Sclerotia Formation Research Articles

Identification of SclB, a Zn(II)2Cys6 transcription factor involved in sclerotium formation in Aspergillus niger

Certain Aspergillus species such as Aspergillus flavus and A. parasiticus are well known for the formation of sclerotia. These developmental structures are thought to act as survival structures during adverse environmental conditions but are also a prerequisite for sexual reproduction. We previously described an A. niger mutant (scl-2) which formed sclerotium-like structures, suggesting a possible first stage of sexual development in this species. Several lines of evidence presented in this study support the previous conclusion that the sclerotium-like structures of scl-2 are indeed sclerotia. These included the observations that: (i) safranin staining of the sclerotia-like structures produced by the scl-2 mutant showed the typical cellular structure of a sclerotium; (ii) metabolite analysis revealed specific production of indoloterpenes, which have previously been connected to sclerotium formation; (iii) formation of the sclerotium-like structures is dependent on a functional NADPH complex, as shown for other fungi forming sclerotia. The mutation in scl-2 responsible for sclerotium formation was identified using parasexual crossing and bulk segregant analysis followed by high throughput sequencing and subsequent complementation analysis. The scl-2 strain contains a mutation that introduces a stop codon in the putative DNA binding domain of a previously uncharacterized Zn(II)2Cys6 type transcription factor (An08g07710). Targeted deletion of this transcription factor (sclB) confirmed its role as a repressor of sclerotial formation and in the promotion of asexual reproduction in A. niger. Finally, a genome-wide transcriptomic comparison of RNA extracted from sclerotia versus mycelia revealed major differences in gene expression. Induction of genes related to indoloterpene synthesis was confirmed and also let to the identification of a gene cluster essential for the production of aurasperones during sclerotium formation. Expression analysis of genes encoding other secondary metabolites, cell wall related genes, transcription factors, and genes related to reproductive processes identified many interesting candidate genes to further understand the regulation and biosynthesis of sclerotia in A. niger. The newly identified SclB transcription factor acts as a repressor of sclerotium formation and manipulation of sclB may represent a first prerequisite step towards engineering A. niger strains capable of sexual reproduction. This will provide exciting opportunities for further strain improvement in relation to protein or metabolite production in A. niger.

The MAP kinase AflSlt2 modulates aflatoxin biosynthesis and peanut infection in the fungus Aspergillus flavus

Aflatoxin contamination in food and feed products has been brought into sharp focus over the last few decades in the world. However, there is no effective strategy for solving the problem thus far. Therefore, basic research on the aflatoxin-producer Aspergillus flavus is an urgent need. The vital role of mitogen-activated protein kinases (MAPKs) in signal transduction has been documented in various pathogenic fungi, but their functions in A. flavus have rarely been investigated. Herein, we characterized the detailed function of one of these MAPKs, AflSlt2. Targeted deletion of AflSlt2 gene indicates that this kinase is required for vegetative growth, conidia generation, and sclerotium formation. The analysis of AflSlt2 deletion mutant revealed hypersensitivity to cell wall-damaging chemicals and resistance against hydrogen peroxide. Interestingly, the ability of the ΔAflSlt2 mutant to generate aflatoxins in medium was significantly increased compared to wild type. However, a pathogenicity assay indicated that the ΔAflSlt2 mutant was deficient in peanut infection. Site-directed mutation study uncovered that the function of AflSlt2 was dependent on the phosphorylated residues (Thr-186 and Tyr-188) within the activation loop and the phosphotransfer residue (Lys-52) within the subdomain II. Interestingly, an autophosphorylation mutant of AflSlt2 (AflSlt2R66S) displayed wild type-like phenotypes. Bringing these observations together, we propose that Slt2-MAPK pathway is involved in development, stress response, aflatoxin biosynthesis, and pathogenicity in A. flavus. This study may be useful to unveil the regulation mechanism of aflatoxin biosynthesis and provide strategy to control A. flavus contamination.

Efficacy of different fungicides and weedicides against Macrophomina phaseolina (Tassi) Goid causing groundnut root rot

In this study, different systemic, non-systemic, combination fungicides and weedicides at four different concentration were evaluated in vitro against groundnut root rot pathogen Macrophomina phaseolina. Among the non-systemic fungicides, Mancozeb 75% WP and Propineb 70% WP were found most effective and showed (99.98 and 99.96 per cent) mycelia growth inhibition at all concentrations. Whereas in systemic fungicides, Carbendazim 50% WP were found most effective with (94.24 per cent) mean mycelial growth inhibition. Among different combination fungicides tested, Carboxin 37.5% + Thiram 37.5% WP were found most effective with mean mycelial growth inhibition of (97.16 per cent).. In case of weedicides tested, Quizalofo-p-pethyle 5% EC found as most effective inhibiting the mycelial growth of M. phaseolina at all concentrations with (90.92 per cent) mean mycelial growth inhibition. Sclerotial formation was also absent in all this treatments.

Role of Purpureocillium lilacinum Cultural Filtrate in Controlling Onion White Rot

Onion white rot is one of the most destructive diseases threat onion production in Egypt. The present study assessed the effect of Purpureocillium lilacinum culture filtrates in vitro and in vivo conditions on Stromatinia cepivora at 75%concentration. Bioagent culture filtrates inhibited the mycelial growth, sclerotial formation and myceliogenic germination of S. cepivora by 82.59, 100 and 93.33%, respectively. The culture filtrates at 75% increased cell membrane permeability of S. cepivora compared to the control. The in vitro analysis revealed that 92.5% of the sclerotia were lost its activity in the soil when treated with filtrates and significantly decreased the disease incidence and severity by 95.10 and 98.30%, respectively under greenhouse conditions. However, soil previously infested with S. cepivora sclerotia and handled continuously with bioagent culture filtrate for 6 months before onion cultivation undoubtedly decreased the disease incidence and severity. During 6 months after field culture filtrates treatment, more than 80 and 60% of the sclerotia failed to emergence in trial I and II plots experiment, respectively. After one-year culture filtrate treatment in consecutive onion field crop cultivation, filtrates were more efficient than control in decreasing white rot incidence attendant with low inoculum density trial field (77.67sclerotia/kg of soil). Consequently, a decrease in white rot incidence resulting promotion to vegetative parameters of culture filtrate treated onion plants in pots and increase growth and bulb yield productivity in field. Decreasing disease incidence and severity was associated with increase activities of antioxidant enzymes polyphenol oxidase, peroxidase and chitinase by application of culture filtrates.

The Methyltransferase AflSet1 Is Involved in Fungal Morphogenesis, AFB1 Biosynthesis, and Virulence of Aspergillus flavus.

The filament fungal pathogen, Aspergillus flavus, spreads worldwide and contaminates several important crops. Histone posttranslational modifications are deeply involved in fungal development and virulence, but the biological function of the histone methyltransferase AflSet1 in A. flavus is still unknown. In the study, Aflset1 deletion strain was constructed through homologous recombination, and it was found that AflSet1 up-regulates hyphae growth, and promotes conidiation by sporulation regulation genes: abaA and brlA. It was also found that AflSet1 involves in sclerotia formation and AFB1 biosynthesis via sclerotia related transcriptional factors and orthodox AFB1 synthesis pathway, respectively. Crop models revealed that AflSet1 plays critical roles in colonization and AFB1 production on crop kernels. Lipase activity analysis suggested that AflSet1 affects fungal virulence to crops via digestive enzymes. Stresses tests revealed that AflSet1 is deeply involved in fungal resistance against osmotic, oxidative and cell membrane stress. The preparation of N_SET, SET domain deletion mutants and H988K mutant revealed that both domains play critical roles in fungal development and AFB1 production, and that H988 is very important in executing biological functions on morphogenesis and AFB1 synthesis. Subcellular location analysis revealed that AflSet1 is stably accumulated in nuclei in both spore germination and hyphae growth stages, even under the stress of SDS. Through immunoblot analysis, it was found that AflSet1 methylates H3K4me2 and me3 as well as H3K9me2. This study provides a solid evidence to discover the biological functions of histone methyltransferase in pathogenic fungi.

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.

Antimicrobial activities of Trichoderma atroviride against common bean seed-borne Macrophomina phaseolina and Rhizoctonia solani

ABSTRACT Common bean (Phaseolus vulgaris L.) is the most consumed legume worldwide, with a commercial value surpassing that of all other legume crops combined. Seed-borne fungi cause at least 10% loss in common bean. Standard blotter (SB) and agar plate (AP) techniques were used to identify seed-borne fungi associated with common bean seeds. Thirty-two fungal species comprising 23 genera were detected. Among the fungal pathogens recovered, Macrophomina phaseolina and Rhizoctonia solani were predominant with a higher recovery using the AP technique than SB technique. Antagonistic activities of six Trichoderma isolates associated with healthy common bean seeds were evaluated against both pathogens, M. phaseolina and R. solani through a dual culture assay. Of them, Trichoderma isolate T22 remarkably suppressed the growth of both pathogens (72.42% and 68%, respectively), 5 days after inoculation. Both pathogens were completely overgrown by T22 mycelia, and sclerotia formation by the two pathogens was lacking compared to the control plates, after 8 days. The T22 isolate was morphologically identified as T. atroviride. The scanning electron microscopy (SEM) confirmed the mycoparasitism of T. atroviride T22 against M. phaseolina and R. solani, resulting in mycelium collapse of both pathogens. A total of nine unique volatile compounds of T. atroviride T22 filtrate were identified by GC-MS technique, representing 75.03% of the total components. Among them, 5-hydroxymethylfurfural (46.11%), and 4 H-pyran-4-one, 2,3-dihydro-3,5-dihydroxy-6-methyl- (15.66%) were detected as major components, while furfural (3.14%), hexadecanoic acid, 2-hydroxy-1-(hydroxymethyl) ethyl ester (2.81%), 1,2-cyclopentanedione (2.79%), and 2-furanmethanol (2.09%) were found to be minor compounds. Our results confirmed that T. atroviride T22 can be used as an effective biocontrol agent against M. phaseolina and R. solani, which are pathogens on common beans and many other crops.

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.

Inhibitory effect of Fungastop and Bion against carrot soft rot caused by Sclerotinia sclerotiorum

This study was conducted to assess likely inhibitory effects of Fungastop™ and Acibenzolar-S-Methyl (Bion®) against carrot rot caused by Sclerotinia sclerotiorum during postharvest phase. The results showed that Fungastop was able to markedly decrease mycelial growth and sclerotia formation of three isolates of the pathogen. In addition, Fungastop significantly reduced myceliogenic and carpogenic germination of sclerotia in the three tested isolates of S. sclerotiorum. Fungastop showed high antifungal efficacy against carrot rot in vivo as carrots were inoculated with the pathogen 2.5 h after treatment with Bion and Fungastop. However inhibitory effect of Bion against the disease was higher than Fungastop over time. The enzymatic tests revealed that Bion was able to significantly enhance levels of enzyme activities related to disease resistance including β-N-acetyl hexosaminidase, endochitinase, chitin 1,4-β-chitobiosidase and β −1,3-glucanase six days after treatment. In addition, activities of phenylalanine ammonia lyase and peroxidase were markedly increased four days after treatment. Bion showed higher effect in increasing enzyme activities related to disease resistance than Fungastop. The treatment with Fungastop and Bion enhanced relative conductivity of S. sclerotiorum mycelia over 160 min compared with control. The treatment with Fungastop increased the cell membrane permeability of mycelia more than Bion.

AflSte20 Regulates Morphogenesis, Stress Response, and Aflatoxin Biosynthesis of Aspergillus flavus.

Various signaling pathways in filamentous fungi help cells receive and respond to environmental information. Previous studies have shown that the mitogen-activated protein kinase (MAPK) pathway is phosphorylation-dependent and activated by different kinase proteins. Serine/threonine kinase plays a very important role in the MAPK pathway. In this study, we selected the serine/threonine kinase AflSte20 in Aspergillus flavus for functional study. By constructing Aflste20 knockout mutants and complemented strains, it was proven that the Aflste20 knockout mutant (ΔAflste20) showed a significant decrease in growth, sporogenesis, sclerotinogenesis, virulence, and infection compared to the WT (wild type) and complemented strain (ΔAflste20C). Further research indicated that ΔAflste20 has more sensitivity characteristics than WT and ΔAflste20C under various stimuli such as osmotic stress and other types of environmental stresses. Above all, our study showed that the mitogen-activated kinase AflSte20 plays an important role in the growth, conidia production, stress response and sclerotia formation, as well as aflatoxin biosynthesis, in A. flavus.

Phenotypic and genotypic characterization of single isolate-derived monoascospore strains of Sclerotinia sclerotiorum from common bean

White mold is a highly destructive disease caused by Sclerotinia sclerotiorum, a polyphagous fungus that infects several hosts including common bean grown that is widely cultivated in Brazil. The pathogenic populations are known to exhibit genetic variability but studies rarely used single ascospore-derived strains. In this study, 12 monoascospore strains originated from a same isolate (UFLA 54), but from different apothecia, were compared among them and with the mother strain with regards: days to first sclerotium formation, mycelial growth rate mycelial compatibility groups, microsatellite markers and mating type locus (MAT1–1 and MAT1–2). Their pathogenicity was assessed on three common bean lines. Cluster analysis of growth rates data separated UFLA 54 from all monoascospore strains, which shared same alleles in the microsatellite locus. Inversion of MAT1–1 region was found for both UFLA 54 and half of monoascospore strains. Aggressiveness of the monoascospore strains was not consistent across common bean lines. Our study reveals significant variability of S. sclerotiorum at the single isolate level.

Function of crzA in Fungal Development and Aflatoxin Production in Aspergillus flavus.

The calcineurin pathway is an important signaling cascade for growth, sexual development, stress response, and pathogenicity in fungi. In this study, we investigated the function of CrzA, a key transcription factor of the calcineurin pathway, in an aflatoxin-producing fungus Aspergillus flavus (A. flavus). To examine the role of the crzA gene, crzA deletion mutant strains in A. flavus were constructed and their phenotypes, including fungal growth, spore formation, and sclerotial formation, were examined. Absence of crzA results in decreased colony growth, the number of conidia, and sclerocia production. The crzA-deficient mutant strains were more susceptible to osmotic pressure and cell wall stress than control or complemented strains. Moreover, deletion of crzA results in a reduction in aflatoxin production. Taken together, these results demonstrate that CrzA is important for differentiation and mycotoxin production in A. flavus.

PRIMER REPORTE DE Colletotrichum coccodes EN FRUTOS DE TOMATE (Solanum lycopersicum) EN MÉXICO

Anthracnose, caused by Colletotrichum coccodes, affects solanaceae and cucurbits. In Mexico this pathogen has been reported in potato (Solanum tuberosum L.) cultivation, but not in tomato (Solanum lycopersicum). In 2015, the presence of symptom of deep-set lesions was observed with 7 % incidence in tomato fruits sampled in open fields in the state of Morelos, Mexico. The objective of this study was to identify the causal agent of anthracnose symptom of the fruit by morphological and molecular characteristics. The collected material was disinfested and seeded in PDA (potato-dextrose-agar) culture medium and agar-V8 juice, from sunken lesions in which formation of sclerotia, mushrooms, cylindrical conidia, hyalines, without, septa of 3.16 to 4.16 μm wide and 12.5 to 17.2 μm long with lobed appressoria. In molecular characterization, the sequences obtained with the ITS5/ITS4 primers had 100 % similarity with Collectotrichum coccodes. The pathogenicity tests were carried out on healthy fruits with or without a wound and the lesion size was evaluated every 48 h. The initial symptoms appeared at 72 h and at 8 d they presented lesions from 0.5 to 3.25 cm in diameter. The Tukey test detected significant differences between inoculation methods. This is the first report of Collectotrichum coccodes that affects tomato fruits in Mexico.

The Formaldehyde Dehydrogenase SsFdh1 Is Regulated by and Functionally Cooperates with the GATA Transcription Factor SsNsd1 in Sclerotinia sclerotiorum.

GATA transcription factors (TFs) are common eukaryotic regulators, and glutathione-dependent formaldehyde dehydrogenases (GD-FDH) are ubiquitous enzymes with formaldehyde detoxification activity. In this study, the formaldehyde dehydrogenase Sclerotinia sclerotiorum Fdh1 (SsFdh1) was first characterized as an interacting partner of a GATA TF, SsNsd1, in S. sclerotiorum Genetic analysis reveals that SsFdh1 functions in formaldehyde detoxification, nitrogen metabolism, sclerotium development, and pathogenicity. Both SsNsd1 and SsFdh1 harbor typical zinc finger motifs with conserved cysteine residues. SsNsd1 regulates SsFdh1 in two distinct manners. SsNsd1 directly binds to GATA-box DNA in the promoter region of Ssfdh1; SsNsd1 associates with SsFdh1 through disulfide bonds formed by conserved Cys residues. The SsNsd1-SsFdh1 interaction and nuclear translocation were found to prevent efficient binding of SsNsd1 to GATA-box DNA. Site-directed point mutation of these Cys residues influences the SsNsd1-SsFdh1 interaction and SsNsd1 DNA binding capacity. SsFdh1 is regulated by and functions jointly with the SsNsd1 factor, providing new insights into the complex transcriptional regulatory mechanisms of GATA factors.IMPORTANCE S. sclerotiorum is a pathogenic fungus with sclerotium and infection cushion development, making S. sclerotiorum one of the most challenging agricultural pathogens with no effective control method. We identified important sclerotium and compound appressorium formation determinants, SsNsd1 and SsFdh1, and investigated their regulatory mechanism at the molecular level. SsNsd1 and SsFdh1 are zinc finger motif-containing proteins and associate with each other in the nucleus. On other hand, SsNsd1, as a GATA transcription factor, directly binds to GATA-box DNA in the promoter region of Ssfdh1 The SsNsd1-SsFdh1 interaction and nuclear translocation were found to prevent efficient binding of SsNsd1 to GATA-box DNA. Our results provide insights into the role of the GATA transcription factor and its regulation of formaldehyde dehydrogenase in stress resistance, fungal sclerotium and compound appressorium development, and pathogenicity.

Selenium reduces the pathogenicity of Sclerotinia sclerotiorum by inhibiting sclerotial formation and germination

Sclerotinia sclerotiorum (S. sclerotiorum) is a devastating fungal pathogen with worldwide distribution, and threatened the agro-ecological safety in the long term. To control the damage caused by Sclerotinia diseases, as well as consider the fungicide resistance and chemical residues, strategy of which plant nutritional regulation, as an eco-friendly approach, is gaining much significance. Selenium (Se), as a beneficial microelement for plant, has been manifested to be effective in inhibiting the mycelial growth of S.sclerotiorum in our previous study. In the present study, we observed that Se (both selenate and selenite) inhibited the formation of sclerotia, which is an important life form in the disease cycle of S. sclerotiorum. And the inhibition ratios of number of sclerotia in treatments of Se(VI)5.0 and Se(IV)5.0 were 54.55% and 43.84%, respectively; the inhibition ratios of weight of sclerotia in treatments of Se(VI)5.0 and Se(IV)5.0 were 42.29% and 25.67%, respectively. Results suggested that Se inhibited mycelial growth, severely damaged sclerotial ultrastructure, reduced the capacity of acid production, decreased superoxide dismutase (SOD) and catalase (CAT) activities, increased the content of hydrogen peroxide (H2O2) and superoxide anion (O2−) in mycelium, and all of these resulted in the reduction in sclerotial formation. Further studies revealed that Se application in medium increased Se concentration in sclerotia and thus inhibited sclerotial germination. Moreover, the pathogenicity of mycelia germinating from sclerotia that pretreated with Se, decreased significantly to rape leaves. These findings broadened our understanding of Se application in plant protection, as well as provided evidences for developing environment-friendly fungicide for S. sclerotiorum control.

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

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. 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. 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.

Molecular and structural basis of nucleoside diphosphate kinase–mediated regulation of spore and sclerotia development in the fungus Aspergillus flavus

The fundamental biological function of nucleoside diphosphate kinase (NDK) is to catalyze the reversible exchange of the γ-phosphate between nucleoside triphosphate (NTP) and nucleoside diphosphate (NDP). This kinase also has functions that extend beyond its canonically defined enzymatic role as a phosphotransferase. However, the role of NDK in filamentous fungi, especially in Aspergillus flavus (A. flavus), is not yet known. Here we report that A. flavus has two NDK-encoding gene copies as assessed by qPCR. Using gene-knockout and complementation experiments, we found that AfNDK regulates spore and sclerotia development and is involved in plant virulence as assessed in corn and peanut seed-based assays. An antifungal test with the inhibitor azidothymidine suppressed AfNDK activity in vitro and prevented spore production and sclerotia formation in A. flavus, confirming AfNDK's regulatory functions. Crystallographic analysis of AfNDK, coupled with site-directed mutagenesis experiments, revealed three residues (Arg-104, His-117, and Asp-120) as key sites that contribute to spore and sclerotia development. These results not only enrich our knowledge of the regulatory role of this important protein in A. flavus, but also provide insights into the prevention of A. flavus infection in plants and seeds, as well as into the structural features relevant for future antifungal drug development.

The HosA Histone Deacetylase Regulates Aflatoxin Biosynthesis Through Direct Regulation of Aflatoxin Cluster Genes.

Histone deacetylases (HDACs) always function as corepressors and sometimes as coactivators in the regulation of fungal development and secondary metabolite production. However, the mechanism through which HDACs play positive roles in secondary metabolite production is still unknown. Here, classical HDAC enzymes were identified and analyzed in Aspergillus flavus, a fungus that produces one of the most carcinogenic secondary metabolites, aflatoxin B1 (AFB1). Characterization of the HDACs revealed that a class I family HDAC, HosA, played crucial roles in growth, reproduction, the oxidative stress response, AFB1 biosynthesis, and pathogenicity. To a lesser extent, a class II family HDAC, HdaA, was also involved in sclerotia formation and AFB1 biosynthesis. An in vitro analysis of HosA revealed that its HDAC activity was considerably diminished at nanomolar concentrations of trichostatin A. Notably, chromatin immunoprecipitation experiments indicated that HosA bound directly to AFB1 biosynthesis cluster genes to regulate their expression. Finally, we found that a transcriptional regulator, SinA, interacts with HosA to regulate fungal development and AFB1 biosynthesis. Overall, our results reveal a novel mechanism by which classical HDACs mediate the induction of secondary metabolite genes in fungi.

Evaluation of Biomass and Chitin Production of Morchella Mushrooms Grown on Starch-Based Substrates.

Morchella sp. is one of the most expensive mushrooms with a high nutritional profile. In this study, the polysaccharide content of Morchella species was investigated. Specifically, mycelium growth rate, biomass production, sclerotia formation, and glucosamine and total polysaccharides content of six Morchella species grown on a starch-based media were evaluated. Submerged fermentations in potato dextrose broth resulted in a glucosamine content of around 3.0%. In solid-state fermentations (SSF), using potato dextrose agar, a high linear growth rate (20.6 mm/day) was determined. Increased glucosamine and total polysaccharides content were observed after the formation of sclerotia. Biomass and glucosamine content were correlated, and the equations were used for the indirect estimation of biomass in SSF with agro-industrial starch-based materials. Wheat grains (WG), potato peels (PP), and a mixture of 1:1 of them (WG–PP) were evaluated as substrates. Results showed that the highest growth rate of 9.05 mm/day was determined on WG and the maximum biomass yield (407 mg/g) on WG–PP. The total polysaccharide content reached up to 18.4% of dried biomass in WG–PP. The results of the present study proved encouraging for the efficient bioconversion of potato and other starch-based agro-industrial waste streams to morel biomass and sclerotia eliciting nutritional and bioactive value.

First Report of Colletotrichum liriopes Causing Leaf Spots on Liriope spicata in China

Liriope spicata (Thunb.) Lour. is an evergreen herb, belonging to the genus Liriope, family Liliaceae. As a traditional Chinese medicine, L. spicata is widely cultured in China, Korea, and Japan for ornamental and medicinal purposes. In May 2018, 80 to 90% of L. spicata leaves were observed with leaf spots on the campus of Qingdao Agricultural University. The diseased leaves had oval or irregularly shaped spots with dark brown edges and yellow halos. Some lesions were observed with black acervuli with setae. One fungus was isolated consistently by placing surface-sterilized tissue pieces from the edge of the leaf spots on potato dextrose agar. The resulting colonies were initially whitish and then turned gray after 4 days of incubation at 25°C, and salmon pink spore masses without setae were also observed. Conidia were one-celled (17 to 24 × 2 to 5 μm), hyaline, and falcate. Based on the colony and conidial morphology, the isolate fungus was identified as Colletotrichum liriopes (Damm et al. 2009; Trigiano 2013). The sexual stage and sclerotia formation in the culture were not observed. Sequences of the internal transcribed spacer region of rDNA (GenBank accession no. MK131716), chitin synthase gene (MK144289), β-tubulin gene (MK144290), glyceraldehyde-3-phosphate dehydrogenase gene (MK144291), and calmodulin gene (MK144292) were amplified and analyzed from two single-spore isolates, Ls-CL-01 and Ls-CL-02 (Damm et al. 2009; Weir 2012; White et al. 1990). Because the sequences amplified from Ls-CL-02 were identical to those of Ls-CL-01, only one set of sequences was submitted to GenBank. These sequences had 99 to 100% nucleotide identity to that of C. liriopes (MG543776.1, GU228294.1, KC244160.1, KY995371.1, and HM582017.1, respectively) in GenBank. In addition, MK131716, MK144289, MK144290, and MK144291 had 99% nucleotide identity to that of C. liriopes ex-type CBS 119444 (GU227804, GU228294, GU228098, and GU228196) (Damm et al. 2009). The pathogenicity test was performed using the spray inoculation method. Spores were harvested from 14-day culture plates of Ls-CL-01 and Ls-CL-02 using sterilized distilled water with 0.025% of Tween 20 (Sigma-Aldrich). The spore suspension was adjusted to a final concentration of 5 × 10⁶ spores/ml and was sprayed over five healthy plants, and sterilized water with 0.025% of Tween 20 was used as a control treatment. The inoculated plants were incubated at 25°C and 90% relative humidity with 12-h/12-h dark/light for 3 days and then moved to normal conditions (12-h/12-h dark/light, 20% relative humidity) for 2 days. The experiment was repeated twice. After 5 days of inoculation, approximately 50% of leaves of the inoculated plants (all 15 plants) showed symptoms similar to those previously observed on the original leaves, whereas the control leaves remained asymptomatic. Three isolates were recovered from inoculated leaves. They were identical to isolate Ls-CL-01 in colonial and conidial features. To our knowledge, this is the first report that C. liriopes causes leaf spots on L. spicata. Leaf spots could greatly affect the value of L. spicata; identification of the pathogen provides scientific basis for the disease management.