Influence of Plant Root Exudates, Germ Tube Orientation and Passive Conidia Transport on Biological Control of Fusarium Wilt by Strains of Nonpathogenic Fusarium oxysporum

In earlier studies, biological control of Fusarium wilt of cucumber induced by Fusarium oxysporum f. sp. cucumerinum was demonstrated using nonpathogenic strains C5 and C14 of Fusarium oxysporum. Strain C14 induced resistance and competed for infection sites whether roots were wounded or intact, whereas strain C5 required wounds to achieve biocontrol. In the current work, additional attributes involved in enhanced resistance by nonpathogenic biocontrol agents strains to Fusarium wilt of cucumber and pea were further investigated. In pre-penetration assays, pathogenic formae specials exhibited a significantly higher percentage of spore germination in 4-day-old root exudates of cucumber and pea than nonpathogens. Also, strain C5 exhibited the lowest significant reduction in spore germination in contrast to strain C14 or control. One-day-old cucumber roots injected with strain C14 resulted in significant reduction in germ tube orientation towards the root surface, 48–96 h after inoculation with F. o. cucumerinum spores, whereas strain C5 induced significantly lower spore orientation of the pathogen and only at 72 and 96 h after inoculation. In post-penetration tests, passive transport of microconidia of pathogenic and nonpathogens in stems from base to apex were examined when severed plant roots were immersed in spore suspension. In repeated trials, strain C5, F. o. cucumerinum and F. o. pisi were consistently isolated from stem tissues of both cucumber and pea at increasing heights over a 17 days incubation period. Strain C14 however, was recovered at a maximum translocation distance of 4.6 cm at day 6 and later height of isolation significantly declined thereafter to 1.2 cm at day 17. In pea stem, the decline was even less. Significant induction of resistance to challenge inoculation by the pathogen in cucumber occurred 72 and 96 h after pre-inoculation with biocontrol agents. Nonetheless, strain C14 induced protection as early as 48 h and the maximum resistance was reached at 96 h. The presented data confirm the previous findings that attributes important for nonpathogenic fusaria to induce resistant are: rapid spore germination and orientation in response to root exudate; active root penetration and passive conidia transport in stem to initiate defence reaction without pathogenicity and enough lag period between induction and challenge inoculation. Strain C14 possesses all these qualifications and hence its ability to enhance host resistance is superior than strain C5.     

Fusarium oxysporum f. sp. fatshederae, a new forma specialis causing wilt of x Fatskedera lizei

A Fusarium oxysporum sensu Snyder & Hansen was pathogenic in the vascular tissue and caused a severe wilt and dieback of ×Fatshedera lizei, Fatsia japonica and Hedera helix. The fungus appeared nonpathogenic on plants of 29 species in numerous genera. Benomyl soil drenches were effective in preventing infection. A new forma specialis, Fusarium oxysporum f. sp. fatshederae, f. sp. nov. is proposed for this pathogen.     

Interaction of Fusarium Oxysporum f.sp. Cubense with Pseudomonas Fluorescens Precolonized to Banana Roots

Effect of precolonization of banana cv Neeypovan roots with Pseudomonas fluorescens on infection with Fusarium oxysporum f.sp. cubense was studied. Under in vitro conditions Pseudomonas fluorescens clearly inhibited Fusarium oxysporum f.sp. cubense. Fluorescein isothiocyanate-tagged antibodies raised in a rabbit system for Pseudomonas fluorescens and Fusarium oxysporum f.sp. cubense separately were used to study the spread of both organisms in banana root. It was observed that precolonization with Pseudomonas fluorescens could reduce Fusarium oxysporum f.sp. cubense colonization by 72%, and also correlated with a number of structural changes in the cortical cells, mainly with densely stained amorphous material and polymorphic wall thickenings as revealed by light and electron microscopic studies. Massive depositions of unusual structures at sites of fungal entry was also noticed, which clearly indicated that bacterized root cells were signalled to mobilize a number of defence structures for preventing the spread of pathogen in the tissue. This revised version was published online in July 2006 with corrections to the Cover Date.     

Colonization of Tomato Root by Pathogenic and Nonpathogenic Fusarium oxysporum Strains Inoculated Together and Separately into the Soil

In soil, fungal colonization of plant roots has been traditionally studied by indirect methods such as microbial isolation that do not enable direct observation of infection sites or of interactions between fungal pathogens and their antagonists. Confocal laser scanning microscopy was used to visualize the colonization of tomato roots in heat-treated soil and to observe the interactions between a nonpathogenic strain, Fo47, and a pathogenic strain, Fol8, inoculated onto tomato roots in soil. When inoculated separately, both fungi colonized the entire root surface, with the exception of the apical zone. When both strains were introduced together, they both colonized the root surface and were observed at the same locations. When Fo47 was introduced at a higher concentration than Fol8, it colonized much of the root surface, but hyphae of Fol8 could still be observed at the same location on the root. There was no exclusion of the pathogenic strain by the presence of the nonpathogenic strain. These results are not consistent with the hypothesis that specific infection sites exist on the root for Fusarium oxysporum and instead support the hypothesis that competition occurs for nutrients rather than for infection sites.     

Biological control ofFusarium oxysporum f.sp.cubense in banana by inoculation withPseudomonas fluorescens

Native strains ofPseudomonas fluorescens exhibitedin vitro antibiosis towards isolates of races 1 and 4 ofFusarium oxysporum f.sp.cubense, the Panama wilt pathogen of banana. The seedlings ofMusa balbisiana seedlings treated withP. fluorescens showed less severe wilting and internal discolouration due toF. oxysporum f.sp.cubense infection in greenhouse experiments. In addition to suppressing Panama wilt, bacterized seedlings ofM. balbisiana also showed better root growth and enhanced plant height.     

Suppressiveness of Vermicompost against Fusarium Wilt of Tomato

Vermicompost added to various container media significantly inhibited the infection of tomato plants by Fusarium oxysporum f. sp. lycopersici. The protective effect increased in proportion to the rate of application of vermicompost. Every type of container media amended with this substrate, used in the experiments, were suppressive to the pathogen. Vermicompost lost its activity after heating. Sterilized extracts of vermicompost added to potato dextrose agar stimulated the growth of F. oxysporum mycelium. The results indicate that chemical factors in this substrate had no direct inhibiting effect on the fungus. The total number of micro-organisms and populations of antagonistic bacteria and fungi were significantly higher in vermicompost than in the control peat substrate. A biotic nature is suggested for the suppressiveness of the vermicompost.     

Antagonistic effects of soil bacteria onFusarium oxysporum Schlecht f. sp.dianthi (Prill and Del.) Snyd. and Hans

Summary Fusarium oxysporum f. sp.dianthi, pathogenic on carnation plants is very sensitive toBacillus subtilis M51 inhibition.Fusarium oxysporum disease (fusariosis) is prevented for a period of two months after treatment of plants withBacillus subtilis M51. The persistence ofB. subtilis M51, marked for selenomycin resistance (MZ51) and inoculated on the roots of carnation cuttings was studied. Soil used was two types: naturally infested withFusarium oxysporum and free from this pathogen. Bacterial cells presence on the roots was detected by direct plating and the presence of the pathogen in the roots was investigated by histological assays. Evidence gathered by these procedures suggest that plant protection is dependent on the physical presence ofB. subtilis M51 cells on the roots.     

Intraspecific Variation within Populations of Fusarium oxysporum Based on RFLP Analysis of the Intergenic Spacer Region of the rDNA

Appel, D. J., and Gordon, T. R. 1995. Intraspecific variation within populations of Fusarium oxysporum based on RFLP analysis of the intergenic spacer region of the rDNA. Experimental Mycology 19, 120-128. Fifty-six isolates of Fusarium oxysporum, including F. oxysporum f. sp. melonis and nonpathogenic strains, were chosen from a larger collection to represent diversity in vegetative compatibility groups (VCGs), mitochondrial DNA (mtDNA) haplotype, geographic distribution, and virulence. Using PCR, a 2.6-kb fragment including the intergenic spacer (IGS) region of the ribosomal DNA was amplified from each isolate. The enzymes EcoRI, Sau 3A, Cfo1, and Ava1I, cut this fragment differentially, revealing 5, 6, 6, and 7 patterns, respectively. Among the 56 isolates, a total of 13 unique IGS haplotypes was identified. Among most F. o. melonis isolates. IGS haplotype correlated with VCG and mtDNA haplotype, but did not differentiate among races. However, a race 1 isolate found in VCG 0131 shared virulence, mtDNA, and IGS haplotypes characteristic of VCG 0134; this isolate may represent a conversion in VCG from 0134 to 0131. Four nonpathogens shared the pathogen vegetative compatibility phenotypes. One race 1,2 isolate associated with VCG 0134 shared both IGS haplotype and VCG with a nonpathogen, but these isolates did not share the same mtDNA haplotype. Another nonpathogenic isolate shared mtDNA and IGS haplotypes with pathogen group 0131 and may simply be an avirulent mutant of a pathogenic strain. For the other two nonpathogenic isolates, vegetative compatibility indicated a close relationship to the pathogen, but differences in both mtDNA and IGS haplotype suggest otherwise. Overall, the IGS haplotype was more variable among the nonpathogenic F. oxysporum VCGs among which 12 of the 13 IGS haplotypes were found. Nonpathogenic isolates that shared a common mtDNA haplotype, but were associated with different VCGs, often had different IGS haplotypes.     

Effect of bottlegourd seedcoating with antagonists on seedlings,quantum of the pathogen inside the seedlings and population of the soil againstFusarium oxysporum

Bottlegourd seedlings raised from antagonists coated seeds did not show any wilting symptoms and remained completely healthy. The pathogen quantum inside the host tissue was found least. The pathogen propagules per gram of host tissue were restricted and the soil population of theFusarium oxysporum was reduced very much in antagonists introduced soil.     

Antagonistic Effect of Nonpathogenic Fusarium oxysporum Fo47 and Pseudobactin 358 upon Pathogenic Fusarium oxysporum f. sp. dianthi

Pseudobactin production by Pseudomonas putida WCS358 significantly improves biological control of fusarium wilt caused by nonpathogenic Fusarium oxysporum Fo47b10 (P. Lemanceau, P. A. H. M. Bakker, W. J. de Kogel, C. Alabouvette, and B. Schippers, Appl. Environ. Microbiol. 58:2978-2982, 1992). The antagonistic effect of Fo47b10 and purified pseudobactin 358 was studied by using an in vitro bioassay. This bioassay allows studies on interactions among nonpathogenic F. oxysporum Fo47b10, pathogenic F. oxysporum f. sp. dianthi WCS816, and purified pseudobactin 358, the fluorescent siderophore produced by P. putida WCS358. Both nonpathogenic and pathogenic F. oxysporum reduced each other's growth when grown together. However, in these coinoculation experiments, pathogenic F. oxysporum WCS816 was relatively more inhibited in its growth than nonpathogenic F. oxysporum Fo47b10. The antagonism of nonpathogenic F. oxysporum against pathogenic F. oxysporum strongly depends on the ratio of nonpathogenic to pathogenic F. oxysporum densities: the higher this ratio, the stronger the antagonism. This fungal antagonism appears to be mainly associated with the competition for glucose. Pseudobactin 358 reduced the growth of both F. oxysporum strains, whereas ferric pseudobactin 358 did not; antagonism by pseudobactin 358 was then related to competition for iron. However, the pathogenic F. oxysporum strain was more sensitive to this antagonism than the nonpathogenic strain. Pseudobactin 358 reduced the efficiency of glucose metabolism by the fungi. These results suggest that pseudobactin 358 increases the intensity of the antagonism of nonpathogenic F. oxysporum Fo47b10 against pathogenic F. oxysporum WCS816 by making WCS816 more sensitive to the glucose competition by Fo47b10.     

Effect of inoculum density,nitrogen source and saprophytic fungi on Fusarium wilt of Mexican lime

Summary Mexican lime seedlings were inoculated with 0, 500, 1000, 2000, 4000 and 8000 microconidia ofFusarium oxysporum f. sp.citri per gram of potting media. The percent infection and mean disease severity rating increased with increasing inoculum density of the pathogen. In potting mix infested withAspergillus ochraceus, Penicillium funiculosum andTrichoderma harzianum at 5000 conidia per gram 2 weeks prior to infestation withF. oxysporum f. sp.citri at 0, 1000, 4000, and 8000 microconidia per gram,A. ochraceus reduced,P. funiculosum increased andT. harzianum had no effect on disease severity or pathogen population. OnlyP. funiculosum showed antagonistic activityin vitro against the pathogen. Disease severity and pathogen propagule densitites were greater and pH was lower in potting media fertilized with NH₄–N than in media fertilized with NO₃–N.Portion of M. S. thesis submitted by the senior author to the Graduate School, University of Florida, Gainesville. Florida Agricultural Experiment Stations Journal Series No. 4221.     

Effects of temperature and inoculum concentration on infection of narcissus bulbs by Fusarium oxysporum f.sp. narcissi

Chlamydospores of Fusarium oxysporum germinated, and mycelium grew on agar, at 10 but not 8°C. Numbers of chlamydospores needed to initiate disease suggest that the principal sources of infection are within the stock of bulbs and not the soil.     

The effects of Fusarium oxysporum f.sp. lycopersici (Sacc.) Snyder & Hansen on tomato in diphenamid-treated soil

Pre-emergence soil application of the herbicide diphenamid in concentrations exceeding the normal field rate increased the resistance of tomato plants towards infection by the wilt fungus Fusarium oxysporum f.sp. lycopersici. This was detected as significant increases in the percentage emergence of seedlings although growth parameters of the raised seedlings were reduced. Treated plants exhibited no wilt symptoms, although the pathogen maintained its population at detectable levels in the rhizosphere of tomato plants. However, the growth inhibition caused by diphenamid alone was much less than that reported for the combined application of pathogen and herbicide. Growth activities of F. oxysporum f.sp. lycopersici were inhibited by high concentrations of diphenamid in vitro. It is possible that the biodegradation of this herbicide by species such as Aspergillus candidus (present in substantial counts in treated rhizospheres) was one of the causes of increased tolerence of the pathogen to the herbicide in situ.     

Effects of Commercial and Indigenous Microorganisms on Fusarium Wilt Development in Chickpea

The purpose of this research was to determine whetherBacillus subtilis,nonpathogenicFusarium oxysporum,and/orTrichoderma harzianum,applied alone or in combination to chickpea (Cicer arietinumL.) cultivars ‘ICCV 4’ and ‘PV 61’ differing in their levels of resistance to Fusarium wilt, could effectively suppress disease caused by the highly virulent race 5 ofFusarium oxysporumf. sp.ciceris.Seeds of both cultivars were sown in soil amended with the three microbial antagonists, alone or in combination, and 7 days later seedlings were transplanted into soil infested with the pathogen. All three antagonistic microorganisms effectively colonized the roots of both chickpea cultivars, whether alone or in combination, and significantly suppressed Fusarium wilt development. In comparison with the control, the incubation period for the disease was delayed on average about 3 days and the final disease severity index and standardized area under the disease progress curve were reduced significantly between 14 and 33% and 16 and 42%, respectively, by all three microbial antagonists. Final disease incidence only was reduced byB. subtilis(18–25%) or nonpathogenicF. oxysporum(18%). The extent of disease suppression was higher and more consistent in ‘PV 61’ than in ‘ICCV 4’ whether colonized byB. subtilis,nonpathogenicF. oxysporum,orT. harzianum.The combination ofB. subtilis+T. harzianumwas effective in suppressing Fusarium wilt development but it did not differ significantly from treatments with either of these antagonists alone. In contrast, the combination ofB. subtilis+ nonpathogenicF. oxysporumtreatment was not effective but either antagonist alone significantly reduced disease development.     

Induction and accumulation of phytoalexins in cowpea roots infected with a mycorrhizal fungusGlomus fasciculatum and their resistance toFusarium wilt disease

The interaction of a vesicular-arbuscular mycorrhizal fungusGlomus fasciculatum with a wilt-causing soil borne pathogen,Fusarium oxysporum, was studied in cowpea (Vigna unguiculata). It was found that pre-establishment by vesicular-arbuscular mycorrhizal fungus reduced the colonization of the pathogen and the severity of the disease, as determined by reduction in vascular discolouration index. In mycorrhizal plants, the production of phytoalexin compounds was always higher than in the nonmycorrhizal plants. There appeared to be a direct correlation between the concentration of the phytoalexins and the degree of mycorrhizal association. Three different compounds withR _f values of 0.23 (I), 0.17 (II) and 0.11 (III) were obtained from mycorrhizal plants. Similar compounds were also found to be induced by an abiotic elicitor CuSO₄. The first compound was identified as an isoflavonoid, daidzein and the other two remain to be identified. These compounds were checked for their antifungal activityin vitro. The germination of conidial spores ofFusarium oxysporum was strongly inhibited by the compound III than the other two. It is argued that the production of phytoalexin compounds in mycorrhizal plant could be one of the mechanisms imparting tolerance of the plants to wilt disease.     

Detection of cross-reactive antigens shared byFusarium oxysporum andGlycine max by indirect ELISA and their cellular location in root tissues

Pathogenicity test ofFusarium oxysporum on ten cultivars of soybean revealed Soymax and Punjab-1 to be most resistant while JS-2 and UPSM-19 were most susceptible. Antigens were prepared from the roots of all the ten varieties of soybean and the mycelium ofF. oxysporum. Polyclonal antisera were raised against the mycelial suspension ofF. oxysporum and the root antigen of the susceptible cultivar UPSM-19. Cross reactive antigens shared by the host and the pathogen were detected first by immunodiffusion. The immunoglobulin fraction of the antiserum was purified by ammonium sulfate precipitation and DEAE-Sephadex column chromatography. The immunoglobulin fractions were used for detection of cross-reactive antigens by enzyme-linked immunosorbent assay. In enzyme-linked immunosorbent assay, antigens of susceptible cultivars showed higher absorbance values when tested against the purified anti-F. oxysporum antiserum. Antiserum produced against UPSM-19 showed cross-reactivity with the antigens of other cultivars. Indirect staining of antibodies using fluorescein isothiocyanate indicated that in cross-sections of roots of susceptible cultivar (UPSM-19) cross-reactive antigens were concentrated around xylem elements, endodermis and epidermal cells, while in the resistant variety, fluorescence was concentrated mainly around epidermal cells and distributed in the cortical tissues. CRAs were also present in microconidia, macroconidia and chlamydospores of the fungus.     

Effects of supplementary UV-B radiation on development of damping-off in spinach caused by the soil-borne fungusFusarium oxysporum

The effects of UV-B radiation (290–320 nm) on development of damping-off of spinach (Spinacia oleracea) caused by the fungusFusarium oxysporum were examined in a growth cabinet. The incidence of disease greatly increased when experimental plants were grown in visible radiation with supplementary UV-B radiation. This increase was suppressed by increasing the irradiation of visible radiation.Fusarium oxysporum was isolated from the roots of all damping-off plants and the roots of some unwilted plants, indicating that spinach infected with the pathogen did not necessarily suffer from damping-off in 15d. Supplementary UV-B radiation suppressed the increase in growth components such as the number of leaves, the plant height and the fresh weight of aboveground plant parts, but did not affect the fresh weight of roots. The ratio of the number of plants infected with pathogen to the total number of plants was over 80% irrespective of light conditions. It was suggested that the defense response of spinach to this pathogen was greatly influenced by the physiological state of aboveground plant parts resulting from supplementary UV-B radiation.     

Studies on a species of Monosporascus isolated from triticum

Hyphal parasitic behaviour of Fusarium oxysporum on Rhizoctonia solani and consecutive changes during this phenomenon have been investigated and studied. The hyphal parasitism was very commonly recorded between the test fungi. During the course of parasitism coiling, penetration, lysis and formation of chlamydospores by F. oxysporum on R. solani were observed. R. solani is a new host record for the mycoparasite F. oxysporum.     

In vitro selection of alfalfa plants resistant to Fusarium oxysporum f. sp. medicaginis

Summary From two lines of Medicago sativa characterized by a high regeneration capability, calli resistant to culture filtrate of Fusarium oxysporum f. sp. medicaginis have been selected. In these calli regeneration capability was greatly reduced and only one plant per callus was recovered. Regenerated plants have been evaluated for resistance to culture filtrate and for in vivo resistance to the pathogen. Three plants out of eight were resistant to the fungus and a high correlation between resistance to culture filtrate and in vivo resistance was observed.Research work supported by C.N.R., Italy. Special grant I.P.R.A. Subproject 1, paper no. 1468     

Colonization of cucumber plants by the biocontrol fungus Clonostachys rosea f. catenulata

Clonostachys rosea f. catenulata (Gliocladium catenulatum) strain J1446 (formulated as Prestop WP) suppressed Fusarium root and stem rot caused by Fusarium oxysporum f. sp. radicis-cucumerinum (Forc) on cucumber plants grown hydroponically in rockwool medium. Sixty days following application at seeding, the biocontrol agent had proliferated through the rockwool blocks and was present on cucumber roots and the crown region of the stem at populations >1 × 10⁵ CFU/g fresh weight. Scanning electron micrographs showed that C. rosea had rapidly colonized the root surface and was associated with root hairs and epidermal cell junctions. Following transformation of the fungus with Agrobacterium tumefaciens strain AGL-1 containing the hygromycin resistance (hph) and β-glucuronidase (uidA) genes, blue-stained mycelia could be seen growing on the surface and within epidermal and cortical cells of roots, stems and shoots 3 weeks after treatment. Quantification of GUS activity by fluorometric assays showed that fungal biomass was highest in the roots and crown area, while the extent of colonization of upper stems and true leaves was variable. Higher population levels resulted following application to rockwool blocks compared to seed treatment. Application of C. rosea preceding inoculation with Forc significantly reduced pathogen populations on roots compared to plants inoculated with Forc alone. Colonization of infection sites in the root zone reduced pathogen development and disease incidence. Densities of the biocontrol agent appeared to increase in the presence of the pathogen.