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.     

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.     

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.     

Mutation of an arginine biosynthesis gene causes reduced pathogenicity in Fusarium oxysporum f. sp. melonis

Restriction enzyme-mediated integration (REMI) mutagenesis was used to tag genes required for pathogenicity of Fusarium oxysporum f. sp. melonis. Of the 1,129 REMI transformants tested, 13 showed reduced pathogenicity on susceptible melon cultivars. One of the mutants, FMMP95-1, was an arginine auxotroph. Structural analysis of the tagged site in FMMP95-1 identified a gene, designated ARG1, which possibly encodes argininosuccinate lyase, catalyzing the last step for arginine biosynthesis. Complementation of FMMP95-1 with the ARG1 gene caused a recovery in pathogenicity, indicating that arginine auxotrophic mutation causes reduced pathogenicity in this pathogen.     

Race Distribution, Vegetative Compatibility and Pathogenicity of Fusarium oxysporum f. sp. melonis Isolates in Greece

Races and vegetative compatibility groups (VCGs) in Greek isolates of Fusarium oxysporum f. sp. melonis(Fom) were characterized. Three races (0, 2 and 1–2) among 12 isolates tested and two VCGs among 19 isolates tested, were identified. Race 1–2 was the most common and race 1 was not detected. One widespread VCG corresponded to a VCG previously reported from Israel (coded 0138), and included seven isolates of races 0 and 1–2. The other VCG, which was unclassified, included four isolates of races 0, 2 and 1–2. The latter VCG was detected only in a specific melon‐growing location of Evros. The remaining eight isolates tested for VCG did not show positive reactions with other isolates, with each other or with the testers of VCGs 0135 or 0138, although they produced complementary mutants. Using two inoculation methods, the local cv. ‘Golden Head’ was found susceptible to all known Fom races, and especially to race 1–2. These results show the presence of more than one VCG and the widespread distribution of the race 1–2, in Greece.     

Purification and characterization of a β-glucosidase from the phytopathogenic fungus Fusarium oxysporum f. sp. melonis

T.M. ALCONADA AND M.J. MARTÍNEZ. 1996. Fusarium oxysporum f. sp. melonis produces cellulase and β-glucosidase activities in a medium with glucose and avicel as carbon source. A β-glucosidase from this crude material was purified by gel filtration and ion exchange chromatography successively. This enzyme is a unique band of protein in SDS-PAGE and isoelectric focussing. It had a molecular weight of 66000 and a pI of 5. Using p -nitrophenyl-β-D-glucopyranoside as substrate β-glucosidase shows a K _m of 210 μmol 1^-1, an optimum pH of 5.5 and an optimum reaction temperature of 60°C, being stable in a pH range of 5–7 for 48 h at room temperature.     

Molecular detection of Fusarium oxysporum f. sp. niveum and Mycosphaerella melonis in infected plant tissues and soil

We developed two species-specific PCR assays for rapid and accurate detection of the pathogenic fungi Fusarium oxysporum f. sp. niveum and Mycosphaerella melonis in diseased plant tissues and soil. Based on differences in internal transcribed spacer (ITS) sequences of Fusarium spp. and Mycosphaerella spp., two pairs of species-specific primers, Fn-1/Fn-2 and Mn-1/Mn-2, were synthesized. After screening 24 isolates of F. oxysporum f. sp. niveum, 22 isolates of M. melonis, and 72 isolates from the Ascomycota, Basidiomycota, Deuteromycota, and Oomycota, the Fn-1/Fn-2 primers amplified only a single PCR band of approximately 320 bp from F. oxysporum f. sp.niveum, and the Mn-1/Mn-2 primers yielded a PCR product of approximately 420 bp from M. melonis. The detection sensitivity with primers Fn-1/Fn-2 and Mn-1/Mn-2 was 1fg of genomic DNA. Using ITS1/ITS4 as the first-round primers, combined with either Fn-1/Fn-2 and or Mn-1/Mn-2, two nested PCR procedures were developed, and the detection sensitivity increased 1000-fold to 1ag. The detection sensitivity for the soil pathogens was 100-microconidia/g soil. A duplex PCR method, combining primers Fn-1/Fn-2 and Mn-1/Mn-2, was used to detect F. oxysporum f. sp. niveum and M. melonis in plant tissues infected by the pathogens. Real-time fluorescent quantitative PCR assays were developed to detect and monitor the pathogens directly in soil samples. The PCR-based methods developed here could simplify both plant disease diagnosis and pathogen monitoring as well as guide plant disease management.     

Immunolocalization of a class III chitinase in two muskmelon cultivars reacting differently to Fusarium oxysporum f. sp. melonis

Fusarium oxysporum f. sp. melonis is a highly specialized fungus that attacks the root system of melon (Cucumis melo L.). In this work the presence of a class III chitinase was examined by immunological techniques in the root and stem base of a susceptible (cv. Galia) and a resistant (cv. Bredor) melon during the infection process. By immunolocalization it was not possible to detect the constitutive presence of class III chitinase in any of the cultivars. However, the immunolabelling appeared in the root tissues of both cultivars as a consequence of wounding and of infection by F. oxysporum f. sp. melonis. Distinct patterns of chitinase detection were observed in the roots of the two cultivars as the infection progressed. Furthermore, by western blotting distinct class III chitinase isoforms were detected, which responded differently to the F. oxysporum f. sp. melonis infection. Our results strongly indicate that a relationship exists between class III chitinase and melon resistance to Fusarium infection, and that the resistance is associated with certain isoforms of this enzyme.     

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.     

Genetic Diversity and Pathogenicity of Fusarium oxysporum f.sp. melonis Races from Different Areas of Italy

Fusarium wilt caused by Fusarium oxysporum f.sp. melonis (FOM) is a devastating disease of melon worldwide. Pathogenicity tests performed with F. oxysporum isolates obtained from Italian melon‐growing areas allowed to identify thirty‐four FOM isolates and the presence of all four races. The aims of this work were to examine genetic relatedness among FOM isolates by race determination and to perform phylogenetic analyses of identified FOM races including also other formae speciales of F. oxysporum of cucurbits. Results showed that FOM race 1,2 was the most numerous with a total of eighteen isolates, while six and nine isolates were identified as race 0 and 1, respectively, and just one isolate was assigned to race 2. Phylogenetic analysis was performed by random amplified polymorphic DNA (RAPD) profiling and by translation elongation factor‐1α (TEF‐1α) sequencing. The analysis of RAPD profiles separated FOM races into two distinct clades. Clade 1, which included races 0, 1 and 1,2, was further divided into ‘subclade a’ which grouped almost all race 1,2 isolates, and into ‘subclade b’ which included race 0 and 1 isolates. Clade 2 comprised only race 2 isolates. The phylogenetic analysis based on TEF‐1α separated FOM from the other formae speciales of F. oxysporum. Also with TEF‐1α analysis, FOM races 0, 1 and 1,2 isolates grouped in one single clade clearly separated from FOM race 2 isolates which grouped closer to F. oxysporum f.sp. cucumerinum. RAPD technique was more effective than TEF‐1α in differentiating FOM race 1,2 isolates from those belonging to the closely related races 0 and 1. Both phylogenetic analyses supported the close relationship between the three different FOM races which might imply the derivation from one another and the different origin of FOM race 2.     

Purification and characterization of an extracellular endo-1,4-β-xylanase from Fusarium oxysporum f. sp. melonis

Abstract Fusarium oxysporum f. sp. melonis produces extracellular endo-1,4-β-xylanase and β-xylosidase when grown in shaken culture at 26°C in a mineral salts medium containing oat spelt xylan and glucose as carbon sources. Endo-1,4-β-xylanase was purified 251 times from 5-day-old culture filtrates, by Sephacryl S-200, ion exchange and gel filtration HPLC. The purified sample yielded a single band in SDS polyacrylamide gels with a molecular mass of 80 kDa on electrophoretic mobility and 83 kDa by gel filtration behavior. High activity of the endo-1,4-β-xylanase against xylan was observed between 5 and 8 pH, and between 40 and 60°C, the optimum pH and temperature being 5.0 and 50°C, respectively. Kinetic properties of the enzyme are similar to those of other fungal xylanases, showing high affinity towards oat spelt xylan with a K _m of 1 mM expressed as xylose equivalent.     

Molecular differences distinguish clonal lineages within East African populations of Fusarium oxysporum f.sp. cubense

Molecular approaches for the assessment of intraspecific diversity within an economically important plant pathogen were compared with traditional physiological methods (vegetative compatibility testing). The vegetative compatibility groups (VCGs) of 14 isolates of Fusarium oxysporum f.sp. cubense (FOC) from Kenya were first assessed using nitrate non-utilizing mutants. Nine of these isolates, from different areas of the country, were compatible with one or more of VCGs 0124, 0125, 0128 and 01220, i.e. they formed a single clonal lineage. Three isolates, all originating from the banana growing district of Kisii, were compatible with the VCG 01212 and formed a second distinct clonal lineage. Mutants could not be recovered from one isolate (62) and two isolates (27 and 30) were not vegetatively compatible with any of the VCG testers and may represent two novel VCGs. Polymerase chain reaction (PCR) fingerprinting, especially when using the M13 derived primer, was found to produce banding patterns that correlated with clonal lineage and also distinguished isolates 27 and 30 when analysed by unweighted pair group method analysis and principle co-ordinate analysis. This approach also distinguished FOC from F. oxysporum IMI350438 isolated from Triticum sp. and from isolates of Colletotrichum gloeosporioides . Total protein profiles were analysed by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and although clonal lineages were not separated, isolates 27 and 30 were again distinguishable and FOC produced a different profile to F. oxysporum (IMI 350438) and C. gloeosporioides.     

Tomatinase from Fusarium oxysporum f. sp. lycopersici defines a new class of saponinases.

Plants produce a variety of secondary metabolites, many of which have antifungal activity. Saponins are plant glycosides that may provide a preformed chemical barrier against phytopathogenic fungi. Fusarium oxysporum f. sp. lycopersici and other tomato pathogens produce extracellular enzymes known as tomatinases, which deglycosylate alpha-tomatine to yield less toxic derivatives. We have cloned and characterized the cDNA and genomic DNA encoding tomatinase from the vascular pathogen of tomato F. oxysporum f. sp. lycopersici. This gene encodes a protein (FoTom1) with no amino acid sequence homology to any previously described saponinase, including tomatinase from Septoria lycopersici. Although FoTom1 is related to family 10 glycosyl hydrolases, which include mainly xylanases, it has no detectable xylanase activity. We have overexpressed and purified the protein with a bacterial heterologous system. The purified enzyme is active and cleaves alpha-tomatine into the less toxic compounds tomatidine and lycotetraose. Tomatinase from F. oxysporum f. sp. lycopersici is encoded by a single gene whose expression is induced by alpha-tomatine. This expression is fully repressed in the presence of glucose, which is consistent with the presence of two putative CREA binding sites in the promoter region of the tomatinase gene. The tomatinase gene is expressed in planta in both roots and stems throughout the entire disease cycle of F. oxysporum f. sp. lycopersici.     

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.     

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。