Azaphilones biosynthesis complements the defence mechanism of Trichoderma guizhouense against oxidative stress

Filamentous fungi are known as producers of a large array of diverse secondary metabolites (SMs) that aid in securing their environmental niche. Here, we demonstrated that the SMs have an additional role in fungal defence against other fungi: Trichoderma guizhouense, a mycoparasite, is able to antagonize Fusarium oxysporum f. sp. cubense race 4 (Foc4) by forming aerial hyphae that kill the host with hydrogen peroxide. At the same time, a gene cluster comprising two polyketide synthases is strongly expressed. Using functional genetics, we characterized this cluster and identified its products as azaphilones (termed as trigazaphilones). The trigazaphilones were found lacking of antifungal toxicity but exhibited high radical scavenging activities. The antioxidant property of trigazaphilones was in vivo functional under various tested conditions of oxidative stress. Thus, we conclude that the biosynthesis of trigazaphilones serves as a complementary antioxidant mechanism and defends T. guizhouense against the hydrogen peroxide that it produces to combat other fungi like Foc4.     

Autophagy contributes to regulation of nuclear dynamics during vegetative growth and hyphal fusion in Fusarium oxysporum

In the fungal pathogen Fusarium oxysporum, vegetative hyphal fusion triggers nuclear mitotic division in the invading hypha followed by migration of a nucleus into the receptor hypha and degradation of the resident nucleus. Here we examined the role of autophagy in fusion-induced nuclear degradation. A search of the F. oxysporum genome database for autophagy pathway components identified putative orthologs of 16 core autophagy-related (ATG) genes in yeast, including the ubiquitin-like protein Atg8, which is required for the formation of autophagosomal membranes. F. oxysporum Foatg8Δ mutants were generated in a strain harboring H1-cherry fluorescent protein (ChFP)-labeled nuclei to facilitate analysis of nuclear dynamics. The Foatg8Δ mutants did not show MDC-positive staining in contrast to the wild type and the FoATG8-complemented (cFoATG8) strain, suggesting that FoAtg8 is required for autophagy in F. oxysporum. The Foatg8Δ strains displayed reduced rates of hyphal growth, conidiation, and fusion, and were significantly attenuated in virulence on tomato plants and in the nonvertebrate animal host Galleria mellonella. In contrast to wild-type hyphae, which are almost exclusively composed of uninucleated hyphal compartments, the hyphae of the Foatg8Δ mutants contained a significant fraction of hyphal compartments with 2 or more nuclei. The increase in the number of nuclei per hyphal compartment was particularly evident after hyphal fusion events. Time-lapse microscopy analyses revealed abnormal mitotic patterns during vegetative growth in the Foatg8Δ mutants. Our results suggest that autophagy mediates nuclear degradation after hyphal fusion and has a general function in the control of nuclear distribution in F. oxysporum.     

The pleiotropic functions of intracellular hydrophobins in aerial hyphae and fungal spores

Higher fungi can rapidly produce large numbers of spores suitable for aerial dispersal. The efficiency of the dispersal and spore resilience to abiotic stresses correlate with their hydrophobicity provided by the unique amphiphilic and superior surface-active proteins–hydrophobins (HFBs)–that self-assemble at hydrophobic/hydrophilic interfaces and thus modulate surface properties. Using the HFB-enriched mold Trichoderma (Hypocreales, Ascomycota) and the HFB-free yeast Pichia pastoris (Saccharomycetales, Ascomycota), we revealed that the rapid release of HFBs by aerial hyphae shortly prior to conidiation is associated with their intracellular accumulation in vacuoles and/or lipid-enriched organelles. The occasional internalization of the latter organelles in vacuoles can provide the hydrophobic/hydrophilic interface for the assembly of HFB layers and thus result in the formation of HFB-enriched vesicles and vacuolar multicisternal structures (VMSs) putatively lined up by HFBs. These HFB-enriched vesicles and VMSs can become fused in large tonoplast-like organelles or move to the periplasm for secretion. The tonoplast-like structures can contribute to the maintenance of turgor pressure in aerial hyphae supporting the erection of sporogenic structures (e.g., conidiophores) and provide intracellular force to squeeze out HFB-enriched vesicles and VMSs from the periplasm through the cell wall. We also show that the secretion of HFBs occurs prior to the conidiation and reveal that the even spore coating of HFBs deposited in the extracellular matrix requires microscopic water droplets that can be either guttated by the hyphae or obtained from the environment. Furthermore, we demonstrate that at least one HFB, HFB4 in T. guizhouense, is produced and secreted by wetted spores. We show that this protein possibly controls spore dormancy and contributes to the water sensing mechanism required for the detection of germination conditions. Thus, intracellular HFBs have a range of pleiotropic functions in aerial hyphae and spores and are essential for fungal development and fitness.     

Mycoparsitic behaviour ofFusarium oxysporum Schlechtendahl towardsAspergillus luchuensis Inui

Summary Mycoparasitic behaviour ofFusarium oxysporum Schlechtendahl towardsAspergillus luchuensis Inui was studiedin vitro. Growth of the hyphae ofF. oxysporum inside the hyphae and conidiophores ofA. luchuensis and formation of resting bodies ofF. oxysporum in conidiophores and hyphae ofA. luchuensis were observed. Bursting of the host hypha attacked by the parasitic hyphae was also noticed.     

Autophagy During Conidiation and Conidial Germination in Filamentous Fungi

Filamentous fungi form aerial hyphae on solid medium, and some of these differentiate into conidiophores for asexual sporulation (conidiation). In the filamentous deuteromycete, Aspergillus oryzae, aerial hyphae are formed from the foot cells and some differentiate into conidiophores, which are composed of vesicles, phialides and conidia. Recently, we isolated the yeast ATG8 gene homologue Aoatg8 from A. oryzae, and visualized autophagy by the expression of an EGFP (enhanced green fluorescent protein)–AoAtg8 fusion protein and DsRed2 protein in this fungus. Furthermore, by constructing the Aoatg8 deletion and conditional mutants, we demonstrated that autophagy functions during the process of differentiation of aerial hyphae, conidiation and conidial germination in A. oryzae. Here, we discuss the contribution of autophagy towards the differentiation and germination processes in filamentous fungi.

Addendum to:

Functional Analysis of the ATG8 Homologue Aoatg8 and Role of Autophagy in Differentiation and Germination in Aspergillus oryzae

T. Kikuma, M. Ohneda, M. Arioka and K. Kitamoto

Eukaryot Cell 2006; 5:1328-36     

Enzymatic Activities of Trametes versicolor and Pleurotus eryngii Implicated in Biocontrol of Fusarium oxysporum f. sp. lycopersici

The growth of the phytopathogenic fungus Fusarium oxysporum f. sp. lycopersici race 2 (FOL 2) was observed in dual culture with two soil fungi as biocontrol agents, Trametes versicolor and Pleurotus eryngii. In both cases, an interaction zone with the pathogen was found with the Fusarium’s hyphae becoming free of cytoplasmic content. The enzymatic complex of fungi, studied as biocontrol agents, showed β-(1,3)-glucanase activity, and no other important glucanase activities were noted in all of the media studied. As the principal components of F. oxysporum cell walls are glucans, the results of the positive attack on the cell walls of FOL 2 by the T. versicolor and P. eryngii enzymatic complex demonstrated the contribution of glucanases in the degradation of the hyphal cell walls of F. oxysporum. The lack of cellulase and xylanase activities (acting on plant cell wall polysaccharides) in T. versicolor makes this species a better alternative for the potential control of diseases caused by Fusarium spp.     

Microscopic observations on the interaction of the mycoparasite Verticillium biguttatum with Rhizoctonia solani and other soil-borne fungi

The mycoparasitic interactions of Verticillium biguttatum with Rhizoctonia solani and with a variety of other soil-borne fungi were investigated in dual cultures. V. biguttatum interacted with various soil fungi by appressed growth along the host hyphae and infrequent penetrations. Intracellular growth and subsequent sporulation, however, only occurred with R. solani, a few binucleate Rhizoctonia and Ceratobasidium spp., and Sclerotinia sclerotiorum. Effective mycoparasitism on sclerotia was restricted to those belonging to R. solani.Electron-microscopic observations revealed that V. biguttatum can penetrate the host cell with infection tubes. This process is probably mediated by enzymatic hydrolysis of the cell wall. Subsequently, trophic hyphae develop within the host cytoplasm, ultimately resulting in death of the host cell.     

Control of pathogenic fungi on Panax notoginseng by volatile oils from the food ingredients Allium sativum and Foeniculum vulgare

To screen natural drugs with strong inhibitory effects against pathogenic fungi related to P. notoginseng, the antifungal activities of garlic and fennel EOs were studied by targeting P. notoginseng disease-associated fungi, and the possible action mechanisms of garlic and fennel EOs as plant fungicides were preliminarily discussed. At present, the antifungal mechanism of EOs has not been fully established. Therefore, understanding the antifungal mechanism of plant EOs is helpful to address P. notoginseng diseases continuous cropping disease-related obstacles and other agricultural cultivation problems. First, the Oxford cup method and chessboard were used to confirm that the EOs and oxamyl had a significant inhibitory effect on the growth of Fusarium oxysporum. F. oxysporum is the main pathogen causing root rot of P. notoginseng and the preliminary study on the antifungal mechanisms of the EOs against F. oxysporum showed that the inhibition of EOs mainly affects cell membrane permeability and cell processes and affects the enzyme activities of micro-organism, to achieve antifungal effects. Finally, an in vivo model verified that both two EOs could significantly inhibit the occurrence of root rot caused by F. oxysporum.     

大豆根瘤内抗棉花枯萎病菌株的筛选及其防病试验

【目的】采用优良抗病性内生菌资源来控制棉花枯萎病是一种有效的措施。本研究从大豆根瘤中筛选棉花枯萎病拮抗性内生细菌,探索其对棉花枯萎病菌丝的抑制作用和代表菌株特性,为发掘和应用防病、抗逆优良菌株提供理论基础。【方法】采用对峙法和代谢液培养法对大豆根瘤内生细菌进行棉花枯萎病菌抑菌性筛选,显微观察法研究筛选菌株引起病原菌菌丝变化,通过菌株培养特征、理化特性和16S r DNA序列同源性分析确定菌株系统发育地位,比色法测定DD174耐盐碱性,盆栽试验验证防病效果。【结果】经复筛和代谢液试验有5株拮抗性较强菌株,被作用病原菌菌丝畸形、细胞壁消失、自溶,菌丝基部加粗、分支增多,呈树根状;菌丝被菌苔包埋而溶解、断裂,菌丝末端球形膨大。对棉花枯萎病菌的抑制作用主要通过菌体产生胞外代谢物发挥作用。菌株DD174、DD176和DD179最相似菌株分别为Bacillus oceanisediminis H2T(GQ292772)和B.thuringiensis ATCC 10792T(AF290545),菌株DD165和DD166最相似菌株均为Stenotrophomonas maltophilia LMG 958T(X95923)。DD174能耐受6%盐浓度,p H 10生长良好,具有一定耐盐碱能力。DD174处理组防治效果达76.32%,其他防效均在62%以上,可作为棉花枯萎病的生防菌株资源。【结论】大豆根瘤内存在棉花枯萎病内生拮抗细菌,其中有些菌株具有一定耐盐碱能力,对棉花枯萎病病原菌及病害有一定抑菌和防病作用。     

A pathogenesis related protein, AhPR10 from peanut: an insight of its mode of antifungal activity

A pathogenesis related protein (AhPR10) is identified from a clone of 6-day old Arachis hypogaea L. (peanut) cDNA library. The clone expressed as a ∼20 kDa protein in E. coli. Nucleotide sequence derived amino acid sequence of the coding region shows its homology with PR10 proteins having Betv1 domain and P loop motif. Recombinant AhPR10 has ribonuclease activity, and antifungal activity against the peanut pathogens Fusarium oxysporum and Rhizoctonia solani. Mutant protein AhPR10-K54N where lys54 is mutated to asn54 loses its ribonuclease and antifungal activities. FITC labeled AhPR10 and AhPR10-K54N are internalized by hyphae of F. oxysporum and R. solani but the later protein does not inhibit the fungal growth. This suggests that the ribonuclease function of AhPR10 is essential for its antifungal activity. Energy and temperature dependent internalization of AhPR10 into sensitive fungal hyphae indicate that internalization of the protein occurs through active uptake.Electronic Supplementary Material Supplementary material is available to authorised users in the online version of this article at .The nucleotide sequence of AhPR10 reported in this paper is submitted to NCBI Nucleotide Sequence Database under the Accession number AY726607.     

Autophagy contributes to regulation of nuclear dynamics during vegetative growth and hyphal fusion in Fusarium oxysporum

In the fungal pathogen Fusarium oxysporum, vegetative hyphal fusion triggers nuclear mitotic division in the invading hypha followed by migration of a nucleus into the receptor hypha and degradation of the resident nucleus. Here we examined the role of autophagy in fusion-induced nuclear degradation. A search of the F. oxysporum genome database for autophagy pathway components identified putative orthologs of 16 core autophagy-related (ATG) genes in yeast, including the ubiquitin-like protein Atg8, which is required for the formation of autophagosomal membranes. F. oxysporum Foatg8Δ mutants were generated in a strain harboring H1-cherry fluorescent protein (ChFP)-labeled nuclei to facilitate analysis of nuclear dynamics. The Foatg8Δ mutants did not show MDC-positive staining in contrast to the wild type and the FoATG8-complemented (cFoATG8) strain, suggesting that FoAtg8 is required for autophagy in F. oxysporum. The Foatg8Δ strains displayed reduced rates of hyphal growth, conidiation, and fusion, and were significantly attenuated in virulence on tomato plants and in the nonvertebrate animal host Galleria mellonella. In contrast to wild-type hyphae, which are almost exclusively composed of uninucleated hyphal compartments, the hyphae of the Foatg8Δ mutants contained a significant fraction of hyphal compartments with 2 or more nuclei. The increase in the number of nuclei per hyphal compartment was particularly evident after hyphal fusion events. Time-lapse microscopy analyses revealed abnormal mitotic patterns during vegetative growth in the Foatg8Δ mutants. Our results suggest that autophagy mediates nuclear degradation after hyphal fusion and has a general function in the control of nuclear distribution in F. oxysporum.     

Bikaverin and fusaric acid from Fusarium oxysporum show antioomycete activity against Phytophthora infestans

Aims: To isolate and identify antioomycete substances from Fusarium oxysporum EF119 against Phytophthora infestans and to investigate their antimicrobial activities against various plant pathogenic bacteria, oomycetes and true fungi. Methods and Results: Two antioomycete substances were isolated from liquid cultures of F. oxysporum EF119, which shows a potent disease control efficacy against tomato late blight caused by P. infestans. They were identified as bikaverin and fusaric acid by mass and nuclear magnetic resonance spectral analyses. They inhibited the mycelial growth of plant pathogenic oomycetes and fungi. Fusaric acid also effectively suppressed the cell growth of various plant pathogenic bacteria, but bikaverin was virtually inactive. Treatment with bikaverin at 300 μg ml^?1 suppressed the development of tomato late blight by 71%. Fusaric acid provided effective control against tomato late blight and wheat leaf rust over 67% at concentrations more than 100 μg ml^?1. Conclusions: Both bikaverin and fusaric acid showed in vitro and in vivo antioomycete activity against P. infestans. Significance and Impact of the Study: Fusarium oxysporum EF119 producing both bikaverin and fusaric acid may be used as a biocontrol agent against tomato late blight caused by P. infestans.     

Transgenic tomato plants expressing a wheat endochitinase gene demonstrate enhanced resistance to <Emphasis Type="Italic">Fusarium oxysporum</Emphasis> f. sp. <Emphasis Type="Italic">lycopersici</Emphasis>

Various chitinases have been shown to inhibit the growth of fungal pathogens in in vitro as well as in planta conditions. chi194, a wheat chitinases gene encoding a 33-kDa chitinase protein, was overexpressed in tomato plants (cv. Pusa Ruby) under the control of maize ubiquitin 1 promoter. The integration of transgene in tomato plants was confirmed with polymerase chain reaction (PCR) and Southern blot analysis. The inheritance of the transgene in T₁ and T₂ generations were shown by molecular analysis and the hygromycin sensitivity test. The broad range of chitinase activity was observed among the transgenic lines in T₀ and a similar range was retained in the T₁ and T₂ generations. Most importantly, the transgenic tomato lines with high chitinase activity were found to be highly resistant to the fungal pathogen Fusarium oxysporum f. sp. lycopersici. Thus, the results demonstrated that the expression of the wheat endochitinase chi194 in tomato plants confers resistance against Fusarium wilt disease caused by the fungal pathogen Fusarium oxysporum f. sp. lycopersici.     

Myrothins A–F from Endophytic Fungus Myrothecium sp. BS-31 Harbored in Panax notoginseng

Endophytic fungi play important roles for host's stress tolerance including invasion by pathogenic microbes. Small molecules are common weapons in the microbe–microbe interactions. Panax notoginseng is a widely used traditional Chinese medicinal plant and harbors many endophytes, some exert functions against pathogens. Here, we report six new compounds named myrothins A–F ( 1 – 6 ) produced by Myrothecium sp. BS-31, an endophyte isolated from P. notoginseng, and their antifungal activities against pathogenic fungi causing host root-rot disease. Their structures were elucidated with analysis of spectroscopic data including 1D and 2D NMR, HR-ESI-MS. Myrothins B ( 2 ) and E ( 5 ) showed the weak activity against Fusarium oxysporum and Phoma herbarum, and myrothins F ( 6 ) showed weak activity against F. oxysporum.     

Biocontrol Efficacy of Bacillus subtilis Strains Isolated from Cow Dung Against Postharvest Yam (Dioscorea rotundata L.) Pathogens

The biocontrol potential of Bacillus subtilis isolated from cow dung microflora was investigated in vitro and in vivo against two postharvest yam pathogenic fungi, Fusarium oxysporum and Botryodiplodia theobromae. B. subtilis strains inhibited the growth of F. oxysporum and B. theobromae in vitro in liquid medium in the range of 49.3–56.6% and in solid medium in the range of 31.0–36.0%, in comparison to the corresponding growth of fungi without bacterial inoculation. The interaction between B. subtilis CM1 and F. oxysporum was also studied by scanning electron microscopy. Chitinase production was demonstrated in vitro when B. subtilis was grown in the presence of colloidal chitin as the sole carbon source in a liquid medium. In vivo study showed that B. subtilis strains inhibited the growth of fungi (F. oxysporum and B. thobromae) up to 83% in wound cavities of yam tubers.