Suppression of plant wilt diseases by nonpathogenic Fusarium oxysporum Fo47 combined with actinomycete strains

In order to build integrated strains with superior growth-promoting and disease-suppression effects, the biological control efficacy of Fo47 solid agents combined with actinomycetes strains toward Fusarium oxysporum and Verticillium dahliae were investigated in experiments on watermelon, cotton and eggplant. Five actinomycetes strains were prepared by solid fermentation. The count of viable solid agents, initially with propagules at 10⁷–10¹¹ CFU/g, slowly decreased after being stored one year at room temperature. After being inoculated into sterile soil for 50 days, the viable count of strain Fo47 remained at a stable level. The suppressive effects of Fo47 combined with strain QLP12 on Fusarium wilt on watermelon and cotton, and Verticillium wilt on eggplant, reaching 58.47%, 50.73% and 58.82%, respectively. This was significantly better than the single strain Fo47 alone, and growth of these treated plants and the colonisation rate of Fo47 were increased substantially as well. These results indicate that solid integrated agents with a high viability count and superior stability in soil could increase disease suppression and promote plant growth by synergy with different strains. The increased suppression obtained by Fo47 combined with actinomycete strains was not due to a simple addition of different mechanisms of biocontrol agents. By being intelligently integrated, these combinations increase disease suppression and provide the best biocontrol effect.     

Production of a Mixed Inoculum of Fusarium oxysporum Fo47 and Pseudomonas fluorescens C7 to Control Fusarium Diseases

Strains of non-pathogenic Fusarium oxysporum and Pseudomonas fluorescens are effective biocontrol agents against Fusarium diseases. Use of strain Fo47 of F. oxysporum in combination with strain C7 of P. fluorescens improves disease control in comparison to application of Fo47 alone. To develop a product based on a combination of these two strains, it would be beneficial to produce and formulate both organisms together. When an irradiated peat was inoculated with these strains either alone or in combination, they grew actively and reached similar maximum population densities irrespective of the initial inoculum concentration (1×10³ and 1×10⁶ g^-1 of peat) and mode of inoculation. These populations survived well with only a slight decrease in population densities after 11 months storage at 25°C. A bioassay was performed to evaluate peat and microbial suspension inocula of C7 and Fo47 alone or in combination against the flax pathogen, F. oxysporum f. sp. lini. Biocontrol activity of the peat-produced inocula was as effective as the suspensions. There was no dose-response relationship for C7, and when C7 was used as a mixed inoculum with Fo47, the biocontrol activity was always greater than that provided by Fo47 alone. This process could be used to produce an effective mixed inoculum with a shelf life of one year or more.     

Effect of pseudobactin 358 production by Pseudomonas putida WCS358 on suppression of fusarium wilt of carnations by nonpathogenic Fusarium oxysporum Fo47.

Nonpathogenic Fusarium oxysporum Fo47b10 combined with Pseudomonas putida WCS358 efficiently suppressed fusarium wilt of carnations grown in soilless culture. This suppression was significantly higher than that obtained by inoculation of either antagonistic microorganism alone. The increased suppression obtained by Fo47b10 combined with WCS358 only occurred when Fo47b10 was introduced at a density high enough (at least 10 times higher than that of the pathogen) to be efficient on its own. P. putida WCS358 had no effect on disease severity when inoculated on its own but significantly improved the control achieved with nonpathogenic F. oxysporum Fo47b10. In contrast, a siderophore-negative mutant of WCS358 had no effect on disease severity even in the presence of Fo47b10. Since the densities of both bacterial strains at the root level were similar, the difference between the wild-type WCS358 and the siderophore-negative mutant with regard to the control of fusarium wilt was related to the production of pseudobactin 358. The production of pseudobactin 358 appeared to be responsible for the increased suppression by Fo47b10 combined with WCS358 relative to that with Fo47b10 alone.     

Effect of pseudobactin 358 production by Pseudomonas putida WCS358 on suppression of fusarium wilt of carnations by nonpathogenic Fusarium oxysporum Fo47.

Nonpathogenic Fusarium oxysporum Fo47b10 combined with Pseudomonas putida WCS358 efficiently suppressed fusarium wilt of carnations grown in soilless culture. This suppression was significantly higher than that obtained by inoculation of either antagonistic microorganism alone. The increased suppression obtained by Fo47b10 combined with WCS358 only occurred when Fo47b10 was introduced at a density high enough (at least 10 times higher than that of the pathogen) to be efficient on its own. P. putida WCS358 had no effect on disease severity when inoculated on its own but significantly improved the control achieved with nonpathogenic F. oxysporum Fo47b10. In contrast, a siderophore-negative mutant of WCS358 had no effect on disease severity even in the presence of Fo47b10. Since the densities of both bacterial strains at the root level were similar, the difference between the wild-type WCS358 and the siderophore-negative mutant with regard to the control of fusarium wilt was related to the production of pseudobactin 358. The production of pseudobactin 358 appeared to be responsible for the increased suppression by Fo47b10 combined with WCS358 relative to that with Fo47b10 alone.     

Oviposition response of scarabaeids: does ‘mother knows best’ about rainfall variability and soil moisture?

Abstract.  The effect of soil moisture on ovipositional preference is studied for four melolonthine scarabaeids, Holotrichia reynaudi , Holotrichia serrata and Dermolepida albohirtum , which are endemic to the semiarid tropics of India ( Holotrichia spp.) and Australia, and Heteronyx piceus , which is endemic to the temperate and subtropical regions of Australia. As predicted by the preference-performance hypothesis, the three tropically adapted species show little or no ovipositional preference for soil moistures between permanent wilting point (−1500 kPa) and field capacity (−10 kPa) under either choice or no-choice conditions in their endemic soils, whereas H.   piceus shows a clear preference to oviposit in drier soils (−1500 to −200 kPa). The ovipositional soil-moisture preferences of D.   albohirtum and H.   serrata are much narrower in geographically adjacent non-endemic soils (between −1500 to −200 kPa) than in their endemic soils. An analysis of daily rainfall determined the rainfall variability within the endemic areas of each species. The semiarid tropics are highly variable, with rainfall at the time of oviposition being a poor predictor of the future rain required for successful larval development, whereas the rainfall in the subtropics is much more reliable. The absence of a clearly preferred soil moisture for oviposition in the tropically adapted species appears to be a life-history strategy that allows these species to live in environments where rainfall is highly variable, and soil moisture at time of oviposition is a poor predictor of future environmental suitability for plant growth, and thus larval survival.     

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.     

Development of a strain-specific real-time PCR assay for the detection and quantification of the biological control agent Fo47 in root tissues

Being able to identify specifically a biological control agent at the strain level is not the only requirement set by regulations (EC)1107/2009, it is also necessary to study the interactions of the agent with the plant and the pathogen in the rhizosphere. Fo47 is a soil-borne strain of Fusarium oxysporum which has the capacity to protect several plant species against the pathogenic formae speciales of F. oxysporum inducing wilts. A strain-specific sequence-characterized amplified region marker has been designed which makes it possible to distinguish Fo47 from other strains of F. oxysporum. In addition, a real-time PCR assay has been developed to quantify Fo47 in root tissues. The proposed assay has been validated by following the dynamics of root colonization of tomato plants grown in soil infested with Fo47. Results showed that with the method it is possible to quantify Fo47 in roots in the absence or presence of the pathogen and in the absence or in presence of the native microbial communities.     

Effects of soil frost on nitrogen net mineralization, soil solution chemistry and seepage losses in a temperate forest soil

Freezing and thawing may alter element turnover and solute fluxes in soils by changing physical and biological soil properties. We simulated soil frost in replicated snow removal plots in a mountainous Norway spruce stand in the Fichtelgebirge area, Germany, and investigated N net mineralization, solute concentrations and fluxes of dissolved organic carbon (DOC) and of mineral ions (NH₄⁺, NO₃⁻, Na⁺, K⁺, Ca²⁺, Mg²⁺). At the snow removal plots the minimum soil temperature was −5 °C at 5 cm depth, while the control plots were covered by snow and experienced no soil frost. The soil frost lasted for about 3 months and penetrated the soil to about 15 cm depth. In the 3 months after thawing, the in situ N net mineralization in the forest floor and upper mineral soil was not affected by soil frost. In late summer, NO₃⁻ concentrations increased in forest floor percolates and soil solutions at 20 cm soil depth in the snow removal plots relative to the control. The increase lasted for about 2–4 months at a time of low seepage water fluxes. Soil frost did not affect DOC concentrations and radiocarbon signatures of DOC. No specific frost effect was observed for K⁺, Ca²⁺ and Mg²⁺ in soil solutions, however, the Na⁺ concentrations in the upper mineral soil increased. In the 12 months following snowmelt, the solute fluxes of N, DOC, and mineral ions were not influenced by the previous soil frost at any depth. Our experiment did not support the hypothesis that moderate soil frost triggers solute losses of N, DOC, and mineral ions from temperate forest soils.     

Visualization of interactions between a pathogenic and a beneficial Fusarium strain during biocontrol of tomato foot and root rot

The soilborne fungus Fusarium oxysporum f. sp. radicis-lycopersici causes tomato foot and root rot (TFRR), which can be controlled by the addition of the nonpathogenic fungus F. oxysporum Fo47 to the soil. To improve our understanding of the interactions between the two Fusarium strains on tomato roots during biocontrol, the fungi were labeled using different autofluorescent proteins as markers and subsequently visualized using confocal laser scanning microscopy. The results were as follows. i) An at least 50-fold excess of Fo47over F. oxysporum f. sp. radicis-lycopersici was required to obtain control of TFRR. ii) When seedlings were planted in sand infested with spores of a single fungus, Fo47 hyphae attached to the root earlier than those of F. oxysporum f. sp. radicis-lycopersici. iii) Subsequent root colonization by F. oxysporum f. sp. radicis-lycopersici was faster and to a larger extent than that by Fo47. iv) Under disease-controlling conditions, colonization of tomato roots by the pathogenic fungus was significantly reduced. v) When the inoculum concentration of Fo47 was increased, root colonization by the pathogen was arrested at the stage of initial attachment to the root. vi) The percentage of spores of Fo47 that germinates in tomato root exudate in vitro is higher than that of the pathogen F. oxysporum f. sp. radicis-lycopersici. Based on these results, the mechanisms by which Fo47 controls TFRR are discussed in terms of i) rate of spore germination and competition for nutrients before the two fungi reach the rhizoplane; ii) competition for initial sites of attachment, intercellular junctions, and nutrients on the tomato root surface; and iii) inducing systemic resistance.     

Tomato root colonization by fluorescent-tagged pathogenic and protective strains of Fusarium oxysporum in hydroponic culture differs from root colonization in soil

The colonization process of tomato roots inoculated separately or/and simultaneously by a pathogenic Fusarium oxysporum f. sp. lycopersici strain Fol8 and the protective F. oxysporum strain Fo47, genetically tagged with the red and green fluorescent protein genes, respectively, was studied in a hydroponic culture. Plants were coinoculated with Fol8 and Fo47 at two conidial concentration ratios of 1/1 and 1/100, in which biological control was not effective or effective, respectively. First observation of fungi on root was possible 48 h after inoculation at a high inoculum level and 5 days post inoculation at the lower concentration of inoculum. The pattern of root colonization was similar for both strains with the initial development of hyphal network on the upper part of taproot, followed by the growth of hyphae towards the elongation zone, lateral roots and root apices. Finally, the whole elongation zone and root apex were invaded by both strains but no specific infection sites were observed. When coinoculated, both strains could grow very closely or even at the same spot on the root surface. At the nonprotective ratio, Fol8 was the successful colonizer, but application of Fo47 at a concentration 100 times >Fol8 delayed vessel colonization by the pathogen.     

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.     

Harvester ant nests, soil biota and soil chemistry

Many ant species accumulate organic debris in the vicinity of their nests. These organic materials should provide a rich resource base for the soil biota. We examined the effect of harvester ant nests (Pogonomyrmex barbatus) on the soil community and soil chemistry. Ant nest soils supported 30-fold higher densities of microarthropods and 5-fold higher densities of protozoa than surrounding, control soils. The relative abundances of the major groups of protozoa differed as well: amoebae and ciliates were relatively overrepresented, and flagellates underrepresented, in ant nest versus control soils. Densities of bacteria and fungi were similar in the two soil types. Concentrations of nitrate, ammonium, phosphorus, and potassium were significantly higher in ant nest soils, while concentrations of magnesium, calcium, and water were similar in nest and control soils. Ant nest soils were marginally more acidic than controls. The results demonstrate that P. barbatus nests constitute a significant source of spatial heterogeneity in soil biota and soil chemistry in arid grasslands. Received: 17 March 1997 / Accepted: 10 June 1997     

Persistence of Shiga toxin-producing Escherichia coli O26 in various manure-amended soil types

Aims:  To evaluate the behaviour of Shiga toxin-producing Escherichia coli (STEC) O26 strains inoculated in manure-amended soils under in vitro conditions.
Methods and Results:  Four green fluorescent protein (GFP)-labelled STEC O26 strains were inoculated in duplicate (at 10⁶ CFU g⁻¹) in three different manure-amended soil types, including two loam soils (A and B) and one clay loam soil (C), and two incubation temperatures (4 and 20°C) were tested. STEC counts and soil physical parameters were periodically monitored. STEC O26 cells were able to persist during extended periods in soil even in the presence of low moisture levels, i.e. less than 0·08 g H₂O g⁻¹ dry soil. At 4 and 20°C, STEC could be detected in soil A for 288 and 196 days, respectively, and in soils B and C for at least 365 days postinoculation at both temperatures. The ambient temperature (i.e. 20°C) was significantly associated with the highest STEC count decline in all soils tested.
Conclusions:  The temperature and soil properties appear to be contributory factors affecting the long-term survival of STEC O26 in manure-amended soils.
Significance and Impact of the Study:  This study provides useful information regarding the ecology of STEC O26 in manure-amended soils and may have implications for land and waste management.     

Survival of genetically modified Pseudomonas fluorescens introduced into subtropical soil microcosms

Abstract A genetically modified strain of Pseudomonas fluorescens and its parent showed grossly similar decline rates following introduction into subtropical clay and sandy soils. In unplanted clay soit at pH 6.9 and 25°C, population densities declined progressively from about 10⁸ to 10³ colony forming units (cfu) g⁻¹ dry soil over 75 days, but in unplanted sandy soil the introduced populations could not be detected after 25 days. In clay soil at pH 8.7 or 4.7, or at environmental temperature, decay rates were enhanced as compared to those at pH 6.9 and 25°C. Counts of introduced strains in clay bulk soil and in rhizosphere and rhizoplane of maize suggested that the introduced bacteria competed well with the native bacteria, and colonized the roots at about 10⁶ cfu g⁻¹ dry root at 25°C, over 20 days. However, rhizoplane colonization was lower at environmental temperature. The decay rate of both strains was slower in planted than in unplanted sandy soil. The population densities in the rhizosphere and rhizoplane in the sandy soil were significantly lower than those in the clay soil. Both introduced strains colonized the maize roots in both soils, using seeds coated with bacteria in 1% carboxymethyl cellulose. Introduced cells were localized at different sites along the roots of plants developing in clay soil, with higher densities in the original (near the seeds) and root hair zones as compared to the intermediate zones. No significant difference was observed between the extent of root colonization of the genetically modified strain and its parent.     

Management of soil microbial communities to enhance populations of Fusarium graminearum-antagonists in soil

Fusarium head blight (FHB), incited by Fusarium graminearum Schwabe is one of the most devastating diseases of wheat. Primary inoculum generated on crop residue is the driving force of FHB epidemics. Fusarium survival on crop residues is affected by soil microbial antagonists. The incorporation of green manures has been shown to increase the density and diversity of microbes in soils, particularly the density and the pathogen-inhibitory activity of specific bacteria and fungi. Evidence of increased streptomycete populations in soil as a response to green manure incorporation, and their negative effect on the survival of Fusarium oxysporum Schlechtendahl in soil, suggests their potential use to reduce the survival of related pathogens. There is, however, no precedent for the use of green manures to promote indigenous streptomycete populations to control FHB. This study investigated the use of green manures (sorghum–sudangrass hybrid [Sorghum bicolor (L.) Moench–S. bicolor (L.) Moench var. sudanense (Piper)] and common buckwheat [Fagopyrum esculentum (Moench)]) for reducing F. graminearum survival in association with wheat residues. Soil bacterial density, streptomycete density and the density and inhibitory activity of F. graminearum-antagonists were monitored from planting until 3 and 6 months following the incorporation of green manures in greenhouse and field experiments, respectively. The decomposition of wheat residues and survival of Fusarium in residues was also assessed. The use of green manures did not statistically impact the survival of F. graminearum in wheat residue. However, green manures promoted the development of higher densities and antagonistic abilities of F. graminearum-antagonists in soils. Additionally, streptomycete densities and F. graminearum-antagonist densities were significantly and positively correlated with reduced survival of Fusarium. The results of our study suggest that the use of green manures can enhance populations of indigenous soil microorganisms antagonistic to the survival of F. graminearum in wheat residue.     

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.     

Fusarium Species From Plant Debris Associated With Soils From Maize Production Areas in the Transkei Region of South Africa

Fusarium species were isolated from plant debris in soil samples collected from cultivated maize fields and from undisturbed grasslands in two areas of the Transkei region. A total of 1205 Fusarium isolates were recovered from 27 soil samples. Fifteen Fusarium species were recovered from plant debris from Bizana soils and 13 Fusarium species from plant debris from Centane soils. The two dominant Fusarium species in both areas were F. oxysporum and F. equiseti. Very few isolates of F. moniliforme and F. subglutinans were recovered, but both of these species had significantly higher relative densities in cultivated soils than in undisturbed soils. This revised version was published online in June 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.     

Changes in topsoil carbon stock in the Tibetan grasslands between the 1980s and 2004

Climate warming is likely inducing carbon loss from soils of northern ecosystems, but little evidence comes from large-scale observations. Here we used data from a repeated soil survey and remote sensing vegetation index to explore changes in soil organic carbon (SOC) stock on the Tibetan Plateau during the past two decades. Our results showed that SOC stock in the top 30 cm depth in alpine grasslands on the plateau amounted to 4.4 Pg C (1 Pg=10¹⁵ g), with an overall average of 3.9 kg C m⁻². SOC changes during 1980s–2004 were estimated at −0.6 g C m⁻² yr⁻¹, ranging from −36.5 to 35.8 g C m⁻² yr⁻¹ at 95% confidence, indicating that SOC stock in the Tibetan alpine grasslands remained relatively stable over the sampling periods. Our findings are nonconsistent with previous reports of loss of soil C in grassland ecosystems due to the accelerated decomposition with warming. In the case of the alpine grasslands on the Tibetan Plateau studied here, we speculate that increased rates of decomposition as soils warmed during the last two decades may have been compensated by increased soil C inputs due to increased grass productivity. These results suggest that soil C stock in terrestrial ecosystems may respond differently to climate change depending on ecosystem type, regional climate pattern, and intensity of human disturbance.     

Colonization of Flax Roots and Early Physiological Responses of Flax Cells Inoculated with Pathogenic and Nonpathogenic Strains of Fusarium oxysporum

Fusarium oxysporum includes nonpathogenic strains and pathogenic strains that can induce necrosis or tracheomycosis in plants. The objective of this study was to compare the abilities of a pathogenic strain (Foln3) and a nonpathogenic strain (Fo47) to colonize flax roots and to induce early physiological responses in flax cell culture suspensions. Both strains colonized the outer cortex of the root; however, plant defense reactions, i.e., the presence of wall appositions, osmiophilic material, and collapsed cells, were less frequent and less intense in a root colonized by Foln3 than by Fo47. Early physiological responses were measured in flax cell suspensions confronted with germinated microconidia of both strains. Both pathogenic (Foln3) and nonpathogenic strains (Fo47) triggered transient H₂O₂ production in the first few minutes of the interaction, but the nonpathogenic strain also induced a second burst 3 h postinoculation. Ca²⁺ influx was more intense in cells inoculated with Fo47 than in cells inoculated with Foln3. Similarly, alkalinization of the extracellular medium was higher with Fo47 than with Foln3. Inoculation of the fungi into flax cell suspensions induced cell death 10 to 20 h postinoculation, with a higher percentage of dead cells observed with Fo47 than with Foln3 beginning at 14 h. This is the first report showing that early physiological responses of flax cells can be used to distinguish pathogenic and nonpathogenic strains of the soil-borne fungus F. oxysporum.