Characterization of the gene encoding pisatin demethylase (FoPDA1) in Fusarium oxysporum

The pea pathogen Fusarium oxysporum f. sp. pisi is able to detoxify pisatin produced as a defense response by pea, and the gene encoding this detoxification mechanism, FoPDA1, was 82% identical to the cytochrome P450 pisatin demethylase PDA1 gene in Nectria haematococca. A survey of F. oxysporum f. sp. pisi isolates demonstrated that, as in N. haematococca, the PDA gene of F. oxysporum f. sp. pisi is generally located on a small chromosome. In N. haematococca, PDA1 is in a cluster of pea pathogenicity (PEP) genes. Homologs of these PEP genes also were found in the F. oxysporum f. sp. pisi isolates, and PEP1 and PEP5 were sometimes located on the same small chromosomes as the FoPDA1 homologs. Transforming FoPDA1 into a pda(?) F. oxysporum f. sp. lini isolate conferred pda activity and promoted pathogenicity on pea to some transformants. Different hybridization patterns of FoPDA1 were found in F. oxysporum f. sp. pisi but these did not correlate with the races of the fungus, suggesting that races within this forma specialis arose independently of FoPDA1. FoPDA1 also was present in the formae speciales lini, glycines, and dianthi of F. oxysporum but they had mutations resulting in nonfunctional proteins. However, an active FoPDA1 was present in F. oxysporum f. sp. phaseoli and it was virulent on pea. Despite their evolutionary distance, the amino acid sequences of FoPDA1 of F. oxysporum f. sp. pisi and F. oxysporum f. sp. phaseoli revealed only six amino acid differences, consistent with a horizontal gene transfer event accounting for the origin of these genes.     

FsFKS1, the 1,3-beta-glucan synthase from the caspofungin-resistant fungus Fusarium solani

The cell wall, a mesh of carbohydrates and proteins, shapes and protects the fungal cell. The enzyme responsible for the synthesis of one of the main components of the fungal wall, 1,3-beta-glucan synthase, is targeted by the antifungal caspofungin acetate (CFA). Clinical isolates of Candida albicans and Aspergillus fumigatus are much more sensitive to CFA than clinical isolates of Fusarium species. To better understand CFA resistance in Fusarium species, we cloned and sequenced FsFKS1, which encodes the Fusarium solani f. sp. pisi beta(1,3)-D-glucan synthase, used RNA interference to reduce its expression and complemented deletion of the essential fks gene of the CFA-sensitive fungus A. fumigatus with FsFKS1. Reduction of the FsFKS1 message in F. solani f. sp. pisi reduced spore viability and caused lysis of spores and hyphae, consistent with cell wall defects. Compensating for the loss of A. fumigatus fks1 with FsFKS1 caused only a modest increase in the tolerance of A. fumigatus for CFA. Our results suggest that FsFKS1 is required for the proper construction of F. solani cell walls and that the resistance of F. solani to CFA is at best only partially due to resistance of the FsFKS1 enzyme to this antifungal agent.     

sti35, a stress-responsive gene in Fusarium spp.

A stress-induced mRNA was identified in the phytopathogenic fungus Fusarium oxysporum f. sp. cucumerinum. Treatment of the fungus with ethanol resulted in the induction of a major mRNA species encoding a protein of approximate Mr 37,000. A full-length cDNA clone of the induced message was obtained. RNA blot analysis indicated that the mRNA was induced by various other stresses, including treatment with copper(II) chloride and heat (37 degrees C). However, it was not greatly induced by treatment with phaseollinisoflavan, an antifungal isoflavonoid produced by Phaseolus vulgaris (French bean). In contrast, phaseollinisoflavan induced the homologous mRNA in the related bean pathogen Fusarium solani f. sp. phaseoli. A genomic clone of the F. solani f. sp. phaseoli gene was obtained, and both this and the cDNA clone from F. oxysporum f. sp. cucumerinum were sequenced. The latter indicated an open reading frame of 320 codons encoding a 34,556-dalton polypeptide. The corresponding reading frame in F. solani f. sp. phaseoli was 324 codons, 89% identical to the F. oxysporum f. sp. cucumerium sequence, and was interrupted by a short intron. The gene was designated sti35 (stress-inducible mRNA). Although computer homology searches were negative, the cloned gene was observed to cross-hybridize to DNAs of other filamentous fungi, Saccharomyces cerevisiae, and soybean. Thus, sti35 appears to be a common gene among a variety of eucaryotes.     

Morphological and Molecular Characterization of Fusarium solani Isolates

Fusarium solani is a species complex (FSSC) containing isolates that cause diseases in important crops such as root and fruit rot of Cucurbita spp., root and stem rot of pea, sudden death syndrome of soybean, foot rot of bean and dry rot of potato tubers during storage. Based on host range tests, F. solani were subdivided into different formae specialis (f. sp.) and varieties, while DNA sequences of 28S rDNA, internally transcribed spacers (ITS) rDNA and elongation factor (EF-1α) distinguished the ' F. solani complex' in 50 subspecific lineages. In this study we characterized, by cultural, morphological and molecular criteria, 34 isolates of F. solani obtained from potato, other crops and soil. The 34 isolates in the FSSC showed wide variability for their cultural, morphological and molecular traits. The wide variability observed with amplified fragment-length polymorphism (AFLP) and mini-microsatellite analyses is in agreement with the polymorphism observed, in a previous study, within FSSC. Nine of 34 isolates in the FSSC, classified as F. solani var. coeruleum , were morphologically distinguishable from the other F. solani isolates but they were distributed in different clusters; moreover, the nine isolates showed instability of the coeruleum pigmentation of the colonies, supporting the ambiguity of the taxa of this variety of F. solani. Using sequence data from ITS plus 5.8S rDNA region, the isolates were classified into different clades. In particular eight isolates were classified into a well-supported clade including F. solani f. sp . pisi , nine into a clade including only isolates of F. solani f. sp . radicicola and four into a clade including F. solani f. sp . cucurbitae , but this classification could not be used if is not in agreement with host specificity. Two of the nine F. solani var. coeruleum isolates were phylogenetically distinct from all the other FSSC strains.     

Genome-Wide Comparative Analysis of pogo-Like Transposable Elements in Different Fusarium Species

The recent availability of genome sequences of four different Fusarium species offers the opportunity to perform extensive comparative analyses, in particular of repeated sequences. In a recent work, the overall content of such sequences in the genomes of three phylogenetically related Fusarium species, F. graminearum, F. verticillioides, and F. oxysporum f. sp. lycopersici has been estimated. In this study, we present an exhaustive characterization of pogo-like elements, named Fots, in four Fusarium genomes. Overall 10 Fot and two Fot-related miniature inverted-repeat transposable element families were identified, revealing a diversification of multiple lineages of pogo-like elements, some of which accompanied by a gain of introns. This analysis also showed that such elements are present in an unusual high proportion in the genomes of F. oxysporum f. sp. lycopersici and Nectria haematococca (anamorph F. solani f. sp. pisi) in contrast with most other fungal genomes in which retroelements are the most represented. Interestingly, our analysis showed that the most numerous Fot families all contain potentially active or mobilisable copies, thus conferring a mutagenic potential of these transposable elements and consequently a role in strain adaptation and genome evolution. This role is strongly reinforced when examining their genomic distribution which is clearly biased with a high proportion (more than 80%) located on strain- or species-specific regions enriched in genes involved in pathogenicity and/or adaptation. Finally, the different reproductive characteristics of the four Fusarium species allowed us to investigate the impact of the process of repeat-induced point mutations on the expansion and diversification of Fot elements.     

Induction of cutinolytic esterase activity during saprophytic growth of cucurbit pathogens, Fusarium solani f. sp. cucurbitae races one and two (Nectria haematococca MPI and MPV, respectively)

Cutins from fruit of Cucurbita maxima and Cucurbita moschata cultivars, apple and a C(16) alcohol (hexadecanol) were used to induce cutinolytic esterase activity during saprophytic growth of strains of the two cucurbit pathogens, Fusarium solani f. sp. cucurbitae, race 1 (Nectria haematococca mating population (MPI) and F. solani f. sp. cucurbitae, race 2 (MPV). Four strains of MPV and 11 strains of MPI were were included in the study. Although we were primarily interested in the two cucurbit pathogens (MPI and MPV), six strains of the pea pathogen F. solani f. sp. pisi (MPVI) were included to provide a comparison since most of the knowledge on cutinase activity in N. haematococca has come from a study of that group. Cutinolytic esterase was induced in all strains from both MPV and MPVI but was not detected in any of the 11 strains from MPI regardless of the induction conditions. The amount of cutinolytic esterase activity induced in the MPV strains differed according to the strain and both the source and the amount of cutin used in the induction medium. Information on the influence of cutin source and pH on the induction of cutinolytic esterase activity during saprophytic growth of strains from MPV demonstrates that the gene is regulated differently from that in MPVI.     

Induced Resistance in Cucumbers against Fusarium oxysporum f. sp. cucumerinum and Rhizoctonia solani AG2–2 by Infection of the Cotyledons

Localized infection in cucumber cotyledons with Colletotrichum lagenarium induced resistance against infection after challenge inoculation with Rhizoctonia solani AG2–2 and Fusarium oxysporum f. sp. cucumerinum in the roots. The plants were unprotected in soil that was infested heavily with R. solani or in contact with the mycelium, and induced resistance was not observed. Wounding of the root also negated the effect of induced resistance to F. oxysporum .     

Effects of Light and of Fusarium solani on Synthesis and Activity of Phenylalanine Ammonia-Lyase in Peas

Phenylalanine ammonia-lyase was purified from peas, and a specific antiserum against the enzyme was produced in rabbits. The antiserum was used to study the first 8 hours of the phenylalanine ammonia-lyase activity response in two different organs of the pea from different developmental stages and in response to two different stimuli. Etiolated seedlings were pulse-labeled with l-[(35)S]methionine after either no light exposure or after specific periods of irradiation with blue light. Immature pods were pulse labeled with mixed l-[(3)H]amino acids after specific time periods following inoculation of the pod endocarp surfaces with macroconidia of Fusarium solani. Immunoprecipitates isolated from extracts of each group were analyzed with sodium dodecyl sulfate disc gel electrophoresis and were found to contain a radioactive protein with an electrophoretic mobility identical to that of the phenylalanine ammonia-lyase subunit (M(r) 81,000). The radioactivity contained in the subunit band was interpreted as being due to de novo synthesis of the enzyme. The net rate of phenylalanine ammonia-lyase labeling, found to be initially low in both tissue types, rose dramatically, peaking at approximately a six- to ten-fold greater level at 4 hours after the beginning of the stimulus. Thereafter, the rate of labeling declined slowly. Inoculation with F. solani f. sp. pisi, a true pathogen of peas, caused a fifty per cent greater rate of peak labeling than did inoculation with a nonpathogen, F. solani f. sp. phaseoli. The time profile of the changing rate of labeling correlates with the changing activity level of the enzyme which peaks at 12 hours after the onset of the stimulus. The data presented favor a model which explains the changing activity of phenylalanine ammonia-lyase as being due to a changing rate of synthesis or degradation (or both) of the enzyme rather than due to the activation of a preformed zymogen.     

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.     

A robust identification and detection assay to discriminate the cucumber pathogens Fusarium oxysporum f. sp. cucumerinum and f. sp. radicis-cucumerinum

The fungal species Fusarium oxysporum is a ubiquitous inhabitant of soils worldwide that includes pathogenic as well as non-pathogenic or even beneficial strains. Pathogenic strains are characterized by a high degree of host specificity and strains that infect the same host range are organized in so-called formae speciales. Strains for which no host plant has been identified are believed to be non-pathogenic strains. Therefore, identification below the species level is highly desired. However, the genetic basis of host specificity and virulence in F. oxysporum is so far unknown. In this study, a robust random-amplified polymorphic DNA (RAPD) marker-based assay was developed to specifically detect and identify the economically important cucumber pathogens F. oxysporum f. sp. cucumerinum and F. oxysporum f. sp. radicis-cucumerinum. While the F. oxysporum radicis-cucumerinum strains were found to cluster in a separate clade based on elongation factor-1alpha phylogeny, strains belonging to F. oxysporum f. sp. cucumerinum were found to be genetically more diverse. This is reflected in the observation that specificity testing of the identified markers using a broad collection of F. oxysporum strains with all known vegetative compatibility groups of the target formae speciales, as well as representative strains belonging to other formae speciales, resulted in two cross-reactions for the F. oxysporum f. sp. cucumerimum marker. However, no cross-reactions were observed for the F. oxysporum f. sp. radicis-cucumerimum marker. This F. oxysporum f. sp. radicis-cucumerimum marker shows homology to Folyt1, a transposable element identified in the tomato pathogen F. oxysporum f. sp. lycopersici and may possibly play a role in host-range specificity in the target forma specialis. The markers were implemented in a DNA array that enabled parallel and sensitive detection and identification of the pathogens in complex samples from diverse origins.     

AM真菌和镰刀菌对西瓜根系几种酶活性的影响*

在温室盆栽条件下研究了丛枝菌根(Arbuscular Mycorrhiza, AM)真菌Glomus versiforme和西瓜枯萎镰刀菌Fusarium oxysporum f.sp. niveum对西瓜根系中过氧化物酶(POD)、苯丙氨酸解氨酶(PAL)、β-1,3-葡聚糖酶和几丁质酶活性的影响。结果表明,接种AM真菌的西瓜根系中4种酶的活性均高于对照,先接种G. versiforme,后接种F. oxysporum f.sp. niveum处理的4种酶的活性均高于只接种F. oxysporum f.sp. niveum 的处理,且酶的活性峰值出现较早。表明接种G. versiforme 能预先诱导这4种酶的产生,提高其活性,从而提高西瓜对F. oxysporum f.sp. niveum侵染的抗性。接种G. versiforme的感枯萎病西瓜品种“郑杂5号”酶的增加幅度大于抗病品种“京欣1号”的接种处理,说明G. versiforme对提高感病西瓜品种酶活性的作用更大。     

Cutinase gene disruption in Fusarium solani f sp pisi decreases its virulence on pea.

Fusarium solani f sp pisi (Nectria haematococca) isolate 77-2-3 with one cutinase gene produced 10 to 20% of the cutinase produced by isolate T-8 that has multiple cutinase genes, whereas cutinase gene-disrupted mutant 77-102 of isolate 77-2-3 did not produce cutinase. On the surface of pea stem segments, lesion formation was most frequent and most severe with T-8, less frequent and less severe with 77-2-3, and much less frequent and much milder with the gene-disrupted mutant. Microscopic examination of the lesions caused by the mutant strongly suggest that it penetrated the host mostly via the stomata. In seedling assays, 77-2-3 caused severe lesions on every seedling and stunted growth, whereas the mutant showed very mild lesions on one-third of the seedlings with no stunting. Thus, cutinase gene disruption resulted in a significant decrease in the pathogenicity of F. s. pisi on pea.     

香蕉上的镰孢菌种类及其系统发育关系(英文)

镰孢菌属真菌是香蕉上的重要病原菌,主要引起香蕉枯萎病以及香蕉冠腐病,在我国已明确引起香蕉枯萎病的病原为尖孢镰孢古巴专化型 Fusarium oxysporum f. sp. cubense(FOC)1号和4号生理小种,但是引起香蕉冠腐病的镰孢菌种类还未明确。为了解香蕉上镰孢菌在种间及种内水平上的多样性,2008–2011 年间作者从华南地区不同的水果市场及香蕉果园采集香蕉样品90份,分离得到143株镰孢菌。通过形态学观察及基于 EF-1α基因的系统进化分析鉴定出10种镰孢菌,即F. oxysporum、F. solani、F. camptoceras、F. pallidoroseum、F. stiloides、F. chlamydosporum、F.verticillioides、F. proliferatum、F. concentricum、F. sacchari,以及藤仓赤霉复合种(Gibberella fujikuroi species complex,GFC)中 3 个未定名的类群。轮纹镰孢 F. concentricum 及甘蔗镰孢 F.sacchari 是香蕉果实中最常见种,前菌为我国首次报道,后菌是首次报道与香蕉有关。对从香蕉上分离的藤仓赤霉复合种(GFC)及尖孢镰孢复合种(FOSC)的EF-1α序列进行了系统发育分析,其GFC中的27个菌株组成的单系群可分为7个不同的亚群,分别为 F.verticillioides、F. proliferatum、F. concentricum、F. sacchari 以及3个没有描述过的菌系 Fusarium sp. 1、Fusarium sp.2和 Fusarium sp.3;FOSC中的50个菌株形成2大分枝共12个谱系,分离自我国华南地区的21株尖孢镰孢形成7个谱系,其中 13株已知的香蕉枯萎病病原菌分布在3个谱系中,我国大陆的香蕉枯萎病病原菌菌株与来源于台湾地区及东南亚的菌株亲缘关系较近,FOC1号生理小种的遗传分化大于4号生理小种,FOC 1号生理小种与分离自香蕉果实上的尖孢镰孢菌的亲缘关系比与FOC 4号生理小种的亲缘关系更近。研究结果表明,我国香蕉上存在着丰富的镰孢菌种类,而且种内遗传多样性丰富。     

Effect of dsRNA-containing and dsRNA-free hypovirulent isolates of Fusarium oxysporum on severity of Fusarium seedling disease of soybean in naturally infested soil

In the sandy soils of eastern Virginia, soybean seedlings are colonized by hypovirulent and virulent isolates of Fusarium oxysporum and F. solani. Our objectives were to determine if prior inoculation of soybean seeds with hypovirulent F. oxysporum isolates reduced severity of seedling disease in naturally infested soil, and to determine if there was an association between the presence of dsRNA mycovirus and hypovirulence in isolates of F. oxysporum and F. solani from soybean plants. The presence of dsRNA was not associated with hypovirulence in F. oxysporum since some hypovirulent isolates contained dsRNA while other hypovirulent isolates did not. Furthermore, of six dsRNA-containing F. oxysporum isolates, three were hypovirulent and three were virulent. Four segments of dsRNA, with sizes of 4.0, 3.1, 2.7 and 2.2 kb were detected in extracts of all six F. oxysporum isolates. No hypovirulent or dsRNA-containing of F. solani isolates were found. Prior inoculation of cv. Essex soybean seeds with conidia of dsRNA-free hypovirulent F. oxysporum isolates significantly (P < 0.05) reduced disease severity on cotyledons and hypocotyls, and increased the rate of seedling emergence in field soil, compared to control plants. No significant (P > 0.05) differences were found between dsRNA-containing and dsRNA-free hypovirulent F. oxysporum isolates in their effects on reducing disease severity. Hypovirulent isolates that colonize soybean tissues may play a role in reducing Fusarium seedling disease of soybean in natural soils.     

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.     

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.     

The mitochondrial genome of Fusarium oxysporum

Physical and genetic maps have been constructed for mtDNA from strains of the fungus Fusarium oxysporum representing three pathogenically specialized forms. All three mtDNA maps are circular. Their sizes are 45 kb for F. oxysporum f.sp. raphani and 52 kb for both F. oxysporum f.sp. conglutinans and F. oxysporum f.sp. matthioli. The genetic loci for cytochrome b, the mitochondrial 25S ribosomal RNA and cytochrome oxidase subunit II, have been identified and are similarly arranged on the three genomes.     

Antibiotic activity of the pyrenocines

Pyrenocine A, a phytotoxin produced by Pyrenochaeta terrestris (Hansen) Gorenz, Walker and Larson, possesses general antibiotic activity against plants, fungi, and bacteria. Effective doses for 50% inhibition (ED50s) are 4 micrograms/mL for onion seedling elongation; 14, 20, 20, and 25 micrograms/mL for the germination of asexual spores of Fusarium oxysporum f. sp. cepae, Fusarium solani f. sp. pisi, Mucor hiemalis, and Rhizopus stolonifer, respectively. Pyrenocine A also inhibits the linear mycelial growth of both P. terrestris and F. oxysporum with ED50s calculated as 77 and 54 micrograms/mL, respectively. Gram-positive bacteria are more susceptible to pyrenocine A than Gram-negative bacteria. ED50s are estimated as 30, 45, and 200 micrograms/mL for the inhibition of growth of Bacillus subtilis, Staphylococcus aureus, and Escherichia coli, respectively, with Pseudomonas aeruginosa resistant to those concentrations tested. Pyrenocine A acts primarily as a biostatic rather than a biocidal agent with all organisms tested showing some degree of recovery when released from pyrenocine A. Pyrenocines B and C show little antibiotic activity in the bioassays performed.