Sclerotial Development Research Articles

Efficacy of Trichoderma longibrachiatum SC5 Fermentation Filtrate in Inhibiting the Sclerotinia sclerotiorum Growth and Development in Sunflower.

Sclerotinia sclerotiorum is a destructive pathogen responsible for sunflower sclerotinia rot, resulting in substantial yield and economic losses worldwide. Trichoderma species have demonstrated the capacity to inhibit plant pathogen growth through the production of secondary metabolites. However, there are fewer recent studies focusing on the application of Trichoderma metabolites in inhibiting S. sclerotiorum growth and development and controlling sunflower sclerotinia rot disease. Our results showed that five Trichoderma strains (SC5, T6, TN, P6, and TS3) exhibited mycelial growth inhibition higher than 60% in dual culture assays out of the 11 tested strains. The Trichoderma SC5 fermentation filtrate exhibited superior efficacy compared to other strains, achieving a 94.65% inhibition rate of mycelial growth on S. sclerotiorum, 96% inhibition of myceliogenic germination of sclerotia, and 81.05% reduction in the oxalic acid content of S. sclerotiorum, while significantly increasing the cell membrane permeability. In addition, the Trichoderma SC5 fermentation filtrate significantly decreased the activities of polygalacturonase and pectin methyl-galacturonic enzymes and even caused S. sclerotiorum hyphae to swell, branch, twist, lyse, and inhibited the production and development of sclerotia. Moreover, the Trichoderma SC5 fermentation filtrate downregulated genes expression that associated with the growth and infection of S. sclerotiorum. The control efficacies of the protective and curative activities of the Trichoderma SC5 fermentation filtrate were 95.45% and 75.36%, respectively, on detached sunflower leaves at a concentration of 8 mg/mL. Finally, the Trichoderma SC5 was identified as Trichoderma longibrachiatum through morphological and phylogenetic analysis. Our research indicates that the T. longibrachiatum SC5 can be considered a promising biological control candidate against S. sclerotiorum and controlling the sunflower sclerotinia rot disease, both in vitro and in vivo.

RNA interference‐mediated targeting of monooxygenase SsMNO1 for controlling Sclerotinia stem rot caused by Sclerotinia sclerotiorum

AbstractBACKGROUNDSclerotinia sclerotiorum is a devastating fungal pathogen that poses a threat to a variety of economically important crops. Owing to the lack of highly resistant cultivars and the prolonged survival of sclerotia, effective control of Sclerotinia diseases remains challenging. RNA interference (RNAi) agents targeting essential active transcripts of genes associated with the development and virulence of pathogens are a valuable and promising disease control method.RESULTSOur finding suggested that a flavin adenine dinucleotide (FAD)‐dependent monooxygenase gene SsMNO1 plays pivotal roles in the hyphal growth, sclerotial development, and virulence of S. sclerotiorum, rendering it a potential target for RNAi‐mediated management of S. sclerotiorum. The external application of double‐stranded RNA (dsRNA) targeting SsMNO1 inhibited sclerotial development in artificial media and plant tissues. Furthermore, dsRNA significantly reduced the hyphal virulence of S. sclerotiorum in host plants by interfering with SsMNO1 expression. The inhibitory activity persisted for over 1 week on the surface of Brassica napus. Artificial small interfering RNA (siRNA) targeting SsMNO1 also exhibited inhibitory effects. Transgenic Arabidopsis thaliana plants expressing SsMNO1 hairpin RNAi constructs showed increased resistance to S. sclerotiorum infection. Notably, the total RNA extracts from SsMNO1‐RNAi plants also reduced the hyphal virulence in Brassica napus.CONCLUSIONSTherefore, RNAi agents targeting SsMNO1 have dual effects on sclerotial development and hyphal virulence, rendering it an ideal target for controlling diseases caused by S. sclerotiorum. © 2024 Society of Chemical Industry.

A novel protein elicitor (Cs08297) from Ciboria shiraiana enhances plant disease resistance.

Ciboria shiraiana is a necrotrophic fungus that causes mulberry sclerotinia disease resulting in huge economic losses in agriculture. During infection, the fungus uses immunity elicitors to induce plant tissue necrosis that could facilitate its colonization on plants. However, the key elicitors and immune mechanisms remain unclear in C. shiraiana. Herein, a novel elicitor Cs08297 secreted by C. shiraiana was identified, and it was found to target the apoplast in plants to induce cell death. Cs08297 is a cysteine-rich protein unique to C. shiraiana, and cysteine residues in Cs08297 were crucial for its ability to induce cell death. Cs08297 induced a series of defence responses in Nicotiana benthamiana, including the burst of reactive oxygen species (ROS), callose deposition, and activation of defence-related genes. Cs08297 induced-cell death was mediated by leucine-rich repeat (LRR) receptor-like kinases BAK1 and SOBIR1. Purified His-tagged Cs08297-thioredoxin fusion protein triggered cell death in different plants and enhanced plant resistance to diseases. Cs08297 was necessary for sclerotial development, oxidative-stress adaptation, and cell wall integrity but negatively regulated virulence of C. shiraiana. In conclusion, our results revealed that Cs08297 is a novel fungal elicitor in fungi inducing plant immunity. Furthermore, its potential to enhance plant resistance provides a new target to control agricultural diseases biologically.

Role of Flavohemoglobins in the Development and Aflatoxin Biosynthesis of Aspergillus flavus.

Aspergillus flavus is notorious for contaminating food with its secondary metabolite-highly carcinogenic aflatoxins. In this study, we found that exogenous nitric oxide (NO) donor could influence aflatoxin production in A. flavus. Flavohemoglobins (FHbs) are vital functional units in maintaining nitric oxide (NO) homeostasis and are crucial for normal cell function. To investigate whether endogenous NO changes affect aflatoxin biosynthesis, two FHbs, FHbA and FHbB, were identified in this study. FHbA was confirmed as the main protein to maintain NO homeostasis, as its absence led to a significant increase in intracellular NO levels and heightened sensitivity to SNP stress. Dramatically, FHbA deletion retarded aflatoxin production. In addition, FHbA played important roles in mycelial growth, conidial germination, and sclerotial development, and response to oxidative stress and high-temperature stress. Although FHbB did not significantly impact the cellular NO level, it was also involved in sclerotial development, aflatoxin synthesis, and stress response. Our findings provide a new perspective for studying the regulatory mechanism of the development and secondary mechanism in A. flavus.

HacA, a key transcription factor for the unfolded protein response, is required for fungal development, aflatoxin biosynthesis and pathogenicity of Aspergillus flavus

Aspergillus flavus is a fungus notorious for contaminating food and feed with aflatoxins. As a saprophytic fungus, it secretes large amounts of enzymes to access nutrients, making endoplasmic reticulum (ER) homeostasis important for protein folding and secretion. The role of HacA, a key transcription factor in the unfolded protein response pathway, remains poorly understood in A. flavus. In this study, the hacA gene in A. flavus was knockout. Results showed that the absence of hacA led to a decreased pathogenicity of the strain, as it failed to colonize intact maize kernels. This may be due to retarded vegetable growth, especially the abnormal development of swollen tips and shorter hyphal septa. Deletion of hacA also hindered conidiogenesis and sclerotial development. Notably, the mutant strain failed to produce aflatoxin B1. Moreover, compared to the wild type, the mutant strain showed increased sensitivity to ER stress inducer such as Dithiothreitol (DTT), and heat stress. It also displayed heightened sensitivity to other environmental stresses, including cell wall, osmotic, and pH stresses. Further transcriptomic analysis revealed the involvement of the hacA in numerous biological processes, including filamentous growth, asexual reproduction, mycotoxin biosynthetic process, signal transduction, budding cell apical bud growth, invasive filamentous growth, response to stimulus, and so on. Taken together, HacA plays a vital role in fungal development, pathogenicity and aflatoxins biosynthesis. This highlights the potential of targeting hacA as a novel approach for early prevention of A. flavus contamination.

The Transcription Factor SsZNC1 Mediates Virulence, Sclerotial Development, and Osmotic Stress Response in Sclerotinia sclerotiorum.

Sclerotinia sclerotiorum is a fungal pathogen with a broad range of hosts, which can cause diseases and pose a great threat to many crops. Fungal-specific Zn2Cys6 transcription factors (TFs) constitute a large family prevalent among plant pathogens. However, the function of Zn2Cys6 TFs remains largely unknown. In this study, we identified and characterized SsZNC1, a Zn2Cys6 TF in S. sclerotiorum, which is involved in virulence, sclerotial development, and osmotic stress response. The expression of SsZNC1 was significantly up-regulated in the early stages of S. sclerotiorum infection on Arabidopsis leaves. The target deletion of SsZNC1 resulted in reduced virulence on Arabidopsis and oilseed rape. In addition, sclerotial development ability and growth ability under hyperosmotic conditions of SsZNC1 knockout transformants were reduced. A transcriptomic analysis unveiled its regulatory role in key cellular functions, including cellulose catabolic process, methyltransferase activity, and virulence, etc. Together, our results indicated that SsZNC1, a core regulatory gene involved in virulence, sclerotial development and stress response, provides new insight into the transcription regulation and pathogenesis of S. sclerotiorum.

Accumulation of aluminium and arsenic in Cenococcum geophilum sclerotia from forest soil affected by mining smoke

ABSTRACT Aluminium toxicity may increase under low soil pH, and an increase in arsenic concentration in the soil may inhibit inorganic P uptake and influence fungal growth. This study investigated metals/metalloids accumulation in Cenococcum geophilum sclerotia collected from highly polluted forest soils affected by mining activities and smoke hazards. The Al and As concentrations in sclerotia were 11,700 ± 823 and 10.0 ± 1.29 mg kg−1, respectively. TOF-SIMS ion mapping confirmed the predominant presence of Al as Al oxalate, acetate, and hydroxides, and the segregation of As showed higher intensities for organic As ions than inorganic As. Ion fragments of Al and As compounds were widely observed in sclerotial medulla and were generally elevated towards the central part coexisting with phosphoric acid ions. The mechanism of Al and As accumulation has been discussed to involve two biotransformation pathways in terms of sclerotial development using 14C dating. Because the sclerotia were significantly older than the historical smoke hazard and Al and As concentrations in sclerotia were high regardless of age, their accumulation were more likely to be promoted in the mature stage. This study provides insight into the contribution of ectomycorrhizal fungi and their sclerotia to soil ecosystems under metals/metalloids toxicity.

A MAP kinase cascade broadly regulates the lifestyle of Sclerotinia sclerotiorum and can be targeted by HIGS for disease control.

Sclerotinia sclerotiorum causes white mold or stem rot in a wide range of economically important plants, bringing significant yield losses worldwide. Control of this pathogen is difficult as its resting structure sclerotia can survive in soil for years, and no Resistance genes have been identified in S. sclerotiorum hosts. Host-induced gene silencing (HIGS) has shown promising effects in controlling many fungal pathogens, including S. sclerotiorum. However, better molecular genetic understanding of signaling pathways involved in its development and pathogenicity is needed to provide effective HIGS gene targets. Here, by employing a forward genetic screen, we characterized an evolutionarily conserved mitogen-activated protein kinase (MAPK) cascade in S. sclerotiorum, consisting of SsSte50-SsSte11-SsSte7-Smk1, which controls mycelial growth, sclerotia development, compound appressoria formation, virulence, and hyphal fusion. Moreover, disruption of the putative downstream transcription factor SsSte12 led to normal sclerotia but deformed appressoria and attenuated host penetration, as well as impaired apothecia formation, suggestive of diverged regulation downstream of the MAPK cascade. Most importantly, targeting SsSte50 using host-expressed double-stranded RNA resulted in largely reduced virulence of S. sclerotiorum on both Nicotiana benthamiana leaves and transgenic Arabidopsis thaliana plants. Therefore, this MAPK signaling cascade is generally needed for its growth, development, and pathogenesis and can serve as ideal HIGS targets for mitigating economic damages caused by S. sclerotiorum infection.

De Novo RNA Sequencing and Transcriptome Analysis of Sclerotium rolfsii Gene Expression during Sclerotium Development.

Sclerotium rolfsii is a destructive soil-borne fungal pathogen that causes stem rot in cultivated plants. However, little is known about the genetic basis of sclerotium development. In this study, we conducted de novo sequencing of genes from three different stages of S. rolfsii (mycelia, early sclerotium formation, and late sclerotium formation) using Illumina HiSeqTM 4000. We then determined differentially expressed genes (DEGs) across the three stages and annotated gene functions. STEM and weighted gene-co-expression network analysis were used to cluster DEGs with similar expression patterns. Our analysis yielded an average of 25,957,621 clean reads per sample (22,913,500-28,988,848). We identified 8929, 8453, and 3744 DEGs between sclerotium developmental stages 1 versus 2, 1 versus 3, and 2 versus 3, respectively. Additionally, four significantly altered gene expression profiles involved 220 genes related to sclerotium formation, and two modules were positively correlated with early and late sclerotium formation. These results were supported by the outcomes of qPCR and RNA-sequencing conducted on six genes. This is the first study to provide a gene expression map during sclerotial development in S. rolfsii, which can be used to reduce the re-infection ability of this pathogen and provide new insights into the scientific prevention and control of the disease. This study also provides a useful resource for further research on the genomics of S. rolfsii.

A Novel Light-Responsive Gene BcCfaS Regulates the Photomorphogenesis and Virulence of Botrytis cinerea via Lipid Metabolism

Light is a fundamental environmental factor for living organisms on earth—not only as a primary energy source but also as an informational signal. In fungi, light can be used as an indicator for both time and space to control important physiological and morphological responses. Botrytis cinerea (B. cinerea) is a devastating phytopathogenic fungus that exploits light cues to optimize virulence and the balance between conidiation and sclerotia development, thereby improving its dispersal and survival in ecosystems. However, the components and mechanisms underlying these processes remain obscure. Here, we identify a novel light-signaling component in B. cinerea, BcCfaS, which encodes a putative cyclopropane fatty-acyl-phospholipid synthase. BcCfaS is strongly induced by light at the transcriptional level and plays a crucial role in regulating photomorphogenesis. Deletion of BcCfaS results in reduced vegetative growth, altered colony morphology, impaired sclerotial development, and enhanced conidiation in a light-dependent manner. Moreover, the mutant exhibits serious defects in stress response and virulence on the host. Based on a lipidomics analysis, a number of previously unknown fungal lipids and many BcCfaS-regulated lipids are identified in B. cinerea, including several novel phospholipids and fatty acids. Importantly, we find that BcCfaS controls conidiation and sclerotial development by positively regulating methyl jasmonate (MeJA) synthesis to activate the transcription of light-signaling components, revealing for the first time the metabolic base of photomorphogenesis in fungi. Thus, we propose that BcCfaS serves as an integration node for light and lipid metabolism, thereby providing a regulatory mechanism by which fungi adapt their development to a changing light environment. These new findings provide an important target for antifungal design to prevent and control fungal disease.

RAS signalling genes can be used as host-induced gene silencing targets to control fungal diseases caused by Sclerotinia sclerotiorum and Botrytis cinerea.

Sclerotinia sclerotiorum causes white mold (also called stem rot, Sclerotinia blight, etc.) in many economically important plants. It is a notorious soilborne fungal pathogen due to its wide host range and ability to survive in soil for long periods of time as sclerotia. Although host-induced gene silencing (HIGS) was recently demonstrated to be an effective method for controlling white mold, limited gene targets are available. Here, using a forward genetics approach, we identified a RAS-GTPase activating protein, SsGAP1, which plays essential roles in sclerotia formation, compound appressoria production and virulence. In parallel, as revealed by our knockout analysis, the SsGAP1 ortholog in Botrytis cinerea, BcGAP1, plays similar roles in fungal development and virulence. By knocking down SsRAS1 and SsRAS2, we also revealed that both SsRAS1 and SsRAS2 are required for vegetative growth, sclerotia development, compound appressoria production and virulence in S. sclerotiorum. Due to the major roles these RAS signalling components play in Sclerotiniaceae biology, they can be used as HIGS targets to control diseases caused by both S. sclerotiorum and B. cinerea. Indeed, when we introduced HIGS constructs targeting SsGAP1, SsRAS1 and SsRAS2 in Nicotiana benthamiana and Arabidopsis thaliana, we observed reduced virulence. Taken together, our forward genetics gene discovery pipeline in S. sclerotiorum is highly effective in identifying novel HIGS targets to control S. sclerotiorum and B. cinerea.

SsCak1 Regulates Growth and Pathogenicity in Sclerotinia sclerotiorum.

Sclerotinia sclerotiorum is a devastating fungal pathogen that causes severe crop losses worldwide. It is of vital importance to understand its pathogenic mechanism for disease control. Through a forward genetic screen combined with next-generation sequencing, a putative protein kinase, SsCak1, was found to be involved in the growth and pathogenicity of S. sclerotiorum. Knockout and complementation experiments confirmed that deletions in SsCak1 caused defects in mycelium and sclerotia development, as well as appressoria formation and host penetration, leading to complete loss of virulence. These findings suggest that SsCak1 is essential for the growth, development, and pathogenicity of S. sclerotiorum. Therefore, SsCak1 could serve as a potential target for the control of S. sclerotiorum infection through host-induced gene silencing (HIGS), which could increase crop resistance to the pathogen.

Mycovirus-induced hypovirulence in notorious fungi Sclerotinia: a comprehensive review.

Members of the genus Sclerotinia are notorious plant pathogens with a diverse host range that includes many important crops. A huge number of mycoviruses have been identified in this genus; some of these viruses are reported to have a hypovirulent effect on the fitness of their fungal hosts. These mycoviruses are important to researchers from a biocontrol perspective which was first implemented against fungal diseases in 1990. In this review, we have presented the data of all hypovirulent mycoviruses infecting Sclerotinia sclerotiorum isolates. The data of hypovirulent mycoviruses ranges from 1992 to 2023. Currently, mycoviruses belonging to 17 different families, including (+) ssRNA, (-ssRNA), dsRNA, and ssDNA viruses, have been reported from this genus. Advances in studies had shown a changed expression of certain host genes (responsible for cell cycle regulation, DNA replication, repair pathways, ubiquitin proteolysis, gene silencing, methylation, pathogenesis-related, sclerotial development, carbohydrate metabolism, and oxalic acid biosynthesis) during the course of mycoviral infection, which were termed differentially expressed genes (DEGs). Together, research on fungal viruses and hypovirulence in Sclerotinia species can deepen our understanding of the cellular processes that affect how virulence manifests in these phytopathogenic fungi and increase the potential of mycoviruses as a distinct mode of biological control. Furthermore, the gathered data can also be used for in-silico analysis, which includes finding the signature sites [e.g., hypovirus papain-like protease (HPP) domain, "CCHH" motif, specific stem-loop structures, p29 motif as in CHV1, A-rich sequence, CA-rich sequences as in MoV1, GCU motif as in RnMBV1, Core motifs in hypovirus-associated RNA elements (HAREs) as in CHV1] that are possibly responsible for hypovirulence in mycoviruses.

Sclerotinia sclerotiorum Agglutinin Modulates Sclerotial Development, Pathogenicity and Response to Abiotic and Biotic Stresses in Different Manners.

Sclerotinia sclerotiorum is an important plant pathogenic fungus of many crops. Our previous study identified the S. sclerotiorum agglutinin (SSA) that can be partially degraded by the serine protease CmSp1 from the mycoparasite Coniothyrium minitans. However, the biological functions of SSA in the pathogenicity of S. sclerotiorum and in its response to infection by C. minitans, as well as to environmental stresses, remain unknown. In this study, SSA disruption and complementary mutants were generated for characterization of its biological functions. Both the wild-type (WT) of S. sclerotiorum and the mutants were compared for growth and sclerotial formation on potato dextrose agar (PDA) and autoclaved carrot slices (ACS), for pathogenicity on oilseed rape, as well as for susceptibility to chemical stresses (NaCl, KCl, CaCl2, sorbitol, mannitol, sucrose, sodium dodecyl sulfate, H2O2) and to the mycoparasitism of C. minitans. The disruption mutants (ΔSSA-175, ΔSSA-178, ΔSSA-225) did not differ from the WT and the complementary mutant ΔSSA-178C in mycelial growth. However, compared to the WT and ΔSSA-178C, the disruption mutants formed immature sclerotia on PDA, and produced less but larger sclerotia on ACS; they became less sensitive to the eight investigated chemical stresses, but more aggressive in infecting leaves of oilseed rape, and more susceptible to mycoparasitism by C. minitans. These results suggest that SSA positively regulates sclerotial development and resistance to C. minitans mycoparasitism, but negatively regulates pathogenicity and resistance to chemical stresses.

Nitrate reductase is required for sclerotial development and virulence of Sclerotinia sclerotiorum.

Sclerotinia sclerotiorum, the causal agent of Sclerotinia stem rot (SSR) on more than 450 plant species, is a notorious fungal pathogen. Nitrate reductase (NR) is required for nitrate assimilation that mediates the reduction of nitrate to nitrite and is the major enzymatic source for NO production in fungi. To explore the possible effects of nitrate reductase SsNR on the development, stress response, and virulence of S. sclerotiorum, RNA interference (RNAi) of SsNR was performed. The results showed that SsNR-silenced mutants showed abnormity in mycelia growth, sclerotia formation, infection cushion formation, reduced virulence on rapeseed and soybean with decreased oxalic acid production. Furthermore SsNR-silenced mutants are more sensitive to abiotic stresses such as Congo Red, SDS, H2O2, and NaCl. Importantly, the expression levels of pathogenicity-related genes SsGgt1, SsSac1, and SsSmk3 are down-regulated in SsNR-silenced mutants, while SsCyp is up-regulated. In summary, phenotypic changes in the gene silenced mutants indicate that SsNR plays important roles in the mycelia growth, sclerotia development, stress response and fungal virulence of S. sclerotiorum.

First Report of Rose Bent Neck Caused by Sclerotinia sclerotiorum on Commercial Cut Roses (Rosa hybrida L.)

Cut roses are highly valuable ornamentals and their profitability depends on the flower postharvest performance. Bent neck symptoms in roses make them unmarketable and are typically related to physiological disorders, bacteria accumulation in the vase solution, and Botrytis cinerea infection. Unusual bent neck symptoms were observed in 4.7% of ‘Orange Crush’ roses from two commercial shipments, resulting in complete flower collapse. This research was aimed to determine the causal agent of the bent neck symptoms. Following incubation in a humid chamber, symptomatic roses evolved in water-soaked lesions with the presence of white mycelium and sclerotia development. Fungal isolations and molecular characterization were performed and Sclerotinia sclerotiorum was identified as the causal agent of rose bent neck. Therefore, when bent neck symptoms are observed, S. sclerotiorum incidence should be considered to avoid possible outbreaks.

Glutamine Synthetase Contributes to the Regulation of Growth, Conidiation, Sclerotia Development, and Resistance to Oxidative Stress in the Fungus Aspergillus flavus.

The basic biological function of glutamine synthetase (Gs) is to catalyze the conversion of ammonium and glutamate to glutamine. This synthetase also performs other biological functions. However, the roles of Gs in fungi, especially in filamentous fungi, are not fully understood. Here, we found that conditional disruption of glutamine synthetase (AflGsA) gene expression in Aspergillus flavus by using a xylose promoter leads to a complete glutamine deficiency. Supplementation of glutamine could restore the nutritional deficiency caused by AflGsA expression deficiency. Additionally, by using the xylose promoter for the downregulation of AflgsA expression, we found that AflGsA regulates spore and sclerotic development by regulating the transcriptional levels of sporulation genes abaA and brlA and the sclerotic generation genes nsdC and nsdD, respectively. In addition, AflGsA was found to maintain the balance of reactive oxygen species (ROS) and to aid in resisting oxidative stress. AflGsA is also involved in the regulation of light signals through the production of glutamine. The results also showed that the recombinant AflGsA had glutamine synthetase activity in vitro and required the assistance of metal ions. The inhibitor molecule L-α-aminoadipic acid suppressed the activity of rAflGsA in vitro and disrupted the morphogenesis of spores, sclerotia, and colonies in A. flavus. These results provide a mechanistic link between nutrition metabolism and glutamine synthetase in A. flavus and suggest a strategy for the prevention of fungal infection.

Characterization of Transcriptional Responses to Genomovirus Infection of the White Mold Fungus, Sclerotinia sclerotiorum.

Soybean leaf-associated gemygorvirus-1 (SlaGemV−1) is a CRESS-DNA virus classified in the family Genomoviridae, which causes hypovirulence and abolishes sclerotia formation in infected fungal pathogens under the family Sclerotiniaceae. To investigate the mechanisms involved in the induction of hypovirulence, RNA-Seq was compared between virus-free and SlaGemV−1-infected Sclerotinia sclerotiorum strain DK3. Overall, 4639 genes were differentially expressed, with 50.5% up regulated and 49.5% down regulated genes. GO enrichments suggest changes in integral membrane components and transmission electron microscopy images reveal virus-like particles localized near the inner cell membrane. Differential gene expression analysis focused on genes responsible for cell cycle and DNA replication and repair pathways, ubiquitin proteolysis, gene silencing, methylation, pathogenesis-related, sclerotial development, carbohydrate metabolism, and oxalic acid biosynthesis. Carbohydrate metabolism showed the most changes, with two glycoside hydrolase genes being the most down regulated by −2396.1- and −648.6-fold. Genes relating to pathogenesis showed consistent down regulation with the greatest being SsNep1, SsSSVP1, and Endo2 showing, −4555-, −14.7-, and −12.3-fold changes. The cell cycle and DNA replication/repair pathways were almost entirely up regulated including a putative cyclin and separase being up regulated 8.3- and 5.2-fold. The oxalate decarboxylase genes necessary for oxalic acid catabolism and oxalic acid precursor biosynthesis genes and its metabolism show down regulations of −17.2- and −12.1-fold changes. Sclerotial formation genes also appear differentially regulated including a melanin biosynthesis gene Pks1 and a sclerotia formation gene Sl2 with fold changes of 3.8 and −2.9.

The Necrotroph Botrytis cinerea BcSpd1 Plays a Key Role in Modulating Both Fungal Pathogenic Factors and Plant Disease Development.

Botrytis cinerea is a necrotrophic microbe that causes gray mold disease in a broad range of hosts. In the present study, we conducted molecular microbiology and transcriptomic analyses of the host–B. cinerea interaction to investigate the plant defense response and fungal pathogenicity. Upon B. cinerea infection, plant defense responses changed from activation to repression; thus, the expression of many defense genes decreased in Arabidopsis thaliana. B. cinerea Zn(II)2Cys6 transcription factor BcSpd1 was involved in the suppression of plant defense as ΔBcSpd1 altered wild-type B05.10 virulence by recovering part of the defense responses at the early infection stage. BcSpd1 affected genes involved in the fungal sclerotium development, infection cushion formation, biosynthesis of melanin, and change in environmental pH values, which were reported to influence fungal virulence. Specifically, BcSpd1 bound to the promoter of the gene encoding quercetin dioxygenase (BcQdo) and positively affected the gene expression, which was involved in catalyzing antifungal flavonoid degradation. This study indicates BcSpd1 plays a key role in the necrotrophic microbe B. cinerea virulence toward plants by regulating pathogenicity-related compounds and thereby suppressing early plant defense.

The C2H2 Transcription Factor SsZFH1 Regulates the Size, Number, and Development of Apothecia in Sclerotinia sclerotiorum.

Sclerotinia sclerotiorum is a notorious phytopathogenic Ascomycota fungus with a host range of >600 plant species worldwide. This homothallic Leotiomycetes species reproduces sexually through a multicellular apothecium that produces and releases ascospores. These ascospores serve as the primary inoculum source for disease initiation in the majority of S. sclerotiorum disease cycles. The regulation of apothecium development for this pathogen and other apothecium-producing fungi remains largely unknown. Here, we report that a C2H2 transcription factor, SsZFH1 (zinc finger homologous protein), is necessary for the proper development and maturation of sclerotia and apothecia in S. sclerotiorum and is required for the normal growth rate of hyphae. Furthermore, ΔSszfh1 strains exhibit decreased H2O2 accumulation in hyphae, increased melanin deposition, and enhanced tolerance to H2O2 in the process of vegetative growth and sclerotia formation. Infection assays on common bean leaves, with thin cuticles, and soybean and tomato leaves, with thick cuticles, suggest that the deletion of Sszfh1 slows the mycelial growth rate, which in turn affects the expansion of leaf lesions. Collectively, our results provide novel insights into a major fungal factor mediating maturation of apothecia with additional effects on hyphae and sclerotia development.