Characterization of Fusarium oxysporum f.sp. cepae from Onion in Turkey Based on Vegetative Compatibility and rDNA RFLP Analysis

Seventy‐five isolates of Fusarium oxysporum f.sp. cepae, the causal agent of basal plate rot on onion, were obtained from seven provinces of Turkey. The isolates were characterized by vegetative compatibility grouping (VCGs) and restriction fragment length polymorphism (RFLP) analysis of the nuclear ribosomal DNA intergenic spacer region (IGS). Forty‐eight vegetative compatibility groups were found, each containing a single isolate. Only one isolate formed strong heterokaryons with the reference isolates of VCG 0423. Five isolates were heterokaryon self‐incompatible. Restriction fragment analysis with six different enzymes revealed 13 IGS types among 75 F. oxysporum isolates from Turkey as well as 16 reference isolates from Colorado, USA. The majority of single‐member VCGs produced identical RFLP banding patterns with minor deviations, considerably different from those of the reference VCG isolates. These results suggested that isolates of F. oxysporum f.sp. cepae in Turkey derived from distinct clonal lineages and mutations at one or more vegetative compatibility loci restrict heterokaryon formation.     

Identification of pathogenicity‐related genes in Fusarium oxysporum f. sp. cepae

Pathogenic isolates of Fusarium oxysporum, distinguished as formae speciales (f. spp.) on the basis of their host specificity, cause crown rots, root rots and vascular wilts on many important crops worldwide. Fusarium oxysporum f. sp. cepae (FOC) is particularly problematic to onion growers worldwide and is increasing in prevalence in the UK. We characterized 31 F. oxysporum isolates collected from UK onions using pathogenicity tests, sequencing of housekeeping genes and identification of effectors. In onion seedling and bulb tests, 21 isolates were pathogenic and 10 were non‐pathogenic. The molecular characterization of these isolates, and 21 additional isolates comprising other f. spp. and different Fusarium species, was carried out by sequencing three housekeeping genes. A concatenated tree separated the F. oxysporum isolates into six clades, but did not distinguish between pathogenic and non‐pathogenic isolates. Ten putative effectors were identified within FOC, including seven Secreted In Xylem (SIX) genes first reported in F. oxysporum f. sp. lycopersici. Two highly homologous proteins with signal peptides and RxLR motifs (CRX1/CRX2) and a gene with no previously characterized domains (C5) were also identified. The presence/absence of nine of these genes was strongly related to pathogenicity against onion and all were shown to be expressed in planta. Different SIX gene complements were identified in other f. spp., but none were identified in three other Fusarium species from onion. Although the FOC SIX genes had a high level of homology with other f. spp., there were clear differences in sequences which were unique to FOC, whereas CRX1 and C5 genes appear to be largely FOC specific.     

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.     

Pectic Enzyme Production by Fusarium oxysporum f. sp, ccpac: Induction by Cell Walls from Different Parts of Onion Bulbs at Different Growth Stages

Fusarium oxysporum f. sp. cepae produced significantly different amounts of pectic enzymes when grown on cell walls from morphologically different parts of onion bulbs. Cell walls from stem plate tissue of both tolerant and susceptible onion genotypes allowed a rapid and high production of both exo-polygalacturonase and endo-pectin-frans-eliminase. Bulb scale cell walls from susceptible genotypes induced synthesis of these enzymes at much lower rates and levels, whereas bulb scale cell walls from tolerant genotypes gave poor induction of pectic enzyme synthesis. Leaf sheath cell walls from both susceptible and tolerant genotypes were poor inducers of enzyme synthesis. Enzyme induction by cell walls from leaf sheaths and bulb scales of tolerant genotypes increased dramatically during ageing. Differences in pectic enzyme accumulation on cell walls were not related to fungal growth. These patterns of enzyme induction could help to explain susceptibility or tolerance of bulb scale and leaf sheath tissue of the different genotypes.     

Pectic activities from Fusarium oxysporum f. sp. melonis: Purification and characterization of an exopolygalacturonase

Abstract: Fusarium oxysporum f. sp. melonis produced an extracellular enzymic mixture with high pectic activities, (at least an exopolygalacturonase, an endopolygalacturonase and two lyases) in a medium with glucose and pectin as carbon sources. An exopolygalacturonase from this crude enzyme preparation was purified 23.8 times by Sephadex G-200 and ion-exchange HPLC. It had a K _m of 6 mM, a M _r of 58 000, a p I of 6.4, optimum pH of 5 and was stable in the 3.5–6.5 pH range. This enzyme preferentially hydrolysed polygalacturonic acid, showing only 5% activity on pectin, and did not exhibit the activity of an endoenzyme.     

Induction of defence-related enzymes in onion by combined application of fungal and bacterial biocontrol agents against Fusarium oxysporum f. sp. cepae

Basal rot disease of onion is a major problem in different onion growing regions of Tamil Nadu, India. Fungal and bacterial cultures were isolated and tested their efficiency against Fusarium oxysporum f. sp. cepae under in vitro conditions. Effective bacterial and fungal antagonists were tested alone and in combinations for the control of F. oxysporum f. sp. cepae in glasshouse experiments. Defence-related enzymes such as peroxidase, polyphenol oxidase and phenylalanine ammonia-lyase were induced and accumulated in onion treated with fungal and bacterial antagonists. Defence-related enzymes were significantly higher in onion pretreated with consortial formulation of Pf12 + Pf27 + TH3 at 5 days after the challenge inoculation with F. oxysporum f. sp. cepae and gave resistance to onion against basal rot disease.     

Genetic and virulence variation in Fusarium oxysporum f. sp. cepae causing root and basal rot of common onion in Iran

Root and basal rot of common onion (Allium cepae L.) caused by Fusarium oxysporum f. sp. cepae is one of the most important diseases causing tremendous losses in onion‐growing areas worldwide. In this study, random amplified polymorphic DNA (RAPD), intersimple sequence repeats (ISSR) and virulence studies were conducted to analyse 26 F. oxysporum f. sp. cepae isolates obtained from the main onion‐growing regions of Iran, including Fars, Azerbaijan and Isfahan states. Cluster analysis using UPGMA method for both RAPD and ISSR markers revealed no clear grouping of the isolates obtained from different geographical regions, and the isolates were observed to derive probably from the same clonal lineage. Pathogenicity test indicated that all F. oxysporum f. sp. cepae isolates were pathogenic on onion; however, virulence variability was observed among the isolates. The grouping based on virulence variability was not correlated with the results of RAPD and ISSR analyses.     

Production of monoclonal antibodies to Fusarium oxysporum f.sp. cubense race 4

The production of monoclonal antibodies (mab) to Fusarium oxysporum f.sp. cubense (Foc ) race 4 is described. Heat-killed conidia of this fungus were toxic to female Balb/c mice, but this toxic reaction was not found with fractionated hyphal walls. A simple and reproducible enzyme immunoassay using a standard 9 cm polystyrene Petri dish as a solid phase was devised for screening culture supernatant fluids. Sixteen stable hybridoma clones secreting mabs of the IgM class were isolated by fusing splenic lymphocytes from immunized female Balb/c mice with P3-NSl-Ag4-l mouse myeloma cells. Monoclonal antibodies produced by eight of the 16 hybridoma clones were selected and the specificity of the mabs was determined by an indirect immunofluorescence test. Of the eight mabs, only one displayed an exceptionally high degree of specificity to the thick-walled chlamydospores of Foc race 4. This specific reactivity allowed differentiation of Foc race 4 from other races.     

Mineral Uptake in Fusarium oxysporum f. sp. vasinfectum

A method for growing Fusarium oxysporum, a mycelial fungus, and a technique for its use in mineral uptake studies have been described. Some general characteristics of the uptake process were determined. The fungus, grown for 54 hours, was found to take up as much K as 15 to 20 meq/100 g dry weight in 2 to 4 hours from a solution of 5 meq/l KCl. Approximately 3 to 5 meq of this uptake was readily removed by a CaCl₂ rinse. The uptake was only slightly sensitive to pH over the range of 4 to 9. Below pH 4 uptake dropped rapidly. The age of the culture appeared to be the dominant factor in determining the rate of uptake. In contrast to other fungi, the presence of glucose during uptake was detrimental to K uptake. Conditions unfavorable for metabolic activity as low temperature, anaerobiosis, or the presence of DNP markedly reduced the uptake rate. Although the fungus took up Na from single salt solutions nearly as well as K, the latter ion was much preferred in mixtures of the two ions. The organism showed no significant metabolic uptake of Ca or Cl. During uptake from KCl solutions, the organic acid content increased. The increase, chiefly in succinic acid and to a lesser extent in acetic and citric acids, amounted to about half the K uptake. The remainder of the K taken up was correlated with a roughly equivalent efflux of cellular Mg.     

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.     

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.     

Analysis of genetic variability of Fusarium oxysporum f. sp. cepae the causal agent of basal rot on onion using RAPD markers

Genetic variability among isolates of Fusarium oxysporum f. sp. cepae was obtained from different onion-growing areas of Tamil Nadu, India. Random amplified polymorphic DNA (RAPD) analysis was carried out using 12 random primers, each of them consisting of 10 base pairs. Four out of the 12 primers were differentiated between some of the tested F. oxysporum f. sp. cepae isolates. Analysis of the genetic coefficient matrix derived from the scores of RAPD profile showed that minimum and maximum per cent similarities among the F. oxysporum f. sp. cepae isolates were in the range of 14–85%. Cluster analysis, using the unweighted pair-group method with arithmetic average, clearly separated the isolates into two clusters (A and B) confirming the genetic diversity among the isolates of F. oxysporum f. sp. cepae from onion.     

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。     

Biological Control of Fusarium oxysporum f.sp. asparagi by Applying Non-pathogenic Isolates of F . oxysporum

Root rot severity of asparagus plants grown in sterilized field soil inoculated with Fusarium oxysporum f . sp . asparagi (Foa) was reduced by more than 50% when the soil was precolo nized by each of 13 non - pathogenic (np) isolates of F. oxysporum originating from asparagus roots or field soils . In a greenhouse experiment , application of six np isolates to naturally infested field soil was followed by a 23 - 49% decrease of disease severity , depending on the isolate . One of them , Fo47 originating from Fusarium suppressive soil in France , was applied to field plots infested with Foa . Foa root rot was not suppressed in asparagus plants grown for 1 year in these plots . Pathogenic and np isolates extensively colonized the root surface and isolates of both types infected the roots of asparagus plants grown in sterilized field soil , with significant differences among the np isolates . Inoculation of sterilized field soil with np isolates reduced germination of Foa chlamydospores by 43 - 64% depending on the isolate used . It is concluded that np isolates of F. oxysporum can suppress asparagus root rot caused by Foa in naturally infested field soil . The differences for root colonization capacity among the np isolates imply that selection for this trait might reveal isolates that perform better under field conditions .     

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.