Characterization of two novel fusariviruses co-infecting a single isolate of phytopathogenic fungus Botrytis cinerea.

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.

Botrytis cinerea Transcription Factor BcXyr1 Regulates (Hemi-)Cellulase Production and Fungal Virulence.

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 Δbcxyr1 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.

Identification of a novel laccase gene EuLAC1 and its potential resistance against Botrytis cinerea.

In this study, a novel laccase gene, EuLAC1, was cloned from Eucommia ulmoides Oliver (E. ulmoides). An overexpression vector harboring the EuLAC1 was constructed and introduced into the tobacco (Nicotiana tabacum cv. Xanthi). The laccase activity, resistance to Botrytis cinerea (B. cinerea) and lignin level in wild-type and transgenic plants were thereafter investigated. Interestingly, the transgenic tobacco displayed a significantly higher laccase activity and resistance to gray mold as compared to the wild-type tobacco. Additionally, the lignin contents in the leaves and stems of the transgenic tobacco were significantly higher in comparison to the wild-type tobacco. Scanning electron microscopy was used to observe the cross sections of wild-type and transgenic tobacco stems and it was noted that the cell wall near the xylem catheter of the transgenic tobacco was substantially thicker and the outline clearer than that of the wild-type. Thus, the EuLAC1 gene can significantly increase laccase activity and lignin content in tobacco, leading to an increase in the physical defenses, thereby increasing tobacco resistance to gray mold.

Botrytis cinerea BcSSP2 protein is a late infection phase, cytotoxic effector.

Botrytis cinerea is a broad-host-range necrotrophic phytopathogen responsible for serious diseases in leading crops. To facilitate infection, B. cinerea secretes a large number of effectors that induce plant cell death. In screening secretome data of B. cinerea during infection stage, we identified a phytotoxic protein (BcSSP2) that can also induce immune resistance in plants. BcSSP2 is a small, cysteine-rich protein without any known domains. Transient expression of BcSSP2 in leaves caused chlorosis that intensifies with time and eventually leads to death. Point mutations in eight of 10 cysteine residues abolished phytotoxicity, but residual toxic activity remained after heating treatment, suggesting contribution of unknown epitopes to protein phytotoxicity. The expression of bcssp2 was low during the first 36 h after inoculation and increased sharply upon transition to late infection stage. Deletion of bcssp2 did not cause statistically significant changes in lesions size on bean and tobacco leaves. Further analyses indicated that the phytotoxicity of BcSSP2 is negatively regulated by the receptor-like kinases BAK1 and SOBIR1. Collectively, our findings show that BcSSP2 is an effector protein that toxifies the host cells, but is also recognized by the plant immune system.

Arabidopsis CHROMATIN REMODELING 19 acts as a transcriptional repressor and contributes to plant pathogen resistance.

Chromatin remodelers act in an ATP-dependent manner to modulate chromatin structure and thus genome function. Here, we report that the Arabidopsis (Arabidopsis thaliana) remodeler CHROMATIN REMODELING19 (CHR19) is enriched in gene body regions, and its depletion causes massive changes in nucleosome position and occupancy in the genome. Consistent with these changes, an in vitro assay verified that CHR19 can utilize ATP to slide nucleosomes. A variety of inducible genes, including several important genes in the salicylic acid (SA) and jasmonic acid (JA) pathways, were transcriptionally upregulated in the chr19 mutant under normal growth conditions, indicative of a role of CHR19 in transcriptional repression. In addition, the chr19 mutation triggered higher susceptibility to the JA pathway-defended necrotrophic fungal pathogen Botrytis cinerea, but did not affect the growth of the SA pathway-defended hemibiotrophic bacterial pathogen Pseudomonas syringae pv. tomato DC3000. Expression of CHR19 was tissue-specific and inhibited specifically by SA treatment. Such inhibition significantly decreased the local chromatin enrichment of CHR19 at the associated SA pathway genes, which resulted in their full activation upon SA treatment. Overall, our findings clarify CHR19 to be a novel regulator acting at the chromatin level to impact the transcription of genes underlying plant resistance to different pathogens.

Identification of the Viral Determinant of Hypovirulence and Host Range in Sclerotiniaceae of a Genomovirus Reconstructed from the Plant Metagenome.

Uncharacterized viral genomes that encode circular replication-associated proteins of single-stranded DNA viruses have been discovered by metagenomics/metatranscriptomics approaches. Some of these novel viruses are classified in the newly formed family Genomoviridae. Here, we determined the host range of a novel genomovirus, SlaGemV-1, through the transfection of Sclerotinia sclerotiorum with infectious clones. Inoculating with the rescued virions, we further transfected Botrytis cinerea and Monilinia fructicola, two economically important members of the family Sclerotiniaceae, and Fusarium oxysporum. SlaGemV-1 causes hypovirulence in S. sclerotiorum, B. cinerea, and M. fructicola. SlaGemV-1 also replicates in Spodoptera frugiperda insect cells but not in Caenorhabditis elegans or plants. By expressing viral genes separately through site-specific integration, the replication protein alone was sufficient to cause debilitation. Our study is the first to demonstrate the reconstruction of a metagenomically discovered genomovirus without known hosts with the potential of inducing hypovirulence, and the infectious clone allows for studying mechanisms of genomovirus-host interactions that are conserved across genera. IMPORTANCE Little is known about the exact host range of widespread genomoviruses. The genome of soybean leaf-associated gemygorvirus-1 (SlaGemV-1) was originally assembled from a metagenomic/metatranscriptomic study without known hosts. Here, we rescued SlaGemV-1 and found that it could infect three important plant-pathogenic fungi and fall armyworm (S. frugiperda Sf9) insect cells but not a model nematode, C. elegans, or model plant species. Most importantly, SlaGemV-1 shows promise for inducing hypovirulence of the tested fungal species in the family Sclerotiniaceae, including Sclerotinia sclerotiorum, Botrytis cinerea, and Monilinia fructicola. The viral determinant of hypovirulence was further identified as replication initiation protein. As a proof of concept, we demonstrate that viromes discovered in plant metagenomes can be a valuable genetic resource when novel viruses are rescued and characterized for their host range.

Population Genomics Reveals Molecular Determinants of Specialization to Tomato in the Polyphagous Fungal Pathogen Botrytis cinerea in France.

Many fungal plant pathogens encompass multiple populations specialized on different plant species. Understanding the factors underlying pathogen adaptation to their hosts is a major challenge of evolutionary microbiology, and it should help to prevent the emergence of new specialized pathogens on novel hosts. Previous studies have shown that French populations of the gray mold pathogen Botrytis cinerea parasitizing tomato and grapevine are differentiated from each other, and have higher aggressiveness on their host of origin than on other hosts, indicating some degree of host specialization in this polyphagous pathogen. Here, we aimed at identifying the genomic features underlying the specialization of B. cinerea populations to tomato and grapevine. Based on whole genome sequences of 32 isolates, we confirmed the subdivision of B. cinerea pathogens into two genetic clusters on grapevine and another, single cluster on tomato. Levels of genetic variation in the different clusters were similar, suggesting that the tomato-specific cluster has not recently emerged following a bottleneck. Using genome scans for selective sweeps and divergent selection, tests of positive selection based on polymorphism and divergence at synonymous and nonsynonymous sites, and analyses of presence and absence variation, we identified several candidate genes that represent possible determinants of host specialization in the tomato-associated population. This work deepens our understanding of the genomic changes underlying the specialization of fungal pathogen populations.

CcPmk1 is a regulator of pathogenicity in Cytospora chrysosperma and can be used as a potential target for disease control.

Fus3/Kss1, also known as Pmk1 in several pathogenic fungi, is a component of the mitogen-activated protein kinase (MAPK) signalling pathway that functions as a regulator in fungal development, stress response, mating, and pathogenicity. Cytospora chrysosperma, a notorious woody plant-pathogenic fungus, causes canker disease in many species, and its Pmk1 homolog, CcPmk1, is required for fungal development and pathogenicity. However, the global regulation network of CcPmk1 is still unclear. In this study, we compared transcriptional analysis between a CcPmk1 deletion mutant and the wild type during the simulated infection process. A subset of transcription factor genes and putative effector genes were significantly down-regulated in the CcPmk1 deletion mutant, which might be important for fungal pathogenicity. Additionally, many tandem genes were found to be regulated by CcPmk1. Eleven out of 68 core secondary metabolism biosynthesis genes and several gene clusters were significantly down-regulated in the CcPmk1 deletion mutant. GO annotation of down-regulated genes showed that the ribosome biosynthesis-related processes were over-represented in the CcPmk1 deletion mutant. Comparison of the CcPmk1-regulated genes with the Pmk1-regulated genes from Magnaporthe oryzae revealed only a few overlapping regulated genes in both CcPmk1 and Pmk1, while the enrichment GO terms in the ribosome biosynthesis-related processes were also found. Subsequently, we calculated that in vitro feeding artificial small interference RNAs of CcPmk1 could silence the target gene, resulting in inhibited fungal growth. Furthermore, silencing of BcPmk1 in Botrytis cinerea with conserved CcPmk1 and BcPmk1 fragments could significantly compromise fungal virulence using the virus-induced gene silencing system in Nicotiana benthamiana. These results suggest that CcPmk1 functions as a regulator of pathogenicity and can potentially be designed as a target for broad-spectrum disease control, but unintended effects on nonpathogenic fungi need to be avoided.

Characterization of the Populations of Botrytis cinerea Infecting Plastic Tunnel-Grown Strawberry and Tomato in the Hubei Province of China.

A total of 707 isolates of Botrytis were collected from plastic tunnel-grown strawberry and tomato in the Hubei province of China. They were identified based on the specific molecular markers. Diversity of the B. cinerea (Bc) isolates was evaluated by typing the transposable elements (Boty, Flipper) and the mating types (MAT1-1, MAT1-2), as well as by determining virulence on tobacco (Nicotiana benthamiana) and fenhexamid sensitivity in agar medium. The results showed that 706 isolates (99.9%) were Bc and 1 isolate (0.1%) was B. pseudocinerea. The Bc isolates (n = 706) were classified into four transposable element types, Vacuma (3.1%), Boty (9.6%), Flipper (18.4%), and Transposa (68.8%). The strawberry and tomato subpopulations of Bc had significantly different (P < 0.05) compositions of the four transposable element types. The overall ratio of MAT1-1 to MAT1-2 deviated from 1:1 (n = 706; P = 0.0002), and MAT1-2 (56.9%) predominated over MAT1-1 (43.1%). In 7 of 12 geographic subpopulations, the ratio of MAT1-1 to MAT1-2 matched 1:1; however, in the remaining five geographic subpopulations, the ratio of MAT1-1 to MAT1-2 did not match 1:1. Results of the biological characterizations showed that most Bc isolates were highly sensitive or sensitive to fenhexamid, and the majority of Bc isolates were highly virulent or virulent on tobacco. Moreover, the relationship between genetic diversity and biological characteristics was analyzed. The results achieved during this study are helpful for understanding of the populations of B. cinerea.

Defects in the Ferroxidase That Participates in the Reductive Iron Assimilation System Results in Hypervirulence in Botrytis Cinerea.

The plant pathogen Botrytis cinerea is responsible for gray-mold disease, which infects a wide variety of species. The outcome of this host-pathogen interaction, a result of the interplay between plant defense and fungal virulence pathways, can be modulated by various environmental factors. Among these, iron availability and acquisition play a crucial role in diverse biological functions. How B. cinerea obtains iron, an essential micronutrient, during infection is unknown. We set out to determine the role of the reductive iron assimilation (RIA) system during B. cinerea infection. This system comprises the BcFET1 ferroxidase, which belongs to the multicopper oxidase (MCO) family of proteins, and the BcFTR1 membrane-bound iron permease. Gene knockout and complementation studies revealed that, compared to the wild type, the bcfet1 mutant displays delayed conidiation, iron-dependent sclerotium production, and significantly reduced whole-cell iron content. Remarkably, this mutant exhibited a hypervirulence phenotype, whereas the bcftr1 mutant presents normal virulence and unaffected whole-cell iron levels and developmental programs. Interestingly, while in iron-starved plants wild-type B. cinerea produced slightly reduced necrotic lesions, the hypervirulence phenotype of the bcfet1 mutant is no longer observed in iron-deprived plants. This suggests that B. cinerea bcfet1 knockout mutants require plant-derived iron to achieve larger necrotic lesions, whereas in planta analyses of reactive oxygen species (ROS) revealed increased ROS levels only for infections caused by the bcfet1 mutant. These results suggest that increased ROS production, under an iron sufficiency environment, at least partly underlie the observed infection phenotype in this mutant.IMPORTANCE The plant-pathogenic fungus B. cinerea causes enormous economic losses, estimated at anywhere between $10 billion and $100 billion worldwide, under both pre- and postharvest conditions. Here, we present the characterization of a loss-of-function mutant in a component involved in iron acquisition that displays hypervirulence. While in different microbial systems iron uptake mechanisms appear to be critical to achieve full pathogenic potential, we found that the absence of the ferroxidase that is part of the reductive iron assimilation system leads to hypervirulence in this fungus. This is an unusual and rather underrepresented phenotype, which can be modulated by iron levels in the plant and provides an unexpected link between iron acquisition, reactive oxygen species (ROS) production, and pathogenesis in the Botrytis-plant interaction.

Transcriptome analysis of Botrytis cinerea in response to tea tree oil and its two characteristic components.

Tea tree oil (TTO) and its two characteristic components (terpinen-4-ol and 1,8-cineole) have been shown to inhibit Botrytis cinerea growth. In this study, we conducted a transcriptome analysis to determine the effects of TTO and its characteristic components, alone and in combination, against B. cinerea. Most differentially expressed genes (DEGs) from B. cinerea cells treated with terpinen-4-ol participated in the biosynthesis of secondary metabolites, and the metabolism of amino acids, carbohydrates, and lipids. All treatments containing terpinen-4-ol potentially induced mitochondrial dysfunction and oxidative stress. These were further confirmed by the decreased activities of several enzymes (e.g., succinate dehydrogenase (SDH), malate dehydrogenase (MDH), α-ketoglutarate dehydrogenase (α-KGDH), isocitrate dehydrogenase (ICDH)), the increased activities of certain enzymes (e.g., catalase (CAT), peroxidase (POD), superoxide dismutase (SOD)), and increased content of hydrogen peroxide (H2O2). 1,8-Cineole mainly affected DEGs involved in genetic information processing, resulting in cell death. This study provides insight into the molecular mechanism of B. cinerea inhibition by TTO, and explains the synergistic effect of terpinen-4-ol and 1,8-cineole on B. cinerea.

The H3K4 demethylase Jar1 orchestrates ROS production and expression of pathogenesis-related genes to facilitate Botrytis cinerea virulence.

Histone 3 Lysine 4 (H3K4) demethylation is ubiquitous in organisms, however the roles of H3K4 demethylase JARID1(Jar1)/KDM5 in fungal development and pathogenesis remain largely unexplored. Here, we demonstrate that Jar1/KDM5 in Botrytis cinerea, the grey mould fungus, plays a crucial role in these processes. The BcJAR1 gene was deleted and its roles in fungal development and pathogenesis were investigated using approaches including genetics, molecular/cell biology, pathogenicity and transcriptomic profiling. BcJar1 regulates H3K4me3 and both H3K4me2 and H3K4me3 methylation levels during vegetative and pathogenic development, respectively. Loss of BcJAR1 impairs conidiation, appressorium formation and stress adaptation; abolishes infection cushion (IC) formation and virulence, but promotes sclerotium production in the ΔBcjar1 mutants. BcJar1 controls reactive oxygen species (ROS) production and proper assembly of Sep4, a core septin protein and virulence determinant, to initiate infection structure (IFS) formation and host penetration. Exogenous cAMP partially restored the mutant appressorium, but not IC, formation. BcJar1 orchestrates global expression of genes for ROS production, stress response, carbohydrate transmembrane transport, secondary metabolites, etc., which may be required for conidiation, IFS formation, host penetration and virulence of the pathogen. Our work systematically elucidates BcJar1 functions and provides novel insights into Jar1/KDM5-mediated H3K4 demethylation in regulating fungal development and pathogenesis.

Comparative quantitative proteomics of osmotic signal transduction mutants in Botrytis cinerea explain mutant phenotypes and highlight interaction with cAMP and Ca2+ signalling pathways.

Signal transduction (ST) is essential for rapid adaptive responses to changing environmental conditions. It acts through rapid post-translational modifications of signalling proteins and downstream effectors that regulate the activity and/or subcellular localisation of target proteins, or the expression of downstream genes. We have performed a quantitative, comparative proteomics study of ST mutants in the phytopathogenic fungus Botrytis cinerea during axenic growth under non-stressed conditions to decipher the roles of two kinases of the hyper-osmolarity pathway in B. cinerea physiology. We studied the mutants of the sensor histidine kinase Bos1 and of the MAP kinase Sak1. Label-free shotgun proteomics detected 2425 proteins, 628 differentially abundant between mutants and wild-type, 270 common to both mutants, indicating independent and shared regulatory functions for both kinases. Gene ontology analysis showed significant changes in functional categories that may explain in vitro growth and virulence defects of both mutants (secondary metabolism enzymes, lytic enzymes, proteins linked to osmotic, oxidative and cell wall stress). The proteome data also highlight a new link between Sak1 MAPK, cAMP and Ca2+ signalling. This study reveals the potential of proteomic analyses of signal transduction mutants to decipher their biological functions. TEXT-VULGARISATION: The fungus Botrytis cinerea is responsible for grey mold disease of hundreds of plant species. During infection, the fungus has to face important changes of its environment. Adaptation to these changing environmental conditions involves proteins of such called signal transduction pathways that regulate the production, activity or localisation of cellular components, mainly proteins. While the components of such signal transduction pathways are well known, their role globally understood, the precise impact on protein production remains unknown. In this study we have analysed and compared the global protein content of two Botrytis cinerea signal transduction mutants - both avirulent - to the pathogenic parental strain. The data of 628 differential proteins between mutants and wild-type, showed significant changes in proteins related to plant infection (secondary metabolism enzymes, lytic enzymes, proteins linked to osmotic, oxidative and cell wall stress) that may explain the virulence defects of both mutants. Moreover, we observed intracellular accumulation of secreted proteins in one of the mutants suggesting a potential secretion defect.

Ethylene and Benzaldehyde Emitted from Postharvest Tomatoes Inhibit Botrytis cinerea via Binding to G-Protein Coupled Receptors and Transmitting with cAMP-Signal Pathway of the Fungus.

Tomato storage conditions are difficult largely due to Botrytis cinerea infection which causes gray mold disease. However, the effects of the volatile organic compounds (VOCs) emitted by postharvest tomatoes on this fungus remain unclear. We analyzed the effects of tomato-emitted VOCs on B. cinerea pathogenicity, germination, and hyphal growth with bioassay, predicted the causative active compounds by principle component analysis, identified G-protein-coupled receptors (GPCRs) which captured chemical signals in the B. cinerea genome by stimulating molecular docking, tested the binding affinities of these receptors for the active compounds by fluorescence binding competition assay, and identified an associated signaling pathway by RNA interfere. The VOCs emitted by postharvest tomatoes inhibited B. cinerea; ethylene and benzaldehyde were the active compounds causing this effect. One of the identified GPCRs in B. cinerea, BcGPR3, bound tightly to both active compounds. Two genes associated with the cAMP signaling pathway (BcRcn1 and BcCnA) were downregulated in wild-type B. cinerea exposed to the active compounds, as well as in the ΔBcgpr3 B. cinerea mutant. Exposure to postharvest tomato VOCs reduces B. cinerea pathogenicity due to ethylene and benzaldehyde volatiles. The BcGPR3 protein is inactivated by the active compounds, and thus fails to transmit signals to the cAMP pathway, thereby inhibiting B. cinerea.

The polyphagous plant pathogenic fungus Botrytis cinerea encompasses host-specialized and generalist populations.

The host plant is often the main variable explaining population structure in fungal plant pathogens, because specialization contributes to reduce gene flow between populations associated with different hosts. Previous population genetic analysis revealed that French populations of the grey mould pathogen Botrytis cinerea were structured by hosts tomato and grapevine, suggesting host specialization in this highly polyphagous pathogen. However, these findings raised questions about the magnitude of this specialization and the possibility of specialization to other hosts. Here we report specialization of B. cinerea populations to tomato and grapevine hosts but not to other tested plants. Population genetic analysis revealed two pathogen clusters associated with tomato and grapevine, while the other clusters co-occurred on hydrangea, strawberry and bramble. Measurements of quantitative pathogenicity were consistent with host specialization of populations found on tomato, and to a lesser extent, populations found on grapevine. Pathogen populations from hydrangea and strawberry appeared to be generalist, while populations from bramble may be weakly specialized. Our results suggest that the polyphagous B. cinerea is more accurately described as a collection of generalist and specialist individuals in populations. This work opens new perspectives for grey mould management, while suggesting spatial optimization of crop organization within agricultural landscapes.

A case report of pulmonary Botrytis sp. infection in an apparently healthy individual.

BACKGROUND: Botrytis species are well known fungal pathogens of various plants but have not been reported as human pathogens, except as allergenic precipitants of asthma and hypersensitivity pneumonitis. CASE PRESENTATION: The asymptomatic patient was referred because of a nodule revealed by chest X-ray. Computed tomography (CT) showed a cavitary nodule in the right upper lobe of the lung. He underwent wedge resection of the nodule, which revealed necrotizing granulomas and a fungus ball containing Y-shaped filamentous fungi, which was confirmed histopathologically. Culture of the specimen yielded white to grayish cotton-like colonies with black sclerotia. We performed multilocus gene sequence analyses including three single-copy nuclear DNA genes encoding glyceraldehyde-3-phosphate dehydrogenase, heat-shock protein 60, and DNA-dependent RNA polymerase subunit II. The analyses revealed that the isolate was most similar to Botrytis elliptica. To date, the pulmonary Botrytis sp. infection has not recurred after lung resection and the patient did not require any additional medication. CONCLUSIONS: We report the first case of an immunocompetent patient with pulmonary Botrytis sp. infection, which has not recurred after lung resection without any additional medication. Precise evaluation is necessary for the diagnosis of pulmonary Botrytis infection because it is indistinguishable from other filamentous fungi both radiologically and by histopathology. The etiology and pathophysiology of pulmonary Botrytis infection remains unclear. Further accumulation and analysis of Botrytis cases is warranted.

NADPH Oxidase Is Crucial for the Cellular Redox Homeostasis in Fungal Pathogen Botrytis cinerea.

During interactions, both plants and pathogens produce reactive oxygen species (ROS). Plants generate ROS for defense induction, while pathogens synthesize ROS for growth, sporulation, and virulence. NADPH oxidase (NOX) complex in the plasma membrane represents a main protein complex for ROS production in pathogens. Although NOX plays a crucial role in pathogenicity of pathogens, the underlying molecular mechanisms of NOX, especially the proteins regulated by NOX, remain largely unknown. Here, we applied an iodoacetyl tandem mass tag-based redox proteomic assay to investigate the protein redox dynamics in deletion mutant of bcnoxR, which encodes a regulatory subunit of NOX in the fungal pathogen Botrytis cinerea. In total, 214 unique peptidyl cysteine (Cys) thiols from 168 proteins were identified and quantified in both the wild type and ∆bcnoxR mutant. The Cys thiols in the ∆bcnoxR mutant were generally more oxidized than those in the wild type, suggesting that BcNoxR is essential for maintaining the equilibrium of the redox state in B. cinerea. Site-specific thiol oxidation analysis indicated that 142 peptides containing the oxidized thiols changed abundance significantly in the ∆bcnoxR mutant. Proteins containing these differential peptides are classified into various functional categories. Functional analysis revealed that one of these proteins, 6-phosphate dehydrogenase, played roles in oxidative stress response and pathogenesis of B. cinerea. These results provide insight into the potential target proteins and the ROS signal transduction pathway regulated by NOX.

Botrytis polyphyllae: A New Botrytis Species Causing Gray Mold on Paris polyphylla.

Paris polyphylla is an important perennial medicinal plant in China. A disease similar to gray mold on P. polyphylla occurred at the seedling stage in March 2016 and 2017 in Tengchong city, Yunnan Province of China. The disease resulted in up to 50% mortality in serious cases. Isolates from diseased plants grew 10.6 mm/day at 20°C on PDA. After 21 days, sclerotia were spherical to elliptical (0.4-2.5 × 0.3-1.8 mm). Conidia from diseased tissues were hyaline to pale brown, long, ovoid, unicellular, and measured 15.1-24.5 × 8.8-13.4 µm; conidiophores were 526-1,064 ×12-15 µm. Isolates did not form conidiophores or conidia on PDA or MYA. A phylogenetic analysis based on G3PDH, RPB2, and HSP60 sequence data supported assignment of three representative isolates as a new species of Botrytis. Based on morphological, phylogenetic characteristics and Koch's Postulates, the causal agent of gray mold on P. polyphylla was identified as a novel species, Botrytis polyphyllae.

Involvement of the cysteine protease BcAtg4 in development and virulence of Botrytis cinerea.

Autophagy serves as a survival mechanism against starvation and has been reported to be important for cell growth and differentiation in eukaryotes. Here, we investigated the function of a cysteine protease BcAtg4 in the gray mold fungus Botrytis cinerea. Yeast complementation experiments revealed that Bcatg4 can functionally replace the counterpart of yeast. Subcellular localization exhibited that BcAtg4 diffused in cytoplasm at different developmental stages. Targeted gene deletion of Bcatg4 (ΔBcatg4) led to autophagy blocking and a significant retardation in growth and conidiation. In addition, ΔBcatg4 failed to form sclerotia. Infection tests demonstrated that ΔBcatg4 was severely attenuated in virulence on different host plant tissues. All of the phenotypic defects were restored by reintroducing an intact copy of Bcatg4 into ΔBcatg4. These results indicate that Bcatg4 plays multiple roles in the developmental processes and pathogenesis of B. cinerea.

Characterization of Botrytis cinerea isolates collected on pepper in Southern Turkey by using molecular markers, fungicide resistance genes and virulence assay.

Botrytis cinerea is a polyphagous fungal pathogen causing gray mold disease. Moreover, it is one of the most destructive infections of small fruit crops such as pepper (Capsicum annnum L.). C. sativum is a species belonging to the Solanaceae family and Turkey is one of the main producers in the World. In the present work, aiming to obtain information useful for pest management, fifty B. cinerea isolates collected from Turkey and a reference isolate (B05.10) were characterized using molecular markers and fungicide resistance genes. Morphological and molecular (ITS1-ITS4) identification of B. cinerea isolates, the degree of virulence and mating types were determined. Since one or several allelic mutations in the histidine kinase (Bos1) and ß-tubulin genes generally confer the resistance to fungicides, the sequences of these target genes were investigated in the selected isolates, which allowed the identification of two different haplotypes. Mating types were also determined by PCR assays using primer specific for MAT1-1 alpha gene (MAT1-1-1) and MAT1-2 HMG (MAT1-2-1) of B. cinerea. Twenty-two out of 50 isolates (44%) were MAT1-2, while 38% were MAT1-1. Interestingly, out of whole studied samples, 9 isolates (18%) were heterokaryotic or mixed colonies. In addition, cluster and population structure analyses identified five main groups and two genetic pools, respectively, underlining a good level of variability in the analysed panel. The results highlighted the presence of remarkable genetic diversity in B. cinerea isolates collected in a crucial economical area for pepper cultivation in Turkey and the data will be beneficial in view of future gray mold disease management.