Stepwise Evolution of Races in Fusarium oxysporum f. sp. ciceris Inferred from Fingerprinting with Repetitive DNA Sequences

ABSTRACT Plant pathogens often exhibit variation in virulence, the ability to cause disease on host plants with specific resistance, evident from the diversity of races observed within pathogen species. The evolution of races in asexual fungal pathogens has been hypothesized to occur in a stepwise fashion, in which mutations to virulence accumulate sequentially in clonal lineages, resulting in races capable of overcoming multiple host plant resistance genes or multiple resistant cultivars. In this study, we demonstrate a simple stepwise pattern of race evolution in Fusarium oxysporum f. sp. ciceris, the fungus that causes Fusarium wilt of chickpeas. The inferred intraspecific phylogeny of races in this fungus, based on DNA fingerprinting with repetitive sequences, shows that each of the eight races forms a monophyletic lineage. By mapping virulence to each differential cultivar (used for defining races) onto the inferred phylogeny, we show that virulence has been acquired in a simple stepwise pattern, with few parallel gains or losses. Such a clear pattern of stepwise evolution of races, to our knowledge, has not been demonstrated previously for other pathogens based on analyses of field populations. We speculate that in other systems the stepwise pattern is obscured by parallel gains or losses of virulence caused by higher mutation rates and selection by widespread deployment of resistant cultivars. Although chickpea cultivars resistant to Fusarium wilt are available, their deployment has not been extensive and the stepwise acquisition of virulence is still clearly evident.     

Evolution of virulence in Fusarium oxysporum f. sp. vasinfectum using serial passage assays through susceptible cotton

Fifty strains of Fusarium oxysporum, recovered from rhizosphere soil around native Gossypium species and found to be mildly virulent on cotton (Gossypium hirsutum), were used to assay the propensity for evolution of virulence using serial passage assays through cotton. Only one lineage A strain, 2613, successfully completed 10 successive passages, while all others lost the ability to cause foliar disease symptoms at various stages during this process. Based on 46 amplified fragment length polymorphism (AFLP) markers generated with four EcoRI x MseI primer combinations, mutants were identified in offspring isolates from strain 2613 regardless of whether serial passages occurred in cotton or on water agar, suggesting the occurrence of spontaneous mutations. Significantly increased virulence was observed in the offspring isolates generated on cotton, while no increasing virulence was found in those obtained on water agar, suggesting that the evolution of virulence in F. oxysporum f. sp. vasinfectum is associated with the presence of cotton. No clear correlation was observed between the AFLP mutations and increased virulence in this study.     

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).     

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。     

不同提取条件对葡萄叶提取物抑制两种病原菌活性的影响

以黄瓜枯萎病菌和辣椒枯萎病菌作为供试菌种,采用生长速率法对葡萄叶不同溶剂提取物和不同提取方法提取物的抑菌活性进行测定。结果表明,葡萄叶不同溶剂提取物对供试菌种均表现出抗菌效果,并筛选出最佳提取溶剂是95%的乙醇,而最佳提取条件是提取温度40 ℃、提取时间和方式是超声波提取30 min,提取料液比1∶8(g/mL)。     

High Genetic Diversity Among Strains of Fusarium oxysporum f. sp. vasinfectum from Cotton in Ivory Coast

ABSTRACT Seventeen isolates of Fusarium oxysporum f. sp. vasinfectum from the Ivory Coast were characterized using vegetative compatibility group (VCG), restriction fragment length polymorphism of the ribosomal inter-genic spacer region (IGS), and mating type (MAT) idiomorph, and compared with a worldwide collection of the pathogen containing all available reference strains. Some of the isolates were identical to known reference strains for all three traits, whereas others had previously unknown varieties of IGS and (possibly) VCG. One or the other MAT idiomorph was present in each of the new isolates and the reference strains. The new isolates and reference strains were grouped based upon the three traits. Strains from the Ivory Coast were found in 7 of 11 groups detected, suggesting multiple sources for Fusarium wilt in the country. Despite the presence of both MAT idiomorphs among isolates, no evidence for recombination was found.     

棉花枯萎病拮抗放线菌K5的分离与鉴定

从扬州地区土壤中分离到207株放线菌,以棉花枯萎病菌为目标菌,采用抑菌带法测定,筛选到9株有拮抗活性菌株,占所分离总数的4.3%.其中K5对棉花枯萎病菌的拮抗作用最强,抑茜带宽为8mm,同时对其它病菌也有一定的拮抗作用.盆栽试验结果表明菌株K5对棉花枯萎病防治效果达46.5%,与多菌灵相当.通过对菌株K5的形态观察、培养特征、生理生化反应和16S rDNA测定,将其鉴定为玫瑰产色链霉菌Streptomyces roseochromogenus.     

Detection of Fusarium oxysporum f.sp. vasinfectum in cotton tissue by polymerase chain reaction

A polymerase chain reaction assay was developed for the detection of Fusarium oxysporum f.sp. vasinfectum (FOV), a serious wilt pathogen of cotton in many parts of the world. Based on small nucleotide differences in internal transcribed spacer sequences between 18S, 5.8S and 28S ribosomal DNAs, primers Fov1 (5'-CCCCTGTGAACATACCTTACT-3') and Fov 2 (5'-ACCAGTAACGAGGGTTTTACT-3') were selected. These primers unambiguously amplified a 400-bp DNA fragment of all the FOV isolates tested (from Angola, Brazil, China and the USA) but did not amplify any other isolates of mycoflora associated with cotton, such as F. moniliforme , Verticillium albo-atrum , V. dahliae , Aspergillus sp., F. oxysporum , F. sambucinum or F. solani . A control PCR assay was developed employing the universal primer pair ITS1 and ITS2 which amplified a fragment of approximately 220 bp from all isolates tested. This control assay demonstrated that all fungal DNAs were readily amplifiable, thus confirming that the lack of amplification with Fov1 and Fov2 primers was a result of primer specificity and not of other possible causes, such as DNA degradation or the presence of PCR inhibitors. The assay was effective on samples from the stems, leaves, roots and calli, and from plant tissues both with and without symptoms. This detection system proved to be accurate and sensitive and could aid not only diagnosis but also disease monitoring and forecasting.     

棉花枯萎病菌新生理型菌株的分子鉴定

 Fusarium oxysporum f.sp. vasinfectum(Fov)已报道有8个生理小种和一个澳大利亚生理型,在我国只报道有3、7、8号小种。前期研究发现,我国还有一个与3、7、8号小种具有较大遗传差异的Fov新生理型。本研究通过对3、7、8号小种和新生理型菌株的翻译延伸因子(EF-1α)基因序列比对分析,发现该新生理型菌株具有特异性碱基序列。根据这些特异性碱基序列,设计了一对针对新生理型菌株的特异性引物,并证明该引物可以特异性检测Fov新生理型菌株。利用该引物对采自河北省主要植棉区的77株Fov菌株进行筛选,鉴定出2株潜在的新生理型菌株。从这2个Fov菌株中克隆出EF-1α和β-tubulin基因序列,通过构建系统发育树,证明这2个Fov菌株为新生理型菌株,而且与澳大利亚生理型菌株具有较近的亲缘关系。本研究建立的分子检测方法可用于棉花枯萎病菌新生理型菌株的鉴定。     

我国棉花枯萎菌生理小种特异性DNA扩增片段的筛选及序列测定

 利用RAPD技术,以随机引物对我国棉花枯萎菌的3个生理小种(3、7、8号小种)共26个菌株进行PCR扩增,从产生的140个RAPD分子标记中寻找到了不同小种的特征性条带,O PF-10₅₁₃(3号小种)、OPF-08₃₇₁(7号小种)及OPF-12₇₀₃(8号小种)。将其纯化后克隆到pGEM-TEasy质粒载体上,并获得了DNA特异性片段的核酸序列。     

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     

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.     

Biological control of Fusarium oxysporum f. sp. lycopersici

Fungi known to produce lytic enzymes were used in an attempt to control wilt of tomato caused by Fusarium oxysporum f. sp. lycopersici (FOL). Some of the fungal species (Penicillium oxalicum, Penicillium purpurogenum and Aspergillus nidulans) damaged hyphae of FOL in vitro and reduced the numbers of microconidia in the soil. Treatments with fungi did not result in a reduction in either chlamydospores of FOL in soil or populations of FOL in the rhizosphere of tomato. P. oxalicum was the most effective agent of biocontrol, and it reduced disease severity in both non-autoclaved (20% decrease) and sterile soil. In sterile soil, P. oxalicum reduced disease with different levels of severity (27% decrease at high levels and 50% decrease at low levels). Disease control by A. nidulans and P purpurogenum was only achieved when disease severity was low in sterile soil (55% and 45%, respectively).     

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.     

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。     

Dispersal of Formulations of Fusarium oxysporum f. sp. erythroxyli and F. oxysporum f. sp. melonis by Ants

ABSTRACT A natural epidemic of Fusarium wilt on coca (Erythroxylum coca) in Peru prompted the suggestion of possibly using the pathogen Fusarium oxysporum f. sp. erythroxyli as a mycoherbicide against this narcotic plant. During field trials conducted in Kauai, HI, to test the pathogenicity of the coca wilt pathogen, ants were observed removing formulations from test plots. While removal of formulations by ants was considered detrimental with respect to conducting field tests, ant removal was considered potentially beneficial in disseminating the mycoherbicide. Thus, research was initiated to assess the ability of formulation additives to alter the preference of ants for the formulated mycoherbicide. In Hawaii, preference of indigenous ants for removing formulations was tested using three different food bases (rice, rice plus canola oil, and wheat flour [gluten]). Similar tests were conducted at Beltsville, MD, using F. oxysporum f. sp. melonis, in which the formulation based on wheat flour was replaced by a formulation based on canola meal. Formulations based on wheat were preferred by ants in both locations; up to 90% of the wheat plus rice flour granules (C-6) and the wheat gluten plus kaolin granules (pesta) were removed within 24 h, while only 20% of those containing rice without oils were taken. However, when either canola, sunflower (Maryland only), or olive oil was added to the rice formulation, up to 90% of the granules were taken. The formulation based on canola meal was less attractive to ants, as only 65% of the granules were removed within a period of 24 h. Ants showed no preference with respect to presence or absence of fungal biomass. To alter the attractiveness of the C-6 formulation to ants, C-6 was amended with three natural products. Canna and tansy leaves were added to C-6 at a ratio of 1:5 (wt/wt), while chili powder was added at 1:25 or 1:2.5 (wt/wt). Canna, tansy, and the higher rate of chili powder significantly reduced the number of C-6 granules removed by ants. Canna and tansy leaves affected neither germination nor sporulation of the mycoherbicide, while the high concentration of chili powder reduced viability of propagules in the formulation. More F. oxysporum f. sp. erythroxyli-type colonies were recovered from inside ant nests (9 cm depth) than from nest surfaces, indicating that ants may distribute the mycoherbicide in the soil profile. Ants passively carried propagules of F. oxysporum f. sp. erythroxyli outside their bodies, as well as either very closely adhering to the outside or within their bodies.     

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.