The decrotonylase FoSir5 facilitates mitochondrial metabolic state switching in conidial germination of Fusarium oxysporum

Fusarium oxysporum is one of the most important pathogenic fungi with a broad range of plant and animal hosts. The first key step of its infection cycle is conidial germination, but there is limited information available on the molecular events supporting this process. We show here that germination is accompanied by a sharp decrease in expression of FoSir5, an ortholog of the human lysine deacetylase SIRT5. We observe that FoSir5 decrotonylates a subunit of the fungal pyruvate dehydrogenase complex (FoDLAT) at K148, resulting in inhibition of the activity of the complex in mitochondria. Moreover, FoSir5 decrotonylates histone H3K18, leading to a downregulation of transcripts encoding enzymes of aerobic respiration pathways. Thus, the activity of FoSir5 coordinates regulation in different organelles to steer metabolic flux through respiration. As ATP content is positively related to fungal germination, we propose that FoSir5 negatively modulates conidial germination in F. oxysporum through its metabolic impact. These findings provide insights into the multifaceted roles of decrotonylation, catalysed by FoSir5, that support conidial germination in F. oxysporum.

The decrotonylase FoSir5 facilitates mitochondrial metabolic state switching in conidial germination of Fusarium oxysporum

Fusarium oxysporum is one of the most important pathogenic fungi with a broad range of plant and animal hosts. The first key step of its infection cycle is conidial germination, but there is limited information available on the molecular events supporting this process. We show here that germination is accompanied by a sharp decrease in expression of FoSir5, an ortholog of the human lysine deacetylase SIRT5. We observe that FoSir5 decrotonylates a subunit of the fungal pyruvate dehydrogenase complex (FoDLAT) at K148, resulting in inhibition of the activity of the complex in mitochondria. Moreover, FoSir5 decrotonylates histone H3K18, leading to a downregulation of transcripts encoding enzymes of aerobic respiration pathways. Thus, the activity of FoSir5 coordinates regulation in different organelles to steer metabolic flux through respiration. As ATP content is positively related to fungal germination, we propose that FoSir5 negatively modulates conidial germination in F. oxysporum through its metabolic impact. These findings provide insights into the multifaceted roles of decrotonylation, catalysed by FoSir5, that support conidial germination in F. oxysporum.

Mating-type genes control sexual reproduction, conidial germination and virulence in Cochliobolus lunatus

Cochliobolus lunatus (anamorph: Curvularia lunata) is a major pathogenic fungus that causes the Curvularia leaf spot of maize. ClMAT1-1-1 and ClMAT1-2-1, the C. lunatus orthologs of Cochliobolus heterostrophus ChMAT1-1-1 and ChMAT1-2-1, were investigated in the present study to uncover their functions in C. lunatus. Southern blot analysis showed that these mating-type MAT genes exist in the C. lunatus genome as a single copy. ClMAT1-1-1 and ClMAT1-2-1 were knocked out and complemented to generate ΔClmat1-1-1 and ΔClmat1-2-1, ΔClmat1-1-1-C and ΔClmat1-2-1-C, respectively. The mutant strains had defective sexual development and failed to produce pseudothecia. There were no significant differences in growth rate or conidia production between the mutant and wild-type strains. However, the aerial mycelia and mycelial dry weight of ΔClmat1-1-1 and ΔClmat1-2-1 were lower than that of wild type, suggesting that MAT genes affect asexual development. ClMAT genes were involved in the responses to cell wall integrity and osmotic adaptation. ΔClmat1-2-1 had a lower conidial germination rate than the wild-type strain CX-3. The virulence of ΔClmat1-2-1 and ΔClmat1-1-1 was also reduced compared to the wild type. Complementary strains could restore all the phenotypes.

Bacterial Inhibition on Beauveria bassiana Contributes to Microbiota Stability in Delia antiqua

Given the multiple roles of associated microbiota in improving animal host fitness in a microbial environment, increasing numbers of researchers have focused on how the associated microbiota keeps stable under complex environmental factors, especially some biological ones. Recent studies show that associated microbiota interacts with pathogenic microbes. However, whether and how the interaction would influence microbiota stability is limitedly investigated. Based on the interaction among Delia antiqua, its associated microbiota, and one pathogen Beauveria bassiana, the associated microbiota's response to the pathogen was determined in this study. Besides, the underlying mechanism for the response was also preliminarily investigated. Results showed that B. bassiana neither infect D. antiqua larvae nor did it colonize inside the associated microbiota, and both the bacterial and fungal microbiota kept stable during the interaction. Further experiments showed that bacterial microbiota almost completely inhibited conidial germination and mycelial growth of B. bassiana during its invasion, while fungal microbiota did not inhibit conidial germination and mycelial growth of B. bassiana. According to the above results, individual dominant bacterial species were isolated, and their inhibition on conidial germination and mycelial growth of B. bassiana was reconfirmed. Thus, these results indicated that bacterial instead of fungal microbiota blocked B. bassiana conidia and stabilized the associated microbiota of D. antiqua larvae during B. bassiana invasion. The findings deepened the understanding of the role of associated microbiota–pathogen microbe interaction in maintaining microbiota stability. They may also contribute to the development of novel biological control agents and pest management strategies.

Functional Analysis of Keto-Acid Reductoisomerase ILVC in the Entomopathogenic Fungus Metarhizium robertsii

Ketol-acid reductoisomerase (ILVC) is the second enzyme in the branched-chain amino acid (BCAA) biosynthesis, which regulates many physiological activities in a variety of organisms from bacteria to fungi and plants. In this work, function mechanisms of ILVC in Metarhizium robertsii Metchnikoff (Hypocreales: Clavicipitaceae) were explored with site-directed mutagenesis, reductase activity assays and transcriptomics analysis. The reductase activity assays showed that ILVC from phytopathogenic fungi exhibited significantly higher activities than those from entomopathogenic fungi but lower than those from yeast. Site-directed mutagenesis and enzymatic activities of MrILVC with different active-site mutants (Arg-113, Ser-118, Asp-152, Asp-260, and Glu-264) confirmed that active sites of MrILVC are conserved with plant and bacterial ILVCs. Deleting MrilvC causes the complete failures of vegetative growth and conidial germination, feeding with branched-chain amino acids (BCAAs) recovers the fungal growth but not conidial germination, while both characteristics are restored when supplemented with yeast extract. Compared to ΔMrilvC cultured in czapek agar (CZA), plenty of genes involved in the biosynthesis of antibiotics and amino acids were up- or down-regulated in the wild type or ΔMrilvC feeding with either BCAAs or yeast extract. Further analysis showed some genes, such as catalase A, participate in mycelial growth and conidial germination was down-regulated in ΔMrilvC from CZA, revealing that MrILVC might control the fungal development by gene regulation and BCAAs or yeast extract could play partial roles of MrILVC. This study will advance our understanding of ILVC function mechanisms in fungi.

Assessment of Plant Extracts and their In vitro Efficacy against Potato Early Blight Incited by Alternaria solani

Botanicals obtained from the plants are well known for the suppression of inimical plant pathogens. The present study explores the efficacy of five locally available plant extracts for their antifungal activity against the early blight of potato incited by Alternaria solani. The extracts include Datura stramonium, Allium sativum, Azadirachta indica, Eucalyptus globulus, and Lantana camara. All extracts reduced mycelial growth and conidial germination of A. solani. In vitro studies showed that extracts obtained from A. sativum and A. indica have significant inhibition of mycelial growth of A. solani (88.80 and 86.62 percent) at 20 percent concentration. Higher concentrations of A. sativum extract caused a higher reduction of A. solani radial growth on potato dextrose agar medium. Extracts obtained from A. sativum and A. indica at 20 percent concentration, were found most effective for inhibition of conidial germination (85.50 and 80.04 percent) respectively of A. solani. Observations by scanning electron microscope (SEM) showed dramatic alteration in A. solani hyphae collapsed and spores shrinked when treated with extract of A. sativum at a 20 per cent concentration. The qualitative and quantitative analysis of various phytochemicals like flavonoids, alkaloids, saponins, tannins, steroids, terpenoids, glycosides, and phenols was showed A. sativum extract better than all the other plant extracts. Observation also revealed that 20 percent concentration of garlic extract has potential to inhibit to A. solani.

On the virulence of two Beauveria bassiana strains against the fall webworm, Hyphantria cunea (Durry) (Lepidoptera: Erebidae), larvae and their biological properties in relation to different abiotic factors

Background The genus Beauveria is frequently used as a mycoinsecticides in many countries to control insect pests in agriculture, it is being very effective against the fall webworm, Hyphantria cunea (Durry) (Lepidoptera: Erebidae), which is a pest of trees in forests and orchards. Multiple abiotic factors during fungal growth are well known to influence mycelial growth and several physiological adaptations in the conidia produced. Results In this study, the pathogenicity of the Beauveria bassiana strains Bb10331 and Bb7725 against H. cunea was evaluated. Peptone potato dextrose agar (PPDA) was used as the medium and colony diameter, conidiation capacity, conidial germination rate were directly affected by relative humidity (RH), illumination, and the ambient pH. The LC50 values of Bb10331 and Bb7725 to H. cunea were 4.72 × 106 and 3.28 × 106 conidia·ml−1, respectively, after 120 h post treatments, while their corresponding LT50 values were 71.13 and 74.54 h at the concentration of 1 × 108 conidia/ml. The Bb7725 had a conidial germination rate than did Bb10331 at the same RH. The two strains grew faster under a dark:light (D:L) photoperiod of 12:12 h, and this particular light condition was also most suitable for their conidia production. The optimum pH for the growth and conidiation of the two strains was approximately 7.0. Conclusions Both strains are promising for pest control, possessing effective virulence against H. cunea, but this is slightly stronger in Bb7725 than Bb10331. The values of abiotic factors apt to promote the biological properties of each B. bassiana were different.

The Msn2 Transcription Factor Regulates Acaricidal Virulence in the Fungal Pathogen Beauveria bassiana

Beauveria bassiana holds promise as a feasible biological control agent for tick control. The B. bassiana stress–response transcription factor Msn2 is known to contribute to fungal growth, conidiogenesis, stress–response and virulence towards insects; however, little is known concerning whether Msn2 is involved in infection across Arthropoda classes. We evaluated the effects of Msn2 on B. bassiana virulence against Rhipicephalus microplus (Acari, Ixodidae) using wild-type, targeted gene knockout (ΔBbmsn2) and complemented mutant (ΔBbmsn2/Bbmsn2) strains. Reproductive parameters of R. microplus engorged females treated topically or by an intra-hemocoel injection of conidial suspensions were assessed. Treated cuticles of engorged females were analyzed by microscopy, and proteolytic activity of B. bassiana on cuticles was assessed. Topically treated engorged females showed high mean larval hatching (>84%) in control and ΔBbmsn2 treatments, whereas treatment with the wild-type or ΔBbmsn2/Bbmsn2 strains resulted in significantly decreased (lowered egg viability) larval hatching. Percent control of R. microplus topically treated with ΔBbmsn2 was lower than in the groups treated with wild-type (56.1%) or ΔBbmsn2/Bbmsn2 strains. However, no differences on reproductive parameters were detected when R. microplus were treated by intra-hemocoel injection using low (800 conidia/tick) doses for all strains tested; R. microplus injected with high doses of wild-type or mutant strains (106 conidia/tick) died before laying eggs (~48 h after treatment). SEM analyses of B. bassiana infection showed similar conidial germination and formation of pseudo-appressoria on tick cuticle. Histological sections of ticks treated with the wild-type or ΔBbmsn2/Bbmsn2 strains showed fungal penetration through the cuticle, and into the tick interior. Hyphae of ΔBbmsn2, however, did not appear to penetrate or breach the tick exocuticle 120 h after treatment. Protease activity was lower on tick cuticles treated with ΔBbmsn2 than those treated with the wild-type or ΔBbmsn2/Bbmsn2 strains. These data show that loss of the Msn2 transcription factor reduced B. bassiana virulence against R. microplus, but did not interfere with conidial germination, appressoria formation or sporulation on tick cadavers, and plays only a minimal role once the cuticle is breached. Our results indicate that the BbMsn2 transcription factor acts mainly during the fungal penetration process and that decreased protease production may be one mechanism that contributes to the inability of the mutant strain to breach the tick cuticle.