Involvement of the Autophagy Protein Atg6 in Development and Virulence in the Gray Mold Fungus Botrytis cinerea

Gray mold caused by Botrytis cinerea is a devastating disease that leads to huge economic losses worldwide. Autophagy is an evolutionarily conserved process that maintains intracellular homeostasis through self-eating. In this study, we identified and characterized the biological function of the autophagy-related protein Atg6 in B. cinerea. Targeted deletion of the BcATG6 gene showed block of autophagy and several phenotypic defects in aspects of mycelial growth, conidiation, sclerotial formation and virulence. All of the phenotypic defects were restored by targeted gene complementation. Taken together, these results suggest that BcAtg6 plays important roles in the regulation of various cellular processes in B. cinerea.

Crop-weather based relation and severity prediction of aerial blight incited by Rhizoctonia solani Kuhn in soybean

Crop-weather based relation and other aspects of aerial blight incited by Rhizoctoniasolani Kuhn were investigated on two mega varieties (JS 335 and JS 97-52, now susceptible) under central Indian conditions during 2017, 2018 and 2019. It was found that aerial severity and sclerotial formation on affected leaves were varied significantly in all three season, and progress of disease was rapid between 63–84 days old crop {full pod (R4) to maturity initiation (R7) stage}.Increasing crop age was also significantly positively correlated with increasing severity (0.732*) and sclerotial formation (0.981**). Mean relative humidity and maximum temperature of current as well as previous week were found to be positively and negatively correlated with increasing severity of aerial blight, respectively. A regression based model with three explanatory variables (Mean RH, Rainfall and Minimum temperature) of current week was found to be most significant in prediction of disease severity (R2=0.946) of aerial blight. Whereas previous week weather variables i.e. rainy days and rainfall were also accounted 71.7 per cent variability (R2 = 0.717) in disease severity. Overall, weekly average maximum temp (27 to 30o C) and mean RH (80 to 90 per cent) in current week coupled with more rainfall and rainy days in previous week were found to be most conducive field condition for rapid progress of aerial blight disease in soybean.

An APSES Transcription Factor Xbp1 Is Required for Sclerotial Development, Appressoria Formation, and Pathogenicity in Ciboria shiraiana

Sclerotinia diseases are important plant fungal diseases that, causes huge economic worldwide losses every year. Ciboria shiraiana is the main pathogen that results in mulberry sclerotia diseases. Sclerotia and appressoria play important roles in long-term pathogen survival and in host infection during life and disease cycles. However, the molecular mechanisms of sclerotial development and appressoria formation in C. shiraiana have not been well studied. Here, an Asm1p, Phd1p, Sok2p, Efg1p and StuAp (APSES)-type transcription factor in C. shiraiana, CsXbp1, involved in sclerotial development and appressoria formation was functionally characterized. Bioinformatics analyses showed that CsXbp1 contained an APSES-type DNA binding domain. The expression levels of CsXbp1 were higher in sclerotia and during later stages of infection. Compared with wild-type strains, hyphal growth was slower, the number and weight of sclerotia were reduced significantly, and appressoria formation was obviously delayed in CsXbp1 RNA interference (RNAi) strains. Moreover, the CsXbp1 RNAi strains showed weakened pathogenicity owing to compound appressoria defects. Tobacco rattle virus-mediated host-induced gene silencing enabled Nicotiana benthamiana to increase its resistance to C. shiraiana by reducing the CsXbp1 transcripts level. Thus, CsXbp1 plays vital roles in sclerotial formation, appressoria formation, and pathogenicity in C. shiraiana. This study provides new insights into the infection mechanisms of C. shiraiana and plant resistance breeding.

Use of transcriptomic profiling to identify candidate genes involved in Polyporus umbellatus sclerotial formation affected by oxalic acid

Polyporus umbellatus is a precious medicinal fungus. Oxalic acid was observed to affect sclerotial formation and sclerotia possessed more medicinal compounds than mycelia. In this study, the transcriptome of P. umbellatus was analysed after the fungus was exposed to various concentrations of oxalic acid. The differentially expressed genes (DEGs) encoding a series of oxidases were upregulated, and reductases were downregulated, in the low-oxalic-acid (Low OA) group compared to the control (No OA) group, while the opposite phenomenon was observed in the high-oxalic-acid (High OA) group. The detection of reactive oxygen species (ROS) in P. umbellatus mycelia was performed visually, and Ca2+ and H2O2 fluxes were measured using non-invasive micro-test technology (NMT). The sclerotial biomass in the Low OA group increased by 66%, however, no sclerotia formed in the High OA group. The ROS fluorescence intensity increased significantly in the Low OA group but decreased considerably in the High OA group. Ca2+ and H2O2 influx significantly increased in the Low OA group, while H2O2 exhibited efflux in the High OA group. A higher level of oxidative stress formed in the Low OA group. Different concentrations of oxalic acid were determined to affect P. umbellatus sclerotial formation in different ways.

Comparative transcriptome analysis of cells from different areas reveals ROS responsive mechanism at sclerotial initiation stage in Morchella importuna

Morels are some of the most highly prized edible and medicinal mushrooms, with great economic and scientific value. Outdoor cultivation has been achieved and expanded on a large scale in China in recent years. Sclerotial formation is one of the most important phases during the morel life cycle, and previous reports indicated that reactive oxygen species (ROS) play an important role. However, ROS response mechanisms at sclerotial initiation (SI) stage are poorly understood. In this study, comparative transcriptome analyses were performed with sclerotial and hyphal cells at different areas in the same plate at SI stage. Gene expression was significantly different at SI stage between sclerotial formation and mycelia growth areas. GO and KEGG analyses indicated more vigorous metabolic characteristics in the hyphae area, while transcription process, DNA repair, and protein processing were enriched in sclerotial cells. Gene expression related to H2O2 production was high in the hyphae area, while expression of H2O2-scavenging genes was high in sclerotial cells, leading to a higher H2O2 concentration in the hyphal region than in the sclerotium. Minor differences were observed in gene expression of H2O2-induced signaling pathway in sclerotial and hyphal cells; however, expression levels of the target genes of transcription factor MSN2, important in the H2O2-induced signaling pathways, were significantly different. MSN2 enhanced stress response regulation in sclerotia by regulating these target genes. Small molecular HSPs were also found upregulated in sclerotial cells. This study indicated that sclerotial cells are more resistant to ROS stress than hyphal cells through transcriptional regulation of related genes.

Characterization of Cultural and Morphological Variability in Rhizoctonia solani Isolates Associated with Root Rot of Ajwain (Trachyspermum ammi L.)

Rhizoctonia solani (teleomorph: Thanatephorus spp.) is a plant pathogenic fungus which had a wide host range. It is best known to cause many diseases in plants such as collar rot, root rot, damping- off, sheath blight, stem canker, web blight and wire stem throughout the world. In vitro study of the various isolates of R. solani for morphological and cultural characters and results revealed that this experiment the various isolates of R. solani differed in colony characters and showed black brown colored cultures with 90.0 mm colony diameter on 7th day of incubation under uniform environmental condition. The highest growth reported was isolates CHIRs-5 and sclerotial formation was recorded in all the isolates of R. solani. Morphology of sclerotia varied from 1.9×1.5 mm of DCHIRs-1 and 1.8×1.5 mm of RUDPRs-2.

Transcriptomic Profiling Identifies Candidate Genes Involved in Polyporus Umbellatus Sclerotial Formation Affected by Oxalic Acid

Polyporus umbellatus is a precious medicinal fungus. The whole transcriptome of P. umbellatus exposed to different concentrations of oxalic acid was performed and analyzed using RNA-seq based on sequencing technology. Reactive oxygen species (ROS) detection of P. umbellatus mycelia was visually conducted and using non-invasive micro-test technology (NMT), the net Ca2+ and H2O2 fluxes of P. umbellatus were measured. Totally, 22,523 unigenes were generated by De novo assembly of reads and there are 1223 differentially expressed genes (DEGs) between the control group and the high oxalic acid complemented (PD_S) group and 459 DEGs between the control group and the low concentration oxalic acid additive (PU_SM) group. The transcriptomic analysis indicated DEGs encoding enzymes related to oxidative stress, energy metabolism and so on. Compared to that of the control group, the biomass of the sclerotia in the PU_SM group increased 66%, however, no sclerotia formed in the PD_S group. The low concentration of oxalic acid could increase Ca2+ and H2O2 influx, while the high concentration of oxalic acid presented slight H2O2 efflux. There is a great significant positive correlation between the net Ca2+ and H2O2 fluxes. Different concentrations of exogenous oxalic acid affected P. umbellatus sclerotial formation in different ways.

The Destructive Fungal Pathogen Botrytis cinerea—Insights from Genes Studied with Mutant Analysis

Botrytis cinerea is one of the most destructive fungal pathogens affecting numerous plant hosts, including many important crop species. As a molecularly under-studied organism, its genome was only sequenced at the beginning of this century and it was recently updated with improved gene annotation and completeness. In this review, we summarize key molecular studies on B. cinerea developmental and pathogenesis processes, specifically on genes studied comprehensively with mutant analysis. Analyses of these studies have unveiled key genes in the biological processes of this pathogen, including hyphal growth, sclerotial formation, conidiation, pathogenicity and melanization. In addition, our synthesis has uncovered gaps in the present knowledge regarding development and virulence mechanisms. We hope this review will serve to enhance the knowledge of the biological mechanisms behind this notorious fungal pathogen.

Mycosynthesis of Silver Nanoparticles Using Screened Trichoderma Isolates and Their Antifungal Activity against Sclerotinia sclerotiorum

To control the disease caused by Sclerotinia sclerotiorum, a total of 15 isolates of the Trichoderma species was screened for the biosynthesis of silver nanoparticles (AgNPs). Among them, the highest yield occurred in the synthesis of AgNPs using a cell-free aqueous filtrate of T.virens HZA14 producing gliotoxin. The synthetic AgNPs were charactered by SEM, EDS, TEM, XRD, and FTIR. Electron microscopy studies revealed that the size of AgNPs ranged from 5–50 nm and had spherical and oval shapes with smooth surfaces. Prepared AgNPs interacted with protein, carbohydrate and heterocyclic compound molecules, and especially, interaction patterns of AgNPs with the gliotoxin molecule were proposed. The antifungal activity assays demonstrated that percentage inhibition of the prepared AgNPs was 100, 93.8 and 100% against hyphal growth, sclerotial formation, and myceliogenic germination of sclerotia at a concentration of 200 μg/mL, respectively. The direct interaction between nanoparticles and fungal cells, including AgNPs’ contact, accumulation, lamellar fragment production and micropore or fissure formation on fungal cell walls, was revealed by SEM and EDS. These will extend our understanding of the mechanisms of AgNPs’ action for preventing diversified fungal disease.