Constitutive activation of TORC1 signalling attenuates virulence in the cross-kingdom fungal pathogen Fusarium oxysporum

The filamentous fungus Fusarium oxysporum causes vascular wilt disease in a wide range of plant species and opportunistic infections in humans. Previous work suggested that invasive growth in this pathogen is controlled by environmental cues such as pH and nutrient status. Here we investigated the role of Target Of Rapamycin Complex 1 (TORC1), a global regulator of eukaryotic cell growth and development. Inactivation of the negative regulator Tuberous Sclerosis Complex 2 (Tsc2), but not constitutive activation of the positive regulator Gtr1, in F. oxysporum resulted in inappropriate activation of TORC1 signalling under nutrient-limiting conditions. The tsc2Δ mutants showed reduced colony growth on minimal medium with different nitrogen sources and increased sensitivity to cell wall or high temperature stress. Furthermore, these mutants were impaired in invasive hyphal growth across cellophane membranes and exhibited a marked decrease in virulence, both on tomato plants and on the invertebrate animal host Galleria mellonella. Importantly, invasive hyphal growth in tsc2Δ strains was rescued by rapamycin-mediated inhibition of TORC1. Collectively, these results reveal a key role of TORC1 signalling in the development and pathogenicity of F. oxysporum and suggest new potential targets for controlling fungal infections.     

Host-specific Fusarium oxysporum Causes Wilt of Jojoba

Jojoba [Simmondsia chinensis (Link) Schneider] plantations in Israel originated from vegetative propagation, planted during 1991–92, have shown symptoms of wilting and subsequent death. Verticillium dahliae was only rarely isolated from these plants and artificial inoculation showed only mild disease symptoms. Fusarium oxysporum caused severe chlorosis, desiccation, defoliation and wilt in leaves of jojoba plants, resulting in plant death. Recovery of the fungus from artificially inoculated stem cuttings and seedlings showed for the first time that F. oxysporum was the primary pathogen. Inoculated cuttings exhibited wilt within 3 weeks, while in seedlings wilt occurred 10–24 weeks after inoculation. Seedlings and cuttings of jojoba which were inoculated with other Fusarium isolates originating from different crops (F. oxysporum f. sp. vasinfectum from cotton, F. oxysporum f. sp. dianthi from carnation, F. oxysporum f. sp. lycopersici from tomato and F. oxysporum f. sp. basilicum from basil) did not develop symptoms. Moreover, cotton, tomato, melon and cucumber seedlings inoculated with several virulent F. oxysporum isolates from jojoba did not show any symptoms of wilt or defoliation. These results indicate a high degree of specificity of the Fusarium isolates from jojoba; therefore, it is suggested that this isolate be defined as F. oxysporum f. sp. simmondsia.     

Two xylanase genes of the vascular wilt pathogen Fusarium oxysporum are differentially expressed during infection of tomato plants

Two genes encoding putative family F xylanases from the tomato vascular wilt pathogen Fusarium oxysporum f.sp. lycopersici have been cloned and sequenced. The two genes, designated xyl2 and xyl3, encode proteins with calculated molecular masses of 33 and 39.3 kDa and isoelectric points of 8.9 and 6.7, respectively. The predicted amino acid sequences show significant homology to other family F xylanases. XYL3 contains a cellulose-binding domain in its N-terminal region. Southern analysis suggested that xyl2 and xyl3 homologs are also present in other formae speciales of F. oxysporum. Both genes were expressed during growth on oat spelt xylan and tomato vascular tissue in vitro. RT-PCR revealed that xyl3 is expressed in roots and in the lower stems of tomato plants infected by F. oxysporum f.sp. lycopersici throughout the whole disease cycle, whereas xyl2 is only expressed during the final stages of disease. Received: 1 June 1998 / Accepted: 25 December 1998     

First record of Fusarium vascular wilt on grapevine in Egypt

During the summer season of 2003 and 2004, wilt syndromes of grapevine leaves (Cv. crimson) and vascular discolouration of roots have been observed in 2-year-old grapevine plants in the field at two sides in Gharbeia Governorate, Egypt. First, symptoms of wilt began on bottom leaves borderline as chlorosis and then these turned to necrotic spots and the leaves died. Wilt symptoms were spread to apical associated with vascular discolouration of roots and stem basal. Routine isolations of discoloured root tissue from diseased plant yielded eight isolates of Fusarium oxysporum Schlechtend only where no other fungi were developed. Microscopic examination revealed the presence of three shapes of microconidia, first is avoid shape non-septate measuring 2.5–3.0 μm × 6–10 μm, second is cylindrical with one septa measuring 2.6 μm × 17.0 μm and third shape also cylindrical with two septate measuring 3.0 μm × 20.0 μm. Macroconidia was rarely with three septate measuring 3.5– 4.0 μm × 35.0–38.0 μm, and chlamydospores were found singly or in pairs or chains. F. oxysporum isolates attacked grapevine plants (Cv. crimson) causing vascular wilt (66.7%) and root-rot syndrome (33.3%). In vitro isolates of F. oxysporum causing wilt of grapevine (Cv. crimson) varied for producing lytic enzymes, i.e. polygalacturonase (PG) and cellulase. The reactions of several grapevines (Cvs.) with a virulent isolate of F. oxysporum indicated the presence of two different symptoms, i.e. vascular wilt only on grapevine plants (Cv. crimson) and root-rot on the other grapevine (Cvs.), i.e. superior, Thompson, King robi and flame seedless. All F. oxysporum isolates caused vascular wilt of grapevine Cv. crimson, successfully reisolated from symptomatic vascular infected tissue and complete identification on the basis of colony, conidia morphology and host range at formae speciales level as F. oxysporum f. sp. herbemontis (Tochetto) Gordan. This is the first report of Fusarium wilt on grapevine in Egypt.     

Effects of treatment with phenylthiosemicarbazide and 4'-chlorophenylthiosemicarbazide on Fusarium wilt of pea and tomato plants

Effects of treatment with phenylthiosemicarbazide (PTS) and its 4′-chloro-derivative (4′-chloro-PTS) on Fusarium wilt of pea and tomato plants were investigated. Depending on pH and availability of oxygen, PTS and 4′-chloro-PTS are converted to their corresponding phenylazothioformamides and phenylazothioformamide-S-oxides, which are the actual fungitoxic compounds. PTS and 4′-chloro-PTS were shown to inhibit growth of Fusarium oxysporum f. pisi and F. oxysporum f. lycopersici in liquid media as well as on agar plates at concentrations of 50–100 mg/1. Inhibition was greater at pH 7 than at pH 5. When administered to pea and tomato plants, both compounds caused severe phytotoxic effects, especially at temperatures favouring Fusarium wilt, thus almost entirely obscuring any protective activity against the diseases. All compounds were strongly adsorbed to loam, but readily released from sand. Neither in pea nor in tomato plants were PTS and 4′-chloro-PTS converted to any fungitoxic substance, not already present in the aqueous solutions administered.     

<Emphasis Type="Italic">Fusarium sambucinum</Emphasis> isolate FS-94 induces resistance against fusarium wilt of tomato via activation and priming of a salicylic acid-dependent signaling system

The wheat rhizosphere-inhabiting nonpathogenic Fusarium sambucinum isolate FS-94 protected tomato from Fusarium wilt (F. oxysporum f. sp. lycopersici) in laboratory experiments. Seed soaking or immersion of seedling roots in a FS-94 spore suspension prior to inoculation with the pathogen delayed the appearance of wilt symptoms and significantly reduced disease severity in plants of a susceptible tomato cultivar. Quantification of fungal ergosterol in infected tomato showed that protection against wilt agent was related to limitation of the pathogen growth in plants exposed to FS-94. Incubation of tomato seedlings in a FS-94 spore suspension for 48 or 72 h led to plant protection and increased the salicylic acid (SA) concentration in their roots, suggesting that this isolate was involved in a plant-mediated mode of action and induced resistance. Soaking tomato seeds in the spore suspension did not induce SA accumulation in seedling roots, but nevertheless resulted in a significant reduction in wilt severity when the seedlings were challenged with the pathogen. In response to pathogen attack, the SA content in susceptible seedlings grown from FS-94-treated seeds started to increase within 1 day and remained elevated for 72 h. This suggests that F. sambucinum isolate FS-94 primed a SA-dependent signaling system in tomato.     

Genetic Diversity of Fusarium oxysporum Populations Isolated from Tomato Plants in Tunisia

Fusarium crown and root rot of tomato (Lycopersicon esculentum) caused by Fusarium oxysporum f. sp. radicis‐lycopersici is a new devastative disease of tomato greenhouse crops in Tunisia. Nothing is known neither about the population of this pathogen in this region, nor about the population of F. oxysporum f. sp. lycopersici the causal agent of Fusarium wilt of tomato. In order to examine the genetic relatedness among the F. oxysporum isolates by intergenic spacer restriction fragment length polymorphism (IGS‐RFLP) analysis and to elucidate the origin of the formae specialesradicis‐lycopersici in Tunisia by looking for genetic similarity of Tunisians isolates with isolates from a foreign source, the genetic diversity among F. oxysporum f. sp. radicis‐lycopersici and F. oxysporum f. sp. lycopersici populations was investigated. A total of 62 isolates of F. oxysporum, obtained from symptomless tomato plants, were characterized using IGS typing and pathogenicity tests on tomato plants. All Fusarium isolates were highly pathogenic on tomato. Fusarium oxysporum f. sp. radicis‐lycopersici isolates were separated into five IGS types. From the 53 F. oxysporum f. sp. radicis‐lycopersici isolates, 34 isolates have the same IGS types (IGS type 25), and the remaining 19 isolates were distributed into four IGS types. However, the only nine isolates of F. oxysporum f. sp. lycopersici have six different IGS types. This difference of diversity between the two formae speciales suggests that F. oxysporum f. sp. radicis‐lycopersici isolates have a foreign origin and may have been accidentally introduced into Tunisia.     

Galleria mellonella as model host for the trans-kingdom pathogen Fusarium oxysporum

Fusarium oxysporum, the causal agent of vascular wilt disease, affects a wide range of plant species and can produce disseminated infections in humans. F. oxysporum f. sp. lycopersici isolate FGSC 9935 causes disease both on tomato plants and immunodepressed mice, making it an ideal model for the comparative analysis of fungal virulence on plant and animal hosts. Here we tested the ability of FGSC 9935 to cause disease in the greater wax moth Galleria mellonella, an invertebrate model host that is widely used for the study of microbial human pathogens. Injection of living but not of heat-killed microconidia into the hemocoel of G. mellonella larvae resulted in dose-dependent killing both at 30 °C and at 37 °C. Fluorescence microscopy of larvae inoculated with a F. oxysporum transformant expressing GFP revealed hyphal proliferation within the hemocoel, interaction with G. mellonella hemocytes, and colonization of the killed insects by the fungus. Fungal gene knockout mutants previously tested in the tomato and immunodepressed mouse systems displayed a good correlation in virulence between the Galleria and the mouse model. Thus, Galleria represents a useful non-vertebrate infection model for studying virulence mechanisms of F. oxysporum on animal hosts.     

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.     

Insight into the molecular requirements for pathogenicity of Fusarium oxysporum f. sp. lycopersici through large-scale insertional mutagenesis

Background  

Fusarium oxysporum f. sp. lycopersici is the causal agent of vascular wilt disease in tomato. In order to gain more insight into the molecular processes in F. oxysporum necessary for pathogenesis and to uncover the genes involved, we used Agrobacterium-mediated insertional mutagenesis to generate 10,290 transformants and screened the transformants for loss or reduction of pathogenicity.     

Role of Bacillus amyloliquefaciens Y1 in the control of Fusarium wilt disease and growth promotion of tomato

The objective of this study was to examine the effects of Bacillus amyloliquefaciens Y1 on the control of Fusarium wilt disease and subsequent improvement in the growth of tomato plants. The Y1 strain strongly inhibited Fusarium oxysporum f. sp. lycopersici in vitro and also produced indole-3-acetic acid (IAA) in both the presence and absence of tryptophan. Over 96% of tomato seeds germinated when treated with either water, tryptone soy broth, or Y1 cultures, whereas root (5.40?cm) and shoot (5.15?cm) lengths were greatest in tomato seedlings treated with Y1 cultures that lacked tryptophan. Three experimental treatments – Black White medium (BW), BW medium with a commercial fungicide (BW?+?F), and Y1 culture inoculated in BW medium (Y1) – were applied to control Fusarium wilt disease under in vivo conditions. Application of Y1 culture and BW?+?F led to significantly lower disease incidence than did BW; moreover, shoot length and fresh and dry weight of both roots and shoots were greater in plants treated with Y1 than in plants treated with either BW or BW?+?F. A similar trend was observed for chitinase and β-1,3-glucanase activities in roots and leaves of tomato plants in all treatment groups over most of the experimental period. Finally, the presence of Y1 in the rhizospheric soils of Y1-treated plants resulted in a significant reduction in the populations of other bacteria. The results of our study demonstrated the effectiveness of Y1 not only in the control of Fusarium wilt disease but also for the enhancement of plant growth in cultivated tomato.     

The transmembrane protein Sho1 cooperates with the mucin Msb2 to regulate invasive growth and plant infection in Fusarium oxysporum

In the vascular wilt pathogen Fusarium oxysporum, the mitogen‐activated protein kinase (MAPK) Fmk1 is essential for plant infection. The mucin‐like membrane protein Msb2 regulates a subset of Fmk1‐dependent functions. Here, we examined the role of the tetraspan transmembrane protein Sho1 as an additional regulator of the Fmk1 pathway and determined its genetic interaction with Msb2. Targeted Δsho1 mutants were generated in wild‐type and Δmsb2 backgrounds to test possible interactions between the two genes. The mutants were examined for hyphal growth under different stress conditions, phosphorylation of the MAPK Fmk1 and an array of Fmk1‐dependent virulence functions. Similar to Msb2, Sho1 was required for the activation of Fmk1 phosphorylation, as well as Fmk1‐dependent gene expression and invasive growth functions, including extracellular pectinolytic activity, cellophane penetration, plant tissue colonization and virulence on tomato plants. Δsho1 mutants were hypersensitive to the cell wall‐perturbing compound Calcofluor White, and this phenotype was exacerbated in the Δmsb2 Δsho1 double mutant. These results highlight that Sho1 and Msb2 have partially overlapping functions upstream of the Fmk1 MAPK cascade, to promote invasive growth and plant infection, as well as cell wall integrity, in F. oxysporum.     

HapX-Mediated Iron Homeostasis Is Essential for Rhizosphere Competence and Virulence of the Soilborne Pathogen Fusarium oxysporum

Soilborne fungal pathogens cause devastating yield losses and are highly persistent and difficult to control. During the infection process, these organisms must cope with limited availability of iron. Here we show that the bZIP protein HapX functions as a key regulator of iron homeostasis and virulence in the vascular wilt fungus Fusarium oxysporum. Deletion of hapX does not affect iron uptake but causes derepression of genes involved in iron-consuming pathways, leading to impaired growth under iron-depleted conditions. F. oxysporum strains lacking HapX are reduced in their capacity to invade and kill tomato (Solanum lycopersicum) plants and immunodepressed mice. The virulence defect of ΔhapX on tomato plants is exacerbated by coinoculation of roots with a biocontrol strain of Pseudomonas putida, but not with a siderophore-deficient mutant, indicating that HapX contributes to iron competition of F. oxysporum in the tomato rhizosphere. These results establish a conserved role for HapX-mediated iron homeostasis in fungal infection of plants and mammals.     

In vitro selection of yellow passion fruit genotypes for resistance to <Emphasis Type="Italic">Fusarium</Emphasis> vascular wilt

Fusarium vascular wilt (caused by Fusarium oxysporum f. sp. passiflorae) is a limiting factor in the cultivation of yellow passion fruit (Passiflora edulis). Since there is no effective and economically viable control available, development of resistant or at least tolerant cultivars are in demand. A number of procedures have been used for the initial selection of plant genotypes resistant to various fungal pathogens by means of a fungal culture filtrate or purified toxin. In this study, seeds and in vitro-grown plantlets of passion fruit were screened with different concentrations of either Fusarium oxysporum f. sp. passiflorae (FOP) culture filtrate (0, 20, 30, 40 or 50%, v/v) or fusaric acid (0.10, 0.20, 0.30 or 0.40 mM) supplemented in Murashige and Skoog (MS) basal media. Subsequently, selected plants were inoculated with a conidial suspension of FOP to assess correlation between in vivo and in vitro responses. In vitro sensitivity to the selective agents and the resistance response to the pathogen were also compared. Root growth was markedly influenced by FA, culture filtrate, and conidial suspension culture treatments. Observations indicated that roots were primary targets for attack by F. oxysporum. Successful in vitro selection of resistant genotypes by both FA and culture filtrate treatments suggested that this strategy was viable for accelerating breeding of passion fruit for resistance to the Fusarium vascular wilt.     

The Nuclear Protein Sge1 of Fusarium oxysporum Is Required for Parasitic Growth

Dimorphism or morphogenic conversion is exploited by several pathogenic fungi and is required for tissue invasion and/or survival in the host. We have identified a homolog of a master regulator of this morphological switch in the plant pathogenic fungus Fusarium oxysporum f. sp. lycopersici. This non-dimorphic fungus causes vascular wilt disease in tomato by penetrating the plant roots and colonizing the vascular tissue. Gene knock-out and complementation studies established that the gene for this putative regulator, SGE1 (SIX Gene Expression 1), is essential for pathogenicity. In addition, microscopic analysis using fluorescent proteins revealed that Sge1 is localized in the nucleus, is not required for root colonization and penetration, but is required for parasitic growth. Furthermore, Sge1 is required for expression of genes encoding effectors that are secreted during infection. We propose that Sge1 is required in F. oxysporum and other non-dimorphic (plant) pathogenic fungi for parasitic growth.     

Two xylanase genes of the vascular wilt pathogen Fusarium oxysporum are differentially expressed during infection of tomato plants

Two genes encoding putative family F xylanases from the tomato vascular wilt pathogen Fusarium oxysporum f.sp. lycopersici have been cloned and sequenced. The two genes, designated xyl2 and xyl3, encode proteins with calculated molecular masses of 33 and 39.3?kDa and isoelectric points of 8.9 and 6.7, respectively. The predicted amino acid sequences show significant homology to other family F xylanases. XYL3 contains a cellulose-binding domain in its N-terminal region. Southern analysis suggested that xyl2 and xyl3 homologs are also present in other formae speciales of F. oxysporum. Both genes were expressed during growth on oat spelt xylan and tomato vascular tissue in vitro. RT-PCR revealed that xyl3 is expressed in roots and in the lower stems of tomato plants infected by F. oxysporum f.sp. lycopersici throughout the whole disease cycle, whereas xyl2 is only expressed during the final stages of disease.