应用PCR-RFLP和巢式PCR检测黄瓜尖镰孢菌

以3株黄瓜尖镰孢菌(Fusarium oxysporum f.sp.cucumarinum)、23株镰孢菌属(Fusariumspp.)真菌和分离自土壤的20株真菌、6株细菌和7株放线菌为材料,采用化学裂解法提取总DNA,进行PCR-RFLP和巢式PCR检测,试验证明PCR-RFLP程序不能完全区分Fusarium属内不同种,而巢式PCR对黄瓜尖镰孢菌具有特异性.运用优化的PCR-RFLP和巢式PCR检测程序对染病黄瓜组织进行了检测,结果表明,两种方法均可在接种发病早期(未显症时)检测出黄瓜枯萎病菌,PCR-RFLP在感病品种接种后3d即可检测到病原菌,而巢式PCR在接种后5d才能检测到病原菌.     

Investigating the bioactivity of cells and cell-free extracts of Streptomyces griseus towards Fusarium oxysporum f. sp. cubense race 4

The antifungal activity of viable cells of Streptomyces griseus (St 4) and its cell-free extracts were investigated against the pathogenic Fusarium oxysporum f. sp. cubense race 4 (FOC race 4), causal agent of wilt disease in bananas. Results from in vitro and soil assays showed cells and cell-free extracts of S. griseus were able to inhibit FOC race 4 with varying degree of success. Antifungal activity was attributed to chitinase and β-1,3-glucanase, detected in both cells and cell-free extracts, which caused lysis of fungal cell wall and inhibited sporulation. Interestingly, β-1,3-glucanase and chitinase activities were significantly higher in cell-free extracts compared to cells, with 8.30 and 5.43 against 7.96 and 4.95 U mL⁻¹, respectively. Application to soil however, showed inoculation using S. griseus cells were more effective in suppressing growth of FOC race 4 than crude extracts, with 6 log₁₀ CFU of FOC race 4 g⁻¹ soil enumerated compared to 7 log₁₀ CFU of FOC race 4 g⁻¹ soil after 20 days. To summarize, this study has shown that cell-free extracts of S. griseus have antifungal properties but may not be suitable for soil application in its current form (liquid suspension). Further investigations on bioformulation may address this limitation.     

Production of Intraspecific Hybrids of Fusarium oxysporum f. sp. radicis-lycoperisici and Fusarium oxysporum f. sp. lycopersici by Protoplast Fusions

The fusion of protoplasts from the cycloheximide-resistant mutant FOL(C) of Fusarium oxysporum f. sp. lycopersici (FOL) and the mycostatin-resistant mutant FORL(M) of F. oxysporum f. sp. radicis-lycopersici (FORL), produced hybrids which expressed significant differences from the parents in their pathogenicity and growth and in the electrophoretic separation patterns of their proteins, enzymes and isoenzymes. The results suggest a transformed genetic basis for these altered expressions and the feasibility of using protoplast fusion technology for examining the biology of pathogenicity genes and for elucidating the disease and virulence potential for new races from within hybridisable taxa of Fusarium spp. Such information would be useful for the design and development of long-term control systems for Fusarium diseases, particularly in breeding programs for disease resistance in crops.     

Dispensable chromosomes in Fusarium oxysporum f. sp. lycopersici

The genomes of many filamentous fungi consist of a ‘core’ part containing conserved genes essential for normal development as well as conditionally dispensable (CD) or lineage‐specific (LS) chromosomes. In the plant‐pathogenic fungus Fusarium oxysporum f. sp. lycopersici, one LS chromosome harbours effector genes that contribute to pathogenicity. We employed flow cytometry to select for events of spontaneous (partial) loss of either the two smallest LS chromosomes or two different core chromosomes. We determined the rate of spontaneous loss of the ‘effector’ LS chromosome in vitro at around 1 in 35 000 spores. In addition, a viable strain was obtained lacking chromosome 12, which is considered to be a part of the core genome. We also isolated strains carrying approximately 1‐Mb deletions in the LS chromosomes and in the dispensable core chromosome. The large core chromosome 1 was never observed to sustain deletions over 200 kb. Whole‐genome sequencing revealed that some of the sites at which the deletions occurred were the same in several independent strains obtained for the two chromosomes tested, indicating the existence of deletion hotspots. For the core chromosome, this deletion hotspot was the site of insertion of the marker used to select for loss events. Loss of the core chromosome did not affect pathogenicity, whereas loss of the effector chromosome led to a complete loss of pathogenicity.     

Race Differentiation in Fusarium oxysporum f.sp. chrysanthemi

The pathogenicity of different isolates of Fusarium oxysporum obtained from plants of Gerbera (Gerbera jamesonii), Chrysanthemum (Chrysanthemum morifolium), Paris daisy (Argyranthemum frutescens) and African daisy (Osteospermum sp.), all in the family Asteraceae, was tested on different cultivars of these hosts, to assess their pathogenicity. The reactions were compared with those of isolates of F. oxysporum f. sp. chrysanthemi and of f.sp. tracheiphilum obtained from the American Type Culture Collection. We found that isolates of F. oxysporum f. sp. chrysanthemi can be distinguished as three physiological races on the basis of their pathogenicity to the panel of differential cultivars. Sequencing of the intergenic spacer (IGS) region of ribosomal DNA (rDNA) and phylogenetic analysis showed that the Fusarium races fell into three phylogenetic groups, which coincided with those observed in pathogenicity tests. Analysis of the IGS sequences revealed a high degree of similarity among strains from Italy and Spain from different host species, suggesting that recent outbreaks in these ornamentals were probably caused by introduction of infected nursery material from a common origin.     

Differential interaction between melon cultivars and race 1.2 of Fusarium oxysporum f.sp. melonis

One hundred and ten melon accessions from different origins were evaluated for resistance to race 1.2 of Fusarium oxysporum f.sp. melonis (Fom). Artificial inoculations were performed and plants were scored for presence or absence of symptoms. The screening revealed that sources of resistance to this fungus are limited. However, a few resistant accessions were found. The accessions 'Kogane Nashi Makuwa', 'C-211' and 'C-40' from Japan provided the highest resistance to both patotypes of race 1.2 of Fom which seems to confirm that the Far East is a rich area in resistances to this race. We also detect an interesting degree of resistance in 'BG-5384', a Portuguese accession that may be classified as belonging to the botanical variety inodorus.     

氨基糖代谢通路影响尖孢镰刀菌古巴专化型厚垣孢子的形成

尖孢镰刀菌古巴专化型Fusarium oxysporum f. sp. cubense（FOC）是威胁香蕉生产的重要土传病原真菌,其厚垣孢子可在土壤中存活多年,是香蕉枯萎病的重要侵染源。为解析该病菌厚垣孢子的形成机制,本研究建立了厚垣孢子的诱导形成体系。通过氨基糖添加试验,证明添加N-乙酰葡糖胺可抑制厚垣孢子的形成;通过对该病菌厚垣孢子形成前期（0h）、初期（24h）、中期（48h）和后期（96h）的转录组分析,发现氨基糖代谢通路中有41个基因的表达水平在厚垣孢子形成过程中发生了变化,其中9个基因与几丁质合成相关,32个基因与糖类化合物的合成和代谢及催化转换相关。本研究首次证明了氨基糖代谢通路与尖孢镰刀菌古巴专化型厚垣孢子形成的相关性。     

Fungus gnats vector Fusarium oxysporum f.sp. radicislycopersici1

Fungus gnat adults transported Fusarium oxysporum f.sp. radicis-lycopersici from Petri dish culture and infected host plants to the roots and hypocotyls of healthy tomato and bean plants. The source of the fungus did not affect the ability of fungus gnats to transport the fungus to healthy hosts. The presence of fungus gnat larvae in media in which young tomato plants were grown did not increase the incidence of plant infection by the pathogen. Fungus gnat adults appear to aid in the dissemination of F. oxysporum f.sp. radicis-lycopersici.     

Fungicidal activity of Ranunculus asiaticus and other weeds against Fusarium oxysporum f.sp. lycopersici

The effects of aqueous extracts of some common weed species against Fusarium oxysporum Schlecht f.sp. lycopersici (the causal agent of tomato wilt disease) were investigated under laboratory conditions. Anagallis foemina L., Cerastium dicotomum L., Falcaria vulgaris L., Ranunculus asiaticus L., Scorpiurus mur-icatus L. and Solanum nigrum L. extracts were the most toxic to the fungus. Further studies on buttercup (Ranunculus asiaticus L.) showed that fresh shoot extract of this species prevented growth of F. oxysporum when incorporated into agar medium. Extracts of different parts of the plant inhibited fungus growth and sporulation, but the fungitoxicity decreased with incubation period with only slight changes in the toxicity of fresh shoot extract. The shoot and fresh parts extracts were more toxic than root and dried tissue extracts. Addition of 0.5 ml fresh shoot or 1 ml fresh root extract to the growing medium significantly reduced fungal colony growth, and the effect was extract concentration dependent. Fresh shoot extract of R. asiaticus added to a liquid medium significantly reduced mycelial dry weight compared with the control, and incorporation of 0.1 g dried shoot or 0.2 g dried roots in the media strongly inhibited fungus growth. Results of a pot experiment showed no harmful effects of R. asiaticus extracts on tomato growth.     

Temperature effect on Fusarium oxysporum f.sp. melonis survival during horticultural waste composting

AIMS: The aim of this work was to study the effect of high temperatures generated during composting process, on the phytopathogen fungus Fusarium oxysporum f.sp. melonis. This investigation was achieved by both in vivo (semipilot-scale composting of horticultural wastes) and in vitro (lab-scale thermal treatments) assays. METHODS AND RESULTS: Vegetable residues infected with F. oxysporum f.sp. melonis were included in compost piles. Studies were conducted in several compost windrows subjected to different treatments. Results showed an effective suppression of persistence and infective capacity, as this process caused complete fungal elimination after 2-3 days of composting. In order to confirm the effect of high temperature during this process, in vitro experiments were carried out. Temperature values of 45, 55 and 65 degrees C were tested. All three treatments caused the elimination of fungal persistence. Treatment at 65 degrees C was especially effective, whereas 45 degrees C eliminated fungal persistence only after 10 days. CONCLUSIONS: The composting process is an excellent alternative for the management of plant wastes after harvesting, as this procedure is able to suppress infective capacity of several harmful phytopathogens such as F. oxysporum f.sp. melonis. SIGNIFICANCE AND IMPACT OF THE STUDY: Fusarium oxysporum f.sp. melonis is a plant pathogen fungus specially important in the province of Almería (south-east Spain), where intensive greenhouse horticulture is very extended. High temperatures reached during composting of horticultural plant wastes ensure the elimination of phytopathogen microorganisms such as F. oxysporum f.sp. melonis from vegetable material, providing an adequate hygienic quality in composts obtained.     

西瓜枯萎病菌的快速检测与鉴定

通过西瓜枯萎病菌与其他专化型枯萎病菌及瓜类几种重要病原菌的比较基因组分析,获得了西瓜枯萎病菌的基因组特异序列。在此基础上,设计出特异引物,筛选可扩增出西瓜枯萎病菌特异性DNA条带的引物。将特异性引物和尖孢镰刀菌专化型的通用引物W106R/W106S结合,建立双重PCR检测体系。该双重PCR检测体系可以在一次PCR反应中快速、准确的检测出西瓜枯萎病菌,为通过分子方法快速鉴定西瓜枯萎病菌提供技术支持。     

Antagonistic Effect of Nonpathogenic Fusarium oxysporum Fo47 and Pseudobactin 358 upon Pathogenic Fusarium oxysporum f. sp. dianthi

Pseudobactin production by Pseudomonas putida WCS358 significantly improves biological control of fusarium wilt caused by nonpathogenic Fusarium oxysporum Fo47b10 (P. Lemanceau, P. A. H. M. Bakker, W. J. de Kogel, C. Alabouvette, and B. Schippers, Appl. Environ. Microbiol. 58:2978-2982, 1992). The antagonistic effect of Fo47b10 and purified pseudobactin 358 was studied by using an in vitro bioassay. This bioassay allows studies on interactions among nonpathogenic F. oxysporum Fo47b10, pathogenic F. oxysporum f. sp. dianthi WCS816, and purified pseudobactin 358, the fluorescent siderophore produced by P. putida WCS358. Both nonpathogenic and pathogenic F. oxysporum reduced each other's growth when grown together. However, in these coinoculation experiments, pathogenic F. oxysporum WCS816 was relatively more inhibited in its growth than nonpathogenic F. oxysporum Fo47b10. The antagonism of nonpathogenic F. oxysporum against pathogenic F. oxysporum strongly depends on the ratio of nonpathogenic to pathogenic F. oxysporum densities: the higher this ratio, the stronger the antagonism. This fungal antagonism appears to be mainly associated with the competition for glucose. Pseudobactin 358 reduced the growth of both F. oxysporum strains, whereas ferric pseudobactin 358 did not; antagonism by pseudobactin 358 was then related to competition for iron. However, the pathogenic F. oxysporum strain was more sensitive to this antagonism than the nonpathogenic strain. Pseudobactin 358 reduced the efficiency of glucose metabolism by the fungi. These results suggest that pseudobactin 358 increases the intensity of the antagonism of nonpathogenic F. oxysporum Fo47b10 against pathogenic F. oxysporum WCS816 by making WCS816 more sensitive to the glucose competition by Fo47b10.     

Purification and characterization of tomatinase from Fusarium oxysporum f. sp. lycopersici.

The antifungal compound alpha-tomatine, present in tomato plants, has been reported to provide a preformed chemical barrier against phytopathogenic fungi. Fusarium oxysporum f. sp. lycopersici, a tomato pathogen, produces an extracellular enzyme inducible by alpha-tomatine. This enzyme, known as tomatinase, catalyzes the hydrolysis of alpha-tomatine into its nonfungitoxic forms, tomatidine and beta-lycotetraose. The maximal tomatinase activity in the fungal culture medium was observed after 48 h of incubation of germinated conidia at an alpha-tomatine concentration of 20 micrograms/ml. The enzymatic activity in the supernatant was concentrated against polyethylene glycol 35,000, and the enzyme was then purified to electrophoretic homogeneity by a procedure that includes preparative isoelectric focusing and preparative gel electrophoresis as main steps. The purification procedure had a yield of 18%, and the protein was purified about 40-fold. Tomatinase was found to be a monomer of 50 kDa by both native gel electrophoresis and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The analytical isoelectric focusing of the native tomatinase showed at least five isoforms with pIs ranging from 4.8 to 5.8. Treatment with N-glycosidase F gave a single protein band of 45 kDa, indicating that the 50-kDa protein was N glycosylated. Tomatinase activity was optimum at 45 to 50 degrees C and at pH 5.5 to 7. The enzyme was stable at acidic pH and temperatures below 50 degrees C. The enzyme had no apparent requirement for cofactors, although Co2+ and Mn2+ produced a slight stimulating effect on tomatinase activity. Kinetic experiments at 30 degrees C gave a K(m) of 1.1 mM for alpha-tomatine and a Vmax of 118 mumol/min/mg. An activation energy of 88 kJ/mol was calculated.     

Antifungal activity of Bacillus amyloliquefaciens NJN-6 volatile compounds against Fusarium oxysporum f. sp. cubense

Bacillus amyloliquefaciens NJN-6 produces volatile compounds (VOCs) that inhibit the growth and spore germination of Fusarium oxysporum f. sp. cubense. Among the total of 36 volatile compounds detected, 11 compounds completely inhibited fungal growth. The antifungal activity of these compounds suggested that VOCs can play important roles over short and long distances in the suppression of Fusarium oxysporum.     

Comparison of two fatty acid analysis protocols to characterize and differentiate Fusarium oxysporum f. sp. lycopersici and F. oxysporum f. sp. radicis-lycopersici

The utility of fatty acid methyl ester (FAME) profiles for characterization and differentiation of isolates of Fusarium oxysporum f. sp. lycopersici and F. oxysporum f. sp. radicis-lycopersici was investigated. Two fatty acid analysis protocols of the normal (MIDI) and a modified MIDI method were used for their utility. Only the modified MIDI method allowed a clear differentiation between F. oxysporum f. sp. lycopersici and F. oxysporum f. sp. radicislycopersici. FAME profiles using the modified MIDI method gave the most consistent and reproducible analyzed fatty acid data. Evaluation of the FAME profiles based on cluster analysis and principal-component analysis revealed that FAME profiles from tested isolates were correlated with the same vegetative compatibility groups (VCGs) compared to the same races in F. oxysporum f. sp. lycopersici. Results indicated that FAME profiles could be an additional tool useful for characterizing isolates and forma species of F. oxysporum obtained from tomato.     

利用荧光蛋白标记西瓜枯萎病菌的过氧化物酶体及细胞核

西瓜枯萎病是一种世界范围的西瓜毁灭性病害,其病原菌为尖孢镰刀菌西瓜专化型(Fusarium oxysporum f.sp.niveum,FON)。研究病原菌生长发育和侵染的机制是解决病害的根本途径。利用荧光蛋白对细胞或细胞器进行标记,是病原菌研究中的重要方法。该研究利用绿色荧光蛋白和红色荧光蛋白对FON的细胞核和过氧化物酶体进行了荧光标记。通过农杆菌介导转化(Agrobacterium tumefaciens-mediated transformation,AtMT),该文将3种不同的荧光定位载体分别导入FON,获得了细胞核红色荧光标记的转化子(潮霉素抗性,含mCherry-H2B融合蛋白),以及过氧化物酶体绿色(潮霉素抗性,含GFP-PTS1融合蛋白)和红色(潮霉素抗性,含DsRED-PTS1融合蛋白)荧光标记的转化子各1种。在标记细胞核的菌株中,菌丝、孢子都可见明亮、圆形的红色荧光点,荧光点与DAPI染色标记的细胞核区域完全重合。在过氧化物酶体标记的菌株中,菌丝、孢子中可见明亮的红色或绿色荧光成小点状分布,符合过氧化物酶体的分布特征,而且在脂类物质诱导的条件下,荧光点的数量明显增加。此外,该文还利用细胞壁荧光染色剂卡氏白对3种荧光蛋白标记菌株进行染色。结果显示,卡氏白染色产生的蓝色荧光与红、绿荧光蛋白的荧光在FON中互不干扰。转化子继代培养和初步分析表明,其表型与野生型无差异,菌株继代后荧光表达稳定、定位明显。该结果为进一步研究FON细胞器动态、生长发育与致病分子机制提供了方法和工具。     

香蕉枯萎菌基因组DNA提取方法的研究

以香蕉枯萎菌菌株为试验材料,在SDS～CTAB法和高盐沉淀法等基础上加以改进,对两种提纯香蕉枯萎菌基因组DNA的方法进行了比较研究。结果表明：高盐沉淀法是适合于香蕉枯萎菌基因组DNA提取的方法。该方法提取的DNA OD260／OD280的比值为1．841,DNA产量为0．81mgDNA／g菌丝体。基因组DNA经琼脂糖凝胶电泳得到一条带型较宽且清晰的DNA谱带,基本无DNA碎带;将提取的DNA直接用于PCR扩增,得到带多而且清晰、整齐、基本无拖尾的RAPD图谱。     

Inoculum sources of Fusarium oxysporum f.sp. asparagi in asparagus production

Fusarium oxysporum f.sp. asparagi (Foa) incites crown and root rot of asparagus which causes early decline of asparagus plantings. The aim of the present study was to identify the main inoculum sources of the pathogen in the Netherlands. As has been reported for foreign seed lots, Dutch seed lots can be infested with Foa at low levels. We found that seed infestation occurs mainly during the seed harvesting process through infested soil adhering to fallen berries. Soil samples from 59 fields without a history of asparagus growing and differing in their distance from asparagus plantings were tested for infestation with Foa, using a bioassay with asparagus as a bait plant. A high correlation was found between the incidence of infestation and proximity to asparagus fields; Foa was found in 69% of the samples from fresh fields in an asparagus production centre, and in only 6% of the samples from fields at a distance of 1 km and more from asparagus fields and outside a production centre. To evaluate planting material as an inoculum source of Foa, 49 lots of one-year-old crowns from 23 nurseries were collected and rated for disease symptoms. Infestation was found to be common with only two lots free of symptomatic plants. Most of the lots had more than 75% of symptomatic plants. Although most of the plants were infested, they showed only slight root rot symptoms. The procedure for production of Foa-free planting material is discussed. Persistence and infestation of asparagus root residues in former asparagus fields was assessed by retrieving the residues from eight former asparagus fields with an asparagus-free period of one to 25 years, and three fields with a standing asparagus crop. Even after an asparagus-free period of 25 yr asparagus root residues were retrieved from soil, although at low levels. Mean population densities of Fusarium spp. declined from 2 times 10⁶ to 1 times 10⁵ colony forming units g^_1 air-dry root tissue during the first 10 years and were still > 10⁴ c.f.u. g“¹ air-dry root tissue 20 to 25 yr after asparagus produced was stopped. The population was dominated by F. oxysporum. Eighty-three of the 112 isolates (74%) of F. oxysporum belonged to the forma specialis asparagi. The proportion of Foa in the population did not decrease in time. It was concluded that persistence of Foa in asparagus root residues is a major reason for its long-term survival.     

Anatomical and biochemical aspects of interaction between roots of chickpea and Fusarium oxysporum f. sp. ciceris race 2

Roots of the susceptible “JG-62” and resistant “WR-315” chickpeas (Cicer arietinum L.) were inoculated with a conidial suspension of Fusarium oxysporum f. sp. ciceris. Anatomical and biochemical studies were carried out in a time-course manner to elucidate the infection process and plant defence reactions. Scanning electron microscope images revealed fungal colonisation in the root hair region. Early occurrence of fungal biofilms associated with the infected “JG-62” root epidermis was also visualised. After 96 h of inoculation, a gradual accumulation of polysaccharide positive deposits was observed in the xylem vessels of the infected “JG-62” roots. Fungal mycelium was observed in the vessel lumen of infected “JG-62” after 22 days of inoculation. Due to fungal invasion during this period, some of the vessels also appeared collapsed in “JG-62”, whereas vessels in “WR-315” remained intact. The host plant defence responses specifically linked to the susceptible interactions were the induction of ascorbate peroxidase, guaiacol peroxidase and superoxide dismutase in roots and shoots.