A Nitrogen Response Pathway Regulates Virulence Functions in Fusarium oxysporum via the Protein Kinase TOR and the bZIP Protein MeaB

During infection, fungal pathogens activate virulence mechanisms, such as host adhesion, penetration and invasive growth. In the vascular wilt fungus Fusarium oxysporum, the mitogen-activated protein kinase Fmk1 is required for plant infection and controls processes such as cellophane penetration, vegetative hyphal fusion, or root adhesion. Here, we show that these virulence-related functions are repressed by the preferred nitrogen source ammonium and restored by treatment with l-methionine sulfoximine or rapamycin, two specific inhibitors of Gln synthetase and the protein kinase TOR, respectively. Deletion of the bZIP protein MeaB also resulted in nitrogen source–independent activation of virulence mechanisms. Activation of these functions did not require the global nitrogen regulator AreA, suggesting that MeaB-mediated repression of virulence functions does not act through inhibition of AreA. Tomato plants (Solanum lycopersicum) supplied with ammonium rather than nitrate showed a significant reduction in vascular wilt symptoms when infected with the wild type but not with the ΔmeaB strain. Nitrogen source also affected invasive growth in the rice blast fungus Magnaporthe oryzae and the wheat head blight pathogen Fusarium graminearum. We propose that a conserved nitrogen-responsive pathway might operate via TOR and MeaB to control virulence in plant pathogenic fungi.     

Fungal Parasitism of Heterodera glycines Eggs as Influenced by Egg Age and Pre-colonization of Cysts by Other Fungi

The objective of this study was to determine the effect of egg age and pre-colonization of cysts by a saprophytic or parasitic fungus on parasitism of Heterodera glycines eggs by other parasitic fungi. In agar and in soil tests, fungi generally parasitized more eggs in early developmental stages than eggs containing a juvenile. The effect of pre-colonization of cysts by a fungus on parasitism of eggs by other fungi depended on the fungi involved. In most cases, pre-colonization of cysts by an unidentified, saprophytic fungal isolate (A-1-24) did not affect parasitism of eggs in the cysts subsequently treated with other fungi. However, pre-colonization of cysts by A-1-24 reduced fungal parasitism of eggs in cysts subsequently treated with Cylindrocarpon destructans isolate 3. In agar tests, pre-colonization of cysts by Chaetomium cochliodes, a saprophytic or weakly parasitic fungus, reduced parasitism of eggs in cysts subsequently treated with Verticillium chlamydosporium Florida isolate, Fusarium oxysporum, Fusarium solani, ARF18, and another sterile fungus. However, in soil tests, pre-colonization of cysts by C. cochliodes had no effect on parasitism of eggs by subsequent fungal parasites. In another test, parasitism of eggs by V. chlamydosporium in cysts was not affected by pre-colonizing fungi C. destructans, F. oxysporum, and F. solani but was reduced by Mortierella sp., Pyrenochaeta terrestris, and C. cochliodes. Parasitism of eggs in cysts by ARF18 was reduced by pre-colonizing fungi C. destructans, F. oxysporum, F. solani, P. terrestris, and C. cochliodes but not Mortierella sp.     

Expression and role of CYP505A1 in pathogenicity of Fusarium oxysporum f. sp. lactucae

BackgroundCytochrome P450 enzymes (CYPs) are monooxygenases present in every domain of life. In fungi CYPs are involved in virulence. Fusarium wilt of lettuce, caused by F. oxysporum f. sp. lactucae, is the most serious disease of lettuce. F. oxysporum f.sp. lactucae MSA35 is an antagonistic fungus. Pathogenic formae specialis of F. oxysporum possess a CYP belonging to the new family CYP505. This enzyme hydroxylates saturated fatty acids that play a role in plant defence.MethodsMolecular tools were adopted to search for cyp505 gene in MSA35 genome. cyp505 gene expression analysis in pathogenic and antagonistic Fusarium was performed. The enzyme was expressed in its recombinant form and used for catalytic reactions with fatty acids, the products of which were characterized by mass spectrometry analysis.ResultsA novel MSA35 self-sufficient CYP505 is differentially expressed in antagonistic and pathogenic F. oxysporum. Its expression is induced by the host plant lettuce in both pathogenesis and antagonism during the early phase of the interaction, while it is silenced during the late phase only in antagonistic Fusarium. Mass-spectrometry investigations proved that CYP505A1 mono-hydroxylates lauric, palmitic and stearic acids.ConclusionsThe ability of CYP505A1 to oxidize fatty acids present in the cortical cell membranes together with its differential expression in its Fusarium antagonistic form point out to the possibility that this enzyme is associated with Fusarium pathogenicity in lettuce.General significanceThe CYP505 clan is present in pathogenic fungal phyla, making CYP505A1 enzyme a putative candidate as a new target for the development of novel antifungal molecules.     

The FRP1 F-box gene has different functions in sexuality, pathogenicity and metabolism in three fungal pathogens

Plant‐pathogenic fungi employ a variety of infection strategies; as a result, fungi probably rely on different sets of proteins for successful infection. The F‐box protein Frp1, only present in filamentous fungi belonging to the Sordariomycetes, Leotiomycetes and Dothideomycetes, is required for nonsugar carbon catabolism and pathogenicity in the root‐infecting fungus Fusarium oxysporum. To assess the role of Frp1 in other plant‐pathogenic fungi, FRP1 deletion mutants were generated in Fusarium graminearum and Botrytis cinerea, and their phenotypes were analysed. Deletion of FgFRP1 in F. graminearum led to impaired infection of barley roots, but not of aerial plant parts. Deletion of BcFRP1 in B. cinerea did not show any effect on pathogenicity. Sexual reproduction, however, was impaired in both F. graminearum and B. cinerea FRP1 deletion mutants. The mutants of all three fungi displayed different phenotypes when grown on an array of carbon sources. The F. oxysporum and B. cinerea deletion mutants showed opposite growth phenotypes on sugar and nonsugar carbon sources. Replacement of FoFRP1 in F. oxysporum with the B. cinerea BcFRP1 resulted in the restoration of pathogenicity, but also in a switch from impaired growth on nonsugar carbon sources to impaired growth on sugar carbon sources. This effect could be ascribed in part to the B. cinerea BcFRP1 promoter sequence. In conclusion, the function of the F‐box protein Frp1, despite its high sequence conservation, is not conserved between different fungi, leading to differential requirements for pathogenicity and carbon source utilization.     

Vegetative hyphal fusion is not essential for plant infection by Fusarium oxysporum

Vegetative hyphal fusion (VHF) is a ubiquitous phenomenon in filamentous fungi whose biological role is poorly understood. In Neurospora crassa, the mitogen-activated protein kinase (MAPK) Mak-2 and the WW domain protein So are required for efficient VHF. A MAPK orthologous to Mak-2, Fmk1, was previously shown to be essential for root penetration and pathogenicity of the vascular wilt fungus Fusarium oxysporum. Here we took a genetic approach to test two hypotheses, that (i) VHF and plant infection have signaling mechanisms in common and (ii) VHF is required for efficient plant infection. F. oxysporum mutants lacking either Fmk1 or Fso1, an orthologue of N. crassa So, were impaired in the fusion of vegetative hyphae and microconidial germ tubes. Δfmk1 Δfso1 double mutants exhibited a more severe fusion phenotype than either single mutant, indicating that the two components function in distinct pathways. Both Δfso1 and Δfmk1 strains were impaired in the formation of hyphal networks on the root surface, a process associated with extensive VHF. The Δfso1 mutants exhibited slightly reduced virulence in tomato fruit infection assays but, in contrast to Δfmk1 strains, were still able to perform functions associated with invasive growth, such as secretion of pectinolytic enzymes or penetration of cellophane sheets, and to infect tomato plants. Thus, although VHF per se is not essential for plant infection, both processes have some signaling components in common, suggesting an evolutionary relationship between the underlying cellular mechanisms.     

Antimicrobial activity of Araucaria cunninghamii Sweet and the chemical constituents of its twigs and leaves

The present study was aimed at the identification of antimicrobial components from Araucaria cunninghamii with an activity-guided purification process. Eight compounds were obtained from the most active n-BuOH fraction and identified as the new compound 4-n-butoxyl-phenylpropanetriol (1), together with seven known compounds (2–8). These compounds were tested for antimicrobial activities against five bacteria and four plant pathogenic fungi. Within the series of compounds tested, compound 2 was the most active, particularly displaying moderate antibacterial activities against Erwinia carotovora and Bacillus subtilis with MICs 7.8 and 15.5 μg/ml. Moreover, this compound exhibited inhibitory activities against four plant pathogenic fungi: Helminthosporium sativum, Rhizoctonia solani, Fusarium oxysporum f. sp. Niveum and Fusarium oxysporum f. sp. Cubense, with EC₅₀ values of 42.3, 90.0, 62.7 and 100.2 μg/ml. To our knowledge, this is the first report that the n-BuOH fraction and compound 2 from A. cunninghamii showed inhibitory activity against plant pathogenic fungi.     

Investigating Agrobacterium-mediated transformation of Verticillium albo-atrum on plant surfaces

Background

Agrobacterium tumefaciens has long been known to transform plant tissue in nature as part of its infection process. This natural mechanism has been utilised over the last few decades in laboratories world wide to genetically manipulate many species of plants. More recently this technology has been successfully applied to non-plant organisms in the laboratory, including fungi, where the plant wound hormone acetosyringone, an inducer of transformation, is supplied exogenously. In the natural environment it is possible that Agrobacterium and fungi may encounter each other at plant wound sites, where acetosyringone would be present, raising the possibility of natural gene transfer from bacterium to fungus.

Methodology/Principal Findings

We investigate this hypothesis through the development of experiments designed to replicate such a situation at a plant wound site. A. tumefaciens harbouring the plasmid pCAMDsRed was co-cultivated with the common plant pathogenic fungus Verticillium albo-atrum on a range of wounded plant tissues. Fungal transformants were obtained from co-cultivation on a range of plant tissue types, demonstrating that plant tissue provides sufficient vir gene inducers to allow A. tumefaciens to transform fungi in planta.

Conclusions/Significance

This work raises interesting questions about whether A. tumefaciens may be able to transform organisms other than plants in nature, or indeed should be considered during GM risk assessments, with further investigations required to determine whether this phenomenon has already occurred in nature.     

Effect of chitosan on hyphal growth and spore germination of plant pathogenic and biocontrol fungi

Aims: To investigate the toxic effect of chitosan on important root pathogenic and biocontrol fungi (nematophagous, entomopathogenic and mycoparasitic). Methods and Results: We have used standard bioassays to investigate the effect of chitosan on colony growth and developed bioassays to test spore germination. The results showed that the root pathogenic and mycoparasitic fungi tested were more sensitive to chitosan than nematophagous and entomopathogenic fungi. Chitosanases (and perhaps related enzymes) are involved in the resistance to chitosan. Two fungi, one sensitive to chitosan, Fusarium oxysporum f. sp. radicis‐lycopersici, and one less sensitive, Pochonia chlamydosporia, were selected for ultrastructural investigations. Transmission electron microscopy revealed differences in the ultrastructural alterations caused by chitosan in the spores of the plant pathogenic fungus and in those of the nematophagous fungus. Confocal laser microscopy showed that Rhodamine‐labelled chitosan enters rapidly into conidia of both fungi, in an energy‐dependent process. Conclusions: Nematophagous and entomopathogenic fungi are rather resistant to the toxic effect of chitosan. Resistance of nematophagous and entomopathogenic fungi to chitosan could be associated with their high extracellular chitosanolytic activity. Furthermore, ultrastructural damage is much more severe in the chitosan sensitive fungus. Significance and impact of the study: The results of this paper suggest that biocontrol fungi tested could be combined with chitosan for biological control of plant pathogens and pests.     

The role of strigolactones during plant interactions with the pathogenic fungus <Emphasis Type="Italic">Fusarium</Emphasis><Emphasis Type="Italic">oxysporum</Emphasis>

Main conclusion

Strigolactones (SLs) do not influence spore germination or hyphal growth of Fusarium oxysporum. Mutant studies revealed no role for SLs but a role for ethylene signalling in defence against this pathogen in pea. Strigolactones (SLs) play important roles both inside the plant as a hormone and outside the plant as a rhizosphere signal in interactions with mycorrhizal fungi and parasitic weeds. What is less well understood is any potential role SLs may play in interactions with disease causing microbes such as pathogenic fungi. In this paper we investigate the influence of SLs on the hemibiotrophic pathogen Fusarium oxysporum f.sp. pisi both directly via their effects on fungal growth and inside the plant through the use of a mutant deficient in SL. Given that various stereoisomers of synthetic and naturally occuring SLs can display different biological activities, we used (+)-GR24, (?)-GR24 and the naturally occurring SL, (+)-strigol, as well as a racemic mixture of 5-deoxystrigol. As a positive control, we examined the influence of a plant mutant with altered ethylene signalling, ein2, on disease development. We found no evidence that SLs influence spore germination or hyphal growth of Fusarium oxysporum and that, while ethylene signalling influences pea susceptibility to this pathogen, SLs do not.

     

Cell Cycle–Mediated Regulation of Plant Infection by the Rice Blast Fungus

To gain entry to plants, many pathogenic fungi develop specialized infection structures called appressoria. Here, we demonstrate that appressorium morphogenesis in the rice blast fungus Magnaporthe oryzae is tightly regulated by the cell cycle. Shortly after a fungus spore lands on the rice (Oryza sativa) leaf surface, a single round of mitosis always occurs in the germ tube. We found that initiation of infection structure development is regulated by a DNA replication-dependent checkpoint. Genetic intervention in DNA synthesis, by conditional mutation of the Never-in-Mitosis 1 gene, prevented germ tubes from developing nascent infection structures. Cellular differentiation of appressoria, however, required entry into mitosis because nimA temperature-sensitive mutants, blocked at mitotic entry, were unable to develop functional appressoria. Arresting the cell cycle after mitotic entry, by conditional inactivation of the Blocked-in-Mitosis 1 gene or expression of stabilized cyclinB-encoding alleles, did not impair appressorium differentiation, but instead prevented these cells from invading plant tissue. When considered together, these data suggest that appressorium-mediated plant infection is coordinated by three distinct cell cycle checkpoints that are necessary for establishment of plant disease.     

Association of Criconemella xenoplax and Fusarium spp. with Root Necrosis and Growth of Peach

Criconemella xenoplax, Fusarium solani, and F. oxysporum caused necrosis of Nemaguard peach feeder roots in greenhouse tests. Root necrosis was more extensive in the presence of either fungus than wtih C. xenoplax alone. Shoot growth and plant height were less for plants inoculated with F. oxysporum or F. solani than for plants inoculated with the fungi plus C. xenoplax. Neither synergistic nor additive effects on root necrosis or plant growth occurred between C. xenoplax and the fungal pathogens.     

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.     

Mycorrhizal fungi induced activation of tomato defense system mitigates Fusarium wilt stress

The fungus Fusarium oxysporum f. sp. lycopersici (FOL) is known to cause vascular wilt on tomato almost over the world. Inoculation of FOL reduced plant growth and increased wilt of tomato. The following study examined the possible role of arbuscular mycorrhizal fungi (AMF) consortium comprising of Rhizophagus intraradices, Funneliformis mosseae and Claroideoglomus etunicatum against FOL in tomato and explored in an inducing plant systemic defense. AMF inoculation reduced the wilt disease within vascular tissue and in vivo production of fusaric acid was observed which may be responsible in reduced wilting. FOL had an antagonistic effect on AMF colonization, reduced the number of spores, arbuscules and vesicles. AMF also inhibited the damage induced by Fusarium wilt through increasing chlorophyll contents along with the activity of phosphate metabolising enzymes (acid and alkaline phosphatases). Moreover, tomato plants with mycorrhizal inoculation showed an increase in the level of antioxidant enzymes including glutathione reductase, catalase, and etc. with an ultimate influence on the elimination of reactive oxygen species. Moreover, rise in phosphatase along with antioxidant enzymatic systems and enhanced photosynthetic performance contributed to induced resistance against FOL in tomato.     

Antifungal potential,chemical composition of Chlorella vulgaris and SEM analysis of morphological changes in Fusarium oxysporum

In pursuit of an environmentally benign fungicide alternative, the current study explored the antifungal activity of Chlorella vulgaris extracts against six plant pathogenic fungi (in vitro). The well diffusion agar method was used to investigate the growth inhibition of Fusarium oxysporum, Fusarium sp., Fusarium solani, A. flavus, A. niger, and A. alternata using the three C. vulgaris extracts viz. methanol (CvME), acetone (CvAE), and diethyl ether (CvDE). Different concentrations of CvDE were also investigated against F. oxysporum. The morphological modifications in F. oxysporum treated with CvDE (5 mg/kg) were studied using SEM and the chemical composition of CvDE was also determined by GC–MS analysis. All extracts, with the exception of A. alternata, were found to be effective in inhibiting the growth of plant pathogenic fungi. The CvDE extract, followed by CvME and CvAE, was found to be efficient against tested fungi. The CvDE was most effective against F. oxysporum with a 73.3% growth inhibition. The effects of various CvDE concentrations on F. oxysporum were found to be dosage dependent. The SEM micrograph revealed that CvDE-treated F. oxysporum had substantially less conidia than the control. The CvDE treatment damaged the mycelial structure as well. Major chemical components detected in CvDE were Heptaldehyde (15.7%), Octadecenoic acid, methyl ester (12.6%), Hexadecanoic acid (12%), 3-Decyn-2-Ol (10.98%), (E)-3,7,11,15-tetramethylhexadec-2-ene (9.76%), heptadecane-1,2,3,4,5-pentol (8.7%), Docosane, 4-methyl (7.28%).     

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.     

Calcium signaling in pathogenic and beneficial plant microbe interactions: What can we learn from the interaction between Piriformospora indica and Arabidopsis thaliana

Elevation of intracellular calcium levels in a plant cell is an early signaling event in many mutualistic and pathogenic plant/microbe interactions. In pathogenic plant/fungus interactions, receptor-mediated cytoplasmic calcium elevations induce defense genes via the activation of ion fluxes at the plasma membrane, an oxidative burst and MAPK activation. Mycorrhizal and beneficial endophytic plant/fungus interactions result in a better plant performance through sequencial cytoplasmic and nuclear calcium elevations. The specificity of the calcium responses depends on the calcium signature, its amplitude, duration, frequency and location, a selective activation of calcium channels in the diverse cellular membranes and the stimulation of calcium-dependent signaling components. Arabidopsis contains more than 100 genes for calcium-binding proteins and channels and the response to pathogens and beneficial fungi relies on a highly specific activation of individual members of these protein families. Genetic tools are required to understand this complex response patterns and the cross talks between the individual calcium-dependent signaling pathways. The beneficial interaction of Arabidopsis with the growth-promoting endophyte Piriformospora indica provides a nice model system to unravel signaling events leading to mutualistic or pathogenic plant/fungus interactions.Key words: Piriformospora indica, calcium, calcium signature, plant/microbe interaction