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Pselliophora is widely distributed in Eurasia and China. To explore the characteristics of the mitogenome of Pselliophora and reveal phylogenetic relationships, the mitogenome of Pselliophora bifascipennis Brunetti, 1911 was sequenced and annotated. This is the first complete mitochondrial genome in this genus. Its mitogenome is 15821 bp in length, containing 13 protein-coding genes, 22 tRNA and 2 rRNA genes. Nucleotide compositions of its whole mitogenome are 39.09% for A, 38.49% for T, 13.42% for C, and 9.01% for G. Consistent with previous observations of Tipulidae species, the mitogenome of Pselliophora bifascipennis is highly conserved in gene size, organization and codon usage, and secondary structures of tRNAs. Most tRNAs have the typical clover-leaf structure. The control region is 1006 bp long with an A + T content of 92.7%. Phylogenetic tree analysis using the sequences of the mitochondrial genomes of Pselliophora bifascipennis and other Tipulidae species showed that Pselliophora bifascipennis is closely related to Tanyptera hebeiensis. These two species are grouped on the same branch, which is in accordance with the traditional morphological classification. The results of this study lay a foundation for screening molecular markers of mitochondrion for molecular identification and genetic structure research in Tipulidae species.

AbstractLibelloides sibiricus (Eversmann, 1850) is widely distributed in China, Korea and eastern Russia. To date, few studies have been conducted on this species, with the exception of morphological taxonomy studies. In this study, we sequenced the complete mitochondrial genome (mitogenome) of Libelloides sibiricus, which is 15,811 bp in length, with an overall A + T content of 74.8%, encoding 2 ribosomal RNA genes, 22 transfer RNA genes, 13 protein-coding genes, and a control region. The gene arrangement and components of L. sibiricus are identical to those of most other Neuropteran species. TAA is utilized as the termination codon for most PCGs and TAG for nd1, however, nd6 and atp6 used the incomplete termination codon TA- and cox1, cox2, nd5, cytb had termination codons consisting of only T–. In addition, we selected all known 59 species of Neuroptera from NCBI, and used Sialis hamata, Sialis melania, Sialis longidens and Sialis jiyuni (Megaloptera: Sialidae) as the outgroup. Phylogenetic analysis suggested that the mitogenome of L. sibiricus was the most closely related to L. macaronius and all the owlflies formed the monophyletic group within the superfamily Myrmeleontoidea.

Abstract Citrus melanose, caused by Diaporthe citri, is one of the most important and widespread fungal diseases of citrus. Previous studies demonstrated that the citrus host was able to trigger the defense response to restrict the spread of D. citri. However, the molecular mechanism underlying this defense response has yet to be elucidated. Here, we used RNA-Seq to explore the gene expression pattern at the early (3 days post infection, dpi) and late (14 dpi) infection stages of citrus leaves in response to D. citri infection, and outlined the differences in transcriptional regulation associated with defense responses. The functional enrichment analysis indicated that the plant cell wall biogenesis was significantly induced at the early infection stage, while the callose deposition response was more active at the late infection stage. CYP83B1 genes of the cytochrome P450 family were extensively induced in the callus deposition-mediated defense response. Remarkably, the gene encoding pectin methylesterase showed the highest upregulation and was only found to be differentially expressed at the late infection stage. Genes involved in the synthesis and regulation of phytoalexin coumarin were effectively activated. F6’H1 and S8H, encoding key enzymes in the biosynthesis of coumarins and their derivatives, were more strongly expressed at the late infection stage than at the early infection stage. Collectively, our study profiled the response pattern of citrus leaves against D. citri infection and provided the transcriptional evidence to support the defense mechanism.

The escalating global population has led to an increased demand for both quantity and quality in food production. Throughout history, plant diseases have posed significant threats to agricultural output by causing substantial food losses annually while also compromising product quality. Accurate identification of pathogens, clarifying the pathogenic mechanism of pathogens, and understanding the interaction between pathogens and hosts are important for the control of plant diseases. This Special Issue, “Research Progress on Pathogenicity of Fungi in Crops”, belongs to the section “Pest and Disease Management” of Agronomy. It contains research papers on the identification and phylogeny of fungal pathogens, the molecular genetics of plant fungal pathogens, the molecular mechanisms of fungal pathogenicity, and the molecular basis of the interaction between fungi and crops. These studies encapsulate efforts to understand disease systems within current genomics, transcriptomics, proteomics, and metabolomics studies, highlighting research findings that could be future targets for crop disease and pest control. The studies presented in this Special Issue promote the progress of fungal pathogenicity research in crops and provide a scientific basis for future disease control, which is of great significance for sustainable agricultural development and global food security.

ABSTRACT The tangerine pathotype of Alternaria alternata produces a host-selective toxin termed Alternaria citri toxin (ACT). The molecular mechanisms underlying the global regulation and biosynthesis of ACT remain unknown. In the present study, the function of an APSES transcription factor StuA was investigated. StuA was shown to be required for ACT biosynthesis and fungal virulence. StuA was found, for the first time, to physically interact with a pH-responsive transcription regulator PacC using yeast two-hybrid, bimolecular fluorescence complementation, and GST pull-down assays. Functional analyses revealed that StuA and PacC regulate the expression of genes involved in toxin biosynthesis and virulence. Mutation of stuA via targeted gene deletion or silencing pacC yielded fungal strains that decreased the expression of seven toxin biosynthetic genes (ACCT) and toxin production. EMSA analyses revealed that PacC could bind to the promoters of ACTT6 encoding an enoyl-CoA hydratase and ACTTR encoding an ACT pathway-specific transcription factor. Site-directed mutagenesis of five potential protein kinase A (PKA) phosphorylation sites in StuA revealed that none of the sites was involved in ACT production, indicating that the function of StuA in the regulation of ACT gene expression is not dependent on phosphorylation. Overall, our results confirmed that PacC is one of the key regulators interacting with StuA for the biosynthesis of ACT. Environmental pH may play a decisive role during A. alternata pathogenesis. Our results also revealed a previously unrecognized (StuA-PacC)→ACTTR module for the biosynthesis of ACT in A. alternata. IMPORTANCE In this study, we used Alternaria alternata as a biological model to report the role of StuA in phytopathogenic fungi. Our findings indicated that StuA is required for Alternaria citri toxin (ACT) biosynthesis and fungal virulence. In addition, StuA physically interacts with PacC. Disruption of stuA or pacC led to decreased expression of seven toxin biosynthetic genes (ACCT) and toxin production. PacC could recognize and bind to the promoter regions of ACTT6 and ACTTR. Our results revealed a previously unrecognized (StuA-PacC)→ACTTR module for the biosynthesis of ACT in A. alternata, which also provides a framework for the study of StuA in other fungi.

Green mold, caused by Penicillium digitatum, is the major cause of citrus postharvest decay. Currently, the application of sterol demethylation inhibitor (DMI) fungicide is one of the main control measures to prevent green mold. However, the fungicide-resistance problem in the pathogen P. digitatum is growing. The regulatory mechanism of DMI fungicide resistance in P. digitatum is poorly understood. Here, we first performed transcriptomic analysis of the P. digitatum strain Pdw03 treated with imazalil (IMZ) for 2 and 12 h. A total of 1338 genes were up-regulated and 1635 were down-regulated under IMZ treatment for 2 h compared to control while 1700 were up-regulated and 1661 down-regulated under IMZ treatment for 12 h. The expression of about half of the genes in the ergosterol biosynthesis pathway was affected during IMZ stress. Further analysis identified that 84 of 320 transcription factors (TFs) were differentially expressed at both conditions, making them potential regulators in DMI resistance. To confirm their roles, three differentially expressed TFs were selected to generate disruption mutants using the CRISPR/Cas9 technology. The results showed that two of them had no response to IMZ stress while ∆PdflbC was more sensitive compared with the wild type. However, disruption of PdflbC did not affect the ergosterol content. The defect in IMZ sensitivity of ∆PdflbC was restored by genetic complementation of the mutant with a functional copy of PdflbC. Taken together, our results offer a rich source of information to identify novel regulators in DMI resistance.

BackgroundThe ruminant gastrointestinal contains numerous microbiomes that serve a crucial role in sustaining the host’s productivity and health. In recent times, numerous studies have revealed that variations in influencing factors, including the environment, diet, and host, contribute to the shaping of gastrointestinal microbial adaptation to specific states. Therefore, understanding how host and environmental factors affect gastrointestinal microbes will help to improve the sustainability of ruminant production systems.ResultsBased on a graphical analysis perspective, this study elucidates the microbial topology and robustness of the gastrointestinal of different ruminant species, showing that the microbial network is more resistant to random attacks. The risk of transmission of high-risk metagenome-assembled genome (MAG) was also demonstrated based on a large-scale survey of the distribution of antibiotic resistance genes (ARG) in the microbiota of most types of ecosystems. In addition, an interpretable machine learning framework was developed to study the complex, high-dimensional data of the gastrointestinal microbial genome. The evolution of gastrointestinal microbial adaptations to the environment in ruminants were analyzed and the adaptability changes of microorganisms to different altitudes were identified, including microbial transcriptional repair.ConclusionOur findings indicate that the environment has an impact on the functional features of microbiomes in ruminant. The findings provide a new insight for the future development of microbial resources for the sustainable development in agriculture.

Pistachio bushy top syndrome (PBTS) is a serious problem for pistachio growers in the western United States, but the cause of this disorder remains controversial. Recently, it was proposed that the Rhodococcus species R. fascians and R. corynebacterioides caused PBTS outbreaks in 2011 and 2015. To investigate the association of Rhodococcus spp. with PBTS in California's pistachio-growing region, Rhodococcus-like isolates were collected from diverse hosts and environments, including pistachio nurseries and orchards. Whole genome sequence analysis of 231 isolates revealed their evolutionary relationships and identified six Rhodococcus species. Combined with data on geography and host of origin, the data reveal that Rhodococcus generally, and R. fascians specifically, is ubiquitous in nature, frequently occurring in both symptomatic and asymptomatic pistachio trees and on other woody and native species. Core gene and single nucleotide polymorphism-based phylogenies and pangenome analyses differentiate R. fascians into distinct genotypes. Although we found examples of common genotypes shared between nurseries and orchards, the observed patterns are most consistent with an environmental source of strains and do not support a scenario in which individual nurseries are point sources of Rhodococcus. Moreover, none of the collected strains harbored known virulence genes, calling into question the role of these common environmental bacteria in causing PBTS. [Figure: see text] Copyright © 2022 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Antioxidant molecules play a crucial role in maintaining redox homeostasis, eliminating oxidative damage, and regulating pathogenesis in phytopathogenic fungi. These antioxidants function through a complex regulatory network involving enzymatic scavengers such as the superoxide dismutases (SODs), catalases, thioredoxins, and glutathiones, as well as non-enzymatic molecules. Specifically, the thioredoxin and glutathione systems (AaTrr1, AaTsa1, AaGpx3, and AaGlr1) mediated by NADPH oxidase represent vital thiol antioxidant systems utilized to regulate the detoxification of reactive oxygen species (ROS) in Alternaria alternata. This present study investigated the regulatory roles of AaBemA, a component of the fungal NADPH oxidase (Nox) complex, in the tangerine pathotype of A. alternata. Loss-of-function genetic analysis demonstrated that AaBemA is essential for the accumulation of cellular hydrogen peroxide (H2O2). Mutant strains with defective AaBemA displayed higher sensitivity to H2O2 and the ROS-generating oxidant tert-butyl-hydroperoxide. These phenotypes closely resembled those previously observed in AaNoxB, AaTrr1, AaTsa1, AaGlr1, and AaYap1 mutants, suggesting a potential interconnection among them. Notably, the defective phenotype of ΔAaBemA could be restored through genetic complementation with wildtype AaBemA gene. Subcellular localization of a functional AaBemA fused with green fluorescent protein (GFP) gene under confocal microscope revealed a widespread distribution of green fluorescence in the cytoplasm and cell membrane, indicating high expression of AaBemA during fungal growth in A. alternata. Intriguingly, inactivation of AaBemA did not affect the morphological phenotype and pathogenicity, indicating that the AaBemA is dispensable for the aforementioned phenotypes. To explore the molecular mechanisms underlying the regulation of ROS stress response, we sequenced the whole transcriptomes of A. alternata wildtype and ROS-sensitive mutants (ΔAaBemA, ΔAaNoxB, ΔAaGlr1, ΔAaTrr1, ΔAaTsa1, ΔAaYap1) which displayed considerable sensitivity to oxidants. Comparative transcriptome analysis revealed significant influence on the gene-expression pattern of numerous genes related to glutathione metabolism, cellular oxidant detoxification, cellular response to abiotic stimulus, and cellular response to osmotic stress. The gene-expression data and pathways related to various essential metabolic processes and ROS tolerance enabled us to propose a NADPH oxidase-mediated regulatory network involving NADPH oxidase (AaNoxB, AaBemA), AaYap1, glutaredoxin (AaGPx3, AaGlr1), and thioredoxin systems (AaTrr1, AaTsa1) in responding to ROS stress in A. alternata.

The Isaria cicadae, is well known highly prized medicinal mushroom with great demand in food and pharmaceutical industry. Due to its economic value and therapeutic uses, natural sources of wild I. cicadae are over-exploited and reducing continuously. Therefore, commercial cultivation in controlled environment is an utmost requirement to fulfill the consumer’s demand. Due to the lack of knowledge on fruiting body (synnemata) development and regulation, commercial cultivation is currently in a difficult situation. In the growth cycle of macrofungi, such as mushrooms, light is the main factor affecting growth and development, but so far, specific effects of light on the growth and development of I. cicadae is unknown. In this study, we identified a blue light receptor white-collar-1 (Icwc-1) gene homologue with well-defined functions in morphological development in I. cicadae based on gene knockout technology and transcriptomic analysis. It was found that the Icwc-1 gene significantly affected hyphal growth and fruiting body development. This study confirms that Icwc-1 acts as an upstream regulatory gene that regulates genes associated with fruiting body formation, pigment-forming genes, and related genes for enzyme synthesis. Transcriptome data analysis also found that Icwc-1 affects many important metabolic pathways of I. cicadae, i.e., amino acid metabolism and fatty acid metabolism. The above findings will not only provide a comprehensive understanding about the molecular mechanism of light regulation in I. cicadae, but also provide new insights for future breeding program and improving this functional food production.

Highly efficient genetic transformation technology is greatly beneficial for crop gene function analysis and precision breeding. However, the most commonly used genetic transformation technology for woody plants, mediated by Agrobacterium tumefaciens, is time-consuming and inefficient, which limits its utility for gene function analysis. In this study, a simple, universal, and highly efficient genetic transformation technology mediated by A. rhizogenes K599 is described. This technology can be applied to multiple citrus genotypes, and only 2–8 weeks were required for the entire workflow. Genome-editing experiments were simultaneously conducted using 11 plasmids targeting different genomic positions and all corresponding transformants with the target knocked out were obtained, indicating that A. rhizogenes-mediated genome editing was highly efficient. In addition, the technology is advantageous for investigation of specific genes (such as ACD2) for obtaining “hard-to-get” transgenic root tissue. Furthermore, A. rhizogenes can be used for direct viral vector inoculation on citrus bypassing the requirement for virion enrichment in tobacco, which facilitates virus-induced gene silencing and virus-mediated gene expression. In summary, we established a highly efficient genetic transformation technology bypassing tissue culture in citrus that can be used for genome editing, gene overexpression, and virus-mediated gene function analysis. We anticipate that by reducing the cost, required workload, experimental period, and other technical obstacles, this genetic transformation technology will be a valuable tool for routine investigation of endogenous and exogenous genes in citrus.

The present study provides a comprehensive overview of the Bacillus velezensis strain Htq6, and its potential applications in plant disease control. Htq6 is an endophytic bacterium derived from walnut, which was found to possess a strong inhibitory effect on a wide range of plant pathogenic microorganisms and was identified as a good plant disease control agent. The entire genome of the Bacillus velezensis Htq6 was sequenced, and a comparative genomic analysis was conducted with various Bacillus species in order to better understand the mechanism of the strain’s biological control. At the same time, a new classification result was presented. Additionally, transcriptome analysis was performed to explore the response mechanism of tomato gray mold fungus after treatment with the fermentation liquid of Bacillus velezensis Htq6. The study analyzed the distribution of various secondary metabolite gene clusters in the Bacillus model strains and employed RNA-Seq technology to obtain transcriptome expression profiles. Furthermore, the cell wall, cell membrane, and antioxidant-related genes of Botrytis cinerea were analyzed, providing insight into the antibacterial mechanism of biocontrol bacteria and the stress response mechanism of Botrytis cinerea. The results of the research are promising, and could potentially lead to the development of an effective biocontrol agent for the prevention and control of various plant diseases.

Alternaria brown spot (ABS), caused by Alternaria alternata, is an economically important fungal disease of citrus worldwide. The ABS pathogen A. alternata tangerine pathotype can produce a host-specific ACT toxin, which is regulated by ACT toxin gene cluster located in the conditionally dispensable chromosome (CDC). Previously, we have assembled a draft genome of A. alternata tangerine pathotype strain Z7, which comprises 165 contigs. In this study, we report a chromosome-level genome assembly of A. alternata Z7 through the combination of Oxford Nanopore sequencing and Illumina sequencing technologies. The assembly of A. alternata Z7 had a total size of 34.28 Mb, with a GC content of 51.01% and contig N50 of 3.08 Mb. The genome is encompassed 12,067 protein-coding genes, 34 ribosomal RNAs, and 107 transfer RNAs. Interestingly, A. alternata Z7 is composed of 10 essential chromosomes and 2 CDCs, which is consistent with the experimental evidences of pulsed-field gel electrophoresis. To our best knowledge, this is the first chromosome-level genome assembly of A. alternata. In addition, a database for citrus-related Alternaria genomes has been established to provide public resources for the sequences, annotation and comparative genomics data of Alternaria spp. The improved genome sequence and annotation at the chromosome level is a significant step toward a better understanding of the pathogenicity of A. alternata. The database will be updated regularly whenever the genomes of newly isolated Alternaria spp. are available. The citrus-related Alternaria genomes database is open accessible through the Citrus Fungal Disease Database.[Graphic: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Histone methylation, which is critical for transcriptional regulation and various biological processes in eukaryotes, is a reversible dynamic process regulated by histone methyltransferases (HMTs) and histone demethylases (HDMs). This study determined the function of 5 HMTs (AaDot1, AaHMT1, AaHnrnp, AaSet1, and AaSet2) and 1 HDMs (AaGhd2) in the phytopathogenic fungus Alternaria alternata by analyzing targeted gene deletion mutants. The vegetative growth, conidiation, and pathogenicity of ∆AaSet1 and ∆AaSet2 were severely inhibited indicating that AaSet1 and AaSet2 play critical roles in cell development in A. alternata. Multiple stresses analysis revealed that both AaSet1 and AaSet2 were involved in the adaptation to cell wall interference agents and osmotic stress. Meanwhile, ∆AaSet1 and ∆AaSet2 displayed serious vegetative growth defects in sole carbon source medium, indicating that AaSet1 and AaSet2 play an important role in carbon source utilization. In addition, ∆AaSet2 colony displayed white in color, while the wild-type colony was dark brown, indicating AaSet2 is an essential gene for melanin biosynthesis in A. alternata. AaSet2 was required for the resistance to oxidative stress. On the other hand, all of ∆AaDot1, ∆AaHMT1, and ∆AaGhd2 mutants displayed wild-type phenotype in vegetative growth, multi-stress resistance, pathogenicity, carbon source utilization, and melanin biosynthesis. To explore the regulatory mechanism of AaSet1 and AaSet2, RNA-seq of these mutants and wild-type strain was performed. Phenotypes mentioned above correlated well with the differentially expressed genes in ∆AaSet1 and ∆AaSet2 according to the KEGG and GO enrichment results. Overall, our study provides genetic evidence that defines the central role of HMTs and HDMs in the pathological and biological functions of A. alternata.

The golden–needle mushroom Flammulina filiformis is one of the most precious cultivated edible fungi in the world. Despite recent progress in the study of F. filiformis, there is still a gap in the regulation of the mitochondrial genome during browning, which poses a serious threat to the golden–needle mushroom industry. Comparative transcriptome analysis of two isonuclear alloplasmic strains showed that changes in the mitochondrial genome lead to different gene expression and key biological pathways at different stages in the two isonuclear alloplasmic strains. Furthermore, transcriptome analysis revealed that the mitochondrial genome has a significant role in the regulation of a multitude of critical metabolic pathways relating to the browning of F. filiformis fruiting bodies. Functional enrichment analysis showed that the differentially expressed genes were mainly involved in many vital processes of mitochondria, mitochondrial membrane, and multiple amino acid metabolisms of F. filiformis. Taken together, the current study highlights the crucial role of the mitochondrial genome in the growth of F. filiformis and could be beneficial to genetic breeding of elite varieties of edible fungi.

Host-pathogen interactions are the result of the continuously evolving dynamics of the genomic interphases between pathogens and the host plants. Alternaria brown spot (ABS) caused by the pathogen Alternaria alternata is a serious threat to tangerine production. Although recent studies have made significant advances in the characterization of A. alternata virulence factors, a gap exists in the regulation of virulent genes throughout the course of A. alternata infection on host plants. To gain a better understanding of the dynamic defense transcriptome in Alternaria alternata during Infection, we performed a comparative transcriptome approach. After inoculation on citrus, we found that 2142, 1964, 2359 genes were up-regulated, and 1948, 1434, 1996 genes were down-regulated at 12 hours-post-inoculation (hpi), 24 hpi and 48 hpi, respectively. Among these genes, 1333 genes were up-regulated at three time points, and 1054 genes were down-regulated, indicating that most of the differentially expressed genes at the early stage of infection tended to remain differentially expressed at the later stage of infection. In addition to the genes that are known to be part of the infection network in plant-pathogen interactions, many novel genes related to plant-pathogen interaction were identified. Interestingly, our results indicate that A. alternata is able to rapidly alter its gene expression pattern during infection process, which is vital for the successful colonization of the pathogen. Moreover, this rapid alteration of gene expression is likely to be an adaptive mechanism, enabling the pathogen to quickly respond to any changes in the environment and adapt to the host’s defense system. This ability to modify gene expression quickly in the face of environmental changes could play a critical role in the successful establishment of infection. RT-qPCR analysis confirmed that the expression pattern of nine randomly selected genes from the peroxisome pathway were consistent with the RNA-seq data. Our study provided a comprehensive study of the expression of genes during A. alternata infection of citrus, which may facilitate the understanding of host-plant interactions in A. alternata.

As a well-known edible fungus that generally grows at low temperature, Flammulina velutipes is believed to be able to withstand most undesirable microbes. However, in this study, we discovered and isolated cold-adapted bacteria that developed rapidly and caused soft rot in the stipe of F. velutipes. The pathogen was identified as Mycetocola saprophilus based on morphology, 16S rRNA sequences, and multilocus sequence typing analysis. To further understand the pathogenic mechanisms of soft rot disease in F. velutipes, we sequenced and analyzed the genome of strain JXN-3 using PacBio sequencing. The de-novo assembly of the genome of strain JXN-3 was ∼3.47 Mb, with a GC content of 66.32%. The genome was composed of 3276 genes, of which 3094 were protein coding. Many of the identified protein-coding genes were associated with the synthesis of cell wall-degrading enzymes and antifungal compounds, including chitin deacetylase, chitinase, 1,4-beta-D-glucan glucohydrolase, beta-glucanase, endoglucanase Acf2, exo-beta-1,3-glucanase and bacteriocin. Compared with other members of Mycetocola, we also found a JXN-3-unique gene cluster for synthesizing bacteriocin. These genes may contribute to the pathogenesis of soft rot in F. velutipes stipes. Future studies should further investigate the involvement of these proteins in the incidence and progression of soft rot disease in F. velutipes.

To analyze the genetic structure and genetic diversity of Colletotrichum gloeosporioides as the dominant Colletotrichum species on Citrus, the Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) genetic diversity, including 63 strains isolated and selected from 8 different sites and 5 different citrus species, was studied. A total of 19 GAPDH haplotypes were identified by genetic analysis, and the main haplotype (haplotype 5) was distributed in 28 isolates, mainly from Citrus unchiu Hort. ex Tanaka (WG) and Citrus reticulata Blanco cv. Succosa (BDZ) in Huangyan (HY), Linhai (LH), and Jiande (JD) of Zhejiang province, and Mashui tangerine (MSJ) in Mengshan of Guangxi province (GX). Using the genetic differentiation index, Fst revealed significant genetic differentiation in C. gloeosporioides populations between Jiangxi province (JXGZ) and GX, HY, LH, JD, and Chun’an (CA) of Zhejiang province, and also revealed slightly less genetic differentiation for C. gloeosporioides populations between HY, LH, JD, GX, Shaanxi province (SX), and Quzhou (QZ) of Zhejiang province. In addition, Fst revealed great genetic differentiation between the C. gloeosporioides populations obtained from MSJ and Citrus paradise Macf (PTY), and also revealed weak genetic differentiation between the C. gloeosporioides populations obtained from Citrus sinensis Osbeck (QC), WG, and BDZ. The AMOVA test showed that the levels of genetic differentiation for C. gloeosporioides were 19% and 81% among and within geographic populations, respectively. It also showed that C. gloeosporioides had levels of genetic differentiation among and within host populations of 12% and 88%, respectively. The Mantel test showed that the genetic distance was not linearly correlated with geographical distance and the haplotype phylogenetic analysis showed that C. gloeosporioides from different regions and hosts were scattered in the phylogenetic tree, implying that the genetic differentiation was independent of host variety and geographical origin. We speculated that genetic differentiation may be mainly due to gene mutation, gene recombination, or gene migration within native populations and has nothing to do with natural selection triggered by geography or host variety.

Alternaria brown spot disease is caused by the Alternaria alternata tangerine pathotype, which relies on ACT-toxin for infection. At present, all identified ACT-toxin biosynthesis-related genes are multi-copy genes. In this study, we summarized the advances in important host-specific toxins (HSTs), and listed key genes required for the pathogenicity of the A. alternata tangerine pathotype. Toxin virulence test results revealed that different citrus species displayed distinctly different tolerances to ACT-toxin. The extraction method of ACT-toxin crude extract was described in schematic form to make the method easier to understand. In addition, target gene disruption of two copies of ACTT5 (∆∆ACTT5) displayed significantly reduced virulence, indicating that ACTT5 is essential for the pathogenicity of the A. alternata tangerine pathotype.

Orthaga achatina Butler is an important pest of camphor trees in Asia. The complete mitochondrial genome of O. achatina was sequenced in this study, which was 15,150 bp in size and comprised of 13 protein-coding genes, 22 transfer RNA genes, 2 ribosomal RNA genes, and a control region. Besides, we used a phylogenomic approach to infer evolutionary relationships of O. achatina and 23 Lepidoptera species based on 13 conserved protein sequences of the mitochondrial genome. Our results underline the potential importance of mitochondrial genomes in comparative genomic analyses of Lepidoptera species and provide a robust evolutionary insight across the tree of Lepidoptera insects.

Histone acetylation, which is critical for transcriptional regulation and various biological processes in eukaryotes, is a reversible dynamic process regulated by HATs and HDACs. This study determined the function of 6 histone acetyltransferases (HATs) (Gcn5, RTT109, Elp3, Sas3, Sas2, Nat3) and 6 histone deacetylases (HDACs) (Hos2, Rpd3, Hda1, Hos3, Hst2, Sir2) in the phytopathogenic fungus Alternaria alternata by analyzing targeted gene deletion mutants. Our data provide evidence that HATs and HDACs are both required for mycelium growth, cell development and pathogenicity as many gene deletion mutants (ΔGcn5, ΔRTT109, ΔElp3, ΔSas3, ΔNat3, ΔHos2, and ΔRpd3) displayed reduced growth, conidiation or virulence at varying degrees. In addition, HATs and HDACs are involved in the resistance to multiple stresses such as oxidative stress (Sas3, Gcn5, Elp3, RTT109, Hos2), osmotic stress (Sas3, Gcn5, RTT109, Hos2), cell wall-targeting agents (Sas3, Gcn5, Hos2), and fungicide (Gcn5, Hos2). ΔGcn5, ΔSas3, and ΔHos2 displayed severe growth defects on sole carbon source medium suggesting a vital role of HATs and HDACs in carbon source utilization. More SNPs were generated in ΔGcn5 in comparison to wild-type when they were exposed to ultraviolet ray. Moreover, ΔRTT109, ΔGcn5, and ΔHos2 showed severe defects in resistance to DNA-damaging agents, indicating the critical role of HATs and HDACs in DNA damage repair. These phenotypes correlated well with the differentially expressed genes in ΔGcn5 and ΔHos2 that are essential for carbon sources metabolism, DNA damage repair, ROS detoxification, and asexual development. Furthermore, Gcn5 is required for the acetylation of H3K4. Overall, our study provides genetic evidence to define the central role of HATs and HDACs in the pathological and biological functions of A. alternata.

Isaria cicadae is a famous edible and medicinal fungus in China and Asia. The molecular basis of morphogenesis and synnemal formation needs to be understood in more detail because this is the main source of biomass production in I. cicadae. In the present study, a fruiting body formation-related gene with a glycosylphosphatidylinositol (GPI) anchoring protein (GPI-Ap) gene homolog IcFBR1 was identified by screening random insertion mutants. Targeted deletion of IcFBR1 resulted in abnormal formation of synnemata, impairing aerial hyphae growth and sporulation. The IcFBR1 mutants were defective in the utilization of carbon sources with reduced polysaccharide contents and the regulation of amylase and protease activities. Transcriptome analysis of ΔIcfbr1 showed that IcFBR1 deletion influenced 49 gene ontology terms, including 23 biological processes, 9 molecular functions, and 14 cellular components. IcFBR1 is therefore necessary for regulating synnemal development, secondary metabolism, and nutrient utilization in this important edible and medicinal fungus. This is the first report illustrating that the function of IcFBR1 is associated with the synnemata in I. cicadae.

The mitochondrial genomes have the potential to be ‘molecular clock’ due to its high mutation rate and low DNA recombination rate. Here, we sequenced the complete mitochondrial genome of three strains of Calonectria ilicicola (anamorph Cylindrocladium parasiticum) isolated from diseased peanut showing symptoms of Cylindrocladium black rot (CBR) and two C. ilicicola strains isolated from diseased soybean showing symptoms of Red crown rot (RCR). The complete mitogenome of C. ilicicola ranged from 41,187 bp to 64,421 bp in length, 22–34 protein-coding genes, 19–20 tRNA genes, 2 rRNA genes, and a control region. Besides, we used a phylogenomic approach to infer evolutionary relationships among 591 mitochondrial genomes that span the diversity of 294 fungal species, including C. ilicicola. Our results underline the potential importance of mitochondrial genomes in comparative genomic analyses and provide a robust evolutionary insight across the tree of life.

Cobalt (Co) and copper (Cu) co-exist commonly in the contaminated soils and at excessive levels, they are toxic to plants. However, their joint effect and possible interaction have not been fully addressed. In this work, a hydroponic experiment was performed to investigate the combined effects of Co and Cu on two barley genotypes at transcriptional level by RNA-seq analysis. The results identified 358 genes inclusively expressed in both genotypes under single and combined treatments of Co and Cu, with most of them being related to metal transport, stress response and transcription factor. The combined treatment induced more differently expressed genes (DEGs) than the single treatment, with Yan66, a metal tolerant genotype having more DEGs than Ea52, a sensitive genotype. The pathways associated with anthocyanin biosynthesis, MAPK signaling, glutathione biosynthesis, phenylalanine metabolism, photosynthesis, arginin biosynthesis, fatty acid elongation, and plant hormone signal transduction biosynthesis were induced and inhibited in Yan66 and Ea52, respectively. Furthermore, flavonoid biosynthesis was much more largely enhanced and accordingly more free flavonoid components (naringin, narirutin and neohesperidin) were accumulated in Yan66 than in Ea52. It may be suggested that high tolerance to both Co and Cu in Yan66 is attributed to its high gene regulatory ability.

In the present study, we identified six GATA transcription factors (AaAreA, AaAreB, AaLreA, AaLreB, AaNsdD, and AaSreA) and characterized their functions in response to environmental stress and virulence in the tangerine pathotype of Alternaria alternata. The targeted gene knockout of each of the GATA-coding genes decreased the growth to varying degrees. The mutation of AaAreA, AaAreB, AaLreB, or AaNsdD decreased the conidiation. All the GATA transcription factors were found to be required for tolerance to cumyl hydroperoxide and tert-butyl-hydroperoxide (oxidants) and Congo red (a cell-wall-destructing agent). Pathogenicity assays assessed on detached citrus leaves revealed that mutations of AaAreA, AaLreA, AaLreB, or AaNsdD significantly decreased the fungal virulence. A comparative transcriptome analysis between the ∆AreA mutant and the wild-type strain revealed that the inactivation of AaAreA led to alterations in the expression of genes involved in a number of biological processes, including oxidoreductase activity, amino acid metabolism, and secondary metabolite biogenesis. Taken together, our findings revealed that GATA-coding genes play diverse roles in response to environmental stress and are important regulators involved in fungal development, conidiation, ROS detoxification, as well as pathogenesis. This study, for the first time, systemically underlines the critical role of GATA transcription factors in response to environmental stress and virulence in A. alternata.

The fungal pathogen Diaporthe citri is a major cause of diseases in citrus. One common disease is melanose, responsible for large economic losses to the citrus fruit industry. However, very little is known about the epidemiology and genetic structure of D. citri. In this study, we analyzed 339 isolates from leaves and fruits with melanose symptoms from five provinces in southern China at 14 polymorphic simple sequence repeat (SSR) loci and the mating type idiomorphs. The genetic variations were analyzed at three levels with separate samples: among provinces, among orchards within one county, and among trees within one orchard. The five provincial populations from Fujian, Zhejiang, Jiangxi, Hunan, and Guizhou were significantly differentiated, while limited differences were found among orchards from the same county or among trees from the same orchard. STRUCTURE analysis detected two genetic clusters in the total sample, with different provincial subpopulations showing different frequencies of isolates in these two clusters. Mantel analysis showed significant positive correlation between genetic and geographic distances, consistent with geographic separation as a significant barrier to gene flow in D. citri in China. High levels of genetic diversity were found within individual subpopulations at all three spatial scales of analyses. Interestingly, most subpopulations at all three spatial scales had the two mating types in similar frequencies and with alleles at the 14 SSR loci not significantly different from linkage equilibrium. Indeed, strains with different mating types and different multilocus genotypes were frequently isolated from the same leaves and fruits. The results indicate that sexual reproduction plays an important role in natural populations of D. citri in southern China and that its ascospores likely represent an important contributor to citrus disease.

The necrotrophic fungal pathogen Alternaria alternata attacks many citrus species, causing brown spot disease. Its pathogenic capability depends primarily on the production of host-selective ACT toxin. In the current study a Ste12 transcription factor was characterized to be required for conidial formation and the production of cell-wall-degrading enzymes (CWDEs) in the tangerine pathotype of A. alternata. The Ste12 deficiency strain (ΔSte12) retained wild-type growth, ACT toxin production, and sensitivity to oxidative and osmotic stress. However, pathogenicity tests assayed on detached Dancy leaves revealed a marked reduction in virulence of ΔSte12. Transcriptome and quantitative RT-PCR analyses revealed that many genes associated with Carbohydrate-Active Enzymes (CAZymes) were downregulated in ΔSte12. Two cutinase-coding genes (AaCut3 and AaCut7) regulated by Ste12 were individually and simultaneously inactivated. The AaCut3 or AaCut7 deficiency strain unchanged in cutinase activities and incited wild-type lesions on Dancy leaves. However, the strain carrying an AaCut3 AaCut7 double mutation produced and secreted significantly fewer cutinases and incited smaller necrotic lesions than wild type. Not only is the host-selective toxin (HST) produced by A. alternata required for fungal penetration and lesion formation, but so too are CWDEs required for full virulence. Overall, this study expands our understanding of how A. alternata overcomes citrus physical barriers to carry out successful penetration and colonization.