Evolution of Fusarium oxysporum f. sp. vasinfectum Races Inferred from Multigene Genealogies

ABSTRACT Fusarium wilt of cotton is a serious fungal disease responsible for significant yield losses throughout the world. Evolution of the causal organism Fusarium oxysporum f. sp. vasinfectum, including the eight races described for this specialized form, was studied using multigene genealogies. Partial sequences of translation elongation factor (EF-1alpha), nitrate reductase (NIR), phosphate permase (PHO), and the mitochondrial small subunit (mtSSU) rDNA were sequenced in 28 isolates of F. oxysporum f. sp. vasinfectum selected to represent the global genetic diversity of this forma specialis. Results of a Wilcoxon Signed-Ranks Templeton test indicated that sequences of the four genes could be combined. In addition, using combined data from EF-1alpha and mtSSU rDNA, the phylogenetic origin of F. oxysporum f. sp. vasinfectum within the F. oxysporum complex was evaluated by the Kishino-Hasegawa likelihood test. Results of this test indicated the eight races of F. oxysporum f. sp. vasinfectum appeared to be nonmonophyletic, having at least two independent, or polyphyletic, evolutionary origins. Races 3 and 5 formed a strongly supported clade separate from the other six races. The combined EF-1alpha, NIR, PHO, and mtSSU rDNA sequence data from the 28 isolates of F. oxysporum f. sp. vasinfectum recovered four lineages that correlated with differences in virulence and geographic origin: lineage I contained race 3, mostly from Egypt, and race 5 from Sudan; lineage II contained races 1, 2, and 6 from North and South America and Africa; lineage III contained race 8 from China; and lineage IV contained isolates of races 4 and 7 from India and China, respectively.     

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.     

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).     

Infection of cotton byfusarium oxysporum f.sp.vasinfectum as affected by water stress

Since virulence ofFusarium oxysporum f.sp.vasinfectum (FOV) on cotton (Gossypium hirsutum) is enhanced when the fungus is cultivated in a saline environment, excessively saline water must not be used for the irrigation of cotton. However, the limitations thus placed on the available water resources may lead to conditions of enforced water stress for the plant. The present study investigated whether water stress affects the susceptibility of cotton to FOV. Groups of 2-month-old cotton plants of theFusarium-susceptible Coker 304 and the moderately resistant GSC 20 varieties were maintained without watering for varying periods immediately before or after being inoculated with FOV (15 plants per group, two replications). Watering was suspended for 3, 6, 12 or 24 days before inoculation, and for 3, 6, 12 or 15 days after inoculation. After inoculation the plants were maintained in a controlled environment with a 15,000 lux, 12-h photoperiod, at 28°/24°C D/N, 20% r.h. Xylem water potential was determined in a pressure chamber. Percent infected leaf area and date of onset of wilt were the parameters used to define severity of FOV infection. There was a consistent relation between low water potential in the xylem (-7 and -20 MPa) and severity of infection, particularly when the dry period occurred after inoculation. After exposure to the lowest post-inoculation water potentials, even variety GSC 20, which is normally moderately resistant, exhibited a fairly high percent infected leaf area. This should be taken into account when the cotton grower is faced with water shortages, especially during the period from branching to flower bud break.     

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。     

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.     

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。     

Effect of the herbicides EPTC and linuron on cotton diseases caused by Rhizoctonia solani and Fusarium oxysporum f. sp. vasinfectum

The herbicides EPTC and linuron applied to soil in the field at three different concentrations X₂X, 1X, and 2X of recommended dose) decreased post-emergence, but not pre-emergence daraping-off in cotton incited by Rhizoctonia solani. Both herbicides at the two high concentrations significantly reduced wilt incited by Fusarium oxysporum f. sp. vasinfectum. Both herbicides reduced germination of chlamydospores of Fusarium in natural soil, but not in steamed soil. The 2X concentration of EPTC and linuron reduced the saprophytic activity of R. solani in soil. The effects of EPTC and linuron on post-emergence damping-off and wilt were attributed Ic their ability to suppress the saprophytic ability of R. solani and chlamydospore germinability of Fusarium in soil, respectively.     

Induction of mutants of Fusarium oxysporum f. sp. lycopersici with altered virulence

Mutants ofFusarium oxysporum f. sp.lycopersici were obtained by UV irradiation. The mutants of race 1 and race 2 caused disease symptoms on plants with resistance genes against the corresponding wild type strains. Mutants of race 1 of the pathogen were stable, whereas mutants of race 2 lost the ability to cause disease symptoms in plants carrying the 1–2 resistance gene, after prolonged maintenenance on potato dextrose agar. Mutants of race 1 resembled race 2 in pathogenicity and they were vegetatively compatible with race 2, but no longer with race 1. These results suggest that the isolated strains with an altered virulence pattern have mutations in loci involved in avirulence.     

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.     

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。     

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.     

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.     

一种评价甘蓝枯萎病菌转化子致病力的方法

为建立甘蓝枯萎病菌(Fusarium oxysporumf.sp.conglutinans)转化子致病力评价方法,从接种方法、甘蓝品种、苗龄、病原菌接种体浓度等几方面探索,建立了一种评价甘蓝枯萎病菌转化子致病力差异的体系。结果表明:蘸根法和伤根法均适于甘蓝枯萎病菌转化子致病力的评价,其中伤根法更优;其他适合甘蓝枯萎病菌转化子致病力评价的因素有:甘蓝品种为‘中甘21’,苗龄为三叶期,甘蓝枯萎病菌孢子悬浮液浓度为孢子含量1×106个/mL。该致病力评价方法的建立为甘蓝枯萎病菌转化子致病力衰弱或增强突变体的筛选提供了方法支持,也为下一步尖孢镰刀菌致病机理的解析奠定了基础。     

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).     

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.