A novel botybirnavirus with a unique satellite dsRNA causes latent infection in Didymella theifolia isolated from tea plants.

IMPORTANCE: A novel botybirnavirus, infecting the tea plant pathogen Didymella theifolia and tentatively named Didymella theifolia botybirnavirus 1 (DtBRV1), together with an additional double-stranded RNA (dsRNA), was characterized. DtBRV1 comprises two dsRNAs (1 and 2) encapsidated in isometric virions, while dsRNA3 is a satellite. The satellite represents a unique specimen since it contains a duplicated region and has high similarity to the two botybirnavirus dsRNAs, supporting the notion that it most likely originated from a deficient genomic component. The biological characteristics of DtBRV1 were further determined. With their unique molecular traits, DtBRV1 and its related dsRNA expand our understanding of virus diversity, taxonomy, and evolution.

Development of PCR based SSR markers for Wilsonomyces carpophilus and a PCR based diagnosis protocol for the early detection of shot hole disease in stone fruit crops.

BACKGROUND: The conidial Ascomycota fungus Wilsonomyces carpophilus causing shot hole in stone fruits is a major constraint in the production of stone fruits worldwide. Shothole disease symptoms appear on leaves, fruits, and twigs. Successful isolation of the pathogen from different hosts on synthetic culture medium is a time consuming and tedious procedure for identification of the pathogen based on morpho-cultural characterization. METHODS AND RESULTS: The present research was carried out to develop a successful PCR based early detection protocol for the shot hole disease of stone fruits, viz., peach, plum, apricot, cherry, and almond using the pathogen specific SSR markers developed from the Wilsonomyces carpophilus genome using Genome-wide Microsatellite Analysing Tool package (GMATA) software. Diseased leaf samples of different stone fruits were collected from the SKUAST-K orchard and the pathogen was isolated on potato dextrose agar (PDA) medium and maintained on Asthana and Hawkers' medium with a total of 50 pathogen isolates comprised of 10 isolates each from peach, plum, apricot, cherry and almond. The DNA was extracted from both healthy and infected leaf samples of different stone fruits. The DNA was also extracted from the isolated pathogen cultures (50 isolates). Out of 2851 SSR markers developed, 30 SSRs were used for the successful amplification of DNA extracted from all the 50 pathogen isolates. These SSRs were used for the amplification DNA from shot hole infected leaf samples of different stone fruits, but the amplification was not observed in the control samples (DNA from healthy leaves), thus confirming the detection of this disease directly from the shot hole infected samples using PCR based SSR markers. To our knowledge, this forms the first report of SSR development for the Wilsonomyces carpophilus and their validation for the detection of shot hole disease directly from infected leaves. CONCLUSION: PCR based SSR makers were successfully developed and used for the detection of Wilsonomyces carpophilus causing shot hole disease in stone fruits including almond in nuts for the first time. These SSR markers could successfully detect the pathogen directly from the infected leaves of stone fruits namely peach, plum, apricot and cherry including almond from the nuts.

Enabling Population Biology Studies of Stemphylium vesicarium from Onion with Microsatellites.

Stemphylium leaf blight (SLB), caused by the fungus Stemphylium vesicarium, is dominant within the foliar disease complex affecting onion production in New York (NY). The disease causes premature defoliation and significant reductions in bulb weight and quality. Foliar diseases of onion are usually managed by an intensive fungicide program, but SLB management is complicated by resistance to multiple single-site modes of action. The design of integrated disease management strategies is limited by incomplete knowledge surrounding the dominant sources of S. vesicarium inoculum. To facilitate genomic-based studies of S. vesicarium populations, nine microsatellite markers were developed. The markers were multiplexed into two PCR assays containing four and five fluorescently labeled microsatellite markers. Initial testing of the S. vesicarium isolates found the markers were highly polymorphic and reproducible with an average of 8.2 alleles per locus. The markers were used to characterize 54 S. vesicarium isolates from major NY onion production regions in 2016 (n = 27) and 2018 (n = 27). Fifty-two multilocus genotypes (MLGs) were identified between these populations. Genotypic and allelic diversities were high in both the 2016 and 2018 populations. A greater degree of genetic variation was observed within populations than between years. No distinct pattern of MLGs according to population was identified and some MLGs were closely related between 2016 and 2018. The lack of evidence for linkage among loci also was strongly suggestive of clonal populations with only minor differences between the two populations. These microsatellite markers will be a foundational resource for the testing of hypotheses surrounding the population biology of S. vesicarium and therefore informing disease management.

Identification and Characterization of the Determinants of Copper Resistance in the Acidophilic Fungus Acidomyces richmondensis MEY-1 Using the CRISPR/Cas9 System.

Copper (Cu) homeostasis has not been well documented in filamentous fungi, especially extremophiles. One of the main obstacles impeding their characterization is the lack of a powerful genome-editing tool. In this study, we applied a CRISPR/Cas9 system for efficient targeted gene disruption in the acidophilic fungus Acidomyces richmondensis MEY-1, formerly known as Bispora sp. strain MEY-1. Using this system, we investigated the basis of Cu tolerance in strain MEY-1. This strain has extremely high Cu tolerance among filamentous fungi, and the transcription factor ArAceA (A. richmondensis AceA) has been shown to be involved in this process. The ArAceA deletion mutant (ΔArAceA) exhibits specific growth defects at Cu concentrations of ≥10 mM and is transcriptionally more sensitive to Cu than the wild-type strain. In addition, the putative metallothionein ArCrdA was involved in Cu tolerance only under high Cu concentrations. MEY-1 has no Aspergillus nidulans CrpA homologs, which are targets of AceA-like transcription factors and play a role in Cu tolerance. Instead, we identified the Cu-transporting P-type ATPase ArYgA, homologous to A. nidulans YgA, which was involved in pigmentation rather than Cu tolerance. When the ΔArYgA mutant was grown on medium supplemented with Cu ions, the black color was completely restored. The lack of CrpA homologs in A. richmondensis MEY-1 and its high tolerance to Cu suggest that a novel Cu detoxification mechanism differing from the AceA-CrpA axis exists. IMPORTANCE Filamentous fungi are widely distributed worldwide and play an important ecological role as decomposers. However, the mechanisms of their adaptability to various environments are not fully understood. Various extremely acidophilic filamentous fungi have been isolated from acidic mine drainage (AMD) with extremely low pH and high heavy metal and sulfate concentrations, including A. richmondensis. The lack of genetic engineering tools, particularly genome-editing tools, hinders the study of these acidophilic and heavy metal-resistant fungi at the molecular level. Here, we first applied a CRISPR/Cas9-mediated gene-editing system to A. richmondensis MEY-1. Using this system, we identified and characterized the determinants of Cu resistance in A. richmondensis MEY-1. The conserved roles of the Cu-binding transcription factor ArAceA in Cu tolerance and the Cu-transporting P-type ATPase ArYgA in the Cu-dependent production of pigment were confirmed. Our findings provide insights into the molecular basis of Cu tolerance in the acidophilic fungus A. richmondensis MEY-1. Furthermore, the CRISPR/Cas9 system used here would be a powerful tool for studies of the mechanisms of adaptability of acidophilic fungi to extreme environments.

Antifungal susceptibility of the endophytic fungus Rhinocladiella similis (URM 7800) isolated from the Caatinga dry forest in Brazil.

The present study reports a new occurrence of Rhinocladiella similis isolated as an endophytic fungus in the Caatinga dry tropical forest in Brazil and describes its antifungal susceptibility. The isolate R. similis URM 7800 was obtained from leaves of the medicinal plant Myracrodruon urundeuva. Its morphological characterization was performed on potato dextrose agar medium and molecular analysis using the ITS rDNA sequence. The antifungal susceptibility profile was defined using the Clinical and Laboratory Standards Institute (CLSI) protocol M38-A2. The colony of isolate URM 7800 showed slow growth, with an olivaceous-gray color and powdery mycelium; in microculture, it showed the typical features of R. similis. In the antifungal susceptibility test, isolate URM 7800 showed high minimal inhibitory concentration (MIC) values for amphotericin B (>16 µg/mL), voriconazole (16 µg/mL), terbinafine (>0.5 µg/mL), and caspofungin (>8 µg/mL), among other antifungal drugs. Pathogenic melanized fungi are frequently isolated in environments where humans may be exposed, and these data show that it is essential to know if these isolates possess antifungal resistance.

Bacterial endophytes as indicators of susceptibility to Cercospora Leaf Spot (CLS) disease in Beta vulgaris L.

The fungus Cercospora beticola causes Cercospora Leaf Spot (CLS) of sugar beet (Beta vulgaris L.). Despite the global importance of this disease, durable resistance to CLS has still not been obtained. Therefore, the breeding of tolerant hybrids is a major goal for the sugar beet sector. Although recent studies have suggested that the leaf microbiome composition can offer useful predictors to assist plant breeders, this is an untapped resource in sugar beet breeding efforts. Using Ion GeneStudio S5 technology to sequence amplicons from seven 16S rRNA hypervariable regions, the most recurring endophytes discriminating CLS-symptomatic and symptomless sea beets (Beta vulgaris L.ssp. maritima) were identified. This allowed the design of taxon-specific primer pairs to quantify the abundance of the most representative endophytic species in large naturally occurring populations of sea beet and subsequently in sugar beet breeding genotypes under either CLS symptomless or infection stages using qPCR. Among the screened bacterial genera, Methylobacterium and Mucilaginibacter were found to be significantly (p < 0.05) more abundant in symptomatic sea beets with respect to symptomless. In cultivated sugar beet material under CLS infection, the comparison between resistant and susceptible genotypes confirmed that the susceptible genotypes hosted higher contents of the above-mentioned bacterial genera. These results suggest that the abundance of these species can be correlated with increased sensitivity to CLS disease. This evidence can further prompt novel protocols to assist plant breeding of sugar beet in the pursuit of improved pathogen resistance.

Analysis of endophyte diversity of two Gentiana plants species and the association with secondary metabolite.

BACKGROUND: The influence of external environmental factors on secondary metabolites of medicinal plants has always been studied. However, little is known about the relationships between endophytes and host metabolites, especially the relationship differences between different plant species. Thus, we used high-throughput sequencing methods to compare endophyte diversity from roots of two closely related species, Gentiana officinalis and G. siphonantha, from the same production area, and analyze the association with four secondary metabolites (Gentiopicroside, Loganic acid, Swertiamarine and Sweroside). RESULTS: The fungal and bacteria communities' richness and diversity of G. siphonantha was higher than G. officinalis. Ascomycota and Proteobacteria were dominant fungal and bacterial phylum of the two closely related species. At the genus level, Tetracladium and Cadophora were dominant fungal genus in G. officinalis and G. siphonantha samples, respectively. While Pseudomonas was dominant bacterial genus in two closely related species, with relative abundances were 8.29 and 8.05%, respectively. Spearman analysis showed that the content of loganic acid was significantly positively correlated with endophytic fungi, the content of gentiopicroside, swertiamarine and sweroside were significantly positively correlated with endophytic bacteria in the two related species. PICRUSt and FUNGuild predictive analysis indicated that metabolism and saprotroph was primary function of endophytic bacteria and fungi in the two related species. CONCLUSION: Our results will expand the knowledge on relationships of plant-microbe interactions and offer pivotal information to reveal the role of endophytes in the production of Gentiana plant and its important secondary metabolite.

Endophytic bacterial and fungal community compositions in different organs of ginseng (Panax ginseng).

Panax ginseng (Panax ginseng C. A. Mey.) is a perennial herb of the genus ginseng, which is used as medicine with dried roots and rhizomes. With the deepening of research on ginseng, the chemical components and pharmacological effects of ginseng have gradually been discovered. Endophytes are beneficial to host plants. However, the composition of endophytes in different organs from ginseng is poorly elucidated. The report of ginsenoside production by endophytic microbes isolated from Panax sp., motivated us to explore the endophytic microbial diversity related to the roots, stems, and leaves. In this study, the V5-V7 variable region of endophytic bacteria 16S rRNA gene and V1 variable region of endophytic fungi ITS gene in different organs were analyzed by high-throughput sequencing. The diversity and abundance of endophytic microbes in the three organs are different and are affected by the organs. For example, the most abundant endophytic bacterial genus in roots was Mycobacterium, while, the stems and leaves were Ochrobactrum. Similarly, the fungal endophytes, Coniothyrium and Cladosporium, were also found in high abundance in stems, in comparison to roots and leaves. The Shannon index shows that the diversity of endophytic bacteria in roots is the highest, and the richness of endophytic bacterial was root > stem (p < 0.05). Principal coordinate analysis showed that there were obvious microbial differences among the three groups, and the endophytic bacterial composition of the leaves was closer to that of the roots. This study provides an important reference for the study of endophytic microorganisms in ginseng.

Genomic Analyses of the Fungus Paraconiothyrium sp. Isolated from the Chinese White Wax Scale Insect Reveals Its Symbiotic Character.

The Chinese white wax scale, Ericerus pela, is an insect native to China. It harbors a variety of microbes. The Paraconiothyrium fungus was isolated from E. pela and genome sequenced in this study. A fungal cytotoxicity assay was performed on the Aedes albopictus cell line C6/36. The assembled Paraconiothyrium sp. genome was 39.55 Mb and consisted of 14,174 genes. The coding sequences accounted for 50.75% of the entire genome. Functional pathway analyses showed that Paraconiothyrium sp. possesses complete pathways for the biosynthesis of 20 amino acids, 10 of which E. pela lacks. It also had complementary genes in the vitamin B groups synthesis pathways. Secondary metabolism prediction showed many gene clusters that produce polyketide. Additionally, a large number of genes associated with 'reduced virulence' in the genome were annotated with the Pathogen-Host Interaction database. A total of 651 genes encoding carbohydrate-active enzymes were predicted to be mostly involved in plant polysaccharide degradation. Pan-specific genomic analyses showed that genes unique to Paraconiothyrium sp. were enriched in the pathways related to amino acid metabolism and secondary metabolism. GO annotation analysis yielded similar results. The top COG categories were 'carbohydrate transport and metabolism', 'lipid transport and metabolism', and 'secondary metabolite biosynthesis, transport and catabolism'. Phylogenetic analyses based on gene family and pan genes showed that Paraconiothyrium sp is clustered together with species from the Didymosphaeriaceae family. A multi-locus sequence analysis showed that it converged with the same branch as P. brasiliense and they formed one group with fungi from the Paraconiothyrium genus. To validate the in vitro toxicity of Paraconiothyrium sp., a cytotoxicity assay was performed. The results showed that medium-cultured Paraconiothyrium sp. had no harmful effect on cell viability. No toxins were secreted by the fungus during growth. Our results imply that Paraconiothyrium sp. may establish a symbiotic relationship with the host to supply complementary nutrition to E. pela.

Biosynthetic Potential of the Endophytic Fungus Helotiales sp. BL73 Revealed via Compound Identification and Genome Mining.

Endophytic fungi have been recognized as prolific producers of chemically diverse secondary metabolites. In this work, we describe a new representative of the order Helotiales isolated from the medicinal plant Bergenia pacumbis. Several bioactive secondary metabolites were produced by this Helotiales sp. BL 73 isolate grown on rice medium, including cochlioquinones and isofusidienols. Sequencing and analysis of the approximately 59-Mb genome revealed at least 77 secondary metabolite biosynthesis gene clusters, of which several could be associated with detected compounds or linked to previously reported molecules. Four terpene synthase genes identified in the BL73 genome were codon optimized and expressed, together with farnesyl-, geranyl-, and geranylgeranyl-pyrophosphate synthases, in Streptomyces spp. An analysis of recombinant strains revealed the production of linalool and its oxidized form, terpenoids typically associated with plants, as well as a yet unidentified terpenoid. This study demonstrates the importance of a complex approach to the investigation of the biosynthetic potential of endophytic fungi using both conventional methods and genome mining. IMPORTANCE Endophytic fungi represent an as yet underexplored source of secondary metabolites, of which some may have industrial and medical applications. We isolated a slow-growing fungus belonging to the order Helotiales from the traditional medicinal plant Bergenia pacumbis and characterized its potential to biosynthesize secondary metabolites. We used cultivation of the isolate with a subsequent analysis of compounds produced, bioinformatics-based mining of the genome, and heterologous expression of several terpene synthase genes. Our study revealed that this Helotiales isolate has enormous potential to produce structurally diverse natural products, including polyketides, nonribosomally synthesized peptides, terpenoids, and ribosomally synthesized and posttranslationally modified peptides (RiPPs). Identification of meroterpenoids and xanthones, along with establishing a link between these molecules and their putative biosynthetic genes, sets the stage for investigation of the respective biosynthetic pathways. The heterologous production of terpenoids suggests that this approach can be used for the discovery of new compounds belonging to this chemical class using Streptomyces bacteria as hosts.

Identification and Characterization of WRKY41, a Gene Conferring Resistance to Powdery Mildew in Wild Tomato (Solanum habrochaites) LA1777.

WRKYs, a large family of transcription factors, are involved in plant response to biotic and abiotic stresses, but the role of them in tomato resistance to Oidium neolycopersici is still unclear. In this study, we evaluate the role of WRKYs in powdery mildew-resistant wild tomato (Solanum habrochaites) LA1777 defense against O. neolycopersici strain lz (On-lz) using a combination of omics, classical plant pathology- and cell biology-based approaches. A total of 27 WRKYs, belonging to group I, II, and III, were identified as differentially expressed genes in LA1777 against On-lz. It was found that expression of ShWRKY41 was increased after Pseudomonas syringae pv. tomato (Pst) DC3000, On-lz and Botrytiscinerea B05 inoculation or ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC) treatment. GUS staining of ShWRKY41 promoter indicated that the expression of ShWRKY41 could be induced by SA and ethylene. Furthermore, ShWRKY41 gene silencing reduced the resistance to On-lz infection by decreasing the generation of H2O2 and HR in LA1777 seedlings. Overall, our research suggests that ShWRKY41 plays a positive role in defense activation and host resistance to O. neolycopersici in wild tomato (S. habrochaites) LA1777.

Functional Annotation of circRNAs in Tea Leaves After Infection by the Tea Leaf Spot Pathogen, Lasiodiplodia theobromae.

Tea leaf spot, caused by Lasiodiplodia theobromae, is an important disease that can seriously decrease the production and quality of tea (Camellia sinensis (L.) O. Kuntze) leaves. The analysis of circular RNA (circRNA) in tea leaves after infection by the pathogen could improve understanding about the mechanism of host-pathogen interactions. In this study, high-performance sequencing of circRNA from C. sinensis Fuding-dabaicha leaves that had been infected with L. theobromae was conducted using the Illumina HiSeq 4000 platform. In total, 192 and 153 differentially expressed circRNAs from tea leaves were significantly up- and downregulated, respectively, after infection with L. theobromae. A gene ontology analysis indicated that the differentially expressed circRNA-hosting genes for DNA binding were significantly enriched. The genes with significantly differential expressions that were annotated in the specified database (S genes) were σ factor E isoform 1, triacylglycerol lipase SDP1, DNA-directed RNA polymerase III subunit 2, WRKY transcription factor WRKY24, and regulator of nonsense transcripts 1 homolog. A Kyoto Encyclopedia of Genes and Genomes analysis indicated that the significantly enriched circRNA-hosting genes involved in the plant-pathogen interaction pathway were Calmodulin-domain protein kinase 5 isoform 1, probable WRKY transcription factor 33, U-box domain-containing protein 35, probable inactive receptor-like protein kinase At3g56050, WRKY transcription factor WRKY24, mitogen-activated protein kinase kinase kinase YODA, SGT1, and protein DGS1. Functional annotation of circRNAs in tea leaves infected by L. theobromae will provide a valuable resource for future research on host-pathogen interactions.

Isolation and characterization of endophytic fungi having plant growth promotion traits that biosynthesizes bacosides and withanolides under in vitro conditions.

Endophytes are regarded with immense potentials in terms of plant growth promoting (PGP) elicitors and mimicking secondary metabolites of medicinal importance. Here in the present study, we explored Bacopa monnieri plants to isolate, identify fungal endophytes with PGP elicitation potentials, and investigate secretion of secondary metabolites such as bacoside and withanolide content under in vitro conditions. Three fungal endophytes isolated (out of 40 saponin producing isolates) from leaves of B. monnieri were examined for in vitro biosynthesis of bacosides. On morphological, biochemical, and molecular identification (ITS gene sequencing), the isolated strains SUBL33, SUBL51, and SUBL206 were identified as Nigrospora oryzae (MH071153), Alternaria alternata (MH071155), and Aspergillus terreus (MH071154) respectively. Among these strains, SUBL33 produced highest quantity of Bacoside A3 (4093 µg mL-1), Jujubogenin isomer of Bacopasaponin C (65,339 µg mL-1), and Bacopasaponin C (1325 µg mL-1) while Bacopaside II (13,030 µg mL-1) was produced by SUBL51 maximally. Moreover, these aforementioned strains also produced detectable concentration of withanolides-Withaferrin A, Withanolide A (480 µg mL-1), and Withanolide B (1024 µg mL-1) respectively. However, Withanolide A was not detected in the secondary metabolites of strain SUBL51. To best of our knowledge, the present study is first reports of Nigrospora oryzae as an endophyte in B. monnieri with potentials of biosynthesis of economically important phytomolecules under in vitro conditions.

Stemphylium Leaf Blight of Welsh Onion (Allium fistulosum): An Emerging Disease in Sanxing, Taiwan.

Welsh onion (Allium fistulosum L.) is one of the main and oldest vegetable crops grown in Taiwan. A severe epidemic of leaf blight in Welsh onion caused by a Stemphylium-like pathogen was found in Sanxing, Taiwan, from 2018 to 2020. However, correct species identification, biology, and control of Stemphylium leaf blight (SLB) of Welsh onion are not well-established. Therefore, the main objective of this study was to investigate the causal agent of SLB in Sanxing and evaluate the in vitro sensitivity of Stemphylium-like pathogen to commonly used fungicides. A phylogenetic analysis based on combining the internal transcribed spacer (ITS) region and glyceraldedyhe-3-phosphate dehydrogenase (gapdh) and calmodulin (cmdA) gene sequences together with morphological features identified that S. vesicarium is associated with SLB in Sanxing. When inoculated onto Welsh onion leaves, the isolates caused symptoms identical to those observed in the field; therefore, S. vesicarium was reisolated and Koch's postulates were confirmed. We observed a higher incidence of SLB symptoms on the oldest leaves compared with younger leaves. The maximum and minimum temperatures for in vitro mycelial growth and conidial germination (%) of S. vesicarium were 20 to 30°C and 5°C, respectively. Sixteen fungicides were tested for their effectiveness to reduce the mycelial growth and conidial germination of S. vesicarium in vitro. Boscalid plus pyraclostrobin, fluopyram, fluxapyroxad, and fluxapyroxad plus pyraclostrobin were highly effective at reducing mycelial growth and conidial germination in S. vesicarium. However, strobilurin fungicides (azoxystrobin and kresoxim-methyl) commonly used in Welsh onion production in Sanxing were ineffective. This study discusses the emergence of SLB caused by S. vesicarium in the foliar disease complex affecting Welsh onion and the management of the disease using fungicides with different modes of action in Taiwan. The research will support the sustainable management of SLB in Sanxing, Taiwan; however, further field assessments of the fungicides are warranted.

Gaeumannomyces nanograminis, sp. nov., a hyphopodiate fungus identified from diseased roots of ultradwarf bermudagrass in the United States.

The genus Gaeumannomyces (Magnaporthaceae, Magnaporthales, Sordariomycetes, Ascomycota) includes root-infecting pathogens, saprobes, and endophytes. Morphological, biological, and phylogenetic analyses were employed to identify fungal isolates derived from turfgrass roots colonized with ectotrophic, dark runner hyphae. Phylogenetic trees for partial sequences of the 18S nuc rDNA, ITS1-5.8S-ITS2 nuc rDNA internal transcribed spacer, and 28S nuc rDNA regions and of the minichromosome maintenance complex 7 (MCM7), largest subunit of RNA polymerase II (RPB1), and translation elongation factor 1-alpha (TEF1) genes were obtained via maximum likelihood and Bayesian methods. Our isolates consistently formed a distinct and highly supported clade within Gaeumannomyces. Common and distinctive biological and morphological characters reinforced these findings. Additionally, we conducted pathogenicity evaluations and demonstrated the ability of this fungus to colonize roots of ultradwarf bermudagrass (Cynodon dactylon (L.) Pers. × C. transvaalensis Burtt-Davey), its native host, via ectotrophic, dark runner hyphae, causing disease symptoms including root discoloration and reduced root and shoot mass. Altogether, our discoveries enabled recognition and description of a new species, Gaeumannomyces nanograminis, associated with rotted roots of ultradwarf bermudagrass.

High-Quality Genome Assembly of Pseudopestalotiopsis theae, the Pathogenic Fungus Causing Tea Gray Blight.

Tea gray blight is one of the most serious foliar diseases of tea tree, caused by the plant-pathogenic fungus Pseudopestalotiopsis theae, which can affect production and quality of tea worldwide. We generated a highly contiguous, 50.41-Mbp genome assembly (N50 = 1.30 Mbp) of P. theae strain CYF27 by combining PacBio long-read and Illumina short-read sequencing technologies. We identified a total of 15,626 gene models, of which 1,038 genes encode putative secreted proteins. The high-quality genome assembly and annotation resource reported here will be useful for the study of fungal infection mechanisms and pathogen-host interaction.

ONT-Based Draft Genome Assembly and Annotation of Alternaria atra.

Species of Alternaria (phylum Ascomycota, family Pleosporaceae) are known as serious plant pathogens, causing major losses on a wide range of crops. Alternaria atra (previously known as Ulocladium atrum) can grow as a saprophyte on many hosts and causes Ulocladium blight on potato. It has been reported that it can also be used as a biocontrol agent against Botrytis cinerea. Here, we present a scaffold-level reference genome assembly for A. atra. The assembly contains 43 scaffolds with a total length of 39.62 Mbp, with scaffold N50 of 3,893,166 bp, L50 of 4, and the longest 10 scaffolds containing 89.9% of the assembled data. RNA-sequencing-guided gene prediction using BRAKER resulted in 12,173 protein-coding genes with their functional annotation. This first high-quality reference genome assembly and annotation for A. atra can be used as a resource for studying evolution in the highly complicated Alternaria genus and might help in understanding the mechanisms defining its role as pathogen or biocontrol agent.[Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY 4.0 International license.

Diversity and Spatiotemporal Distribution of Fungal Endophytes Associated with Salvia multicaulis.

Salvia multicaulis has been an important medicinal plant in Iran and several East Asian countries for hundreds of years. Because of growing demand, overharvesting of wild S. multicaulis has endangered its wild populations. Endophytes are well known for protecting wild plant populations against biotic and abiotic stresses, especially under harsh situations, as well as for their plant growth enhancement activities. Since no information was on endophyte biology in S. multicaulis, here we aimed at analyzing diversity and spatiotemporal distribution of fungal endophytes associating S. multicaulis in their main wild habitats in Iran, i.e., Qazvin, Alborz and Mazandaran provinces. A total of 153 fungal endophytes were isolated and identified according to their morphology and ribosomal ITS rDNA sequences. As results indicated Ascomycota dominated in colonizing S. multicaulis with a relative frequency (RF) of 96.77%, comprising of Eurotiomycetes (RF: 40.5%), Sordariomycetes (RF: 33.9%) and Dothideomycetes (RF: 20.5%). Mucoromycota, comprised the rest of endophytes (RF: 5.23%). The entire fungal microbiome was classified into nine genera including Fusarium (25.5%), Penicillium (21.5%), Aspergillus (17.0%), Alternaria (15.5%), Colletotrichum (5.2%), Rhizopus (5.2%), Macrophomina (4.5%), Trichoderma (3.25%) and Nodulisporium (2.0%). Analyses of different diversity indices indicated significant correlations with tissue type, sampling locations and season of recovery. Almost 43% of fungal endophytes were recovered at Mazandaran, Kojur; 35.4% at Qazvin, Barajin Forest Park; 30.1% at Alborz, Taleqan; and 21% at Alborz, Mahdasht. The highest overall endophyte recovery was in summer (36.8%), followed by spring (31.6%), autumn (21%), and winter (10.5%). In total, the number of endophytes recovered from roots (91) was higher than those of stems (32) and leaves (30), especially during autumn and winter. Accordingly, we conclude that Ascomycota are the major endophytic fungi colonizing S. multicaulis, and that sampling location, tissue type and season can affect the fungal endophyte composition of this medicinal plant. This knowledge could be further applied in protection and health management of this endangered species.