Quantitative comparison of Fusarium oxysporum competitiveness in relation to carbon utilization

Abstract The competitive saprophytic interaction between pathogenic and non-pathogenic strains of Fusarium oxysporum was investigated by studying their ability to competitively colonize sterilized soil. It was demonstrated that carbon was the first limiting substrate of Fusarium oxysporum growth in sterilized soil. Moreover, the efficiency with which glucose was utilized in the formation of propagules varied from one strain to another. Results of competitiion experiments showed that a linear relationship existed between the ratio of inoculum densities at the plateau (carrying capacity) and the ratio of inoculum densities incorporated into non-amended sterilized soil. The slope of the regression line indicated the competitiveness index, i.e. the competitiveness of a non-pathogenic strain in relation to that of a pathogenic strain. This parameter could be related to the yield coefficient of the strains in glucose-amended soil.     

Regulation of Fusarium oxysporum population introduced into soil: the amoebal predation hypothesis

Abstract Inoculation of fungi into soil has been suggested for biological control of plant diseases. The aim of our work was to test the ability of protozoa to reduce the density of introduced fungal populations. The survival of Fusarium oxysporum in non-sterile soil was studied after introduction at densities of: 1 × 10⁴, 1 × 10⁶ and 5 × 10⁷ cfu/g soil. The dynamics of protozoa were also followed. The fungal populations remained close to the initial inoculation densities and did not induce the growth of indigenous protozoa. A bacterial population ( Enterobacter aerogenes ) was used to promote and stimulate the predatory activity of amoebae. Then, after simultaneous inoculation with bacteria and fungi, the density of protozoa increased but this had no effect on the fungal population, although some amoebae are able to feed on small fungal propagules such as conidia. The physiological state of Fusarium in soil and intraspecific competition seem to be more important in regulating introduced fungal populations than amoebal predation. We conclude that the regulation of bacterial and fungal populations in soil depend on different mechanisms.     

Monoxenic in vitro production and colonization potential of AM fungus Glomus intraradices

The paper reports the establishment of mycorrhizal infection of a non-mycorrhizal Ri-T-DNA transformed carrot root when co-cultured with a surface sterilized sweet potato root segment colonized by arbuscular mycorrhizal (AM) fungus G. intraradices on minimal M medium. Extensive fungal hyphal emergence from each cut end of the mycorrhizal sweet potato root piece was observed in one week old cultures. These hyphae caused infection on contacting the transformed-carrot- root segment and produced many hyphae and spores both inside and outside the zone of the root after 6 week of growth. Axenically produced fungal propagules proliferated on the surface of fresh minimal M medium when sub-cultured without any root segment. On repeated sub-culturing, these propagules did not lose their ability to grow and produced many juvenile small spore-like vesicles during the non-symbiotic phase. Although these spores were morphologically and anatomically similar to their soil borne counter parts, they were much smaller. When placed in the vicinity of a fresh hairy root on the minimal medium or a Sudan grass seedling in sand culture, the axenically produced AM fungal propagules caused root infection, but the infection characteristics were significantly different to the original culture in terms of shape (spherical vs oval) and size (20 microm vs 45 microm) of the intraradical vesicles, and absence of 'H' branches. Sudan grass seedlings inoculated with the axenically cultured fungus showed significantly (P < 0.05) higher dry weights plant'. When compared to the plants inoculated with sand cultures, the growth parameters and the percentage infection were not significantly different. However, when both sources of inocula were used together, a synergistic effect on plant growth as well as root infection was observed.     

Biological Control of Fusarium oxysporum f.sp. asparagi by Applying Non-pathogenic Isolates of F . oxysporum

Root rot severity of asparagus plants grown in sterilized field soil inoculated with Fusarium oxysporum f . sp . asparagi (Foa) was reduced by more than 50% when the soil was precolo nized by each of 13 non - pathogenic (np) isolates of F. oxysporum originating from asparagus roots or field soils . In a greenhouse experiment , application of six np isolates to naturally infested field soil was followed by a 23 - 49% decrease of disease severity , depending on the isolate . One of them , Fo47 originating from Fusarium suppressive soil in France , was applied to field plots infested with Foa . Foa root rot was not suppressed in asparagus plants grown for 1 year in these plots . Pathogenic and np isolates extensively colonized the root surface and isolates of both types infected the roots of asparagus plants grown in sterilized field soil , with significant differences among the np isolates . Inoculation of sterilized field soil with np isolates reduced germination of Foa chlamydospores by 43 - 64% depending on the isolate used . It is concluded that np isolates of F. oxysporum can suppress asparagus root rot caused by Foa in naturally infested field soil . The differences for root colonization capacity among the np isolates imply that selection for this trait might reveal isolates that perform better under field conditions .     

高耐砷真菌的分离及其耐砷能力

从湖南石门县及郴州市重金属矿周边地区采集的6个砷污染土壤样品中,初步分离得到13株具有较强耐砷能力的真菌菌株,其中3株真菌的耐砷能力最强,经形态与分子鉴定分别为微紫青霉(Penicillin janthinellum)、尖孢镰刀菌(Fusarium oxysporum)和棘孢木霉(Trichoderma asperellum).培养试验表明：微紫青霉、尖孢镰刀菌和棘孢木霉分别在30000 mg·L^-1、30000 mg·L^-1和20000 mg·L^-1的砷胁迫平板中表现出很好的菌落生长状况;在砷浓度分别为0～50、0～50和0～80 mg·L^-1的液态培养基中培养2 d后,尖孢镰刀菌、棘孢木霉和微紫青霉的生物量均随砷浓度增加而增加,且在相应最高浓度(50、50、80 mg·L^-1)下的生物量显著高于不加砷的对照.此外,高浓度砷对该3株真菌的产孢能力没有影响.     

Effects on growth and comparison of root tissue colonization patterns of Eucalyptus viminalis by pathogenic and nonpathogenic strains of Fusarium oxysporum

Soilborne pathogens, especially Fusarium oxysporum , are responsible for damping-off and root necrosis in Eucalyptus nurseries. New technologies are increasingly considering strategies for plant disease control other than chemical fungicides. Among these, natural fungal antagonists, which are colonizers of the root cortex, are potential biocontrol agents. An in vitro system was used: (1) to test the pathogenic effects of F. oxysporum strain Foeu1 which was recovered from a forest nursery soil; (2) to explore the potential of the nonpathogenic F. oxysporum strain Fo47, which is known for its efficiency in biological control, to suppress damping-off of Eucalyptus seedlings; (3) to compare the patterns of root colonization and host response to invasion by the two Fusarium strains inoculated separately in a time-course study. Root inoculation of E. viminalis with F. oxysporum strain Foeu1 caused damping-off in young seedlings in vitro , whilst disease symptoms were not visible in plants inoculated with F. oxysporum strain Fo47 or when both strains (Foeu1 + Fo47) were inoculated simultaneously. Each strain showed similarities in patterns of root tissue colonization, and in the processes of root penetration and initial colonization. Differential effects on root tissue were observed with fungal development within the cortex: ingress of strain Foeu1 was accompanied by severe host-cell alterations whilst no tissue damage occurred with development of strain Fo47.     

Possibilities of the use of vinasses in the control of fungi phytopathogens

The purpose of this research was to study the biocide effect of three agroindustrial subproducts, concretely sugar beet, sugar cane and wine vinasse. Results from in vitro testing determined that wine vinasse is what shows a 100% capacity to suppress fungal growth with concentrations between 5% and 7% for Fusarium oxysporum f.sp. melonis race 0 and 1, Sclerotinia sclerotiorum, Pythium aphanidermatum and Phytophthora parasitica and 10-15% for F. oxysporum f.sp. radicis-cucumerinum. On the other hand, sugar cane vinasse produced an increase at high concentrations and sugar beet vinasse showed an approximate 100% suppressor effect on fungal growth for only some of the phytopathogens tested: S. sclerotiorum (15%), P. aphanidermatum (7%), P. parasitica (15%) and F. oxysporum f.sp. radicis-cucumerinum (15%). In the soil samples analyzed none of the three vinasse extracts decreased fusaric microbiota, producing an increase in the three samples tested. This would implicitly convey an improvement in soil quality by producing a potential increase in bacterial and fungal microbiota.     

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.     

An Alginate Prill Formulation of Fusarium oxysporum Schlechtend: Fr. f. sp. erythroxyli for Biocontrol of Erythroxylum coca var. coca

A rice alginate prill formulation of isolate EN - 4 of Fusarium oxysporum f . sp . erythroxyli, pathogenic to Erythroxylum coca var . coca (coca) , was evaluated in greenhouse and field studies for its ability to enhance pathogen populations in the soil and cause disease in coca . The formulation was applied to four different soil types in the greenhouse at 33 . 6 kg ha 1 . It enhanced the population of EN - 4 in each soil and most ( > 90%) of the fungal population remained in the upper 5 cm of soil during the 49 - day experiment . When applied in field experiments , the formulation enhanced the population of EN - 4 in the soil . Isolate EN - 4 was present in the upper 7 . 6 cm of soil 28 days after application at populations similar to those in the greenhouse studies (1 103 to 1 104 colony - forming units (CFUs) / g of soil) . Elevated populations of the pathogen (1 102 CFUs / g of soil) were still present in treated soils 229 days after application of the formulation . The areas used for field studies were already infested with the pathogen and typically developed high levels of fusarium wilt within 2 years of planting with coca . The formulated F. oxysporum began having a significant effect on plant death 100 - 200 days after application based on repeated measures analysis . These data suggest that a formulation of F. oxysporum f . sp . erythroxyli which enhances the incidence of fusarium wilt in coca fields can be produced using established techniques .     

Production of volatile organic compounds by an antagonistic strain Paenibacillus polymyxa WR-2 in the presence of root exudates and organic fertilizer and their antifungal activity against Fusarium oxysporum f. sp. niveum

The volatile organic compounds (VOCs) produced by antagonistic microbes have great antifungal potential against soil-borne fungal pathogens. The VOCs produced by Paenibacillus polymyxa strain WR-2 in the presence of root exudates and organic fertilizer were identified and their effects on the growth and spore germination of Fusarium oxysporum f. sp. niveum were evaluated. The VOCs produced by WR-2 inhibited the growth of F. oxysporum by 38%, 36% and 40% in agar medium, sterilized soil and natural soil, respectively. This inhibitory effect was increased to 60%, 58% and 64% with the addition of organic fertilizer in agar medium, sterilized soil and natural soil, respectively. The addition of root exudates did not affect the production of antifungal VOCs by WR-2. The VOCs produced by WR-2 completely inhibited the germination of F. oxysporum spores. Out of 42 identified VOCs, seven VOCs; benzothiazole, benzaldehyde, undecanal, dodecanal, hexadecanal, 2-tridecanone and phenol were found to inhibit the growth of F. oxysporum. The results of these experiments suggest another significance of using organic fertilizer as a carrier material with the biocontrol agents to control soil-borne fungal pathogens.     

Effect of osmotic, alkaline, acid or thermal stresses on the growth and inhibition of Listeria monocytogenes

Five strains of Listeria monocytogenes (a, b, c, d and e) isolated from industrial plants have been subjected to different osmotic, alkaline, acid or thermal stresses. The effects of these treatments on lag-phase (L) and growth rate (mu) of cells in mid-log phase have been followed using an automated optical density monitoring system. Increasing the osmotic pressure by the addition of different amounts of NaCl increased the lag phase and decreased the growth rate. The same phenomena were observed after decreasing the pH of the medium to 5.8, 5.6 or 5.4 by addition of acetic, lactic or hydrochloric acids. The inhibitory effect was: acetic acid > lactic acid > hydrochloric acid. The addition of NaOH to attain pH values of 9.5, 10.0, 10.5 or 11.0 in the medium produced a dramatic increase of the lag phase at pH 10.5 and 11. Growth rates were also decreased while the maximal population increased with high pH values. These effects varied according to strains. Strains d and e were the most resistant to acidic and alkaline stresses, and e was the most affected by the addition of NaCl. A cold shock of 30 min at 0 degree C had limited effects on growth parameters. On the other hand, hyperthermal shocks (55 or 63 degrees C, 30 min) led to similar increased lag phases and to significant increases of the maximal population in all five strains.     

The influence of crop rotation and soil fumigation on a mycorrhizal fungal community associated with soybean

Population densities of mycorrhizal fungal propagules in a western Kentucky field highly productive for soybean were measured by bioassay throughout a soybean production season. The primary experimental variables were crop rotation (soybeans in 1985, then 2 years in corn, milo, fescue, or soybean, then soybean in 1988 on all plots when populations of propagules were determined) and soil fumigation with 67% methyl bromide/33% chloropicrin. Of the 20 species in three genera found, Glomus predominated both in terms of number of species and population densities. Most species of Glomus occurred at higher population densities in rotated plots than in continuous soybean plots. In continuous soybean plots, species of Gigaspora made up a much higher proportion of the mycorrhizal fungal community than in rotated crops. Species richness and diversity were lower, and dominance and equitability higher, in nonfumigated continuous soybean plots than in rotated plots early in the season, but the differences were not present at the end of the season. Soil fumigation killed most propagules in the upper 15 cm of soil, but after production of a crop of soybeans, populations of total propagules and most Glomus spp. recovered to prefumigation densities. However, Gigaspora margarita and Gigaspora gigantea did not recover similarly. Fumigation reduced species richness and diversity and increased dominance, but the effects were ameliorated by the end of the season. Colonization of roots was low during vegetative growth but increased rapidly after the onset of soybean reproduction. There was no evidence for mutualism during the early half of the season, perhaps due to high soil P and low dependency of soybean. Fumigation increased soybean yields. A stable mycorrhizal fungal community appeared to become established with continuous soybean production, and both crop rotation and soil fumigation disrupted the community.     

The effects of sequential inoculation of mixed rumen protozoa on the degradation of orchard grass cell walls by anaerobic fungus Anaeromyces mucronatus 543

The effects of protozoa on the degradation of plant cell walls (CW) during different growth stages of the fungus Anaeromyces mucronatus have been investigated. Since fungi show a marked lag in their in vitro cultures and many protozoa rapidly die during a prolonged incubation time, the effects of protozoa may vary according to the growth phase of the fungi. Therefore, the approach adopted was (i) to inoculate CW with fungus monoculture, (ii) to inoculate CW with fungus-protozoa coculture, or (iii) to sequentially inoculate fungal cultures that had been grown in CW for 24 (initial stage of growth), 48, and 72 h (late stage of growth) with mixed protozoa. When a fungus was associated with protozoa, a growth phase dependent effect was observed. Ruminal protozoa adversely affected the growth and activity when introduced in the initial growth stage of A. mucronatus, but a synergetic interaction was detected when added to late growth stage cultures. Although there is no immediate explanation for these results, the data suggested that protozoa can engulf the fungal zoospores, which are in ruminal fluids and (or) attached to small feed particles, but cannot engulf the fungal thallus that is tightly attached to feed particles by a rhizoidal system. Our data indicated that the protozoa did not influence cellulolysis by the fungi in exponential and (or) stationary phase, but they had a marked inhibitory effect on fungi that were in lag phase. Inhibition during lag phase could result from the protozoal predation of fungal zoospores that had failed to attach to substrates.     

Survival of insect pathogenic and human clinical isolates of Photorhabdus luminescens in previously sterile soil.

Most characterized strains of the bacterium Photorhabdus luminescens are symbionts of entomopathogenic nematodes, whereas other strains have been isolated from human clinical specimens. The ability of P. luminescens strains to survive and grow in soil has received limited attention, with some studies indicating these bacteria have little or no ability to persist in soil. Survival and (or) growth of P. luminescens strains in previously sterilized soil, and examination of different soil amendments on their numbers in soil, have not been previously reported. Entomopathogenic P. luminescens (ATCC 29999) and a human clinical isolate (ATCC 43949) were introduced into a soil that had been sterilized by autoclaving, with or without different soil amendments, and bacterial numbers were estimated over time by viable plate count. In the previously sterilized soil receiving no exogenous amendments, numbers fell drastically over a week's time, followed by an increase in numbers by day 30. Treatments involving the addition of calcium carbonate and gelatin or casamino acids to soil usually resulted in higher bacterial numbers. For some sampling dates and soil treatments, there were statistically significant differences between the numbers of the two bacterial strains recovered from soil. The two strains of P. luminescens used in this study were able to survive and grow after being inoculated into previously sterilized soil.     

Growth of moulds inoculated into commercial mineral water

The growth of mould spores of Penicillium sp. and Cladosporium sp. inoculated in a commercial mineral water product was studied. The strains had been isolated as fungal foreign bodies in commercial mineral waters. In product A, which was not originally sterilized and was contaminated with psychrophilic bacteria, the inoculated mould spores of the strains did not grow; no increases in viable colony counts or beta-glucans concentration in the samples were observed during storage. In a sterilized product A, inoculated spores of the strains grew into visible foreign bodies. The viable colony counts and the beta-glucans concentration in the samples increased during storage. These results showed that in a sterilized mineral water product, mould spores could grow into visible foreign bodies.     

Suppression of Fusarium Colonization of Cotton Roots and Fusarium Wilt by Seed Treatments with Gliocladium virens and Bacillus subtilis

In a growth chamber at 25 C, the fungal antagonist Gliocladium virens colonized tap roots and secondary roots of cotton in non-sterile soil after seed treatment with preparations of G. virens. Colonization of tap roots by G. virens increased over time, and decreased with root depth. Seed treatments with G. virens strains G-4 and G-6 and with Bacillus subtilis strains GB03 and GB07 reduced the colonization of tap roots and secondary roots of cotton seedlings by Fusarium spp. Under greenhouse conditions, the same seed treatments suppressed the incidence and severity of fusarium wilt of cotton in soil infested with Fusarium oxysporum f. sp. vasinfectum and Meloidogyne incognita. Gliotoxin, produced by 'Q-group' strains of G. virens, inhibited F. oxysporum f. sp. vasinfectum in vitro. The seed treatments with G. virens strain G-6 and B. subtilis strains GB03 and GB07 did not affect the reproduction of root-knot nematodes. The results of this study may help to explain why the treatment of cotton seed with biocontrol agents often results in more vigorous and higher yielding plants, and indicate that there is potential for using G. virens and B. subtilis as seed treatments to control fusarium wilt of cotton.     

Auxin signaling and transport promote susceptibility to the root-infecting fungal pathogen Fusarium oxysporum in Arabidopsis

Fusarium oxysporum is a root-infecting fungal pathogen that causes wilt disease on a broad range of plant species, including the model plant Arabidopsis thaliana. Currently, very little is known about the molecular or physiological processes that are activated in the host during infection and the roles these processes play in resistance and susceptibility to F. oxysporum. In this study, we analyzed global gene expression profiles of F. oxysporum-infected Arabidopsis plants. Genes involved in jasmonate biosynthesis as well as jasmonate-dependent defense were coordinately induced by F. oxysporum. Similarly, tryptophan pathway genes, including those involved in both indole-glucosinolate and auxin biosynthesis, were upregulated in both the leaves and the roots of inoculated plants. Analysis of plants expressing the DR5:GUS construct suggested that root auxin homeostasis was altered during F. oxysporum infection. However, Arabidopsis mutants with altered auxin and tryptophan-derived metabolites such as indole-glucosinolates and camalexin did not show an altered resistance to this pathogen. In contrast, several auxin-signaling mutants were more resistant to F. oxysporum. Chemical or genetic alteration of polar auxin transport also conferred increased pathogen resistance. Our results suggest that, similarly to many other pathogenic and nonpathogenic or beneficial soil organisms, F. oxysporum requires components of auxin signaling and transport to colonize the plant more effectively. Potential mechanisms of auxin signaling and transport-mediated F. oxysporum susceptibility are discussed.     

Effect of fusaric acid and phytoanticipins on growth of rhizobacteria and Fusarium oxysporum

Suppression of soilborne diseases by biocontrol agents involves complex interactions among biocontrol agents and the pathogen and between these microorganisms and the plant. In general, these interactions are not well characterized. In this work, we studied (i) the diversity among strains of fluorescent Pseudomonas spp., Bacillus spp., and Paenibacillus sp. for their sensitivity to fusaric acid (FAc) and phytoanticipins from different host plants, (ii) the diversity of pathogenic and nonpathogenic Fusarium oxysporum isolates for their sensitivity to phytoanticipins, and (iii) the influence of FAc on the production of pyoverdine by fluorescent Pseudomonas spp. tolerant to this compound. There was a great diversity in the response of the bacterial strains to FAc; however, as a group, Bacillus spp. and Paenibacillus macerans were much more sensitive to FAc than Pseudomonas spp. FAc also affected production of pyoverdine by FAc-tolerant Pseudomonas spp. strains. Phytoanticipins differed in their effects on microbial growth, and sensitivity to a phytoanticipin varied among bacterial and fungal strains. Biochanin A did not affect growth of bacteria, but coumarin inhibited growth of Pseudomonas spp. strains and had no effect on Bacillus circulans and P. macerans. Conversely, tomatine inhibited growth of B. circulans and P. macerans. Biochanin A and tomatine inhibited growth of three pathogenic isolates of F. oxysporum but increased growth of three nonpathogenic F. oxysporum isolates. Coumarin inhibited growth of all pathogenic and nonpathogenic F. oxysporum isolates. These results are indicative of the complex interactions that can occur among plants, pathogens, and biological control agents in the rhizosphere and on the root surface. Also, these results may help to explain the low efficacy of some combinations of biocontrol agents, as well as the inconsistency in achieving disease suppression under field conditions.