Isolation of differentially expressed genes during interactions between tomato cells and a protective or a non-protective strain of Fusarium oxysporum

Pathogenic isolates of Fusarium oxysporum applied on a non-host plant species, as soil-borne non-pathogenic isolates, are able to protect this plant against pathogenic strains inducing wilts. Several modes of action contribute to the biocontrol activity of these protective strains; however the genetic basis of the biocontrol mechanisms is far from being understood. The aim of this study was to identify genes involved in biocontrol activity of F. oxysporum using an original model made of Fom24, a strain protective on tomato and its mutant rev157 which has lost its protective capacity. A Rapid Subtractive Hybridization (RaSH) approach was chosen to identify genes up-regulated in the protective or in the non-protective interaction when germinated conidia of either Fom24 or rev157 are confronted to tomato cell cultures. A total of 86 up-regulated sequences were generated, 42 and 44 from the protective and the non-protective interaction respectively. Homology searches led to identification of both plant and fungal genes that were grouped according to their putative functions. Among plant genes, those involved in plant response to stresses were the most abundant. Expression profiles of genes homolog to a basic endochitinase, a ferredoxine-NADP(H) reductase (FNR), an ATP synthase and the RPM1-interacting protein 4 (RIN4) were confirmed by Northern blotting. A large proportion of fungal sequences were encoding genes of unknown function; among other, those involved in response to oxidative stress and a gene putatively encoding an enolase are the most promising to further study their potential role in the protective interaction between F. oxysporum and tomato.     

Selective media for <Emphasis Type="Italic">Fusarium oxysporum</Emphasis>

Selective media without pentachloronitrobenzene were developed for quantitative assays of Fusarium oxysporum in soils. Media Fo-G1 and Fo-G2 were effective for naturally infested soils, Fo-W1 and Fo-W2 for wild-type isolates in soils containing a nitrate-nonutilizing (nit) mutant, and Fo-N1 and Fo-N2 for nit mutants. Selective media were made using ammonium citrate dibasic, l-sorbose, econazole nitrate, 25% iminoctadine triacetate solution and 50% tolclofos-methyl wettable powder for soil dilutions of 100-fold or more (Fo-G1, FoW1 and Fo-N1) and 10-fold (Fo-G2, Fo-W2 and Fo-N2). Potassium chlorate was added to Fo-N1 and Fo-N2. The efficacy for selectively isolating F. oxysporum was confirmed using six soils naturally infested with one of six formae speciales of F. oxysporum and with soil dilutions containing conidia of wild-type strains or nit mutants from the six formae speciales. On Fo-G1 and Fo-G2, most colonies of F. oxysporum were compact and round with purplish or reddish pigment in the reverse. Cylindrocarpon sp. formed colonies as large as those of F. oxysporum but were distinguishable by their colony morphology. Other contaminants such as F. solani, F. moniliforme, and Trichoderma were suppressed by medium ingredients and colonies of F. oxysporum. On Fo-W1 and Fo-W2, colony morphology of F. oxysporum and contaminants corresponded to that on Fo-G1 and Fo-G2, although F. oxysporum failed to produce the pigment. On Fo-N1 and Fo-N2, nit mutants formed clear colonies from 100- and 10-fold soil dilutions, respectively, and contaminants seldom formed large colonies.     

Impaired purine biosynthesis affects pathogenicity of <Emphasis Type="Italic">Fusarium oxysporum</Emphasis> f. sp. <Emphasis Type="Italic">melonis</Emphasis>

The vascular wilt pathogen Fusarium oxysporum f. sp. melonis causes worldwide yield losses of muskmelon. In this study, we characterized a UV-induced non-pathogenic mutant (strain 4/4) of F. oxysporum f. sp. melonis, previously identified as a potential biological control agent. During comparative analysis of vegetative growth parameters using different carbon sources, mutant strain 4/4 showed a delay in development and secretion of extracellular enzymes, compared to the wild type strain. Amendments of the growth medium with yeast extract, adenine or hypoxanthine, but not guanine, complemented the growth defect of strain 4/4, as well as secretion and partial activity of cellulases and endopolygalacturonases, indicating that the strain is an adenine auxotroph. Incubation of strain 4/4 conidia in adenine solution, prior to inoculation of muskmelon plants, partially restored pathogenicity to the mutant strain.     

Cloning of the pathogenicity-related gene <Emphasis Type="Italic">FPD1</Emphasis> in <Emphasis Type="Italic">Fusarium oxysporum</Emphasis> f. sp. <Emphasis Type="Italic">lycopersici</Emphasis>

We selected a reduced-pathogenicity mutant of Fusarium oxysporum f. sp. lycopersici, a tomato wilt pathogen, from the transformants generated by restriction enzyme-mediated integration (REMI) transformation. The gene tagged with the plasmid in the mutant was predicted to encode a protein of 321 amino acids and was designated FPD1. Homology search showed its partial similarity to a chloride conductance regulatory protein of Xenopus, suggesting that FPD1 is a transmembrane protein. Although the function of FPD1 has not been identified, it does participate in the pathogenicity of F. oxysporum f. sp. lycopersici because FPD1-deficient mutants reproduced the reduced pathogenicity on tomato.The nucleotide sequence data reported are available in the DDBJ/EMBL/GenBank databases under the accession number AB110097     

Characterization of biotin-autotrophic isolates derived from naturally occurring auxotrophic race 2 isolates of <Emphasis Type="Italic">Fusarium oxysporum</Emphasis> f. sp. <Emphasis Type="Italic">lactucae</Emphasis>

Race 2 isolates of Fusarium oxysporum f. sp. lactucae have been recognized as biotin auxotrophs and consequently have restricted growth on Puhalla's minimal medium (MM), which contains no biotin. Biotin-autotrophic isolates were raised from race 2 isolates through cultural mutation that grew as well on MM as they did on MM supplemented with biotin. These autotrophs were identical to the parental isolates in pathogenicity on race differential cultivars of lettuce (Patriot, Banchu Red Fire, and Costa Rica No. 4), and thus were designated as race 2. A vegetative compatibility test indicated that the autotrophic isolates fell into the same vegetative compatibility group as the parents. Culture filtrates of the autotrophs allowed abundant growth of the parental auxotroph on MM, and, through a competitive enzyme-binding assay, biotin was detected in the culture filtrates. These results suggest that biotin auxotrophy in the natural race 2 isolates has no direct relation to pathogenicity, qualitatively defined as physiological race, or to vegetative compatibility.     

<Emphasis Type="Italic">In vivo</Emphasis> evaluation of essential oils and biocontrol agents combined with heat treatments on basil cv Genovese Gigante seeds against <Emphasis Type="Italic">Fusarium oxysporum</Emphasis> f. sp. <Emphasis Type="Italic">basilici</Emphasis>

Seed treatments with essential oils (from savory and thyme) and biocontrol agents (Pseudomonas spp. and Fusarium oxysporum) have been evaluated in vivo after dry hot air treatments against Fusarium oxysporum f. sp. basilici on basil seeds. The savory and thyme essential oils showed a significant pathogen control activity because of their innate antifungal activity and because of the seed application method, but the dry hot pre-treatment did not show any obvious effect on the performance of the essential oil treatments. The dry heat treatment improved the Pseudomonas seed dressing effect against F.oxysporum f. sp. basilici, and showed important reductions in plant infection and the disease index on the treated seed plants, without any negative effect on seed germination. However, the pathogen control provided by the heat treatments combined with the application of the biocontrol agents never reached the same performance as the chemical treatments considered as the reference. Thus, short dry heat treatments on basil seeds have been shown to be a valid but complementary seed disinfection method against Fusarium wilt.     

Isolation of pathogenicity- and gregatin-deficient mutants of <Emphasis Type="Italic">Phialophora gregata</Emphasis> f. sp. <Emphasis Type="Italic">adzukicola</Emphasis> through <Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis>-mediated transformation

Phialophora gregata f. sp. adzukicola, a causal agent of brown stem rot in adzuki beans, produces phytotoxic compounds: gregatins A, B, C, D, and E. Gregatins A, C, and D cause wilting and vascular browning in adzuki beans, which resemble the disease symptoms. Thus, gregatins are considered to be involved in pathogenicity. However, molecular analyses have not been conducted, and little is known about other pathogenic factors. We sought to isolate nonpathogenic and gregatin-deficient mutants through Agrobacterium tumefaciens-mediated transformation (ATMT) for cloning of pathogenicity-related genes. The co-cultivation of P. gregata and A. tumefaciens for 48 h at 20°C with 200 μM acetosyringone resulted in approximately 80 transformants per 10⁶ conidia. The presence of acetosyringone in the A. tumefaciens pre-cultivation period led to an increase in T-DNA copy number per genome. Of 420 and 110 transformants tested for their pathogenicity and productivity of gregatins, one nonpathogenic and three gregatin-deficient mutants were obtained, respectively. The nonpathogenic mutant produced gregatins, whereas the gregatin-deficient mutants had pathogenicity comparable to the wild-type strain. This is the first report of ATMT of P. gregata. Further analysis of these mutants will help reveal the nature of the pathogenicity of this fungus including the role of gregatin in pathogenesis.     

Distinguishable staining with neutral red for GFP-marked and GFP-nonmarked <Emphasis Type="Italic">Fusarium oxysporum</Emphasis> strains simultaneously colonizing root surfaces

 Green fluorescent protein (GFP)-marked Fusarium oxysporum f. sp. melonis and nonmarked F. oxysporum f. sp. fragariae were stained with neutral red. The neutral red stained vacuoles of the fungi without disturbing GFP fluorescence in the cytoplasm. GFP-marked fungi showed fluorescent hyphae with dark-stained vacuoles, whereas nonmarked fungi were detected as nonfluorescent hyphae with dark-dotted vacuoles. Root colonization by these two fungi was monitored using this method. Microconidia attached similarly to the root surface and elongated vegetative hyphae. Only the pathogenic fungi invaded, causing necrosis at the inoculation site. Thus, the present method enabled us to track simultaneously the various formae speciales of F. oxysporum colonizing the root surface. Received: March 25, 2002 / Accepted: September 27, 2002     

Seed transmission of<Emphasis Type="Italic">Fusarium oxysporum</Emphasis> f.sp.<Emphasis Type="Italic">lactucae</Emphasis>

Twenty-seven seed samples belonging to the lettuce cultivars most frequently grown in Lombardy (northwestern Italy), in an area severely affected by Fusarium wilt of lettuce, were assayed for the presence ofFusarium oxysporum on a Fusarium-selective medium. Isolations were carried out on subsamples of seeds (500 to 1500) belonging to the same seed lots used for sowing, and either unwashed or disinfected in 1% sodium hypochloride. The pathogenicity of the isolates ofF. oxysporum obtained was tested in four trials carried out on lettuce cultivars of the butterhead type, very susceptible to Fusarium wilt. Nine of the 27 samples of seeds obtained from commercial seed lots used for sowing in fields affected by Fusarium wilt were contaminated byF. oxysporum. Among the 16 isolates ofF. oxysporum obtained, only one was isolated from disinfected seeds. Three of the isolates were pathogenic on the tested cultivars of lettuce, exhibiting a level of pathogenicity similar to that of the isolates ofF. oxysporum f.sp.lactucae obtained from infected wilted plants in Italy, USA and Taiwan, used as comparison. The results obtained indicate that lettuce seeds are a potential source of inoculum for Fusarium wilt of lettuce. The possibility of isolatingF. oxysporum f.sp.lactucae, although from a low percent of seeds, supports the hypothesis that the rapid spread of Fusarium wilt of lettuce observed recently in Italy is due to the use of infected propagation material. Measures for prevention and control of the disease are discussed. http://www.phytoparasitica.org posting Dec. 16, 2003.     

Genetic diversity of <Emphasis Type="Italic">Fusarium oxysporum</Emphasis> f. sp. <Emphasis Type="Italic">spinaciae</Emphasis> in Japan based on phylogenetic analyses of rDNA-IGS and <Emphasis Type="Italic">MAT1</Emphasis> sequences

Twenty-eight isolates of Fusarium oxysporum f. sp. spinaciae (FOS; the causal agent of spinach wilt) collected from Japan were assessed for mating type and subjected to phylogenetic analysis. Mating type analysis revealed all isolates to be MAT1-2, suggesting that there is no sexual recombination within the population. Phylogenetic analyses based on nucleotide sequences of the ribosomal DNA intergenic spacer (IGS) and the mating type locus (MAT1) suggested that FOS is polyphyletic. The cluster analysis based on IGS showed four phylogenetic groups (S1–S4) among the isolates. Two distinct lineages, S1 and S3, included FOS isolates both of the vegetative compatibility group (VCG) types, 0330 and 0331, demonstrating that VCG differentiation in FOS may not necessarily reflect the phylogenetic relationships based on IGS and MAT1-2-1.     

Interaction of root colonizing biocontrol agents demonstrates the antagonistic effect against <Emphasis Type="Italic">Fusarium oxysporum</Emphasis> f. sp. <Emphasis Type="Italic">lycopersici</Emphasis> on tomato

Plant growth promoting Bacillus subtilis MSS9 and Bacillus licheniformis MSS14 were isolated from the tomato rhizosphere. These isolates were capable of inhibiting the fungal pathogen, Fusarium oxysporum f. sp. lycopersici causing fusarium wilt in tomato, tested by dual culture method and by mycolytic enzyme production. The isolates have the capacity to form biofilm on the microtitre plate. Scanning electron microscopy revealed good colonization capacity of Bacillus licheniformis MSS14 on tomato plant root as compared to Bacillus subtilis MSS9, pot experiments were also analyzed to study the effects of both rhizobacterial cultures on pathogen development and plant growth. It was observed that MSS14 reduces the incidence of Fusarium oxysporum f. sp. lycopersici in tomato and there was significant increase in vegetative parameters like root length, shoot length, plant wet weight, dry weight and chlorophyll content after which indicates that the root colonization property of the culture MSS14 helps in enhancing the biocontrol capacity against pathogen than that of MSS9.     

Tobacco cultivars vary in induction of systemic resistance against <Emphasis Type="Italic">Cucumber mosaic virus</Emphasis> and growth promotion by <Emphasis Type="Italic">Pseudomonas chlororaphis</Emphasis> O6 and its <Emphasis Type="Italic">gacS</Emphasis> mutant

The colonization of plant roots with certain rhizosphere bacteria promotes plant growth and induces long lasting systemic protection against a broad spectrum of plant pathogens. The role of the global regulator, GacS, in the rhizosphere colonist Pseudomonas chlororaphis O6 in stimulating growth promotion and induced resistance against Cucumber mosaic virus was examined in tobacco. Responses were compared in tobacco cvs Samsun and GX3. Root colonization of Samsun with wild-type O6 and the gacS complemented mutant-elicited reduced viral symptoms and viral titre. On GX3, there was little affect on symptoms when roots were colonized by the wild-type, gacS mutant or complemented mutant but colonization by both the wild-type and the gacS mutant lowered viral titre. Wild-type O6 and the gacS mutant caused plant growth to be maintained in both tobacco cultivars after viral infection, although the affect was stronger with GX3 than Samsun. In contrast, although a chemical inducer, benzothiadiazole, reduced symptoms and viral titre in both cultivars, plant growth was suppressed. Our results indicate rhizobacteria-elicited induced viral resistance without a negative impact on growth but there was a differential response between cultivars. Detailed knowledge regarding the mechanisms inherent to these differences between cultivars requires further investigation.     

A study on the susceptibility of the model legume plant <Emphasis Type="Italic">Medicago truncatula</Emphasis> to the soil-borne pathogen <Emphasis Type="Italic">Fusarium oxysporum</Emphasis>

Fusarium wilt is a soil-borne disease caused by formae specialis of Fusarium oxysporum on a large number of cultivated and wild plants. The susceptibility of the model legume plant Medicago truncatula to Fusarium oxysporum was studied by root-inoculating young plants in a miniaturised hydroponic culture. Among eight tested M. truncatula lines, all were susceptible to F. oxysporum f.sp. medicaginis, the causal agent of Fusarium wilt in alfalfa. However, a tolerant line, F83005.5, and a susceptible line, A17, could be distinguished by scoring the disease index. The fungus was transformed with the GFP marker gene and colonisation of the plant roots was analysed by epifluorescence and confocal microscopy. A slightly atypical pattern of root colonisation was observed, with massive fungal growth in the cortex. Although colonisation was not significantly different between susceptible and tolerant plants, the expression of some defence-related genes showed discrimination between both lines. A study with 10 strains from various host-plants indicated that M. truncatula was a permissive host to F. oxysporum.     

Suppression of Fusarium wilt of spinach with hypovirulent binucleate <Emphasis Type="Italic">Rhizoctonia</Emphasis>

 Four isolates of hypovirulent binucleate Rhizoctonia (HBNR) were evaluated for their ability to control Fusarium wilt of spinach (FWS) caused by Fusarium oxysporum f. sp. spinaciae (FOS). Fourteen-day-old spinach seedlings grown in paper pots with HBNR-amended soil (1% w/w ground barley grain inoculum) were transferred to artificially pathogen-infested soil. Treatments with HBNR isolates significantly (P = 0.05) reduced disease and discoloration severity by 56%–100% and 52%–100%, respectively. The numbers of colony-forming units of FOS per gram fresh weight in petioles or roots were reduced significantly (P = 0.01) in the plants treated with HBNR. HBNR isolates were well reisolated from the roots inside paper pots where they were inoculated, whereas inconsistent colonization of HBNR was recorded from the roots outside paper pots where only pathogen was inoculated. Root extracts from HBNR-treated and pathogen-challenged plants significantly inhibited germination and germling length of FOS. The fresh weight of spinach leaves in the HBNR-treated plants increased significantly (P = 0.01), as much as 53%–63%, over the untreated and pathogen-challenged plants. This is the first report of biocontrol of FWS by HBNR. Received: July 18, 2002 / Accepted: October 22, 2002 Acknowledgments We are grateful to Dr. Komada for providing nonpathogenic Fusarium F13. The senior author (A.M.) thanks the Ministry of Education, Culture, Sports, Science, and Technology (Monbukagakusho) Japan, for financial assistance.     

Induced suppressiveness to<Emphasis Type="Italic">Fusarium oxysporum</Emphasis> f.sp.<Emphasis Type="Italic">radicis lycopersici</Emphasis> in rockwool substrate used in closed soilless systems

Tomatoes grown in soilless systems can be seriously damaged byFusarium oxysporum Schlect f.sp.radicis lycopersici (Forl) causing Fusarium crown and root rot (FCRR). FCRR suppression can be achieved through the use of chemicals, selected substrates, composts and artificially introduced antagonistic microorganisms. This study evaluated the natural capacity of a used rockwool to suppress FCRR infections. New and used rockwool, sampled from closed soilless systems, was either autoclaved or not, either artificially inoculated withForl or not and, finally, sown with tomato seeds cv. ‘Cuore di Bue’. The effects of autoclaved/non-autoclaved and used/new rockwool on FCRR incidence were assessed by evaluating the symptoms of crown rot on the root — shoot transition zone of tomato seedlings. Non-autoclaved and inoculated used rockwool significantly reduced FCRR incidence when compared with non-autoclaved and inoculated new rockwool. Autoclaved and inoculated used rockwool did not suppress FCRR, similarly to new and inoculated rockwool. These findings are in accordance with other research that, on a cucumber/Pythium host/pathogen complex in a closed rockwool soilless system, demonstrated the key role of resident microflora in suppressing the root rot disease. http://www.phytoparasitica.org posting Dec. 8, 2006.     

Induced resistance against Fusarium diseases of <Emphasis Type="Italic">Cymbidium</Emphasis> species by weakly virulent strain HPF-1 (<Emphasis Type="Italic">Fusarium</Emphasis> sp.)

The mechanism by which Fusarium diseases of cymbidium plants are suppressed by a weakly virulent strain HPF-1 of Fusarium sp. was studied. Strain HPF-1 produced microscopic, necrotic local lesions on cymbidium leaves, causing minor damage to palisade tissues at the infection sites. This weakly virulent strain remained near the site of infection and did not develop further. It systemically and nonselectively suppressed some diseases of cymbidium such as yellow spot of leaves caused by Fusarium proliferatum and F. fractiflexum, bulb and root rot caused by F. oxysporum, and dry rot of bulbs and roots caused by F. solani. Because endogenous salicylic acid levels increased in cymbidium leaves inoculated with strain HPF-1, the mechanism of disease suppression is thought to be systemic acquired resistance.     

Effect of antagonistic<Emphasis Type="Italic">Fusarium</Emphasis> spp. and of different commercial biofungicide formulations on Fusarium wilt of lettuce

Five experimental trials were carried out to test different biological control agents against Fusarium wilt of lettuce, cause byFusarium oxysporum f.sp.lactucae. In the presence of a very high disease incidence, the best results in terms of disease control as well as increased growth response were shown byTrichoderma harzianum T 22 (RootShield), which, at 3 gl ⁻¹ of substrate, provided very consistent results.F. oxysporum IF 23 gave good disease control but in two out of five trials reduced the biomass produced. Less consistent, but still significant results were provided byF oxysporium MSA 25, at 3 gl ⁻¹ of substrate, and byTrichoderma viride TV 1. The twoF. oxysporum agents Fo 251/2 and Fo 47 and the mixture ofT. harzianum + T. viride (Remedier) partially reduced disease incidence but were less effective than the above mentioned. Less interesting results were offered byStreptomyces griseoviridis (Mycostop). The results obtained show that biological control can play a role in the management of Fusarium wilt of lettuce.     

Ultrastructural and cell wall modifications during infection of <Emphasis Type="Italic">Eucalyptus viminalis</Emphasis> roots by a pathogenic <Emphasis Type="Italic">Fusarium oxysporum</Emphasis> strain

Fusarium species are soil-borne fungal pathogens that produce a variety of disease symptoms when attacking crop plants. The mode of root colonization of Eucalyptus viminalis seedlings by a pathogenic F. oxyporum strain (Foeu1) at the ultrastructural level and changes in cell wall pectin during host pathogen interactions are described. Root systems of E. viminalis plants were inoculated with F. oxysporum in an in vitro model system. Hyphae of F. oxysporum adhered to the outer epidermal cell walls through fibrillar material, and after penetration they spread into the internal tissues. They developed intercellularly and intracellularly in the root cortex and invaded vascular tissues. Papillae were induced, and the host plasma membrane ruptured in colonized cells, causing rapid host tissue and cell damage. Changes in distribution and occurrence of nonesterified and methyl-esterified pectins were evaluated after root colonization by F. oxysporum using two monoclonal antibodies, JIM 5 and JIM 7, respectively. Nonesterified pectin in control roots was mainly localized in the epidermal cell walls and middle lamellae in parenchymal cortex, whereas methyl-esterified pectin accumulated more in primary cell walls of the cortex and phloem. Decreases in immunodetected nonesterified and methyl-esterified pectins were associated with extensive plant tissue degradation after root colonization by the pathogenic fungus.     

Adsorption of OP<Subscript>1</Subscript>-related bacteriophages requires the gene encoding a TonB-dependent receptor-like protein in <Emphasis Type="Italic">Xanthomonas</Emphasis><Emphasis Type="Italic">oryzae</Emphasis> pv. <Emphasis Type="Italic">oryzae</Emphasis>

Xanthomonas oryzae pv. oryzae strain T7174R is lysed by bacteriophage OP_1h and OP_1h2. Three mutants tolerant to both OP_1h and OP_1h2 were isolated by transposon mutagenesis. The mutants had an insertion of the transposon in XOO1687, which is predicted to encode a TonB-dependent receptor gene. Plasmid pHMIroNB that contained XOO1687 of T7174R was constructed, and the mutant was transformed with the plasmid. The transformant recovered sensitivity to OP_1h and OP_1h2. Electron microscopic analysis demonstrated that OP_1h and OP_1h2 can adsorb to the wild type and the transformant, but they could not adsorb to the phage-tolerant mutant. These results suggest that the TonB-dependent receptor gene relates to adsorption and infection of T7174R by OP_1h2 and OP_1h. Y. Inoue and S. Tsuge have contributed equally to this work.