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Sterol regulatory element-binding proteins (SREBPs) are transcription factors governing various biological processes in fungi, including virulence and fungicide tolerance, by regulating ergosterol biosynthesis and homeostasis. While studied in model fungal species, their role in fungal species used for biocontrol remains elusive. This study delves into the biological and regulatory function of SREBPs in the fungal biocontrol agent (BCA) Clonostachys rosea IK726, with a specific focus on fungicide tolerance and antagonism. Clonostachys rosea genome contains two SREBP coding genes (sre1 and sre2) with distinct characteristics. Deletion of sre1 resulted in mutant strains with pleiotropic phenotypes, including reduced C. rosea growth on medium supplemented with prothioconazole and boscalid fungicides, hypoxia mimicking agent CoCl 2 and cell wall stressor SDS, and altered antagonistic abilities against Botrytis cinerea and Rhizoctonia solani. However, Δsre2 strains showed no significant effect. Consistent with the gene deletion results, overexpression of sre1 in Saccharomyces cerevisiae enhanced tolerance to prothioconazole. The functional differentiation between SRE1 and SRE2 was elucidated by the yeast-two-hybridization assay, which showed an interaction between SREBP cleavage-activating protein (SCAP) and SRE1 but not between SRE2 and SCAP. Transcriptome analysis of the Δsre1 strain unveiled SRE1-mediated expression regulation of genes involved in lipid metabolism, respiration, and xenobiotic tolerance. Notably, genes coding for antimicrobial compounds chitinases and polyketide synthases were downregulated, aligning with the altered antagonism phenotype. This study uncovers the role of SREBPs in fungal BCAs, providing insights for C. rosea IK726 application into integrated pest management strategies., (Copyright © 2024 The Authors. Published by Elsevier GmbH.. All rights reserved.)

Gray mold caused by Botrytis spp. is a common disease on various hosts, including strawberries. In this study, we obtained 59 Botrytis isolates from strawberries from greenhouses in Zhejiang Province, China. Identification of the sampled isolates at species level was performed by a duplex PCR assay method, the result showed that, in Zhejiang, gray mold on strawberry fruits is caused by a complex of Botrytis groups including B. cinerea group N (47.5 %) and B. cinerea group S (52.5 %). The sensitivities of all Botrytis isolates to pyrimethanil were determined based on discriminatory dose method on L-asparagine-based agar medium plate. Our results showed that the isolates obtained from the greenhouses with continuous use of pyrimethanil developed severe resistance to pyrimethanil, and the resistance frequencies of B. cinerea group N and group S isolates were 89.3 % and 41.9 %, respectively. By sequencing, four different resistance-related point mutations were identified in 38 Botrytis isolates: Bcpos5 L412F (16 isolates, 42.1 %), Bcpos5 L412V (14 isolates, 36.8 %), Bcmdl1 E407K (2 isolates, 5.3 %), and Bcpos5 L412S ⸱Bcmdl1 E407K (1 isolate, 2.6 %). The exogenous addition of methionine could not completely alter the resistance of Botrytis isolates to pyrimethanil. In this study, the pyrimethanil resistance in Botrytis isolates was steadily inherited, and compared to the pyrimethanil-sensitive isolates, the resistant mutants exhibited good fitness in sporulation capacity, spore germination rate, and virulence on strawberries. In conclusion, our results provided a description of the genetic structure of Botrytis groups complex on strawberry fruits and reminded growers to focus on the stable pyrimethanil resistance in Botrytis isolates, caused by point mutations in BcPos5 and BcMdl1., Competing Interests: Declaration of competing interest The authors declare no conflicts of interest., (Copyright © 2024. Published by Elsevier Inc.)

Background: Gray mold caused by Botrytis cinerea is one of the most serious diseases affecting strawberry. Succinate dehydrogenase inhibitor (SDHI) fungicides have been used for more than a decade to control the disease. Monitoring resistance and improving in-depth understanding of resistance mechanisms are essential for the control of B. cinerea., Results: In this study, resistance monitoring of a SDHI fungicide boscalid was conducted on B. cinerea isolated from strawberries in Korea during 2020 and 2021, with resistance rates of 76.92% and 72.25%, respectively. In resistant strains, mutations P225F/H and H272R were found in SdhB, with P225F representing the dominant mutation type. Simultaneous mutations G85A, I93V, M158V, and V168I in SdhC were detected in 54.84% of sensitive strains. Sensitivity profiles of different Sdh genotypes of B. cinerea strains to six SDHIs were determined in vitro and in vivo. In addition, the mutation(s) were genetically validated through in situ SdhB (SdhC) expression. Docking assays between SDHIs and AlphaFold model-based Sdh complexes revealed generally consistent patterns with their in vitro phenotypes., Conclusion: Resistance of B. cinerea to SDHI fungicide on strawberry was systematically investigated in this study. Deciphering of SDHI resistance through the genetic transformation system and AlphaFold model-based molecular docking will provide valuable insights into other target site-based fungicide resistance in fungal pathogens. © 2024 The Author(s). Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry., (© 2024 The Author(s). Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.)

Background: Grapes are highly valued for their nutritional and economic benefits, and have been widely studied for their biological attributes such as fruit development, quality formation, and stress resistance. One significant threat to grape quality is gray mold, caused by Botrytis cinerea, which can infect the flowers, fruits, leaves, and stems. The quantitative real-time PCR (qRT-PCR), known for its high sensitivity and quantitative accuracy, is an essential tool for analyzing gene expression related to the pathogenesis of gray mold, thereby providing deeper insights into the disease., Result: In this study, we aim to identify stable internal reference genes crucial for accurate gene expression analysis via qRT-PCR. Utilizing transcriptome data from grapes under various disease stresses, we identified twelve candidate reference genes with consistently high expression levels. The stability of these genes was assessed through delta-CT, geNorm, NormFinder, BestKeeper, and RefFinder analyses after establishing the cycling thresholds (Ct) in different grape varieties treated with Botrytis cinerea., Conclusions: Our findings reveal that VIT-17s0000g02750 and VIT-06s0004g04280 exhibit stable expression and are suitable as new reference genes. This foundational work supports further research into the molecular mechanisms of grape biological processes., (© 2024. The Author(s).)

Background: Botrytis cinerea is a broad-host-range pathogen causing gray mold disease and significant yield losses of numerous crops. However, the mechanisms underlying its rapid invasion and efficient killing of plant cells remain unclear., Results: In this study, we elucidated the dynamics of B. cinerea infection in Arabidopsis thaliana by live cell imaging and dual RNA sequencing. We found extensive transcriptional reprogramming events in both the pathogen and the host, which involved metabolic pathways, signaling cascades, and transcriptional regulation. For the pathogen, we identified 591 candidate effector proteins (CEPs) and comprehensively analyzed their co-expression, sequence similarity, and structural conservation. The results revealed temporal co-regulation patterns of these CEPs, indicating coordinated deployment of effectors during B. cinerea infection. Through functional screening of 48 selected CEPs in Nicotiana benthamiana, we identified 11 cell death-inducing proteins (CDIPs) in B. cinerea., Conclusions: The findings provide important insights into the transcriptional dynamics and effector biology driving B. cinerea pathogenesis. The rapid infection of this pathogen involves the temporal co-regulation of CEPs and the prominent role of CDIPs in host cell death. This work highlights significant changes in gene expression associated with gray mold disease, underscoring the importance of a diverse repertoire of effectors crucial for successful infection., (© 2024. The Author(s).)

Objectives: Botrytis cinerea, the causal agent of gray mold, is a necrotrophic fungus that can infect a wide variety of plant species and plant tissues. During infection, this pathogen modulates the pH of its environment by secreting organic acids or ammonia. Deletion of the gene encoding the pH-responsive transcription factor PacC revealed the importance of this regulator in different steps of the infection process and particularly in the secretion of organics acids, reactive oxygen species and plant cell wall degrading enzymes. This study aimed to identify the genes controlled by this fungus-specific transcription factor when the fungus is placed under acidic or neutral conditions., Data Description: Botrytis cinerea B05.10 and the knock-out BcpacC mutant strains were grown on solid non-buffered medium for 3 days on the surface of cellophane membranes before transfer for 4 h onto the surface of liquid medium buffered at pH 5.0 or 7.0 followed by mycelium collection. After RNA sequencing, differentially expressed genes according to strains or pH conditions were listed. These data will be useful in understanding the adaptation process of B cinerea during plant infection., (© 2024. The Author(s).)

One of the best-known Hungarian products on world wine market is Aszú, which belongs to the family of Tokaj wine specialties and is made from aszú berries. An important condition for the formation of aszú berries is the noble rot of technologically mature grapes, which is caused by Botrytis cinerea. At the same time botrytized sweet wines are produced not only in Hungary, but in many locations of wine-producing areas of Europe as well as in certain wine growing regions of other continents. The determination of botrytization is mostly based on sensory evaluations, which is a highly subjective procedure and largely depends on the training and experience of the evaluator. Currently, the classification of aszú berries (class I and class II) is based only on visual inspection and determination of sugar content. Based on these facts the primary goal of our work was to develop a qPCR assay capable for objective rating and classification of aszú berries. The developed qPCR is highly specific and sensitive as can clearly distinguish between B. cinerea and other filamentous fungi and yeast species occur on grapes. Moreover, it is suitable for categorizing berries colonized by B. cinerea to varying degrees. Thus, the developed qPCR method can be a useful technique for classification of the grape berries into four quality groups: healthy, semi-shrivelled, Aszú Class II and Aszú Class I., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Zn(II)2Cys6 proteins constitute the largest group of fungal-specific transcription factors. However, little is known about their functions in the crop killer Botrytis cinerea . In this work, a T-DNA insertion strain M13448 was identified which was inserted into the Zn(II)2Cys6 TF-encoding gene BcTBS1 . Knockout of BcTBS1 did not affect mycelia growth, appressorium formation, and sclerotium germination, but impaired fungal conidiation, conidial morphogenesis, conidial germination, infection cushion development, and sclerotial formation. Accordingly, Δ Bctbs1 mutants showed reduced virulence in its host plants. Further study proved that BcTBS1 , BCIN_15g03870 , and BCIN_12g06630 were induced by cellulose. Subsequent cellulase activity assays revealed that the loss of BcTBS1 significantly decreased cellulase activity. In addition, we verified that the BCIN_15g03870 and BCIN_12g06630 genes were positive regulated by BcTBS1 by quantitative real-time reverse-transcription-polymerase chain reaction (qRT-PCR). Taken together, these results suggested that BcTBS1 can promote pathogenicity by modulating cellulase-encoding genes that participate in host cellulose degradation.

Mycoviruses are highly genetically diverse and can significantly change their fungal host's phenotype, yet they are generally under-described in genotypic and biological studies. We propose Botrytis cinerea as a model mycovirus system in which to develop a deeper understanding of mycovirus epidemiology including diversity, impact, and the associated cellular biology of the host and virus interaction. Over 100 mycoviruses have been described in this fungal host. B. cinerea is an ideal model fungus for mycovirology as it has highly tractable characteristics-it is easy to culture, has a worldwide distribution, infects a wide range of host plants, can be transformed and gene-edited, and has an existing depth of biological resources including annotated genomes, transcriptomes, and isolates with gene knockouts. Focusing on a model system for mycoviruses will enable the research community to address deep research questions that cannot be answered in a non-systematic manner. Since B. cinerea is a major plant pathogen, new insights may have immediate utility as well as creating new knowledge that complements and extends the knowledge of mycovirus interactions in other fungi, alone or with their respective plant hosts. In this review, we set out some of the critical steps required to develop B. cinerea as a model mycovirus system and how this may be used in the future.

Botrytis cinerea is a notorious pathogen causing pre- and post-harvest spoilage in many economically important crops. Excessive application of site-specific fungicides to control the pathogen has led to the selection of strains possessing target site alterations associated with resistance to these fungicides and/or strains overexpressing efflux transporters associated with multidrug resistance (MDR). MDR in B. cinerea has been correlated with the overexpression of atrB and mfsM2, encoding an ATP-binding cassette (ABC) and a major facilitator superfamily (MFS) transporter, respectively. However, it remains unknown whether other transporters may also contribute to the MDR phenotype. In the current study, the transcriptome of a B. cinerea multidrug-resistant (MDR) field strain was analysed upon exposure to the fungicide fludioxonil, and compared to the B05.10 reference strain. The transcriptome of this field strain displayed significant differences as compared to B05.10, including genes involved in sugar membrane transport, toxin production and virulence. Among the induced genes in the field strain, even before exposure to fludioxonil, were several putatively encoding ABC and MFS transmembrane transporters. Overexpression of a highly induced MFS transporter gene in the B05.10 strain led to an increased tolerance to the fungicides fluopyram and boscalid, indicating an involvement in efflux transport of these compounds. Overall, the data from this study give insights towards better understanding the molecular mechanisms involved in MDR and fitness cost, contributing to the development of more efficient control strategies against this pathogen., (© 2024 The Author(s). Molecular Plant Pathology published by British Society for Plant Pathology and John Wiley & Sons Ltd.)

Botrytis cinerea is a destructive necrotrophic phytopathogen causing overwhelming diseases in more than 1400 plant species, especially fruit crops, resulting in significant economic losses worldwide. The pathogen causes rotting of fruits at both pre-harvest and postharvest stages. Aside from causing gray mold of the mature fruits, the fungus infects leaves, flowers, and seeds, which makes it a notorious phytopathogen. Worldwide, in the majority of fruit crops, B. cinerea causes gray mold. In order to effectively control this pathogen, extensive research has been conducted due to its wide host range and the huge economic losses it causes. It is advantageous to explore detection and diagnosis techniques of B. cinerea to provide the fundamental basis for mitigation strategies. Botrytis cinerea has been identified and quantified in fruit/plant samples at pre- and post-infection levels using various detection techniques including DNA markers, volatile organic compounds, qPCR, chip-digital PCR, and PCR-based nucleic acid sensors. In addition, cultural, physical, chemical, biological, and botanical methods have all been used to combat Botrytis fruit rot. This review discusses research progress made on estimating economic losses, detection and diagnosis, as well as management strategies, including cultural, physical, chemical, and biological studies on B. cinerea along with knowledge gaps and potential areas for future research., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Background: Fludioxonil is a fungicide used to control gray mold. However, the frequency of resistance in the field is low, and highly resistant strains are rarely isolated. The biological fitness of the resistant strain is lower than that of the wild strain. Therefore, the molecular mechanism underlying the decrease in the fitness of the fludioxonil-resistant strain of Botrytis cinerea was explored to provide a theoretical basis for resistance monitoring and management., Results: Transcriptome analysis was performed on five different-point mutant resistant strains of fludioxonil, focusing on mining and screening candidate genes that lead to reduced fitness of the resistant strains and the functional verification of these genes. The differentially expressed genes (DEGs) of the five point-mutation resistant strains intersected with 1869 DEGs. Enrichment analysis showed that three downregulated genes (Bcin05g07030, Bcgad1, and Bcin03g05840) were enriched in multiple metabolic pathways and were downregulated in both domesticated strains. Bcin05g07030 and Bcin03g05840 were involved in mycelial growth and development, pathogenicity, and conidial yield, and negatively regulated oxidative stress and cell wall synthesis. Bcgad1 was involved in mycelial growth and development, conidial yield, oxidative stress, and cell wall synthesis. Furthermore, Bcin05g07030 was involved in osmotic stress and spore germination, whereas Bcin03g05840 and Bcgad1 negatively regulated osmotic stress and cell wall integrity., Conclusion: These results enable us to further understand the molecular mechanism underlying the decrease in the biological fitness of B. cinerea fludioxonil-resistant strains. © 2024 Society of Chemical Industry., (© 2024 Society of Chemical Industry.)

The Zn(II) 2 Cys 6 zinc cluster protein family comprises a subclass of zinc-finger proteins that serve as transcriptional regulators involved in a diverse array of fugal biological processes. However, the roles and mechanisms of the Zn(II) 2 Cys 6 transcription factors in mediating Botrytis cinerea , a necrotrophic fungus that causes gray mold in over 1000 plant species, development and virulence remain obscure. Here, we demonstrate that a novel B. cinerea pathogenicity-associated factor BcFTG1 ( f ungal t ranscription factor containing the G AL4 domain), identified from a virulence-attenuated mutant M20162 from a B. cinerea T-DNA insertion mutant library, plays an important role in oxalic acid (OA) secretion, carbon source absorption and cell wall integrity. Loss of BcFTG1 compromises the ability of the pathogen to secrete OA, absorb carbon sources, maintain cell wall integrity, and promote virulence. Our findings provide novel insights into fungal factors mediating the pathogenesis of the gray mold fungus via regulation of OA secretion, carbon source utilization and cell wall integrity.

Plant diseases caused by fungal pathogens pose a great threat to crop production. Conidiation of fungi is critical for disease epidemics and serves as a promising drug target. Here, we show that deacetylation of the FolTFIIS transcription elongation factor is indispensable for Fusarium oxysporum f. sp. lycopersici (Fol) conidiation. Upon microconidiation, Fol decreases K76 acetylation of FolTFIIS by altering the level of controlling enzymes, allowing for its nuclear translocation by FolIws1. Increased nuclear FolTFIIS enhances the transcription of sporulation-related genes and, consequently, enables microconidia production. Deacetylation of FolTFIIS is also critical for the production of macroconidia and chlamydospores, and its homolog has similar functions in Botrytis cinerea. We identify two FolIws1-targeting chemicals that block the conidiation of Fol and have effective activity against a wide range of pathogenic fungi without harm to the hosts. These findings reveal a conserved mechanism of conidiation regulation and provide candidate agrochemicals for disease management., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

A wide diversity of mycoviruses has been reported from Botrytis species, some with the potential to suppress the pathogenic abilities of this fungus. Considering their importance, this study was devised to find potential hypovirulence-associated mycoviruses found in Botrytis cinerea strains isolated from Pakistani strawberry fields. Here we report the complete genome characterization of two fusariviruses co-infecting a single isolate of phytopathogenic fungus B. cinerea (Kst14a). The viral genomes were sequenced by deep sequencing using total RNA fractions of the Kst14a isolate. The identified viruses were tentatively named Botrytis cinerea fusarivirus 9 (BcFV9) and Botrytis cinerea fusarivirus 3a (BcFV3a). Both viruses had a single-segmented (ssRNA) genome having a size of 6424 and 8370 nucleotides encoding two discontinuous open reading frames (ORFs). ORF-1 of both mycoviruses encodes for a polyprotein having a conserved domain of RNA-dependent RNA polymerase (RdRP) and a helicase domain (Hel) which function in RNA replication, while ORF2 encodes a hypothetical protein with an unknown function, respectively. Phylogenetic analysis indicated that BcFV9 made a clade with the genus Alphafusarivirus and BcFV3a fall in the genus Betafusarivirus in the family Fusariviridae. To our knowledge, this is the first report of two fusariviruses identified in isolates of B. cinerea from Pakistan. Both mycoviruses successfully transfected to a compatible strain of B. cinerea (Mst11). A comparison of virus-free (VF) and virus-infected (VI) isogenic lines showed the presence of these viruses was causing hypovirulence in infected strains. Virus-infected strains also had a small lesion size while testing the pathogenicity via apple assay., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Saponins are plant secondary metabolites comprising glycosylated triterpenoids, steroids or steroidal alkaloids with a broad spectrum of toxicity to microbial pathogens and pest organisms that contribute to basal plant defense to biotic attack. Secretion of glycosyl hydrolases that enzymatically convert saponins into less toxic products was thus far the only mechanism reported to enable fungal pathogens to colonize their saponin-containing host plant(s). We studied the mechanisms that the fungus Botrytis cinerea utilizes to be tolerant to well-characterized, structurally related saponins from tomato and Digitalis purpurea. By gene expression studies, comparative genomics, enzyme assays and testing a large panel of fungal (knockout and complemented) mutants, we unraveled four distinct cellular mechanisms that participate in the mitigation of the toxic activity of these saponins and in virulence on saponin-producing host plants. The enzymatic deglycosylation that we identified is novel and unique to this fungus-saponin combination. The other three tolerance mechanisms operate in the fungal membrane and are mediated by protein families that are widely distributed in the fungal kingdom. We present a spatial and temporal model on how these mechanisms jointly confer tolerance to saponins and discuss the repercussions of these findings for other plant pathogenic fungi, as well as human pathogens., (© 2024. The Author(s).)

Botrytis cinerea is a typical necrotrophic plant pathogenic fungus which can deliberately acidify host tissues and trigger oxidative bursts therein to facilitate its virulence. The white collar complex (WCC), consisting of BcWCL1 and BcWCL2, is recognized as the primary light receptor in B. cinerea . Nevertheless, the specific mechanisms through which the WCC components, particularly BcWCL2 as a GATA transcription factor, control virulence are not yet fully understood. This study demonstrates that deletion of BcWCL2 results in the loss of light-sensitive phenotypic characteristics. Additionally, the Δ bcwcl2 strain exhibits reduced secretion of citrate, delayed infection cushion development, weaker hyphal penetration, and decreased virulence. The application of exogenous citric acid was found to restore infection cushion formation, hyphal penetration, and virulence of the Δ bcwcl2 strain. Transcriptome analysis at 48 h post-inoculation revealed that two citrate synthases, putative citrate transporters, hydrolytic enzymes, and reactive oxygen species scavenging-related genes were down-regulated in Δ bcwcl2 , whereas exogenous citric acid application restored the expression of the above genes involved in the early infection process of Δ bcwcl2 . Moreover, the expression of Bcvel1 , a known regulator of citrate secretion, tissue acidification, and secondary metabolism, was down-regulated in Δ bcwcl2 but not in Δ bcwcl1 . ChIP-qPCR and electrophoretic mobility shift assays revealed that BcWCL2 can bind to the promoter sequences of Bcvel1 . Overexpressing Bcvel1 in Δ bcwcl2 was found to rescue the mutant defects. Collectively, our findings indicate that BcWCL2 regulates the expression of the global regulator Bcvel1 to influence citrate secretion, tissue acidification, redox homeostasis, and virulence of B. cinerea .IMPORTANCEThis study illustrated the significance of the fungal blue light receptor component BcWCL2 protein in regulating citrate secretion in Botrytis cinerea . Unlike BcWCL1, BcWCL2 may contribute to redox homeostasis maintenance during infection cushion formation, ultimately proving to be essential for full virulence. It is also demonstrated that BcWCL2 can regulate the expression of Bcvel1 to influence host tissue acidification, citrate secretion, infection cushion development, and virulence. While the role of organic acids secreted by plant pathogenic fungi in fungus-host interactions has been recognized, this paper revealed the importance, regulatory mechanisms, and key transcription factors that control organic acid secretion. These understanding of the pathogenetic mechanism of plant pathogens can provide valuable insights for developing effective prevention and treatment strategies against fungal diseases., Competing Interests: The authors declare no conflict of interest.

Grey mould caused by Botrytis cinerea is a devastating disease responsible for large losses to agricultural production, and B. cinerea is a necrotrophic model fungal plant pathogen. Membrane proteins are important targets of fungicides and hotspots in the research and development of fungicide products. Wuyiencin affects the permeability and pathogenicity of B. cinerea, parallel reaction monitoring revealed the association of membrane protein Bcsdr2, and the bacteriostatic mechanism of wuyiencin was elucidated. In the present work, we generated and characterised ΔBcsdr2 deletion and complemented mutant B. cinerea strains. The ΔBcsdr2 deletion mutants exhibited biofilm loss and dissolution, and their functional activity was illustrated by reduced necrotic colonisation on strawberry and grape fruits. Targeted deletion of Bcsdr2 also blocked several phenotypic defects in aspects of mycelial growth, conidiation and virulence. All phenotypic defects were restored by targeted gene complementation. The roles of Bcsdr2 in biofilms and pathogenicity were also supported by quantitative real-time RT-PCR results showing that phosphatidylserine decarboxylase synthesis gene Bcpsd and chitin synthase gene BcCHSV II were downregulated in the early stages of infection for the ΔBcsdr2 strain. The results suggest that Bcsdr2 plays important roles in regulating various cellular processes in B. cinerea. KEY POINTS: • The mechanism of wuyiencin inhibits B. cinerea is closely associated with membrane proteins. • Wuyiencin can downregulate the expression of the membrane protein Bcsdr2 in B. cinerea. • Bcsdr2 is involved in regulating B. cinerea virulence, growth and development., (© 2024. The Author(s).)

Background: Currently, industrial fermentation of Botrytis cinerea is a significant source of abscisic acid (ABA). The crucial role of ABA in plants and its wide range of applications in agricultural production have resulted in the constant discovery of new derivatives and analogues. While modifying the ABA synthesis pathway of existing strains to produce ABA derivatives is a viable option, it is hindered by the limited synthesis capacity of these strains, which hinders further development and application., Results: In this study, we knocked out the bcaba4 gene of B. cinerea TB-31 to obtain the 1',4'-trans-ABA-diol producing strain ZX2. We then studied the fermentation broth of the batch-fed fermentation of the ZX2 strain using metabolomic analysis. The results showed significant accumulation of 3-hydroxy-3-methylglutaric acid, mevalonic acid, and mevalonolactone during the fermentation process, indicating potential rate-limiting steps in the 1',4'-trans-ABA-diol synthesis pathway. This may be hindering the flow of the synthetic pathway. Additionally, analysis of the transcript levels of terpene synthesis pathway genes in this strain revealed a correlation between the bchmgr, bcerg12, and bcaba1-3 genes and 1',4'-trans-ABA-diol synthesis. To further increase the yield of 1',4'-trans-ABA-diol, we constructed a pCBg418 plasmid suitable for the Agrobacterium tumefaciens-mediated transformation (ATMT) system and transformed it to obtain a single-gene overexpression strain. We found that overexpression of bchmgr, bcerg12, bcaba1, bcaba2, and bcaba3 genes increased the yield of 1',4'-trans-ABA-diol. The highest yielding ZX2 A3 strain was eventually screened, which produced a 1',4'-trans-ABA-diol concentration of 7.96 mg/g DCW (54.4 mg/L) in 144 h of shake flask fermentation. This represents a 2.1-fold increase compared to the ZX2 strain., Conclusions: We utilized metabolic engineering techniques to alter the ABA-synthesizing strain B. cinerea, resulting in the creation of the mutant strain ZX2, which has the ability to produce 1',4'-trans-ABA-diol. By overexpressing the crucial genes involved in the 1',4'-trans-ABA-diol synthesis pathway in ZX2, we observed a substantial increase in the production of 1',4'-trans-ABA-diol., (© 2024. The Author(s).)

In plants, small RNAs (sRNAs), mainly microRNAs (miRNAs) and small interfering RNAs (siRNAs), have been described as key regulators of plant development, growth, and abiotic and biotic responses. Despite reports indicating the involvement of certain sRNAs in regulating the interaction between Botrytis cinerea (a major necrotrophic fungal phytopathogen) and host plants, there remains a lack of analysis regarding the potential regulatory roles of plant sRNAs during early stages of the interaction despite early immune responses observed then during infection. We present the first transcriptome-wide analysis of small RNA expression on the early interaction between the necrotrophic fungus Botrytis cinerea and the model plant Arabidopsis thaliana. We found that evolutionary conserved A. thaliana miRNAs were the sRNAs that accumulated the most in the presence of B. cinerea. The upregulation of miR167, miR159 and miR319 was of particular interest because these, together with their target transcripts, are involved in the fine regulation of the plant hormone signaling pathways. We also describe that miR173, which triggers the production of secondary siRNAs from TAS1 and TAS2 loci, as well as secondary siRNAs derived from these loci, is upregulated in response to B. cinerea. Thus, at an early stage of the interaction there are transcriptional changes of sRNA-guided silencing pathway genes and of a subset of sRNAs that targeted genes from the PPR gene superfamily, and these may be important mechanisms regulating the interaction between A. thaliana and B. cinerea. This work provides the basis for a better understanding of the regulation mediated by sRNAs during early B. cinerea-plant interaction and may help in the development of more effective strategies for its control., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright: © 2024 Padilla-Padilla et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

The main phytosanitary problem for table grape production in Chile is gray mold caused by the fungus Botrytis cinerea . To manage this issue, the primary method utilized is chemical control. Fludioxonil, a phenylpyrrole, is highly effective in controlling B. cinerea and other plant pathogens. Consistently, there have been no field reports of reduced efficacy of fludioxonil; however, subpopulations with reduced sensitivity to fludioxonil are on the rise globally, as per increasing reports. Our study involved a large-scale evaluation of B. cinerea 's sensitivity to fludioxonil in the Central Valley of Chile's primary table grape production area during the growing seasons from 2015 to 2018. Out of 2,207 isolates, only 1.04% of the isolates ( n = 23) exceeded the sensitivity threshold value of 1 μg/ml. Remarkably, 95.7% are concentrated in a geographic region (Valparaíso Region). Isolates with reduced sensitivity to fludioxonil showed growth comparable with sensitive isolates and even more robust growth under nutritional deficit, temperature, or osmotic stress, suggesting greater environmental adaptation. When table grape detached berries were stored at 0°C, isolates less sensitive to fludioxonil caused larger lesions than sensitive isolates (2.82 mm compared with 1.48 mm). However, the lesions generated by both types of isolates were equivalent at room temperature. This study found no cross-resistance between fludioxonil and fenhexamid, an essential fungicide integrated with fludioxonil in Chilean B. cinerea control programs. All the Chilean isolates with reduced sensitivity to fludioxonil were controlled by the fludioxonil/cyprodinil mixture, a commonly employed form of fludioxonil. The cyprodinil sensitivity in the isolates with reduced sensitivity to fludioxonil explains their low field frequency despite their null fitness penalties. However, the emergence of fludioxonil-resistant isolates inside the Chilean B. cinerea population demands a comprehensive analysis of their genetic bases, accompanied by monitoring tools that allow the permanence of field fludioxonil efficacy., Competing Interests: The author(s) declare no conflict of interest.

Botrytis cinerea is a necrotrophic pathogen that infects across a broad range of plant hosts, including high-impact crop species. Its generalist necrotrophic behavior stems from its ability to detoxify structurally diverse phytoalexins. The current study aims to provide evidence of the ability of B. cinerea to tolerate the sesquiterpenoid phytoalexin rishitin, which is produced by potato and tomato. While the growth of potato pathogens Phytophthora infestans (late blight) and Alternaria solani (early blight) was severely inhibited by rishitin, B. cinerea was tolerant to rishitin. After incubation of rishitin with the mycelia of B. cinerea, it was metabolized to at least six oxidized forms. Structural analysis of these purified rishitin metabolites revealed a variety of oxidative metabolism including hydroxylation at C7 or C12, ketone formation at C5, and dihydroxylation at the 10,11-olefin. Six rishitin metabolites showed reduced toxicity to P. infestans and A. solani, indicating that B. cinerea has at least 5 distinct enzymatic reactions to detoxify rishitin. Four host-specialized phytopathogenic Botrytis species, namely B. elliptica, B. allii, B. squamosa, and B. tulipae also had at least a partial ability to metabolize rishitin as B. cinerea, but their metabolic capacity was significantly weaker than that of B. cinerea. These results suggest that the ability of B. cinerea to rapidly metabolize rishitin through multiple detoxification mechanisms could be critical for its pathogenicity in potato and tomato., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Utilizing bioinformatics tools, this study expands our understanding of secondary metabolism in Botrytis cinerea , identifying novel genes within polyketide synthase (PKS), non-ribosomal peptide synthetase (NRPS), sesquiterpene cyclase (STC), diterpene cyclase (DTC), and dimethylallyltryptophan synthase (DMATS) families. These findings enrich the genetic framework associated with B. cinerea 's pathogenicity and ecological adaptation, offering insights into uncharted metabolic pathways. Significantly, the discovery of previously unannotated genes provides new molecular targets for developing targeted antifungal strategies, promising to enhance crop protection and advance our understanding of fungal biochemistry. This research not only broadens the scope of known secondary metabolites but also opens avenues for future exploration into B. cinerea 's biosynthetic capabilities, potentially leading to novel antifungal compounds. Our work underscores the importance of integrating bioinformatics and genomics for fungal research, paving the way for sustainable agricultural practices by pinpointing precise molecular interventions against B. cinerea . This study sets a foundation for further investigations into the fungus's secondary metabolism, with implications for biotechnology and crop disease management.

The genome sequencing of Botrytis cinerea supplies a general overview of the map of genes involved in secondary metabolite synthesis. B. cinerea genomic data reveals that this phytopathogenic fungus has seven sesquiterpene cyclase ( Bcstc ) genes that encode proteins involved in the farnesyl diphosphate cyclization. Three sesquiterpene cyclases (BcStc1, BcStc5 and BcStc7) are characterized, related to the biosynthesis of botrydial, abscisic acid and (+)-4-epi-eremophilenol, respectively. However, the role of the other four sesquiterpene cyclases (BcStc2, BcStc3, BcStc4 and BcStc6) remains unknown. BcStc3 is a well-conserved protein with homologues in many fungal species, and here, we undertake its functional characterization in the lifecycle of the fungus. A null mutant Δ Bcstc3 and an overexpressed- Bcstc3 transformant (Ov Bcstc3 ) are generated, and both strains show the deregulation of those other sesquiterpene cyclase-encoding genes ( Bcstc1 , Bcstc5 and Bcstc7 ). These results suggest a co-regulation of the expression of the sesquiterpene cyclase gene family in B. cinerea . The phenotypic characterization of both transformants reveals that BcStc3 is involved in oxidative stress tolerance, the production of reactive oxygen species and virulence. The metabolomic analysis allows the isolation of characteristic polyketides and eremophilenols from the secondary metabolism of B. cinerea , although no sesquiterpenes different from those already described are identified.

Succinate dehydrogenase inhibitor (SDHI) fungicides are the most commonly and effectively used class of fungicides for controlling gray mold. Among them, only boscalid has been registered in China for controlling grape gray mold, whereas isofetamid and pydiflumetofen are two new SDHI fungicides that have demonstrated high efficacy against various fungal diseases. However, the sensitivity of Botrytis cinerea isolates from vineyards in China to these three fungicides is currently unknown. In this study, the sensitivity of 55 B. cinerea isolates from vineyards to boscalid, isofetamid, and pydiflumetofen was determined, with the effective concentrations for inhibiting 50% of spore germination (EC 50 ) values ranging from 1.10 to 393, 0.0300 to 42.0, and 0.0990 to 25.5 μg ml -1 , respectively. The resistance frequencies for boscalid, isofetamid, and pydiflumetofen were 60.0, 7.2, and 12.8%, respectively. Three mutations (H272R, H272Y, and P225F) were detected in the SdhB subunit, with H272R being the most prevalent (75.7%), followed by H272Y (16.2%) and P225F (8.1%). All three mutations are associated with resistance to boscalid, and of them, H272R mutants exhibited high resistance. Only P225F and H272Y mutants exhibited resistance to isofetamid and pydiflumetofen, respectively. A weakly positive cross-resistance relationship was observed between boscalid and pydiflumetofen ( r = 0.38, P < 0.05). Additionally, the H272R mutants showed no significant fitness costs, whereas the remaining mutants exhibited reduced mycelial growth (P225F) and sporulation (H272Y and P225F). These results suggest that isofetamid and pydiflumetofen are effective fungicides against B. cinerea in vineyards, but appropriate rotation strategies must be implemented to reduce the selection of existing SDHI-resistant isolates., Competing Interests: The author(s) declare no conflict of interest.

Plant pathogens have frequently shown multidrug resistance (MDR) in the field, often linked to efflux and sometimes metabolism of fungicides. To investigate the potential role of metabolic resistance in B. cinerea strains showing MDR, the azoxystrobin-sensitive strain B05.10 and -resistant strain Bc242 were treated with azoxystrobin. The degradation half-life of azoxystrobin in Bc242 (9.63 days) was shorter than that in B05.10 (28.88 days). Azoxystrobin acid, identified as a metabolite, exhibited significantly lower inhibition rates on colony and conidia (9.34 and 11.98%, respectively) than azoxystrobin. Bc242 exhibited higher expression levels of 34 cytochrome P450s (P450s) and 11 carboxylesterase genes (CarEs) compared to B05.10 according to RNA-seq analysis. The expression of P450 genes Bcin&#95;02g01260 and Bcin&#95;12g06380 , along with the CarEs Bcin&#95;12g06360 in Saccharomyces cerevisiae , resulted in reduced sensitivity to various fungicides, including azoxystrobin, kresoxim-methyl, pyraclostrobin, trifloxystrobin, iprodione, and carbendazim. Thus, the mechanism of B. cinerea MDR is linked to metabolism mediated by the CarE and P450 genes.

Botrytis cinerea is one of the most destructive pathogens worldwide. It can damage over 200 crops, resulting in significant yield and quality losses. Cyclobutrifluram, a new generation of succinate dehydrogenase inhibitors, exhibits excellent inhibitory activity against B. cinerea. However, the baseline sensitivity and resistance of B. cinerea to cyclobutrifluram remains poorly understood. This study was designed to monitor the sensitivity frequency distribution, assess the resistance risk, and clarify the resistance mechanism of B. cinerea to cyclobutrifluram. The baseline sensitivity of B. cinerea isolates to cyclobutrifluram was 0.89 μg/mL. Cyclobutrifluram-resistant B. cinerea populations are present in the field. Six resistant B. cinerea isolates investigated in this study possessed enhanced compound fitness index compared to the sensitive isolates according to mycelial growth, mycelial dry weight, conidiation, conidial germination rate, and pathogenicity. Cyclobutrifluram exhibited no cross-resistance with tebuconazole, fludioxonil, cyprodinil, or iprodione. Sequence alignment revealed that BcSDHB from cyclobutrifluram-resistant B. cinerea isolates had three single substitutions (P225F, N230I, or H272R). Molecular docking verified that these mutations in BcSDHB conferred cyclobutrifluram resistance in B. cinerea. In conclusion, the resistance risk of B. cinerea to cyclobutrifluram is high, and the point mutations in BcSDHB (P225F, N230I, or H272R) confer cyclobutrifluram resistance in B. cinerea. This study provided important insights into cyclobutrifluram resistance in B. cinerea and offered valuable information for monitoring and managing cyclobutrifluram resistance in the future., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Botrytis cinerea is a broad-host-range necrotrophic phytopathogen responsible for serious diseases in leading crops worldwide. The novel sterol 14α-demethylase inhibitor (DMI) pyrisoxazole was recently registered for the control of tomato gray mold caused by B. cinerea in China. One hundred fifty-seven isolates of B. cinerea were collected from tomato greenhouses in 14 cities of Liaoning Province from 2016 to 2021 and examined for sensitivity to pyrisoxazole, with a mean EC 50 value of 0.151 μg/ml. Three highly resistant isolates, XD-5, DG-4, and GQ-3, were screened, and the EC 50 values were 0.734, 0.606, and 0.639 μg/ml with corresponding resistance factors of 12.88, 10.63, and 11.21, respectively. Compared with field-sensitive strains, the highly resistant isolate XD-5 exhibited fitness defects in traits, including mycelial growth, conidial production, and pathogenicity, but DG-4 and GQ-3 did not experience fitness costs. Positive cross-resistance was observed only between pyrisoxazole and the DMIs tebuconazole and prochloraz but not between pyrisoxazole and the non-DMIs iprodione, procymidone, pyrimethanil, fludioxonil, fluazinam, and fluopyram. Sequence alignment of the CYP51 gene indicated that three point mutations were observed in the highly resistant mutant, namely, V24I in XD-5, G461S in GQ-3, and R464K in DG-4. When exposed to pyrisoxazole, the induced expression levels of the ABC transporter AtrD and MFS transporter Mfs1 increased in the resistant isolates compared with those in the sensitive isolates, whereas the expression level of the CYP51 gene did not change significantly. Molecular docking suggested that the G461S and R464K mutations both led to a decrease in the binding energy between CYP51 and pyrisoxazole, whereas no change was found with the V24I mutation. Thus, two point mutations in the CYP51 protein combined with induced expression of the Mfs1 and AtrD genes appeared to mediate the pyrisoxazole resistance of the highly resistant mutants DG-4 and GQ-3, while the overexpression of the Mfs1 and AtrD genes was responsible for the highly resistant mutant XD-5., Competing Interests: The author(s) declare no conflict of interest.

Gray mold caused by Botrytis cinerea is among the 10 most serious fungal diseases worldwide. Fludioxonil is widely used to prevent and control gray mold due to its low toxicity and high efficiency; however, resistance caused by long-term use has become increasingly prominent. Therefore, exploring the resistance mechanism of fungicides provides a theoretical basis for delaying the occurrence of diseases and controlling gray mold. In this study, fludioxonil-resistant strains were obtained through indoor drug domestication, and the mutation sites were determined by sequencing. Strains obtained by site-directed mutagenesis were subjected to biological analysis, and the binding modes of fludioxonil and iprodione to Botrytis cinerea Bos1 BcBos1 were predicted by molecular docking. The results showed that F127S, I365S/N, F127S + I365N, and I376M mutations on the Bos1 protein led to a decrease in the binding energy between the drug and BcBos1 . The A1259T mutation did not lead to a decrease in the binding energy, which was not the cause of drug resistance. The biological fitness of the fludioxonil- and point mutation-resistant strains decreased, and their growth rate, sporulation rate, and pathogenicity decreased significantly. The glycerol content of the sensitive strains was significantly lower than that of the resistant strains and increased significantly after treatment with 0.1 μg/ml of fludioxonil, whereas that of the resistant strains decreased. The osmotic sensitivity of the resistant strains was significantly lower than that of the sensitive strains. Positive cross-resistance was observed between fludioxonil and iprodione. These results will help to understand the resistance mechanism of fludioxonil in Botrytis cinerea more deeply., Competing Interests: The author(s) declare no conflict of interest.

Gray mold caused by Botrytis cinerea severely threatens the yield of ginseng ( Panax ginseng ). Various categories of fungicides have been utilized to control gray mold on this crop. In this study, the resistance of 102 isolates of B. cinerea from 11 commercial ginseng-growing regions in China to fungicides was examined. A total of 32.4% were resistant to boscalid, with EC 50 values that ranged from 12.26 to 235.87 μg/ml, and 94.1% were resistant to pyraclostrobin, with EC 50 values that ranged from 5.88 to 487.72 μg/ml. Except for sdh A and sdh D, the amino acid substitutions of P225F, P225L, N230I, H272Y, and H272R in the sdh B subunit from 24 (4 sensitive [S] and 20 resistant [R]), 5 (1 S and 4 R), 1 (S), 1 (R), and 8 (4 S and 4 R) strains, respectively, and the concurrent amino acid substitutions of G85A + I93V + M158V + V168I in the sdh C subunit from 5 (4 S and 1 R) strains were identified. A G143A substitution in cytochrome b was identified in 96 isolates that were resistant to pyraclostrobin and three that were sensitive to it. The Bcbi-143/144 intron was identified in the other three isolates sensitive to pyraclostrobin, but it was absent in the isolates that harbored the G143A mutation. The results showed that the populations of B. cinerea on ginseng have developed strong resistance to pyraclostrobin. Therefore, it is not recommended to continue using this fungicide to control gray mold on P. ginseng . Boscalid is still effective against most isolates. However, to prevent fungicide resistance, it is recommended to use a mixture of boscalid with other categories of fungicides., Competing Interests: The author(s) declare no conflict of interest.

Botrytis cinerea poses a recurring threat to viticulture, causing significant yield losses each year. The study explored the biocontrol capabilities of commercially used winemaking yeasts as a strategy to manage B. cinerea in grape berries. The winemaking yeast strains-Saccharomyces cerevisiae ES181, Saccharomyces pastorianus KBG6, S. cerevisiae BCS103, Lachancea thermotolerans Omega, and Torulaspora delbrueckii TD291-reduced B. cinerea growth and conidiation in vitro. Furthermore, they demonstrated a decreased disease severity and number of conidia in grape berries. Among these strains, S. cerevisiae BCS103 was the most effective, inducing the expression of the defense-related gene PR4 in berries. Its diffusible compounds and volatile organic compounds also reduced the expression of BcLTF2, a positive regulator of B. cinerea conidiogenesis. The examined winemaking yeast strains, especially S. cerevisiae BCS103, demonstrated effective inhibition of B. cinerea in vitro and in grape berries, influencing key defense genes and reducing BcLTF2 expression, offering potential solutions for disease management in viticulture. The study underscores the promise of commercially available winemaking yeast strains as eco-friendly tools against B. cinerea in viticulture. Leveraging their safety and existing use in winemaking offers a potential avenue for sustainable disease management., (© The Author(s) 2024. Published by Oxford University Press on behalf of Applied Microbiology International.)

Apoptosis-like programmed cell death is associated with fungal development, ageing, pathogenicity and stress responses. Here, to explore the potential of Botrytis cinerea type II inhibitor of apoptosis (IAP) BcBIR1 in elevating the biocontrol efficacy of Coniothyrium minitans, the BcBIR1 gene was heterologously expressed in C. minitans. Results indicated that the strains expressing BcBIR1 had higher rates of conidiation, mycelial growth and biomass growth than the wild-type strain. Moreover, BcBIR1 was found to inhibit apoptosis, indicating its role as an IAP in C. minitans. Under various abiotic stresses, the growth rates of BcBIR1-expressing strains were significantly higher than that of the wild-type strain. Moreover, the conidial survival rate of the BcBIR1-expressing strains treated with ultraviolet irradiation was enhanced. In antifungal activity assay, the culture filtrates of BcBIR1-expressing strains displayed a stronger inhibitory effect on B. cinerea and Sclerotinia sclerotiorum than the wild-type strain. The study also found that BcBIR1 expression increased the mycoparasitism against the sclerotia, but not the hyphae of S. sclerotiorum. Taken together, these results suggest that BcBIR1 enhances vegetative growth, conidiation, anti-apoptosis activity, abiotic stress resistance, antifungal activity and mycoparasitism in C. minitans. As an IAP, BcBIR1 may improve the control capacity of C. minitans against S. sclerotiorum., (© 2024 The Authors. Microbial Biotechnology published by Applied Microbiology International and John Wiley & Sons Ltd.)

Background: Chickpea (Cicer arietinum L.) production is affected by many biotic factors, among them Fusarium wilt caused by Fusarium oxysporum f. sp. ciceri and Botrytis gray mold caused by Botrytis cinerea led to severe losses. As fungicide application is not advisable, biological management is the best alternative for plant protection. The rhizosphere-dwelling antagonistic bacteria are one of the important successful alternative strategy to manage these diseases of chickpea. Rhizosphere dwelling bacteria serve as biocontrol agents by different mechanisms like producing antibiotics, different enzymes, siderophores against pathogens and thereby reducing the growth of pathogens., Results: The present study aimed to isolate rhizospheric bacteria from the soils of different chickpea fields to evaluate biocontrol efficacy of the isolated bacteria to manage Fusarium wilt and Botrytis gray mold in chickpea. A total of 67 bacteria were isolated from chickpea rhizosphere from Bundelkhand region of India. Study revealed the isolated bacteria could reduce the Fusarium oxysporum f. sp. ciceris and Botrytis cinerea infection in chickpea between 17.29 and 75.29%. After screening of all the bacteria for their biocontrol efficacy, 13 most promising bacterial isolates were considered for further study out of which, three bacterial isolates (15d, 9c and 14a) have shown the maximum in vitro antagonistic effects against Fusarium oxysporum f. sp. ciceri and Botrytis cinerea comparable to in vivo effects. However, Isolate (15d) showed highest 87.5% and 82.69% reduction in disease against Fusarium wilt and Botrytis gray mold respectively, under pot condition. Three most potential isolates were characterized at molecular level using 16S rRNA gene and found to be Priestia megaterium (9c and 14a) and Serratia marcescens (15d)., Conclusion: This study identified two native biocontrol agents Priestia megaterium and Serratia marcescens from the rhizospheric soils of Bundelkhand region of India for control of Fusarium wilt, Botrytis gray mold. In future, efforts should be made to further validate the biocontrol agents in conjugation with nanomaterials for enhancing the synergistic effects in managing the fungal diseases in chickpea. This study will definitely enhance our understanding of these bioagents, and to increase their performance by developing effective formulations, application methods, and integrated strategies., (© 2024. The Author(s).)

Pulcherrimin is an iron (III) chelate of pulcherriminic acid that plays a role in antagonistic microbial interactions, iron metabolism, and stress responses. Some bacteria and yeasts produce pulcherriminic acid, but so far, pulcherrimin could not be produced in Saccharomyces cerevisiae. Here, multiple integrations of the Metschnikowia pulcherrima PUL1 and PUL2 genes in the S. cerevisiae genome resulted in red colonies, which indicated pulcherrimin formation. The coloration correlated positively and significantly with the number of PUL1 and PUL2 genes. The presence of pulcherriminic acid was confirmed by mass spectrometry. In vitro competition assays with the plant pathogenic fungus Botrytis caroliana revealed inhibitory activity on conidiation by an engineered, strong pulcherrimin-producing S. cerevisiae strain. We demonstrate that the PUL1 and PUL2 genes from M. pulcherrima, in multiple copies, are sufficient to transfer pulcherrimin production to S. cerevisiae and represent the starting point for engineering and optimizing this biosynthetic pathway in the future., (© The Author(s) 2023. Published by Oxford University Press on behalf of FEMS.)

Next-generation sequencing (NGS) of total RNA has allowed the detection of novel viruses infecting different hosts, such as fungi, increasing our knowledge on virus horizontal transfer events among different hosts, virus diversity, and virus evolution. Here, we describe the detailed protocols for the isolation of the plant pathogenic fungus Botrytis cinerea, from grapevine plants showing symptoms of the mold gray disease, the culture and maintenance of the isolated B. cinerea strains, the extraction of total RNA from B. cinerea strains for NGS, the bioinformatics pipeline designed and followed to detect mycoviruses in the sequenced samples, and the validation of the in silico detected mycoviruses by different approaches., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Small RNAs act as fungal pathogen effectors that silence host target genes to promote infection, a virulence mechanism termed cross-kingdom RNA interference (RNAi). The essential pathogen factors of cross-kingdom small RNA production are largely unknown. We here characterized the RNA-dependent RNA polymerase (RDR)1 in the fungal plant pathogen Botrytis cinerea that is required for pathogenicity and cross-kingdom RNAi. B. cinerea bcrdr1 knockout (ko) mutants exhibited reduced pathogenicity and loss of cross-kingdom small RNAs. We developed a "switch-on" GFP reporter to study cross-kingdom RNAi in real-time within the living plant tissue which highlighted that bcrdr1 ko mutants were compromised in cross-kingdom RNAi. Moreover, blocking seven pathogen cross-kingdom small RNAs by expressing a short-tandem target mimic RNA in transgenic Arabidopsis thaliana led to reduced infection levels of the fungal pathogen B. cinerea and the oomycete pathogen Hyaloperonospora arabidopsidis. These results demonstrate that cross-kingdom RNAi is significant to promote host infection and making pathogen small RNAs an effective target for crop protection., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Cheng et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Background: Family Genomoviridae was recently established, and only a few mycoviruses have been described and characterized, and almost all of them (Sclerotinia sclerotiorum hypovirulence-associated DNA virus 1, Fusarium graminearum gemyptripvirus 1 and Botrytis cinerea gemydayirivirus 1) induced hypovirulence in their host. Botrytis cinerea ssDNA virus 1 (BcssDV1), a tetrasegmented single-stranded DNA virus infecting the fungus Botrytis cinerea, has been molecularly characterized in this work., Methods: BcssDV1 was detected in Spanish and Italian B. cinerea field isolates obtained from grapevine. BcssDV1 variants genomes were molecularly characterized via NGS and Sanger sequencing. Nucleotide and amino acid sequences were used for diversity and phylogenetic analysis. Prediction of protein tertiary structures and putative associated functions were performed by AlphaFold2 and DALI., Results: BcssDV1 is a tetrasegmented single-stranded DNA virus. The mycovirus was composed by four genomic segments of approximately 1.7 Kb each, which are DNA-A, DNA-B, and DNA-C and DNA-D, that coded, respectively, for the rolling-circle replication initiation protein (Rep), capsid protein (CP) and two hypothetical proteins. BcssDV1 was present in several Italian and Spanish regions with high incidence and low variability among the different viral variants. DNA-A and DNA-D were found to be the more conserved genomic segments among variants, while DNA-B and DNA-C segments were shown to be the most variable ones. Tertiary structures of the proteins encoded by each segment suggested specific functions associated with each of them., Conclusions: This study presented the first complete sequencing and characterization of a tetrasegmented ssDNA mycovirus, its incidence in Spain and Italy, its presence in other countries and its high conservation among regions., (© 2023. The Author(s).)

The fungal Gβ-like protein has been reported to be involved in a variety of biological processes, such as mycelial growth, differentiation, conidiation, stress responses and infection. However, molecular mechanisms of the Gβ-like protein in regulating fungal development and pathogenicity are largely unknown. Here, we show that the Gβ-like protein gene Bcgbl1 in the gray mold fungus Botrytis cinerea plays a pivotal role in development and pathogenicity by regulating the mitogen-activated protein (MAP) kinases signaling pathways. The Bcgbl1 deletion mutants were defective in mycelial growth, sclerotial formation, conidiation, macroconidial morphogenesis, plant adhesion, and formation of infection cushions and appressorium-like structures, resulting in a complete loss of pathogenicity. Bcgbl1 interacted with BcSte50, the adapter protein of the cascade of MAP kinase (MAPK). Bcgbl1 mutants had reduced phosphorylation levels of two MAPKs, namely Bmp1 and Bmp3, thereby reducing infection. However, deletion of Bcgbl1 did not affect the intracellular cAMP level, and exogenous cAMP could not restore the defects. Moreover, Bcgbl1 mutants exhibited defects in cell wall integrity and oxidative stress tolerance. Transcriptional profiling revealed that Bcgbl1 plays a global role in regulation of gene expression upon hydrophobic surface induction. We further uncovered that three target genes encoding the hydrophobic surface binding proteins (HsbAs) contributed to the adhesion and virulence of B. cinerea. Overall, these findings suggest that Bcgbl1 had multiple functions and provided new insights for deciphering the Bcgbl1-mediated network for regulating development and pathogenicity of B. cinerea., Competing Interests: The authors have declared that no competing interests exist., (Copyright: This is an open access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.)

Vegetative incompatibility is a fungal allorecognition system characterised by the inability of genetically distinct conspecific fungal strains to form a viable heterokaryon and is controlled by multiple polymorphic loci termed vic (vegetative incompatibility) or het (heterokaryon incompatibility). We have genetically identified and characterised the first vic locus in the economically important, plant-pathogenic, necrotrophic fungus Botrytis cinerea. A bulked segregant approach coupled with whole genome Illumina sequencing of near-isogenic lines of B. cinerea was used to map a vic locus to a 60-kb region of the genome. Within that locus, we identified two adjacent, highly polymorphic open reading frames, Bcvic1 and Bcvic2, which encode predicted proteins that contain domain architectures implicated in vegetative incompatibility in other filamentous fungi. Bcvic1 encodes a predicted protein containing a putative serine esterase domain, a NACHT family of NTPases domain, and several Ankyrin repeats. Bcvic2 encodes a putative syntaxin protein containing a SNARE domain; such proteins typically function in vesicular transport. Deletion of Bcvic1 and Bcvic2 individually had no effect on vegetative incompatibility. However, deletion of the region containing both Bcvic1 and Bcvic2 resulted in mutant lines that were severely restricted in growth and showed loss of vegetative incompatibility. Complementation of these mutants by ectopic expression restored the growth and vegetative incompatibility phenotype, indicating that Bcvic1 and Bcvic2 are controlling vegetative incompatibility at this vic locus., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Gray mold in strawberry is caused by multiple species of Botrytis , including Botrytis cinerea , B. pseudocinerea , B. fragariae , and B. mali . The species B. cinerea and B. fragariae are widespread in production regions of the eastern United States and Germany, and their distinction is important for disease management strategies. Currently, the only way to differentiate these species in field samples is by PCR, which is time consuming, labor intensive, and costly. In this study, a loop-mediated isothermal amplification (LAMP) technique was developed based on species-specific NEP2 gene nucleotide sequences. The designed primer set specifically amplified B. fragariae DNA and no other Botrytis spp. ( B. cinerea , B. mali , and B. pseudocinerea ) or plant pathogens. The LAMP assay was able to amplify fragments from DNA extracted from infected fruit using a rapid DNA extraction protocol, confirming its ability to detect low amounts of B. fragaria DNA from field-infected fruit. In addition, a blind test was performed to identify B. fragariae in 51 samples collected from strawberry fields in the eastern United States using the LAMP technique. The B. fragariae samples were identified with a reliability of 93.5% (29 of 32), and none of the B. cinerea , B. pseudocinerea , or B. mali samples included in the test were amplified in 10 min. Our results show that the LAMP technique is a specific and reliable method for the detection of B. fragariae from infected fruit tissue and can help to control this important disease in the field., Competing Interests: The author(s) declare no conflict of interest.

Despite the increasing number of fungal microRNA-like small RNAs (milRNAs) being identified and reported, profiling of milRNAs in biocontrol fungi and their roles in the mycoparasitism of pathogenic fungi remains limited. Therefore, in this study, we constructed a GFP fluorescence strain to evaluate the critical period of mycoparasitism in the interaction system between T. breve T069 and B. cinerea. The results showed that the early stage of Trichoderma mycoparasitism occurred 12 h after hyphal contact and was characterized by hyphal parallelism, whereas the middle stage lasted 36 h was characterized by wrapping. The late stage of mycoparasitism occurred at 72 h was characterized by the degradation of B. cinerea mycelia. We subsequently identified the sRNAs of T. breve T069 and B. cinerea during the critical period of mycoparasitism using high-throughput sequencing. In ltR1, 45 potential milRNA targets were identified for 243 genes, and 73 milRNAs targeted 733 genes in ltR3. Additionally, to identify potential transboundary miRNAs in T. breve T069, we screened for miRNAs that were exclusively expressed and had precursor structures in the T. breve T069 genome but were absent in the B. cinerea genome. Next, we predicted the target genes of B. cinerea. Our findings showed that 14 potential transboundary milRNAs from T. breve T069 targeted 41 genes in B. cinerea. Notably, cme-MIR164a-p5&#95;1ss17CT can target 15 genes, including Rim15 (BCIN&#95;15g00280), Nop53 (BCIN&#95;12g03770), Skn7 (BCIN&#95;02g08650), and Vel3 (BCIN&#95;03g06410), while ppe-MIR477b-p3&#95;1ss11TC targeted polyketide synthase (BCIN&#95;03g04360, PKS3). The target gene of PC-5p-27397&#95;41 was a non-ribosomal peptide synthetase (BCIN&#95;01g03730, Bcnrps6). PC-3p-0029 (Tri-milR29) targeted chitin synthetase 7. These genes play crucial roles in normal mycelial growth and pathogenicity of B. cinerea. In conclusion, this study highlights the significance of milRNAs in Trichoderma mycoparasitism of B. cinerea. This discovery provides a new strategy for the application of miRNAs in the prevention and treatment of fungal pathogens., Competing Interests: Declaration of Competing Interest The authors declare that there are no conflicts of interest., (Copyright © 2023. Published by Elsevier Inc.)

Botrytis cinerea is a ubiquitous pathogen that can infect at least 200 dicotyledonous plant species including many agriculturally and economically important crops. In Ginseng, the fungus may cause ginseng gray mold disease, causing great economic losses in the ginseng industry. Therefore, the early detection of B. cinerea in the process of ginseng production is necessary for the disease prevention and control of the pathogen's spread. In this study, a polymerase chain reaction-nucleic acid sensor (PCR-NAS) rapid detection technique was established, and it can be used for field detection of B. cinerea through antipollution design and portable integration. The present study showed that the sensitivity of PCR-NAS technology is 10 times higher than that of traditional PCR-electrophoresis, and there is no need for expensive detection equipment or professional technicians. The detection results of nucleic acid sensors can be read by the naked eye in under 3 min. Meanwhile, the technique has high specificity for the detection of B. cinerea . The testing of 50 field samples showed that the detection results of PCR-NAS were consistent with those of the real-time quantitative PCR (qPCR) method. The PCR-NAS technique established in this study can be used as a novel nucleic acid field detection technique, and it has a potential application in the field detection of B. cinerea to achieve early warning of the pathogen infection., Competing Interests: The author(s) declare no conflict of interest.

Bidirectional flow of information shapes the outcome of the host-pathogen interactions and depends on the genetics of each organism. Recent work has begun to use co-transcriptomic studies to shed light on this bidirectional flow, but it is unclear how plastic the co-transcriptome is in response to genetic variation in both the host and pathogen. To study co-transcriptome plasticity, we conducted transcriptomics using natural genetic variation in the pathogen, Botrytis cinerea, and large-effect genetic variation abolishing defense signaling pathways within the host, Arabidopsis thaliana. We show that genetic variation in the pathogen has a greater influence on the co-transcriptome than mutations that abolish defense signaling pathways in the host. Genome-wide association mapping using the pathogens' genetic variation and both organisms' transcriptomes allowed an assessment of how the pathogen modulates plasticity in response to the host. This showed that the differences in both organism's responses were linked to trans-expression quantitative trait loci (eQTL) hotspots within the pathogen's genome. These hotspots control gene sets in either the host or pathogen and show differential allele sensitivity to the host's genetic variation rather than qualitative host specificity. Interestingly, nearly all the trans-eQTL hotspots were unique to the host or pathogen transcriptomes. In this system of differential plasticity, the pathogen mediates the shift in the co-transcriptome more than the host., Competing Interests: Conflicts of interest The author(s) declare no conflict of interest., (© The Author(s) 2023. Published by Oxford University Press on behalf of The Genetics Society of America. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Histone proteins play a critical role in the primary organization of nucleosomes, which is the fundamental unit of chromatin. Among the five types of the histones, histone H3 has multiple variants, and the number differs among the species. Amongst histone H3 variants, centromeric histone H3 (CENH3) is crucial for centromere identification and proper chromosomal segregation during cell division. In the present study, we have identified 17 putative histone H3 genes of Brassica oleracea. Furthermore, we have done a detailed characterization of the CENH3 gene of B. oleracea. We showed that a single CENH3 gene exhibits allelic diversity with at least two alleles and alternative splicing pattern. Also, we have identified a CENH3 gene-specific co-dominant cleaved amplified polymorphic sequence marker SNP34(A/C) to distinguish CENH3 alleles and follow their expression in leaf and flower tissues. The gene structure analysis of the CENH3 gene revealed the conserved 5'-CAGCAG-3' sequence at the intron 3-exon 4 junction in B. oleracea, which serves as an alternative splicing site with one-codon (alanine) addition/deletion. However, this one-codon alternative splicing feature is not conserved in the CENH3 genes of wild allied Brassica species. Our finding suggests that transcriptional complexity and alternative splicing might play a key role in the transcriptional regulation and function of the CENH3 gene in B. oleracea. Altogether, data generated from the present study can serve as a primary information resource and can be used to engineer CENH3 gene towards developing haploid inducer lines in B. oleracea., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Austria, part of Springer Nature.)

Background: Wall associated kinase (WAK) and WAK-like (WAKL) are typical pattern recognition receptors act as the first sentry of plant defense. But little of WAK/WAKL family is known in Rosaceae., Results: In this study, 131 WAK/WAKL genes from apple, peach and strawberry were identified using a bioinformatics approach. Together with 68 RcWAK/RcWAKL in rose, we performed a comparative analysis of 199 WAK/WAKL in four Rosaceae crops. The phylogenetic analysis divided all the WAK/WAKL into five clades. Among them, the cis-elements of Clade II and Clade V promoters were enriched in jasmonic acid (JA) signaling and abiotic stress, respectively. And this can also be verified by the rose transcriptome responding to different hormone treatments. WAK/WAKL families have experienced a considerable proportion of purifying selection during evolution, but still 26 amino acid sites evolved under positive selection, which focused on extracellular conserved domains. WAK/WAKL genes presented collinearity relationship within and between crops, throughout four crops we mined four orthologous groups (OGs). The WAK/WAKL genes in OG1 and OG4 were speculated to involve in plant-Botrytis cinerea interaction, which were validated in rose via VIGS as well as strawberry by qRT-PCR., Conclusions: These results not only provide genetic resources and valuable information for the evolutionary relationship of WAK/WAKL gene family, but also offer a reference for future in-depth studies of Rosaceae WAK/WAKL genes., (© 2023. The Author(s).)

Succinate dehydrogenase inhibitors (SDHIs) are keystone synthetic fungicides used to manage Botrytis cinerea in several hosts. In this study, we investigated the cross-resistance between five new SDHIs (pyraziflumid, isofetamid, benzovindiflupyr, fluxapyroxad, and pydiflumetofen) with commonly used SDHIs boscalid and fluopyram. Different mutations were detected in the sdhB gene in B. cinerea collected from Michigan grapes, and their frequency and EC 50 value were determined. Among 216 B. cinerea boscalid-resistant isolates, five different mutations were detected, including H272R/Y, P225F/H, and N230I, at frequencies of 82.6, 4.3, 11.5, 0.4, and 5.3%, respectively. Five isolates of each genotype were used to screen the cross-resistance of the SDHIs. We classified the resistance profile of our mutants into five patterns. We report that all tested mutants were sensitive to benzovindiflupyr, indicating that it can be used as an effective fungicide against all B. cinerea mutants identified in this study. In addition, fluopyram, pydiflumetofen, and isofetamid can provide effective control according to which type of mutation is present in the field. We also developed and compared two molecular diagnostic tools, rhAMP and TaqMan assays, for rapid detection of SDHI resistance-associated mutants in B. cinerea . We report that the TaqMan assay was more successful than the rhAMP assay in detecting the B. cinerea mutant DNA at ≤10 pg and in a single assay was capable of monitoring two amino acid positions. Our results provide essential information about new SDHIs and provide molecular tools for monitoring SDHI resistance mutations, which will assist in gray mold disease control., Competing Interests: The author(s) declare no conflict of interest.

Biotic stress due to fungal infection is detrimental to the growth and development of chickpea. In our study, two chickpea genotypes viz Cicer pinnatifidum (resistant) and PBG5 (susceptible) were inoculated with (1 × 10 4 spore mL -1 ) of nectrotrophic fungus Botrytis cinerea at seedling stage. These seedlings were evaluated for morphological, ultrastructural, and molecular differences after 3, 5 and 7 days post inoculation (dpi). Visual symptoms were recorded in terms of water-soaked lesions, rotten pods and twigs with fungal colonies. Light and scanning electron microscopy (SEM) revealed the differences in number of stomata, hyphal network and extent of topographical damage in resistant ( C. pinnatifidum ) and susceptible (PBG5) genotypes, which were validated by stomatal index studies done by using fluorescence microscopy in the infection process of B. cinerea in leaves of both chickpea genotypes. In case of control (water inoculated) samples, there were differences in PCR analysis done using five primers for screening the genetic variations between two genotypes. The presence of a Botrytis responsive gene (LrWRKY) of size ~300 bp was observed in uninoculated resistant genotype which might have a role in resistance against Botrytis grey mould. The present investigation provides information about the variation in the infection process of B. cinerea in two genotypes which can be further exploited to develop robust and effective strategies to manage grey mould disease., Competing Interests: Ravinder Kumar is an Academic Editor for PeerJ., (© 2023 Thakur et al.)

Botrytis cinerea is an agriculturally notorious plant-pathogenic fungus with a broad host range. During plant colonization, B. cinerea secretes a wide range of plant-cell-wall-degrading enzymes (PCWDEs) that help in macerating the plant tissue, but their role in pathogenicity has been unclear. Here, we report on the identification of a transcription factor, BcXyr1, that regulates the production of (hemi-)cellulases and is necessary for fungal virulence. Deletion of the bcxyr1 gene led to impaired spore germination and reduced fungal virulence and reactive oxygen species (ROS) production in planta . Secreted proteins collected from the bcxyr1 deletion strain displayed a weaker cell-death-inducing effect than the wild-type secretome when infiltrated to Nicotiana benthamiana leaves. Transcriptome sequencing (RNA-seq) analysis revealed 41 genes with reduced expression in the Δ bcxyr1 mutant compared with those in the wild-type strain, of which half encode secreted proteins that are particularly enriched in carbohydrate-active enzyme (CAZyme)-encoding genes. Among them, we identified a novel putative expansin-like protein that was necessary for fungal virulence, supporting the involvement of BcXyr1 in the regulation of extracellular virulence factors. IMPORTANCE PCWDEs are considered important components of the virulence arsenal of necrotrophic plant pathogens. However, despite intensive research, the role of PCWDEs in the pathogenicity of necrotrophic phytopathogenic fungi remains ambiguous. Here, we demonstrate that the transcription factor BcXyr1 regulates the expression of a specific set of secreted PCWDE-encoding genes and that it is essential for fungal virulence. Furthermore, we identified a BcXyr1-regulated expansin-like gene that is required for fungal virulence. Our findings provide strong evidence for the importance of PCWDEs in the pathogenicity of B. cinerea and highlight specific PCWDEs that might be more important than others.

Botrytis is an important genus of plant pathogens causing pre- and postharvest disease on diverse crops worldwide. This study evaluated Botrytis isolates collected from strawberry, blueberry, and table grape berries in California. Isolates were evaluated for resistance to eight different fungicides, and 60 amplicon markers were sequenced (neutral, species identification, and fungicide resistance associated) distributed across 15 of the 18 B . cinerea chromosomes. Fungicide resistance was common among the populations, with resistance to pyraclostrobin and boscalid being most frequent. Isolates from blueberry had resistance to the least number of fungicides, whereas isolates from strawberry had resistance to the highest number. Host and fungicide resistance-specific population structure explained 12 and 7 to 26%, respectively, of the population variability observed. Fungicide resistance was the major driver for population structure, with select fungicides explaining up to 26% and multiple fungicide resistance explaining 17% of the variability observed. Shared and unique significant single-nucleotide polymorphisms (SNPs) associated with host and fungicide (fluopyram, thiabendazole, pyraclostrobin, and fenhexamid) resistance-associated population structures were identified. Although overlap between host and fungicide resistance SNPs were detected, unique SNPs suggest that both host and fungicide resistance play an important role in Botrytis population structure.

The relations between physical and chemical characteristics (e.g., color, firmness, volatile and non-volatile metabolites) of red ripe strawberry fruit and the natural spoilage caused by Botrytis cinerea were investigated. The spoilage rates differed between genotypes, and this was highly correlated over two successive years. Among seventeen genotypes, a more intense red coloration of the fruit skin was associated with a lower spoilage rate (r = -0.63). Additionally, weakly negative correlations were found between the levels of anthocyanins, ascorbic acid, malic acid and spoilage rates. No clear correlations were found between spoilage rates and soluble sugars, most volatiles, firmness and dry weight percentage. High levels of two volatile compounds, ethyl butanoate (r = 0.55) and 1-hexanol (r = 0.61), were correlated to high spoilage rates. These characteristics may assist strawberry breeders in selecting for genotypes with reduced susceptibility to B. cinerea., (Copyright © 2022. Published by Elsevier Ltd.)