-defence mechanisms. To elucidate the putative np Fo -mediated defence pathways, the effect of salicylic acid (SA) was examined in a split-root system of asparagus where one half of the seedling root system was drenched with SA and the activation of defence responses was measured subsequently on the remaining roots. SA-treated plants exhibited enhanced systemic resistance, with a significant reduction in disease severity of the roots inoculated with Foa , compared with untreated plants. SA activated peroxidase and phenylalanine ammonia-lyase, as well as lignification, upon Foa attack, in a manner similar to that observed with np Fo pretreatment. In addition, application of diphenyleneiodonium, an SA biosynthesis inhibitor, led to failure of np Fo to induce lignin deposition and systemic resistance. Treatment of fungal spores with SA did not affect germination and growth of either np Fo or Foa in in vitro antifungal assays. Production of SA at the site of np Fo infection may be involved in the induction of Foa resistance in asparagus roots.     

Host specificity of Fusarium oxysporum Schlect (isolate PSM 197), a potential mycoherbicide for controlling Striga spp. in West Africa

The ability of Fusarium oxysporum (PSM 197), a potential mycoherbicide for control of Striga hermonthica, to control different Striga species (S. hermonthica, S. asiatica and S. gesneroides) and another parasitic weed, Alectra vogelli, was investigated under glasshouse conditions. Significant reductions in the total number of emerged plants of S. asiatica (91.3%), S. gesneroides (81.8%) and S. hermonthica (94.3%) were achieved in the presence of F. oxysporum (PSM 197). The pathogen only caused a reduction of 8.5% in A. vogelli. This high susceptibility of the three Striga species provides a possible opportunity to control these parasites simultaneously with this mycoherbicide.     

利用香蕉水培系统测定枯萎病病原菌致病力

 为建立一种评价香蕉枯萎病病原菌(Fusarium oxysporum f.sp. cubense)菌株间致病力差异的体系,在改进的香蕉水培系统基础上,对影响香蕉枯萎病菌致病力的接种菌液浓度、类型及处理方式等因素开展分析,同时与不同菌株盆栽测定结果进行比较以验证该方法的可靠性。结果表明:在香蕉水培系统下,将摇培5 d的菌液稀释10倍以上,使孢子初始浓度为1×10⁶ cfu·mL^-1,直接加入该菌液50 mL即可用于致病力评价,且不同菌株用该测定方法与盆栽测定的致病力结果基本一致。该方法的建立为香蕉枯萎病菌致病力的评价提供了快速简便的方法,也为下一步解析尖孢镰刀菌致病相关基因功能和致病机理及抗病品种的选育奠定了基础。     

The potential for Fusarium oxysporum f. sp. fragariae,cause of fusarium wilt of strawberry,to colonize organic matter in soil and persist through anaerobic soil disinfestation

Fusarium wilt of strawberry, caused by Fusarium oxysporum f. sp. fragariae, is a disease of primary concern for strawberry production in many countries. Crop rotation and anaerobic soil disinfestation (ASD) have gained recent interest for their potential to contribute to management of this disease. Both techniques involve incorporation of organic matter into soil, which may be utilized by strains of Fusarium that are competitive saprophytes. We show that F. oxysporum f. sp. fragariae can colonize strawberry, lettuce, raspberry, and broccoli leaf tissues, which are sources of organic matter generated during crop rotation. This pathogen increased in soil population density during ASD treatments that did not become anaerobic, possibly as a result of growth on the organic amendment. However, significant population decreases were observed after ASD treatment when at least 100,000 cumulative reduced mV hours occurred in a 14-day experiment. Post-ASD abundance of F. oxysporum f. sp. fragariae in soil was negatively correlated with cumulative reduced mV hours. The only treatment that consistently caused disinfestation was exposed to a maximum temperature of 22 °C, which indicates there is potential for developing effective ASD treatments in the cool climates where strawberries are grown. Awareness that F. oxysporum f. sp. fragariae can act as a competitive soil saprophyte should be further investigated for its potential to alter disease outcomes where organic amendments are applied.     

The G-protein subunits FGA2 and FGB1 play distinct roles in development and pathogenicity in the banana fungal pathogen Fusarium oxysporum f. sp. cubense

The soil-borne fungus Fusarium oxysporum f. sp. cubense causes banana (Musa spp.) vascular wilt. Here, we examine the roles of G-protein α and β subunit genes fga2 and fgb1 in F. oxysporum development and pathogenicity. Deletion of either or both genes led to increased heat resistance, lower cAMP levels, and enhanced pigmentation, whereas phenotypic defects of colony morphology and reduced conidiation were seen in Δfgb1 and Δfga2/Δfgb1 deletion strains but not in Δfga2. Conversely, Δfgb1 retained greater virulence against banana, suggesting that FGA2 regulates fungal virulence whereas FGB1 modulates both development and virulence, potentially via the cAMP-dependent protein kinase A pathway.