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Sclerotinia stem rot (SSR), caused by the necrotrophic fungal pathogen Sclerotinia sclerotiorum, is a severe disease of broadleaf crops including canola/rapeseed (Brassica napus), leading to significant yield loss in conducive years. Replication of field conditions is challenging in variety disease resistance screening with testing required under a wide range of environmental conditions and at different plant growth stages. We investigated the role of thermal time in disease progression using three sowing times in the growing season, with six commonly grown Australian varieties of canola inoculated with four diverse West Australian isolates of S. sclerotiorum at 30% flowering. Area Under the Disease Progression Stairs (AUDPS), sclerotia production and weight, and seed production were measured. Time of sowing was found to be a crucial factor in explaining differences in AUDPS, stem width, seed production and sclerotia number according to the analysis of variance (P < 0.05), influencing isolate aggressiveness and disease progression. Linear mixed-effect models, regression decision tree models and principal components analysis were also conducted to determine the importance of a range of variables being included in variety screening for resistance. For all these analyses, both thermal time from sowing to 30% flowering when plants were inoculated, as well as thermal time over the 28-day inoculation period, in both canola and S. sclerotiorum, were important in explaining the variation. The study concludes by recommending that thermal time should be included in future SSR prediction risk models. [ABSTRACT FROM AUTHOR]

Pleurotus tuber-regium (Fr.) Sing. can evade oxygen by forming sclerotia under oxidative stress, consequently averting the development of hyperoxidative state, during which the expression level of catalase gene (PtCat) is significantly up-regulated. To investigate the relationship between the catalase gene and sclerotia formation, over-expression and interference strains of the PtCat gene were obtained by Agrobacterium tumefaciens-mediated transformation for phenotypic analysis. In the absence of hydrogen peroxide (H2O2) stress, a minor difference was observed in the mycelial growth rate and the activity of antioxidant enzymes between the over-expression and interference strains. However, when exposed to 1–2 mM H2O2, the colony diameter of the over-expression strain was approximately 2–3× that of the interference strain after 8 days of culturing. The catalase activity of the over-expression strain increased by 1000 U/g under 2 mM H2O2 stress, while the interference strain increased by only 250 U/g. After one month of cultivation, the interference strain formed an oval sclerotium measuring 3.5 cm on the long axis and 2 cm on the short axis, while the over-expression strain did not form sclerotia. Therefore, it is concluded that catalase activity regulates the formation of sclerotia in P. tuber-regium. [ABSTRACT FROM AUTHOR]

Fungal HacA/Hac1 transcription factors play a crucial role in regulating the unfolded protein response (UPR). The UPR helps cells to maintain endoplasmic reticulum (ER) protein homeostasis, which is critical for growth, development, and virulence. The Aspergillus flavus hacA gene encodes a domain rich in basic and acidic amino acids (Bsc) and a basic leucine zipper (bZip) domain, and features a non-conventional intron (Nt20). In this study, CRISPR/Cas9 was utilized to dissect the Bsc-coding, bZip-coding, and Nt20 sequences to elucidate the relationship between genotype and phenotype. In the Bsc and bZip experimental sets, all observed mutations in both coding sequences were in frame, suggesting that out-of-frame mutations are lethal. The survival rate of transformants in the Nt20 experiment set was low, at approximately 7%. Mutations in the intron primarily consisted of out-of-frame insertions and deletions. In addition to the wild-type-like conidial morphology, the mutants exhibited varied colony morphologies, including sclerotial, mixed (conidial and sclerotial), and mycelial morphologies. An ER stress test using dithiothreitol revealed that the sclerotial and mycelial mutants were much more sensitive than the conidial mutants. Additionally, the mycelial mutants were unable to produce aflatoxin but still produced aspergillic acid and kojic acid. RNAi experiments targeting the region encompassing Bsc and bZip indicated that transformant survival rates generally decreased, with a small number of transformants displaying phenotypic changes. Defects in the hacA gene at the DNA and transcript levels affected the survival, growth, and development of A. flavus. Thus, this gene may serve as a promising target for future host-induced gene-silencing strategies aimed at controlling infection and reducing aflatoxin contamination in crops. [ABSTRACT FROM AUTHOR]

P. umbellatus sclerotium is a traditional Chinese medicine that is widely utilized in China, Korea, Japan, and other countries due to its diverse medicinal activities, such as diuretic, antitumor, anticancer, and immune system enhancement effects. Conidia, which are common asexual spores in various fungi, are not universally present in Polyporus species. In this study, the asexual life cycle of P. umbellatus was elucidated. Conidia, i.e. arthorconidia, were produced by both dikaryotic and monokaryotic strains. In the dikaryotic strain, binucleate, uninucleate, and nuclei-free conidia were identified with proportions of 67.9 %, 12.4 %, and 19.7 %, respectively. Conversely, the monokaryotic strain did not produce binucleate conidia. This discrepancy suggests that binucleate spores are heterokaryons, while uninucleate spores are homokaryons. Clamp connections were observed in dikaryotic hyphae, but were absent in monokaryotic hyphae. Monokaryotic strains were obtained from conidia of the dikaryotic strain. Additionally, mating types were determined through pairing tests, and successful crossbreeding occurred between monokaryotic strains derived from conidia and basidiospores from different strains. This study introduced the first crossbreeding strategy for P. umbellatus. • The conidia of Polyporus umbellatus are holoarthric arthrospores. • This is the first report of nuclear pattern study for P. umbellatus conidia. • The asexual life cycle of P. umbellatus was firstly depicted. • Providing the first crossbreeding strategy for P. umbellatus. [ABSTRACT FROM AUTHOR]

Sclerotium root rot, caused by the fungus Sclerotium rolfsii, presents a significant challenge to sugar beet cultivation, particularly in tropical and warmer climates where high temperatures favor pathogen proliferation. This disease is prevalent in southern regions globally, where optimal conditions enable the fungus to produce white cottony mycelium and sclerotia rapidly. These sclerotia can be dispersed by wind or during agricultural activities, integrating into the soil profile and complicating disease management. The pathogen's complex life cycle and broad host range exacerbate management difficulties. Early identification of symptoms is crucial for effective management, emphasizing the need for advanced diagnostic techniques. This study highlights recent advancements in managing Sclerotium root rot, focusing on biotechnological innovations and precision agriculture methods. Techniques such as CRISPR/Cas gene editing, artificial intelligence, satellite farming, and augmented reality offer promising solutions for disease control. CRISPR/Cas technology provides precise genetic modifications to enhance disease resistance in sugar beets. Artificial intelligence and satellite farming enable real-time monitoring and predictive analytics for early detection and management of the disease. Augmented reality tools facilitate farmer education and decision-making through immersive and interactive platforms. The integration of these advanced technologies presents a comprehensive approach to combating Sclerotium root rot, ensuring sustainable sugar beet production in affected regions. This study underscores the importance of leveraging cutting-edge innovations to address the complexities of pathogen management in agriculture. [ABSTRACT FROM AUTHOR]

Claviceps purpurea (Fries) Tulasne is a valuable source of many bioactive metabolites (ergot alkaloids) for the pharmaceutical industry. Selection and cultivation of pharmaceutical ergot Claviceps purpurea needs quick qualitative and quantitative analyses for alkaloid identification and chemotaxonomic strain passportisation; however, express methods can be uninformative for some conditions. We used an alternative method of reversed-phase HPLC-UV (based on formic acid elution) for the identification and determination of concentrations for ergot alkaloids in sclerotias from parasitic cultures of 3 strains of pharmaceutical Claviceps purpurea. Two different types of standard solutions of ergocriptine—sum of the α+β isomers and the ergotoxine mixture (ergocornine +α+β ergocriptine with a known ratio)—allowed us to clearly identify each peak in the analysis of standard solutions and sclerotia samples of parasitic ergot. Parameters of retention time and concentrations were determined for ergotamine (25.4 ± 0.11 min, 0.57 ± 0.04 g/100 g respectively), ergometrine (7.67 ± 0.01 min, 0.02 ± 0.004 g/100 g), ergocristine (27.83 ± 0.01 min, 0.62 ± 0.05 g/100 g), ergocornine (25.61 ± 0.11 min, 0.27 ± 0.04 g/100 g), and α- and β-isomers of ergocriptine (α-ergocriptine – 27.19 ± 0.01 min, 0.17 ± 0.01 g/100 g; β-ergocriptine – 27.42 ± 0.01 min, 0.061 ± 0.05 g/100 g). Ergotamine, ergometrine, ergocristine, ergocornine, α- and β-isomers of ergocriptine were identified and quantitatively determined in sclerotias from different strains of pharmaceutical ergot Claviceps purpurea. [ABSTRACT FROM AUTHOR]

Severe infection of stem rot was observed on plants of Withania somnifera during January and February of 2020 and 2021. Initial symptoms appeared on the plants showing paleness at infected areas, cottony, fluffy white mycelia was observed on the leaves, stem and inflorescence. In advanced stage of infection wilting of stem and defoliation was observed leading to partial or whole plant death. Based on its morphological and molecular traits, the fungus was identified as Sclerotinia sclerotiorum. Pathogenicity test was performed under controlled conditions resulting in similar symptoms to those observed in field. To the best of our knowledge, this is the worldwide first evidence of S. sclerotiorum causing stem rot disease on W. somnifera. [ABSTRACT FROM AUTHOR]

To understand the effect of sclerotial density on tubers, and the method vis-a-vis timing of dehaulming on the development of black scurf, field studies were conducted under artificial epiphytotic conditions. Infection levels of up to 10% tuber coverage by scurf sclerotia (grades 0,1 or 2) did not affect yield, but tubers with more than 10% sclerotial coverage (grade 3 or higher) resulted in significantly lower yields compared to apparently healthy tubers (grade 0). The severity of the scurf was significantly higher when the tubers having up to 5% sclerotial coverage (grade 1) were used for sowing compared to sclerotia-free seeds. Seed tubers with more than 5% sclerotial coverage corresponding to grades 2, 3, and 4 of scurf severity were statistically at par with respect to scurf severity, but these had higher scurf intensity compared to grades 0 and 1 (sclerotial coverage 0–5%). The three test methods of dehaulming viz., cutting the tops, herbicide desiccation, and vine pulling didn't impact the overall tuber yield, but the timing of dehaulming i.e., 7,14,21, and 28 days before harvesting did impact the yield. There was a significant effect of the time gap between vine kill and harvest on the development of black scurf. Dehaulming conducted at 7 days before harvesting produced significantly higher yields than 14, 21, and 28 days before harvesting. The longer the time gap, the more severe was scurf infection. Using herbicide as a method of dehaulming led to more sclerotial formation compared to cutting and pulling. [ABSTRACT FROM AUTHOR]

Controlling the hazard of sclerotia produced by the Sclerotinia sclerotiorum is very complex, and it is urgent to adopt an effective method that is harmonious environmentally to control the disease. Among the six isolates isolated from the rhizosphere of lettuce, the isolate HZA84 demonstrated a high activity in its antagonism towards Sclerotinia sclerotiorum in vitro, and produces siderophore. By amplification of internal transcribed spacer (ITS), translation elongation factor 1-alpha (TEF1-α), and RNA polymerase II subunit (RPB2) genes, the isolate HZA84 was identified as Trichoderma asperellum, which was confirmed by analysis of phylogenetic tree. The Scanning electron microscope monitoring detected that the isolate HZA84 spread over the sclerotial surface, thus, damaging, decomposing, and distorting the globular cells of the outer cortex of the sclerotia. The Real-time polymerase chain reaction (RT-qPCR) analysis disclosed the overexpression of two genes (chit33 and chit37) encoding the endochitinase in addition to one gene (prb1) encoding the proteinase during 4 and 8 days of the parasitism behavior of isolate HZA84 on the sclerotia surface. These enzymes aligned together in the sclerotia destruction by hyperparasitism. On the other hand, the pots trial revealed that spraying of isolate HZA84 reduced the drop disease symptoms of lettuce. The disease severity was decreased by 19.33 and the biocontrol efficiency was increased by 80.67% within the fourth week of inoculation. These findings magnify the unique role of Trichoderma in disrupting the development of plant diseases in sustainable ways. [ABSTRACT FROM AUTHOR]

In recent years, there has been an intensification of weather variability worldwide as a result of climate change. Some regions have been affected by drought, while others have experienced more intense rainfall. The incidence and severity of moldy grain and mycotoxin contamination during the growing and harvesting seasons have increased as a result of these weather conditions. Additionally, torrential rains and wet conditions may cause delays in grain drying, leading to mold growth in the field. In July 2023, a wheat field in Lecco (Lombardy, Italy) was affected by torrential rains that led to the development of the Claviceps fungi. In the field, dark sclerotia were identified on some ears. Wheat ears, kernels, and sclerotia were collected and analyzed by LC-MS/MS at IZSLER, Food Chemical Department, in Bologna. The wheat ears, kernels, and sclerotia were analyzed for 12 ergot alkaloids (EAs) according to (EU) Regulation 2023/915 (ergocornine/ergocorninine; ergocristine/ergocristinine; ergocryptine/ergocryptinine; ergometrine/ergometrinine; ergosine/ergosinine; ergotamine/ergotaminine), after QuEChERS (Z-Sep/C18) purification. The analyzed sclerotia showed significant differences in total alkaloid content that vary between 0.01 and 0.5% (w/w), according to the results of the 2017 EFSA scientific report. EAs detected in sclerotia were up to 4951 mg/kg, in wheat ears up to 33 mg/kg, and in kernels were 1 mg/kg. Additional mycotoxins, including ochratoxin A, deoxynivalenol, zearalenone, fumonisins, T2-HT2 toxins, and aflatoxins, were investigated in wheat kernels after purification with immunoaffinity columns (IAC). The analysis revealed the presence of deoxynivalenol in wheat kernels at a concentration of 2251 µg/kg. It is expected that climate change will increase the frequency of extreme weather events. In order to mitigate the potential risks associated with mycotoxin-producing fungi and to ensure the protection of human health, it is suggested that official controls be implemented in the field. [ABSTRACT FROM AUTHOR]

Background: The Botrytis gray mold (BGM) caused by Botrytis cinerea Pers. ex Fr., has become a major threat for chickpea cultivation in India. In view of monetary significance of this disease and growing its spread into new regions through seed and soil (sclerotia) encouraged us to study variability of this pathogen from diverse regions. Methods: Botrytis cinerea infected chickpea samples were collected from Punjab and Uttarakhand states. Pathogen was isolated using chickpea dextrose agar media. Pathogenicity was proved by inoculation of spore suspension (3 X 105 spores'mL-1) of the fungus on susceptible cultivar. In vitro studies were conducted for observations of morphological and cultural variability of the fungus. The pathogenic fungus was also identified through molecular characterization using ITS primers. Result: The isolates were categorized into different groups based on growth rate of the pathogen on PDA media. Pathogenicity test proved that B. cinerea isolates from Punjab were showed less disease severity scale compared to Uttarakhand state isolates. The B. cinerea fungus prefers PDA for efficient growth and multiplication than Chickpea Dextrose Agar (CDA) media. There was no conspicuous difference in colour of the colony on PDA and CDA media. The colour of the mycelium were initially white or dirty white or greyish and sporulated culture shown to have as grey with profuse mass on the surface of the mycelium. The fungus B. fabae, a different species was also responsible for causing BGM disease which was confirmed through pathogenicity and ITS sequencing. Twenty-eight isolates were studied for diversity in cultural, morphological and molecular level. The diversity in colony colour, growth type and diameter of mycelial growth, sclerotia initiation, number and pattern of sclerotia formation were observed amongst the isolates. This study would help in designing breeding strategy for development of disease resistant cultivars. [ABSTRACT FROM AUTHOR]

APSES (Asm1p, Phd1p, Sok2p, Efg1p, and StuAp) family transcription factors play crucial roles in various biological processes of fungi, however, their functional characterization in phytopathogenic fungi is limited. In this study, we explored the role of SsStuA, a typical APSES transcription factor, in the regulation of cell wall integrity (CWI), sclerotia formation and pathogenicity of Sclerotinia sclerotiorum, which is a globally important plant pathogenic fungus. A deficiency of SsStuA led to abnormal phosphorylation level of SsSmk3, the key gene SsAGM1 for UDP-GlcNAc synthesis was unable to respond to cell wall stress, and decreased tolerance to tebuconazole. In addition, ΔSsStuA was unable to form sclerotia but produced more compound appressoria. Nevertheless, the virulence of ΔSsStuA was significantly reduced due to the deficiency of the invasive hyphal growth and increased susceptibility to hydrogen peroxide. We also revealed that SsStuA could bind to the promoter of catalase family genes which regulate the expression of catalase genes. Furthermore, the level of reactive oxygen species (ROS) accumulation was found to be increased in ΔSsStuA. In summary, SsStuA, as a core transcription factor involved in the CWI pathway and ROS response, is required for vegetative growth, sclerotia formation, fungicide tolerance and the full virulence of S. sclerotiorum. [ABSTRACT FROM AUTHOR]

Velvet (VeA), a light-regulated protein that shuttles between the cytoplasm and the nucleus, serves as a key global regulator of secondary metabolism in various Aspergillus species and plays a pivotal role in controlling multiple developmental processes. The gene vepN was chosen for further investigation through CHIP-seq analysis due to significant alterations in its interaction with VeA under varying conditions. This gene (AFLA_006970) contains a Septin-type guanine nucleotide-binding (G) domain, which has not been previously reported in Aspergillus flavus (A. flavus). The functional role of vepN in A. flavus was elucidated through the creation of a gene knockout mutant and a gene overexpression strain using a well-established dual-crossover recombinational technique. A comparison between the wild type (WT) and the ΔvepN mutant revealed distinct differences in morphology, reproductive capacity, colonization efficiency, and aflatoxin production. The mutant displayed reduced growth rate; dispersion of conidial heads; impaired cell wall integrity; and decreased sclerotia formation, colonization capacity, and aflatoxin levels. Notably, ΔvepN exhibited complete growth inhibition under specific stress conditions, highlighting the essential role of vepN in A. flavus. This study provides evidence that vepN positively influences aflatoxin production, morphological development, and pathogenicity in A. flavus. [ABSTRACT FROM AUTHOR]

Sheath blight (ShB) caused by the soil-borne fungus Rhizoctonia solani AG1-IA is one of the most important diseases of rice in the world, which is also common in some areas of rice cultivation in Iran. The pathogen usually overwinters as sclerotia in soil and plant debries and mycelium in plant debris and seeds. Sclerotia can survive inactive in soil, and rice debris for several years. After transplanting susceptible rice cultivars, the sclerotia germinate, produce mycelium, and infect the lower sheaths of the rice plant. The characteristic symptoms of ShB are green-gray water-soaked lesions, spherical to oval, or irregularly discolored on rice sheath. The lesions are connected, and the center of the lesions becomes gray to light brown with a dark brown-to-red border, by passing time. Factors such as the rice variety, plant density, and growth stage, initial inoculum population of the pathogen, environmental conditions, and plant nutrition affect the disease severity and epidemic. The disease management is difficult, because of the wide host range of the pathogen, its ability to survive for a long time in the soil, and the low level of resistance of rice cultivars to it. Proper management such as avoiding cultivation of dwarf and high tillering cultivars in fields with a history of pathogen presence, seed disinfection, proper plant densities, and optimal use of nitrogen fertilizer, field sanitation, and chemical control can prevent the epidemy and damage of the disease. [ABSTRACT FROM AUTHOR]

It is perceived that sclerotia of the pathogen, in addition to chlamydospores, play an important role in the epidemiology of rice false smut disease. The propagule has been identified only in five rice growing countries of the world, and never recorded in Bangladesh. This study was undertaken to identify the sclerotia, quantify their concentration on smut balls and estimate the time of its formation. Intensive field-by-field monitoring was conducted during 2014, 2015 and 2016 at experimental farm of the Bangladesh Rice Research Institute, Gazipur. After rigorous search, sclerotia of rice false smut pathogen was identified on the 14th of December 2014. The was observed in the later part of crop maturity during the ‘T. Aman' season (early-November to mid-December) on olivaceous greenish-black smut balls, but not on orange smut ball. During the three years study, on average, 30.61±9.79% (± is 95%confidence interval) olivaceous greenish-black smut balls borne sclerotia, the average number being 1.63±0.09 per sclerotia-bearing ball. The ‘threshold temperature' for sclerotia formation was estimated as above 11°C difference in day-night temperature. Consistence presence and high concentration of sclerotia in smut balls signify its role in the epidemiology of the disease in the agroecology of Bangladesh. [Fundam Appl Agric 2023; 8(3.000): 580-589]

Claviceps purpurea causes ergot, a floral disease of major cereal crops, such as winter rye and winter triticale. The dark sclerotia formed by C. purpurea contain numerous toxic ergot alkaloids, which pose a major health risk for humans and animals when ingested. The countermeasures against ergot infection are currently limited to intensive tillage, seed cleaning, crop rotation or integration of optical sorting machines to remove ergot from cereals before processing. However, these practices confer only a minor protective effect and are highly energy-demanding. Thus, more effective and sustainable plant protection measures to combat ergot infections in cereals are needed. The application of Trichoderma spp. as biological control agent has already proven successful against a wide range of phytopathogenic fungi. However, there is only minor scientific evidence about its protective capacity against C. purpurea in cereal crops. In our study, we therefore investigated the antagonistic potential of several Trichoderma isolates against C. purpurea and their ability to confer protection against ergot infection. In initial in vitro tests and confrontation assays, we quantified and compared the production of iron-chelating compounds and hydrolytic enzymes as well as the antagonistic activity against C. purpurea sclerotia and mycelial cultures. Several strains showed high antifungal capacity and growth inhibitory effects towards C. purpurea. Selected Trichoderma strains were then tested for their potential to protect rye (Secale cereale) plants from ergot infection within a greenhouse trial. Rhizosphere-inoculated plants displayed a decreased disease severity compared to plants that had been sprayed with Trichoderma spores above-ground. Gene expression analysis by reverse transcription quantitative PCR also indicated the induction of systemic defense reactions. Overall, our data suggest that individual Trichoderma isolates possess a high antagonistic potential towards C. purpurea, which could be effective by direct mycoparasitism and by inducing systemic plant resistance, and therefore provide important guidance towards the development of Trichoderma treatments of cereals as biocontrol measure against ergot. [ABSTRACT FROM AUTHOR]

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In recent years, there has been an intensification of weather variability worldwide as a result of climate change. Some regions have been affected by drought, while others have experienced more intense rainfall. The incidence and severity of moldy grain and mycotoxin contamination during the growing and harvesting seasons have increased as a result of these weather conditions. Additionally, torrential rains and wet conditions may cause delays in grain drying, leading to mold growth in the field. In July 2023, a wheat field in Lecco (Lombardy, Italy) was affected by torrential rains that led to the development of the Claviceps fungi. In the field, dark sclerotia were identified on some ears. Wheat ears, kernels, and sclerotia were collected and analyzed by LC-MS/MS at IZSLER, Food Chemical Department, in Bologna. The wheat ears, kernels, and sclerotia were analyzed for 12 ergot alkaloids (EAs) according to (EU) Regulation 2023/915 (ergocornine/ergocorninine; ergocristine/ergocristinine; ergocryptine/ergocryptinine; ergometrine/ergometrinine; ergosine/ergosinine; ergotamine/ergotaminine), after QuEChERS (Z-Sep/C18) purification. The analyzed sclerotia showed significant differences in total alkaloid content that vary between 0.01 and 0.5% (w/w), according to the results of the 2017 EFSA scientific report. EAs detected in sclerotia were up to 4951 mg/kg, in wheat ears up to 33 mg/kg, and in kernels were 1 mg/kg. Additional mycotoxins, including ochratoxin A, deoxynivalenol, zearalenone, fumonisins, T2-HT2 toxins, and aflatoxins, were investigated in wheat kernels after purification with immunoaffinity columns (IAC). The analysis revealed the presence of deoxynivalenol in wheat kernels at a concentration of 2251 µg/kg. It is expected that climate change will increase the frequency of extreme weather events. In order to mitigate the potential risks associated with mycotoxin-producing fungi and to ensure the protection of human health, it is suggested that official controls be implemented in the field.

Velvet (VeA), a light-regulated protein that shuttles between the cytoplasm and the nucleus, serves as a key global regulator of secondary metabolism in various Aspergillus species and plays a pivotal role in controlling multiple developmental processes. The gene vepN was chosen for further investigation through CHIP-seq analysis due to significant alterations in its interaction with VeA under varying conditions. This gene (AFLA_006970) contains a Septin-type guanine nucleotide-binding (G) domain, which has not been previously reported in Aspergillus flavus (A. flavus). The functional role of vepN in A. flavus was elucidated through the creation of a gene knockout mutant and a gene overexpression strain using a well-established dual-crossover recombinational technique. A comparison between the wild type (WT) and the ΔvepN mutant revealed distinct differences in morphology, reproductive capacity, colonization efficiency, and aflatoxin production. The mutant displayed reduced growth rate; dispersion of conidial heads; impaired cell wall integrity; and decreased sclerotia formation, colonization capacity, and aflatoxin levels. Notably, ΔvepN exhibited complete growth inhibition under specific stress conditions, highlighting the essential role of vepN in A. flavus. This study provides evidence that vepN positively influences aflatoxin production, morphological development, and pathogenicity in A. flavus.

Members of the fungal genus Morchella are widely known for their important ecological roles and significant economic value. In this study, we used amplicon and genome sequencing to characterize bacterial communities associated with sexual fruiting bodies from wild specimens, as well as vegetative mycelium and sclerotia obtained from Morchella isolates grown in vitro. These investigations included diverse representatives from both Elata and Esculenta Morchella clades. Unique bacterial community compositions were observed across the various structures examined, both within and across individual Morchella isolates or specimens. However, specific bacterial taxa were frequently detected in association with certain structures, providing support for an associated core bacterial community. Bacteria from the genus Pseudomonas and Ralstonia constituted the core bacterial associates of Morchella mycelia and sclerotia, while other genera (e.g., Pedobacter spp., Deviosa spp., and Bradyrhizobium spp.) constituted the core bacterial community of fruiting bodies. Furthermore, the importance of Pseudomonas as a key member of the bacteriome was supported by the isolation of several Pseudomonas strains from mycelia during in vitro cultivation. Four of the six mycelial-derived Pseudomonas isolates shared 16S rDNA sequence identity with amplicon sequences recovered directly from the examined fungal structures. Distinct interaction phenotypes (antagonistic or neutral) were observed in confrontation assays between these bacteria and various Morchella isolates. Genome sequences obtained from these Pseudomonas isolates revealed intriguing differences in gene content and annotated functions, specifically with respect to toxin-antitoxin systems, cell adhesion, chitinases, and insecticidal toxins. These genetic differences correlated with the interaction phenotypes. This study provides evidence that Pseudomonas spp. are frequently associated with Morchella and these associations may greatly impact fungal physiology.

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. [ABSTRACT FROM AUTHOR]

One of the most harmful infections to chickpea plants, Sclerotinia sclerotiorum, causes stem rot and causes financial losses all over the world. Beneath the soil's surface, as sclerotia, the pathogen can live for a very long time alongside the detritus. As a result, the condition is extremely difficult to manage, has nearly no known treatments, and only a few types have been shown to be somewhat successful against the infection. Thus, the primary goal of the research is to investigate the effects of a variety of treatments, including Rhizobium leguminosarum + Pseudomonas fluorescens combination, Trichoderma viride, Saaf (Carbendazim 12%+ Mancozeb 63% WP), Vitavax Power (Carboxin 37.5%+ Thiram 37.5%), Hexaconazole, and Rhizobium leguminosarum + Pseudomonas fluorescens, to investigate their impact on S. sclerotiorum suppression. While maintaining sustainability, bioagents improve the soil's properties and productivity. Fungicides have been found to be effective when bio-agents are not able to control a disease. Therefore, in this investigation, both methods of treatment were used. The results of an experiment conducted in both in vitro and in vivo conditions showed that chemical treatment with Saaf fungicide and seed treatment with T. viride were highly efficient against S. sclerotiorum. [ABSTRACT FROM AUTHOR]

In a previous study, the endophytic Bacillus velezensis NC318 was isolated from the rhizosphere of date palm and showed strong antifungal activity against the soil-borne plant pathogenic fungus, Sclerotium rolfsii Sacc, the causal agent of Southern blight. The potential of the Bacillus genus in the inhibition of plant pathogens is mainly due to the production of certain bioactive compounds. In the present study, secondary metabolites extracted from the cell-free supernatant of strain NC318 showed strong antifungal activity on the mycelial growth and germination of S. rolfsii sclerotia in vitro. With 50 μl of bioactive compounds crude extracts, the mycelial growth inhibition rate was 97% and any germination of sclerotia was reported. Chemical analysis of the secondary metabolite crude extracts performed by high performance liquid chromatography coupled with mass spectrometry (HPLC/MS), revealed that the secreted bioactive compounds belonged to the family of lipopeptides (iturin, fengycin, surfactin), polyketides (bacillaene, macrolactin, difficidin and bacilysin) and siderophores (bacillibactin). These results provide a better understanding of the biocontrol mechanism of the bacteria strain B. velezensis NC318 against the soil fungal pathogens, especially S. rolfsii root rot.

Management of Sclerotinia stem rot (SSR) disease in Brassica napus is heavily reliant on prophylactic fungicide applications at flowering, which often provides inconsistent control depending on timing of ascospore release in the field and environmental conditions. Understanding host response to Sclerotinia sclerotiorum infection is essential for sustainable disease management in the future. This study determined host response of nine B. napus varieties to four aggressive S. sclerotiorum isolates across two years by measuring four disease variables: area under the disease progress stairs (AUDPS), seed production, sclerotia number and average sclerotia weight. Brassica napus varieties varied greatly in their response to the four measured variables, with varieties producing the highest AUDPS not being the same varieties that had the lowest seed production, the highest numbers of sclerotia or heaviest sclerotia. Repeating the experiment over two years using the same varieties and isolates identified the impact of environment on measured disease variables as the most influential factor, highlighting the complexity of disease responses to diverse isolates and host genotypes under different environments. It was recommended that both long-term (such as inoculum production) and short-term (such as seed production) disease outcomes be combined with lesion length measurement (i.e., AUDPS) for future host screening studies. [ABSTRACT FROM AUTHOR]

Background: Sclerotium rolfsii, the causal agent of peanut southern blight, has become increasingly prevalent and harmful in China, causing serious economic losses to the peanut industry. To effectively manage peanut southern blight, this study evaluated the bioactivity of the new‐generation sterol demethylation inhibitor (DMI) fungicide mefentrifluconazole against peanut S. rolfsii. Results: In this study, the DMI fungicide mefentrifluconazole exhibited excellent inhibitory activity against the mycelial growth of S. rolfsii, with a mean EC50 value of 0.21 ± 0.11 mg L−1 and a range of 0.02 to 0.55 mg L−1 for 261 isolates collected from Hebei, Henan and Shandong provinces. Mefentrifluconazole significantly reduced the biomass of mycelia and affected the morphology of hyphae. Although sclerotia were more tolerant to mefentrifluconazole than mycelial growth, mefentrifluconazole greatly inhibited the formation and germination of sclerotia. In addition, sclerotia produced by mefentrifluconazole‐treated mycelia were deficient in nutrients (e.g., protein, carbohydrate and lipid). These results indicated that mefentrifluconazole may reduce the population of S. rolfsii in the following year. In greenhouse experiments, mefentrifluconazole showed control efficacy and good persistence against peanut S. rolfsii. The preventative and curative activities of mefentrifluconazole at 200 mg L−1 against southern blight still reached 95.36% and 60.94%, respectively, after 9 days of application. No correlation was observed for the sensitivity of S. rolfsii to mefentrifluconazole and the tested DMI, quinone outside inhibitor and succinate dehydrogenase inhibitor fungicides. Conclusion: All data indicated that mefentrifluconazole could provide favorable control efficacy against S. rolfsii from peanuts and reduce the infection and population of S. rolfsii in the following year. © 2023 Society of Chemical Industry. [ABSTRACT FROM AUTHOR]

Identifying the sources of fungal inoculum that induces epidemics is instrumental to managing crop health in a more efficient way by implementing prophylactic methods and by using better targeted biocontrol and fungicide applications. For some phytopathogenic fungi, this identification is hampered by the multiplicity of inoculum types. This is the case for Sclerotinia sclerotiorum, the agent of white mould, which can produce ascospores disseminated via the atmosphere, sclerotia stored in the soil and mycelium in plant debris of a large variety of hosts. The present study aims to assess the relative importance of airborne and soilborne inocula in white mould disease development and its persistence across two crop seasons. S. sclerotiorum isolates were collected from air, soil and carrot plants during two crop seasons in south‐western France and genotyped with 16 microsatellite markers. Among the 490 isolates, 162 clonal lineages were identified. The genetic characteristics did not differ significantly among isolates collected from air, soil and carrots. The epidemics of white mould on carrots were initiated either by airborne inoculum or by soilborne inoculum. It also appears that some isolates from airborne inoculum persisted from year to year. [ABSTRACT FROM AUTHOR]

The effect of steam thermotherapy on Botrytis spp. populations in strawberry transplants was evaluated. Tray plants rooted in 0.2 L peat plugs of seasonal flowering cvs. Falco, Sonsation, and Soprano, and everbearing cvs. Favori and Murano were pre-treated with steam at 37 °C for 1 h, followed by 1 h at ambient temperature and air humidity, and then 2 or 4 h steam treatment at 44 °C. Except for one cultivar with a slight reduction in yield, there were no negative effects on plant performance. Compared to untreated transplants, mean incidence of Botrytis on the five cultivars was reduced by 43 and 86% with the 2 and 4 h treatments, respectively. Within cultivars the reduction was significant in 2 and 3 experiments following the 2 and 4 h treatments, respectively. Sclerotia from four different isolates of Botrytis were subjected to treatment including 4 h of steam thermotherapy and subsequently tested for viability. Following 14 days of incubation, 90 to 100% (mean 97%) of treated sclerotia failed to produce mycelial growth compared with untreated sclerotia, which all germinated and produced mycelia. Botrytis isolates recovered from both treated and untreated strawberry transplants were tested for resistance to seven fungicides, including boscalid, fenhexamid, fludioxonil, fluopyram, pyraclostrobin, pyrimethanil and thiophanate-methyl. Multiple fungicide resistance was common; 35.5% of isolates were resistant to fungicides from at least three FRAC groups. Results indicate that steam thermotherapy treatment strongly reduces populations of Botrytis spp., including fungicide-resistant strains, in strawberry transplants with negligible negative impacts on the transplants. [ABSTRACT FROM AUTHOR]

APSES (Asm1p, Phd1p, Sok2p, Efg1p, and StuAp) family transcription factors play crucial roles in various biological processes of fungi, however, their functional characterization in phytopathogenic fungi is limited. In this study, we explored the role of SsStuA, a typical APSES transcription factor, in the regulation of cell wall integrity (CWI), sclerotia formation and pathogenicity of Sclerotinia sclerotiorum, which is a globally important plant pathogenic fungus. A deficiency of SsStuA led to abnormal phosphorylation level of SsSmk3, the key gene SsAGM1 for UDP-GlcNAc synthesis was unable to respond to cell wall stress, and decreased tolerance to tebuconazole. In addition, ΔSsStuA was unable to form sclerotia but produced more compound appressoria. Nevertheless, the virulence of ΔSsStuA was significantly reduced due to the deficiency of the invasive hyphal growth and increased susceptibility to hydrogen peroxide. We also revealed that SsStuA could bind to the promoter of catalase family genes which regulate the expression of catalase genes. Furthermore, the level of reactive oxygen species (ROS) accumulation was found to be increased in ΔSsStuA. In summary, SsStuA, as a core transcription factor involved in the CWI pathway and ROS response, is required for vegetative growth, sclerotia formation, fungicide tolerance and the full virulence of S. sclerotiorum.

AbstractWhite mold (or Sclerotinia stem rot), caused by Sclerotinia species, is a major air, soil, or seed-transmitted disease affecting numerous crops and wild plants. Microscopic or culture-based methods currently available for their detection and identification are time-consuming, laborious, and often erroneous. Therefore, we developed a multiplex quantitative PCR (qPCR) assay for the discrimination, detection, and quantification of DNA collected from each of the three economically relevant Sclerotinia species, namely, S. sclerotiorum, S. minor, and S. nivalis. TaqMan primer/probe combinations specific for each Sclerotinia species were designed based on the gene sequences encoding aspartyl protease. High specificity and sensitivity of each probe were confirmed for sclerotium and soil samples, as well as pure cultures, using simplex and multiplex qPCRs. This multiplex assay could be helpful in detecting and quantifying specific species of Sclerotinia, and therefore, may be valuable for disease diagnosis, forecasting, and management.

Abstract Rice sheath blight pathogen, Rhizoctonia solani, produces numerous sclerotia to overwinter. As a rich source of nutrients in the soil, sclerotia may lead to the change of soil microbiota. For this purpose, we amended the sclerotia of R. solani in soil and analyzed the changes in bacterial microbiota within the soil at different time points. At the phyla level, Proteobacteria, Acidobacteria, Bacteroidetes, Actinobacteria, Chloroflexi and Firmicutes showed varied abundance in the amended soil samples compared to those in the control. An increased abundance of ammonia-oxidizing bacterium (AOB) Nitrosospira and Nitrite oxidizing bacteria (NOB) i.e., Nitrospira was observed, where the latter is reportedly involved in the nitrifier denitrification. Moreover, Thiobacillus, Gemmatimonas, Anaeromyxobacter and Geobacter, the vital players in denitrification, N2O reduction and reductive nitrogen transformation, respectively, depicted enhanced abundance in R. solani sclerotia-amended samples. Furthermore, asymbiotic nitrogen-fixing bacteria, notably, Azotobacter as well as Microvirga and Phenylobacterium with nitrogen-fixing potential also enriched in the amended samples compared to the control. Plant growth promoting bacteria, such as Kribbella, Chitinophaga and Flavisolibacter also enriched in the sclerotia-amended soil. As per our knowledge, this study is of its kind where pathogenic fungal sclerotia activated microbes with a potential role in N transformation and provided clues about the ecological functions of R. solani sclerotia on the stimulation of bacterial genera involved in different processes of N-cycle within the soil in the absence of host plants.

Athelia rolfsii is one of the most destructive and aggressive fungal pathogens worldwide and causes southern blight disease of lettuce. A nonthermal atmospheric-pressure dielectric barrier discharge (DBD) plasma has attracted interest as an alternative control method to chemical usage because of its antimicrobial activity. Exposure of A. rolfsii to DBD plasma for 5, 10, 15, and 20 min resulted in in vitro fungal inhibition of mycelial discs and sclerotia. The results showed that DBD plasma exposure for 10 min completely inhibited fungal growth of mycelial discs, whereas exposure for over 20 min was required to inhibit the hyphal growth of sclerotia. Scanning electron microscopy (SEM) observations of mycelia and sclerotia abnormalities revealed laceration and damage of both mycelia and sclerotia. In addition, disease incidence and severity were reduced in mycelial and sclerotia inoculation following DBD plasma exposure for 15 and 20 min, respectively, compared with the positive control. In conclusion, the DBD plasma demonstrates antifungal activity against A. rolfsii via inhibition of fungal growth and reduction in disease incidence and severity. Therefore, DBD plasma has the potential to be applied in controlling southern blight disease of lettuce. [ABSTRACT FROM AUTHOR]

Clonostachys rosea, an ascomycetous, omnipresent, cellulose-decaying soil fungus, has been reported to be a well-known mycoparasitic biological control agent. In this study, we isolated C. rosea, a mycoparasitic fungus for the first time in India from sclerotia of the notorious plant pathogen Sclerotinia sclerotiorum, causing head rot disease in cabbage. A total of five mycoparasitic fungi were isolated from the sclerotial bodies of S. sclerotiorum (TNAU-CR 01, 02, 03, 04 and 05). All the isolates were tested under morpho-molecular characterization. Among them, TNAU-CR 02 showed the greatest mycelial inhibition of 79.63% over the control. Similarly, the SEM imaging of effective C. rosea isolates indicated the presence of numerous conidia destroying the outer cortex layers of sclerotia. Metabolite fingerprinting of C. rosea TNAU-CR 02 identified 18 chemical compounds using GC-MS analysis. The crude antibiotics of C. rosea TNAU-CR 02 were verified for their antifungal activity against S. sclerotiorum and the results revealed 97.17% mycelial inhibition compared with the control. Similarly, foliar application of TNAU-CR 02 at 5 mL/litre on 30, 45 and 60 days after transplanting showed the lowest disease incidence of 15.1 PDI compared to the control. This discovery expands our understanding of the biology and the dissemination of C. rosea, providing a way for the exploitation of C. rosea against cabbage head rot pathogens. [ABSTRACT FROM AUTHOR]

Sclerotium cepivorum Berk is the etiological agent of white rot disease that affects plants of the genus Allium. This fungus produces resistance structures called sclerotia that are formed by a rolled mycelium with a thick layer of melanin and it can remain dormant for many years in the soil. Current interest in S. cepivorum has arisen from economic losses in Allium crops in the agricultural sector. Melanin is a component that protects the sclerotia from adverse environmental conditions In many organisms, it plays an important role in the infectious process; in S. cepivorum, the pathway by which this component is synthetized is not fully described. By using infrared spectrophotometric assays applied direct to the sclerotia and a melanin extract followed by an NMR analysis and a tricyclazole melanin inhibition experiment, it allowed us to determine the dihydroxynaphthalene (DHN)-melanin pathway by which S. cepivorum performs its melanin synthesis. Moreover, we focused on studying scytalone dehydratase (SDH) as a key enzyme of the DHN-melanin synthesis. We obtained the recombinant SDH enzyme and tested its activity by a zymogram assay. Thereby, the S. cepivorum melanogenic route was established as a DHN pathway.

ABSTRACT The filamentous fungus Aspergillus flavus causes devastating diseases not only to cash crops but also to humans by secreting a series of secondary metabolites called aflatoxins. In the cotranslational or posttranslational process, N-myristoyltransferase (Nmt) is a crucial enzyme that catalyzes the myristate group from myristoyl-coenzyme A (myristoyl-CoA) to the N terminus or internal glycine residue of a protein by forming a covalent bond. Members of the Nmt family execute a diverse range of biological functions across a broad range of fungi. However, the underlying mechanism of AflNmt action in the A. flavus life cycle is unclear, particularly during the growth, development, and secondary metabolic synthesis stages. In the present study, AlfNmt was found to be essential for the development of spore and sclerotia, based on the regulation of the xylose-inducible promoter. AflNmt, located in the cytoplasm of A. flavus, is also involved in modulating aflatoxin (AFB1) in A. flavus, which has not previously been reported in Aspergillus spp. In addition, we purified, characterized, and crystallized the recombinant AflNmt protein (rAflNmt) from the Escherichia coli expression system. Interestingly, the crystal structure of rAlfNmt is moderately different from the models predicted by AlphaFold2 in the N-terminal region, indicating the limitations of machine-learning prediction. In conclusion, these results provide a molecular basis for the functional role of AflNmt in A. flavus and structural insights concerning protein prediction. IMPORTANCE As an opportunistic pathogen, A. flavus causes crop loss due to fungal growth and mycotoxin contamination. Investigating the role of virulence factors during infection and searching for novel drug targets have been popular scientific topics in the field of fungal control. Nmt has become a potential target in some organisms. However, whether Nmt is involved in the developmental stages of A. flavus and aflatoxin synthesis, and whether AlfNmt is an ideal target for structure-based drug design, remains unclear. This study systematically explored and identified the role of AlfNmt in the development of spore and sclerotia, especially in aflatoxin biosynthesis. Moreover, although there is not much difference between the AflNmt model predicted using the AlphaFold2 technique and the structure determined using the X-ray method, current AI prediction models may not be suitable for structure-based drug development. There is still room for further improvements in protein prediction.

Sclerotium rolfsii is one of the most challenging pathogens among fungal phytopathogens. Botanicals and biopesticides projected as alternatives to the synthetic fungicides also don't serve the purpose for S. rolfsii control pertaining to its vast host range and high adaptability. Present project therefore, focused on the preparation of Silver nanoemulsion (AgNE) having optimum stability and bestowed with significant antifungal properties and growth promoting abilities for the mungbean plants. The influence of different AgNE concentrations on growth pattern of S. rolfsii was tested under in vitro and in vivo conditions. Surprisingly, 100 ppm of AgNE could culminate the fungal progression completely by inhibiting both hyphal as well as sclerotial growth in plate and broth assays. Results of in planta application of AgNE at100 ppm on mungbean plants also coincided with the in vitro findings in terms of controlling the pathogen progression; however the internalization of Ag ions was observed in root, stem and leaves. Toxicity indications were apparent in the form of stunted and broadened root growth at 100 ppm AgNE concentration, but the above ground plant parts appeared perfectly normal. In inference, 50 ppm AgNE was found to be the most significant and safe concentration in relation to the phytopathogen control and plant growth promotion. These results revealed the evolving role of AgNE as a promising nanofungicide and nanofertilizer to accelerate overall mungbean productivity. Awaiting complications lies in the stability improvement of AgNE, scaled up production, phytotoxicity studies and their possible applications in several crops and multilocational field trials. [ABSTRACT FROM AUTHOR]

Sclerotinia sclerotiorum is a necrotrophic phytopathogenic fungus that produces sclerotia. Sclerotia are essential components of the survival and disease cycle of this devastating pathogen. In this study, we analyzed comparative transcriptomics of hyphae and sclerotia. A total of 1959 differentially expressed genes, 919 down-regulated and 1040 up-regulated, were identified. Transcriptomes data provide the possibility to precisely comprehend the sclerotia development. We further analyzed the differentially expressed genes (DEGs) in sclerotia to explore the molecular mechanism of sclerotia development, which include ribosome biogenesis and translation, melanin biosynthesis, autophagy and reactivate oxygen metabolism. Among these, the autophagy-related gene SsAtg1 was up-regulated in sclerotia. Atg1 homologs play critical roles in autophagy, a ubiquitous and evolutionarily highly conserved cellular mechanism for turnover of intracellular materials in eukaryotes. Therefore, we investigated the function of SsAtg1 to explore the function of the autophagy pathway in S. sclerotiorum. Deficiency of SsAtg1 inhibited autophagosome accumulation in the vacuoles of nitrogen-starved cells. Notably, ΔSsAtg1 was unable to form sclerotia and displayed defects in vegetative growth under conditions of nutrient restriction. Furthermore, the development and penetration of the compound appressoria in ΔSsAtg1 was abnormal. Pathogenicity analysis showed that SsAtg1 was required for full virulence of S. sclerotiorum. Taken together, these results indicate that SsAtg1 is a core autophagy-related gene that has vital functions in nutrient utilization, sclerotia development and pathogenicity in S. sclerotiorum. [ABSTRACT FROM AUTHOR]

White mold (or Sclerotinia stem rot), caused by Sclerotinia species, is a major air, soil, or seed-transmitted disease affecting numerous crops and wild plants. Microscopic or culture-based methods currently available for their detection and identification are time-consuming, laborious, and often erroneous. Therefore, we developed a multiplex quantitative PCR (qPCR) assay for the discrimination, detection, and quantification of DNA collected from each of the three economically relevant Sclerotinia species, namely, S. sclerotiorum, S. minor, and S. nivalis. TaqMan primer/probe combinations specific for each Sclerotinia species were designed based on the gene sequences encoding aspartyl protease. High specificity and sensitivity of each probe were confirmed for sclerotium and soil samples, as well as pure cultures, using simplex and multiplex qPCRs. This multiplex assay could be helpful in detecting and quantifying specific species of Sclerotinia, and therefore, may be valuable for disease diagnosis, forecasting, and management. [ABSTRACT FROM AUTHOR]

In a previous study, the endophytic Bacillus velezensis NC318 was isolated from the rhizosphere of date palm and showed strong antifungal activity against the soil-borne plant pathogenic fungus, Sclerotium rolfsii Sacc, the causal agent of Southern blight. The potential of the Bacillus genus in the inhibition of plant pathogens is mainly due to the production of certain bioactive compounds. In the present study, secondary metabolites extracted from the cell-free supernatant of strain NC318 showed strong antifungal activity on the mycelial growth and germination of S. rolfsii sclerotia in vitro. With 50 µl of bioactive compounds crude extracts, the mycelial growth inhibition rate was 97% and any germination of sclerotia was reported. Chemical analysis of the secondary metabolite crude extracts performed by high performance liquid chromatography coupled with mass spectrometry (HPLC/MS), revealed that the secreted bioactive compounds belonged to the family of lipopeptides (iturin, fengycin, surfactin), polyketides (bacillaene, macrolactin, difficidin and bacilysin) and siderophores (bacillibactin). These results provide a better understanding of the biocontrol mechanism of the bacteria strain B. velezensis NC318 against the soil fungal pathogens, especially S. rolfsii root rot. [ABSTRACT FROM AUTHOR]