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.     

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.     

Salicylic acid-induced resistance to Fusarium oxysporum f. sp. lycopersici in tomato

We demonstrated that exogenous application of 200 μM salicylic acid through root feeding and foliar spray could induce resistance against Fusarium oxysporum f. sp. Lycopersici (Fol) in tomato. Endogenous accumulation of free salicylic acid in tomato roots was detected by HPLC and identification was confirmed by LC–MS/MS analysis. At 168 h of salicylic acid treatment through roots, the endogenous salicylic acid level in the roots increased to 1477 ng g^?1 FW which was 10 times higher than control plants. Similarly, the salicylic acid content was 1001 ng g^?1 FW at 168 h of treatment by foliar spray, which was 8.7 times higher than control plants. The activities of phenylalanine ammonia lyase (PAL, EC 4.3.1.5) and peroxidase (POD, EC 1.11.1.7) were 5.9 and 4.7 times higher, respectively than the control plants at 168 h of salicylic acid feeding through the roots. The increase in PAL and POD activities was 3.7 and 3.3 times higher, respectively at 168 h of salicylic acid treatments through foliar spray than control plants. The salicylic acid-treated tomato plants challenged with Fol exhibited significantly reduced vascular browning and leaf yellowing wilting. The mycelial growth of Fol was not significantly affected by salicylic acid. Significant increase in basal level of salicylic acid in noninoculated plants indicated that tomato root system might have the capacity to assimilate and distribute salicylic acid throughout the plant. The results indicated that the induced resistance observed in tomato against Fol might be a case of salicylic acid-dependent systemic acquired resistance.     

Properties of spin labelled membranes of Fusarium oxysporum f. sp. lycopersici.

Growth temperature-induced compositional changes in membranes of Fusarium oxysporum provided a test system for study of the relationship between physical properties and composition. Growth at 15 degrees C was characterized by a decrease in phospholipid content relative to sterol content, a shift in phospholipid composition from phosphatidylcholine to phosphatidylethanolamine and a marked enhancement in the amount of polyunsaturated fatty acids in the phospholipid and triglyceride classes. Uptake of a spin labelled analog of stearic acid during growth and subsequent solution of the probe in the membranes allowed estimation of viscosity and molecular order of the membranes of live cells and of isolated membrane preparations. Less than 1/20 of the intracellular label was accessible to sodium ascorbate while none was released by sodium dodecyl sulfate. All of the label in live cells was reduced by in vivo respiratory activity above 20 degrees C but this process could be reversed or avoided by added ferricyanide. A cholestane spin probe was also incorporated into the membranes. The probes were not reduced as readily in isolated membranes and hence fluidity of the membranes could be assessed over a wide temperature range. At low temperatures (-10 degrees C) a nonlethal, liquid-solid phase transition was indicated in isolated membrane lipids while at higher (lethal) temperatures (40-45 degrees C), discontinuities appeared in Arrhenius plots of rotational correlation time. Activation energies for isotropic rotation of the stearate probes in the membranes changed markedly in this temperature range and this effect correlated closely with loss of viability of conidial cells. Correlation times for stearate probes showed little variation with growth temperature nor were any breaks in Arrhenius plots of this parameter detected in the range 0-35 degrees C in whole cells or isolated membranes. The data indicated control of membrane physical properties within close tolerances throughout the physiological temperature range regardless of growth temperature. It was concluded that this homeostatic phenomenon was due to the counteractive effects of sterol/phospholipid ratio, phospholipid composition and fatty acid polyunsaturation since the condensing and fluidizing components of the isolated total membranes vary in a reciprocal manner.     

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.     

Effect of flavonoids on the development of Fusarium oxysporum f. sp. lycopersici

Abstract

In the present study the effect of flavonoid compounds on the germination and fungal growth of the soil-borne tomato pathogen Fusarium oxysporum f. sp. lycopersici was studied. Out of 12 flavonoid compounds only myricetin and luteolin exhibited a low stimulating activity on microconidia germination of Fusarium oxysporum f. sp. lycopersici, whereas the other flavonoids tested were inactive when applied at five different concentrations. In our study the tested flavonoids affect fungal growth differently to microconidia germination. Individual flavonoid concentrations resulted in a small increase of fungal growth, but the lowest flavonoid concentrations showed an inhibiting effect on fungal growth for all flavonoids tested. There is evidence to suggest, that low flavonoid concentrations exhibit slight antimicrobial properties against Fusarium oxysporum f. sp. lycopersici.     

Pantothenate synthetase from Fusarium oxysporum f. sp. lycopersici is induced by alpha-tomatine

The steroidal glycoalkaloid alpha-tomatine which is present in tomato (Lycopersicum sculentum) is assumed to protect the plant against phytopathogenic fungi. We have isolated a gene from the fungal pathogen Fusarium oxysporum f. sp. lycopersici that is induced by this glycoalkaloid. This gene, designated panC, encodes a predicted protein with a molecular mass of 41 kDa that shows a high degree of sequence similarity to pantothenate synthetases from yeast, plants and bacteria. Recombinant PanC protein from F. oxysporum has been over-expressed in Escherichia coli and purified to homogeneity. It shows pantothenate synthetase activity in the presence of D-pantoate, beta-alanine and ATP. The panC gene from F. oxysporum functionally complements an E. coli panC mutant, demonstrating that the PanC protein functions in vivo as a pantothenate synthetase. Southern analysis of F. oxysporum genomic DNA from other formae speciales indicates that there is a single copy of the pantothenate syntethase gene in this fungus. The presence of a STRE consensus sequence (CCCCT) in the promoter region of the gene suggests that the induction of panC may be part of a cellular stress response triggered by alpha-tomatine.     

Characterization of two endopolygalacturonase isozymes produced by Fusarium oxysporum f. sp. lycopersici.

Polygalacturonase (EC 3.2.1.15) produced by Fursarium oxysporum f. sp. lycopersici was purified by chromatography on DEAE-cellulose, CM-cellulose, and hydroxyapatite. The purified enzyme consisted of two electrophoretically distinct "isozymes", that behaved as charge isomers during electrophoresis in several different concentrations of polyacrylamide gel. The two isozymes had similar "endo" modes of action on polygalacturonic acid, as determined by comparison of viscosity reduction, reducing group release, and thin-layer chromatography of oligomeric hydrolysis products. Both isozymes hydrolzyed 5% of the substrate bonds in reaching 50% viscosity reduction. The amino acid compositions of the isozymes were similar and their molecular weights were about 37000 as determined by sedimentation equilibrium. Removal of large amounts of carbohydrate during purification did not affect heat stability of the enzymes. A large proportion of the remaining carbohydrate appeared to be covalently linked to the enzyme protein.     

Purification and characterization of a novel exopolygalacturonase from Fusarium oxysporum f.sp. lycopersici

A novel exopolygalacturonase (EC 3.2.1.15) was purified to apparent homogeneity from cultures of Fusarium oxysporum f.sp. lycopersici on synthetic medium supplemented with polygalacturonic acid, using two steps of purification: preparative isoelectric focusing and cationic exchange chromatography. The enzyme designated PG3 had an apparent M_r of 63 000±3000 Da upon SDS-PAGE and a pI of 7.0. PG3 was active within a broad range of pH from 3.5 to 9. The temperature optimum was 55°C. PG3 hydrolyzed polygalacturonic acid in an exo-manner, as demonstrated by analysis of degradation products. The enzyme was N-glycosylated. The production of PG3 was constitutive at low levels, and synthesis was increased following induction by PGA and partially repressed by glucose.     

Infectivity of Chlamydospores vs Microconidia of Fusarium oxysporum f.sp. lycopersici on Tomato

The effect of nature of inoculum on disease induced by Fusarium oxysporum f.sp. lycopersici on tomato was tested. Chlamydospores produced in soil 30 days after inoculation induced a more severe disease than microconidia indicating a higher inoculum potential of chlamydospores.
The method proposed produces easily an inoculum of F. oxysporum f.sp. lycopersici which infects the plants consistently and induces a relatively high disease severity.     

Identification of virulence genes in Fusarium oxysporum f. sp. lycopersici by large-scale transposon tagging

Forward genetic screens are efficient tools for the dissection of complex biological processes, such as fungal pathogenicity. A transposon tagging system was developed in the vascular wilt fungus Fusarium oxysporum f. sp. lycopersici by inserting the novel modified impala element imp160::gfp upstream of the Aspergillus nidulans niaD gene, followed by transactivation with a constitutively expressed transposase. A collection of 2072 Nia⁺ revertants was obtained from reporter strain T12 and screened for alterations in virulence, using a rapid assay for invasive growth on apple slices. Seven strains exhibited reduced virulence on both apple slices and intact tomato plants. Five of these were true revertants showing the re-insertion of imp160::gfp within or upstream of predicted coding regions, whereas the other two showed either excision without re-insertion or no excision. Linkage between imp160::gfp insertion and virulence phenotype was determined in four transposon-tagged loci using targeted deletion in the wild-type strain. Knockout mutants in one of the genes, FOXG_00016 , displayed significantly reduced virulence, and complementation of the original revertant with the wild-type FOXG_00016 allele fully restored virulence. FOXG_00016 has homology to the velvet gene family of A. nidulans . The high rate of untagged virulence mutations in the T12 reporter strain appears to be associated with increased genetic instability, possibly as a result of the transactivation of endogenous transposable elements by the constitutively expressed transposase.     

The in vitro physiological phenotype of tomato resistance to Fusarium oxysporum f. sp. lycopersici

Summary With the aim of dissecting host-parasite interaction processes in the system Lycopersicon aesculentum-Fusarium oxysporum f. sp. lycopersici we have isolated plant cell mutants having single-step alterations in their defense response. A previous analysis of the physiological phenotypes of mutant cell clones suggested that recognition is the crucial event for active defence, and that polysaccharide content, fungal growth inhibition, peroxidase induction in in vitro dual culture and ion leakage induced by cultural filtrates of the pathogen can be markers of resistance. In this paper we present the results of a similar analysis carried out on cell cultures from one susceptible (Red River), one tolerant (UC 105) and three resistant (Davis UC 82, Heinz, UC 90) tomato cultivars. Our data confirm that the differences in the parameters considered are correlated with resistance versus susceptibility in vivo. Therefore, these parameters can be used for early screening in selection programmes. These data, together with those obtained on isolated cell mutants, suggest that the selection in vitro for altered fungal recognition and/or polysaccharide or callose content may lead to in vivo — resistant genotypes. The data are thoroughly discussed with particular attention paid to the importance of polysaccharides in active defense initiation.     

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.     

Purification and characterization of an exopolygalacturonase produced by Fusarium oxysporum f. sp. radicis lycopersici

Abstract An exopolygalacturonase produced by Fusarium oxysporum f. sp. radicis lycopersici , a fungus that produces root rot, was purified by gel filtration and ion exchange chromatography. It had a M _r 68 K, a pH optimum of 5.6 and an optimum temperature of 60°C. This polygalacturonase was inhibited by calcium ions and had a K _m of 0.64 mM using sodium polypectate as substrate. The exo mode of action of this enzyme was revealed by thin-layer chromatography of hydrolysed substrate.     

Investigations on the mode of action of sodium N-methyldithiocarbamate in Fusarium oxysporum f. sp. lycopersici

Summary A radiorespirometric technique was used to determine the effects of sodium N-methyldithiocarbamate (NaMDC) on uptake and oxidation of glucose by Fusarium oxysporum f. sp. lycopersici. The principal glucose oxidation pathway of the fungus was found to be the pentose cycle. Shifts to other oxidation pathways were not significant even when the fungus was treated with extremely high concentrations of NaMDC (5×10^-2 M). Only when zinc ions were added to NaMDC solutions were shifts in pathway (and a drastic increase in toxicity) noted. The main effect of NaMDC was to delay the complete oxidation of glucose to CO₂. In the concentration range between 0.5 to 10.0×10^-2 M NaMDC dose response curves were bimodal with respect to spore germination, colony development, and CO₂ production; with respect to glucose uptake the curves were unimodal. The bimodal dosage response curves could be divided into a first zone of inhibition, a zone of reversed toxicity, and a second zone of inhibition. Within the first zone of inhibition, colony development proved to be more sensitive to NaMDC than spore germination. In the zone of reversed toxicity the incorporation of carbon into the spores drops sharply, while in the second zone of inhibition a strong inhibition of glucose uptake is the dominating effect. It is indicated that NaMDC interfers with biosynthetic activities rather than enzymes of catabolic pathways.List of abbreviations NaMDC Sodium N-methyldithiocarbamate - NaDMDC Sodium dimethyldithiocarbamate - TMTD Tetramethylthiuram disulfide, [=Bis(dimethylthiocarbamyl)disulfid, Thiram^®] - DMTD N,N-dimethylthiuram disulfide [=Bis-(methylthiocarbamyl)disulfid] - MIT Methylisothiocyanate     

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.     

Resistance in pepper plants induced by Fusarium oxysporum f. sp. lycopersici involves different defence-related genes

Inoculation with Fusarium oxysporum f. sp. lycopersici (FOL) protects pepper plants from subsequent infection with Phytophthora capsici . In the present paper, the level of local and systemic protection achieved by plants induced with FOL was evaluated by quantifying the pathogen biomass and using real-time PCR. Differences in the amount of pathogen were found in stems and roots between FOL-treated and untreated plants, while pathogen biomass could not be detected in leaves of induced plants. Five defence-related genes coding for a PR-1 protein, a β-1,3-glucanase, a chitinase, a peroxidase and a sesquiterpene cyclase were up-regulated 48 h after treatment in all the tissues studied, and maximal mRNAs levels were found in leaves.     

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.