Relationship between antioxidant enzymes and sclerotial formation of Pleurotus tuber-regium under abiotic stress.

In order to explore the relationship between sclerotial formation and antioxidant enzymes under abiotic stresses, the effects of abiotic stresses including temperature, pH value, osmotic pressure, limited nitrogen, and hydrogen peroxide (H2O2) on the activities of antioxidant enzymes, ascorbate peroxidase (APX), superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) in Pleurotus tuber-regium were studied. Meanwhile, the sclerotial formation under these abiotic stress conditions was also investigated. It was found that low temperature, weak alkaline, appropriate osmotic stress, and H2O2 can promote sclerotial formation, and sclerotial formation always tended to occur when the activities of antioxidant enzymes were at a high value. During the prolonged low temperature stress, SOD acted mainly in the early stage of stress, while POD and CAT had higher activity in the middle and late stage. Moreover, the reverse transcription quantitative polymerase chain reaction (RT-qPCR) results showed that SOD.193 and POD.535 were significantly down-regulated in sclerotia, and CAT.1115 and POD.401 were up-regulated instead. These antioxidant enzyme genes played an important role in the sclerotial formation under low temperature stress. It is strongly suggested that antioxidant enzymes and abiotic stresses are closely related to sclerotial formation in P. tuber-regium. KEY POINTS: • Low temperature and H2O2 can promote sclerotial formation. • Sclerotia are more likely to form under high antioxidant enzyme activity. • POD.401, POD.535, SOD.193, and CAT.1115 are important for sclerotial formation.

A Forward Genetic Screen in Sclerotinia sclerotiorum Revealed the Transcriptional Regulation of Its Sclerotial Melanization Pathway.

Most plant fungal pathogens that cause worldwide crop losses are understudied, due to various technical challenges. With the increasing availability of sequenced whole genomes of these non-model fungi, effective genetic analysis methods are highly desirable. Here, we describe a newly developed pipeline, which combines forward genetic screening with high-throughput next-generation sequencing to enable quick gene discovery. We applied this pipeline in the notorious soilborne phytopathogen Sclerotinia sclerotiorum and identified 32 mutants with various developmental and growth deficiencies. Detailed molecular studies of three melanization-deficient mutants provide a proof of concept for the effectiveness of our method. A master transcription factor was found to regulate melanization of sclerotia through the DHN (1,8-dihydroxynaphthalene) melanin biosynthesis pathway. In addition, these mutants revealed that sclerotial melanization is important for sclerotia survival under abiotic stresses, sclerotial surface structure, and sexual reproduction. Foreseeably, this pipeline can be applied to facilitate efficient in-depth studies of other non-model fungal species in the future.[Formula: see text] Copyright © 2022 The Author(s). This is an open access article distributed under the CC BY 4.0 International license.

The Wolfiporia cocos Genome and Transcriptome Shed Light on the Formation of Its Edible and Medicinal Sclerotium.

Wolfiporia cocos (F. A. Wolf) has been praised as a food delicacy and medicine for centuries in China. Here, we present the genome and transcriptome of the Chinese strain CGMCC5.78 of W. cocos. High-confidence functional prediction was made for 9277 genes among the 10,908 total predicted gene models in the W. cocos genome. Up to 2838 differentially expressed genes (DEGs) were identified to be related to sclerotial development by comparing the transcriptomes of mycelial and sclerotial tissues. These DEGs are involved in mating processes, differentiation of fruiting body tissues, and metabolic pathways. A number of genes encoding enzymes and regulatory factors related to polysaccharide and triterpenoid production were strikingly regulated. A potential triterpenoid gene cluster including the signature lanosterol synthase (LSS) gene and its modified components were annotated. In addition, five nonribosomal peptide synthase (NRPS)-like gene clusters, eight polyketide synthase (PKS) gene clusters, and 15 terpene gene clusters were discovered in the genome. The differential expression of the velevt family proteins, transcription factors, carbohydrate-active enzymes, and signaling components indicated their essential roles in the regulation of fungal development and secondary metabolism in W. cocos. These genomic and transcriptomic resources will be valuable for further investigations of the molecular mechanisms controlling sclerotial formation and for its improved medicinal applications.

Characterization of Two Mitochondrial Genomes and Gene Expression Analysis Reveal Clues for Variations, Evolution, and Large-Sclerotium Formation in Medical Fungus Wolfiporia cocos.

Wolfiporia cocos, a precious mushroom with a long history as an edible food and Asian traditional medicine, remains unclear in the genetic mechanism underlying the formation of large sclerotia. Here, two complete circular mitogenomes (BL16, 135,686 bp and MD-104 SS10, 124,842 bp, respectively) were presented in detail first. The salient features in the mitogenomes of W. cocos include an intron in the tRNA (trnQ-UUG2), and an obvious gene rearrangement identified between the two mitogenomes from the widely geographically separated W. cocos strains. Genome comparison and phylogenetic analyses reveal some variations and evolutional characteristics in W. cocos. Whether the mitochondrion is functional in W. cocos sclerotium development was investigated by analyzing the mitogenome synteny of 10 sclerotium-forming fungi and mitochondrial gene expression patterns in different W. cocos sclerotium-developmental stages. Three common homologous genes identified across ten sclerotium-forming fungi were also found to exhibit significant differential expression levels during W. cocos sclerotium development. Most of the mitogenomic genes are not expressed in the mycelial stage but highly expressed in the sclerotium initial or developmental stage. These results indicate that some of mitochondrial genes may play a role in the development of sclerotium in W. cocos, which needs to be further elucidated in future studies. This study will stimulate new ideas on cytoplasmic inheritance of W. cocos and facilitate the research on the role of mitochondria in large sclerotium formation.

A Single Nucleotide Mutation in Adenylate Cyclase Affects Vegetative Growth, Sclerotial Formation and Virulence of Botrytis cinerea.

Botrytis cinerea is a pathogenic fungus that causes gray mold disease in a broad range of crops. The high intraspecific variability of B. cinerea makes control of this fungus very difficult. Here, we isolated a variant B05.10M strain from wild-type B05.10. The B05.10M strain showed serious defects in mycelial growth, spore and sclerotia production, and virulence. Using whole-genome resequencing and site-directed mutagenesis, a single nucleotide mutation in the adenylate cyclase (BAC) gene that results in an amino acid residue (from serine to proline, S1407P) was shown to be the cause of various defects in the B05.10M strain. When we further investigated the effect of S1407 on BAC function, the S1407P mutation in bac showed decreased accumulation of intracellular cyclic AMP (cAMP), and the growth defect could be partially restored by exogenous cAMP, indicating that the S1407P mutation reduced the enzyme activity of BAC. Moreover, the S1407P mutation exhibited decreased spore germination rate and infection cushion formation, and increased sensitivity to cell wall stress, which closely related to fungal development and virulence. Taken together, our study indicates that the S1407 site of bac plays an important role in vegetative growth, sclerotial formation, conidiation and virulence in B. cinerea.

The Subtilisin-Like Protease Bcser2 Affects the Sclerotial Formation, Conidiation and Virulence of Botrytis cinerea.

Botrytis cinerea, a ubiquitous necrotrophic plant-pathogenic fungus, is responsible for grey mold and rot disease in a very wide range of plant species. Subtilisin-like proteases (or subtilases) are a very diverse family of serine proteases present in many organisms and are reported to have a broad spectrum of biological functions. Here, we identified two genes encoding subtilisin-like proteases (Bcser1 and Bcser2) in the genome of B. cinerea, both of which contain an inhibitor I9 domain and a peptidase S8 domain. The expression levels of Bcser1 and Bcser2 increased during the sclerotial forming stage, as well as during a later stage of hyphal infection on Arabidopsis thaliana leaves, but the up-regulation of Bcser1 was significantly higher than that of Bcser2. Interestingly, deletion of Bcser1 had no effect on the fungal development or virulence of B. cinerea. However, deletion of Bcser2 or double deletion of Bcser1 and Bcser2 severely impaired the hyphal growth, sclerotial formation and conidiation of B. cinerea. We also found that ∆Bcser2 and ∆Bcser1/2 could not form complete infection cushions and then lost the ability to infect intact plant leaves of Arabidopsis and tomato but could infect wounded plant tissues. Taken together, our results indicate that the subtilisin-like protease Bcser2 is crucial for the sclerotial formation, conidiation, and virulence of B. cinerea.

Biocontrol effects of Penicillium griseofulvum against monkshood (Aconitum carmichaelii Debx.) root diseases caused by Sclerotium rolfsiii and Fusarium spp.

AIMS: The aims of this study were to investigate the biocontrol effects of Penicillium griseofulvum strain CF3 and its mechanisms against soil-borne root pathogens (Fusarium oxysporum and Sclerotium rolfsii) of the medical plant Aconitum carmichaelii Debx. METHODS AND RESULTS: The effects of P. griseofulvum strain CF3 were evaluated with regard to the hyphal growth of S. rolfsii and F. oxysporum, the sclerotial formation and germination of S. rolfsii and its expression of sclerotia-formation-related genes. A field experiment was conducted to explore how strain CF3 controls the severity of soil-borne diseases, promotes the growth of A. carmichaelii plants and mediates shifts in the culturable rhizosphere microbial populations. The results showed that treatment with a cell-free culture filtrate of strain CF3 considerably inhibited the hyphal growth of both S. rolfsii and F. oxysporum, in addition to limiting the sclerotial formation and germination of S. rolfsii. Three genes related to sclerotial formation (ArsclR, ArnsdD1 and ArnsdD2) were predicted in S. rolfsii and their expression was found suppressed by the CF3 treatment. Field application of the CF3 biocontrol agent in a powder form (1·9 × 1010 conidia per gram of substrate) reduced soil-borne disease severity by 15·0%. The shoot and root growth of A. carmichaelii plants was promoted by 61·6 and 83·1% respectively, as the biocontrol strain massively colonized the rhizosphere soil. The CF3 treatment also markedly reduced the density of some known species harmful to plants while increasing the density of some beneficial species in the rhizosphere soil. SIGNIFICANCE AND IMPACT OF THE STUDY: Genes related to sclerotia formation of S. rolfsii are predicted for the first time and their expression patterns in the presence of P. griseofulvum strain CF3 are evaluated. This comprehensive study provides a candidate fungal biocontrol strain and reveals its potential mechanisms against S. rolfsii and F. oxysporum in A. carmichaelii plants.

A novel Botrytis cinerea-specific gene BcHBF1 enhances virulence of the grey mould fungus via promoting host penetration and invasive hyphal development.

Botrytis cinerea is the causative agent of grey mould on over 1000 plant species and annually causes enormous economic losses worldwide. However, the fungal factors that mediate pathogenesis of the pathogen remain largely unknown. Here, we demonstrate that a novel B. cinerea-specific pathogenicity-associated factor BcHBF1 (hyphal branching-related factor 1), identified from virulence-attenuated mutant M8008 from a B. cinerea T-DNA insertion mutant library, plays an important role in hyphal branching, infection structure formation, sclerotial formation and full virulence of the pathogen. Deletion of BcHBF1 in B. cinerea did not impair radial growth of mycelia, conidiation, conidial germination, osmotic- and oxidative-stress adaptation, as well as cell wall integrity of the ∆Bchbf1 mutant strains. However, loss of BcHBF1 impaired the capability of hyphal branching, appressorium and infection cushion formation, appressorium host penetration and virulence of the pathogen. Moreover, disruption of BcHBF1 altered conidial morphology and dramatically impaired sclerotial formation of the mutant strains. Complementation of BcHBF1 completely rescued all the phenotypic defects of the ∆Bchbf1 mutants. During young hyphal branching, host penetration and early invasive growth of the pathogen, BcHBF1 expression was up-regulated, suggesting that BcHBF1 is required for these processes. Our findings provide novel insights into the fungal factor mediating pathogenesis of the grey mould fungus via regulation of its infection structure formation, host penetration and invasive hyphal branching and growth.

The Notorious Soilborne Pathogenic Fungus Sclerotinia sclerotiorum: An Update on Genes Studied with Mutant Analysis.

Ascomycete Sclerotinia sclerotiorum (Lib.) de Bary is one of the most damaging soilborne fungal pathogens affecting hundreds of plant hosts, including many economically important crops. Its genomic sequence has been available for less than a decade, and it was recently updated with higher completion and better gene annotation. Here, we review key molecular findings on the unique biology and pathogenesis process of S. sclerotiorum, focusing on genes that have been studied in depth using mutant analysis. Analyses of these genes have revealed critical players in the basic biological processes of this unique pathogen, including mycelial growth, appressorium establishment, sclerotial formation, apothecial and ascospore development, and virulence. Additionally, the synthesis has uncovered gaps in the current knowledge regarding this fungus. We hope that this review will serve to build a better current understanding of the biology of this under-studied notorious soilborne pathogenic fungus.

Reactive oxygen species induce sclerotial formation in Morchella importuna.

Morels are some of the most highly prized edible and medicinal mushrooms, and the outdoor cultivation has been achieved in China in recent years. Sclerotial formation is one of the most important phases during the morel life cycle, and the number of sclerotia indicates the spawn quality during cultivation. However, the sclerotial formation and differentiation mechanisms are poorly understood. In this study, the sclerotial formation process of Morchella importuna and the effects of reactive oxygen species on scerotial formation were studied. Scerotial formation was defined as five distinctive phases, hypha early, hyphal growth, sclerotial initiation, development, and maturation. The mycelia in the sclerotium-forming area were swollen, darkened, and dense with sclerotial formation, but hydrogen peroxide accumulated in the region lacking sclerotial formation. The expression of all six genes for superoxide dismutases tested increased with sclerotial maturation. A difference in hydrogen peroxide concentration of 20 mM could promote the sclerotial initiation and induce expression of sod genes. The MAPK signaling pathway was activated, and they passed the signal from an area of high oxidative stress to a low area to initiate sclerotial formation. An understanding of the sclerotial formation mechanisms in M. importuna may help to understand the life cycle and facilitate the fruiting body cultivation.

An atypical forkhead-containing transcription factor SsFKH1 is involved in sclerotial formation and is essential for pathogenicity in Sclerotinia sclerotiorum.

Sclerotinia sclerotiorum (Lib.) de Bary is a necrotrophic plant pathogen with a worldwide distribution. The sclerotia of S. sclerotiorum are pigmented multicellular structures formed from the aggregation of vegetative hyphae. These survival structures play a central role in the life and infection cycles of this pathogen. Here, we characterized an atypical forkhead (FKH)-box-containing protein, SsFKH1, involved in sclerotial development and virulence. To investigate the role of SsFkh1 in S. sclerotiorum, the partial sequence of SsFkh1 was cloned and RNA interference (RNAi)-based gene silencing was employed to alter the expression of SsFkh1. RNA-silenced mutants with significantly reduced SsFkh1 RNA levels exhibited slow hyphal growth and sclerotial developmental defects. In addition, the expression levels of a set of putative melanin biosynthesis-related laccase genes and a polyketide synthase-encoding gene were significantly down-regulated in silenced strains. Disease assays demonstrated that pathogenicity in RNAi-silenced strains was significantly compromised with the development of a smaller infection lesion on tomato leaves. Collectively, the results suggest that SsFkh1 is involved in hyphal growth, virulence and sclerotial formation in S. sclerotiorum.