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Alternaria species (Deuteromycetes, Ascomycota) as ubiquitous fungi and prolific producers of a variety of toxic compounds are a part of microbiomes of plants, humans, and animals, mainly causing disease, allergic reactions, and toxicosis. However, some species have also been reported as endophytic microorganisms with highly bioactive metabolites. Our previous results indicate that potentially endophytic Alternaria species from Cupressaceae produce bioactive metabolites that possibly contribute to plant holobiont's health. Here, a possible mechanism behind this bioactivity is elucidated. As some endophytic fungi are reported to produce cytotoxic taxane diterpenoids, eight potentially endophytic Alternaria isolates from our collection were analyzed for the presence of the key genes of the paclitaxel (Taxol) biosynthetic pathway, i.e., taxadin synthase (ts), 10-deacetylbaccatin III-10-O-acetyltransferase (dbat), and C-13-phenylpropanoid side-chain CoA acyltransferase (bapt). The presence of all genes, i.e., ts, dbat, and bapt, was detected by PCR in six isolates and dbat and bapt in two isolates. Chemical analyses of the fermentation broths by TLC and HPLC chromatography and IR spectroscopy indicated the synthesis of the final product, i.e., paclitaxel. So, we introduce the synthesis of taxane diterpenoids as a possible mechanism by which Alternaria occupies the plant niches and protects the plant holobiont in the presence of competing microorganisms., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Background: Black spot disease in tree peony caused by the fungal necrotroph A. alternata, is a primary limiting factor in the production of the tree peony. The intricate molecular mechanisms underlying the tree peony resistance to A. alternata have not been thoroughly investigated., Results: The present study utilized high-throughput RNA sequencing (RNA-seq) technology to conduct global expression profiling, revealing an intricate network of genes implicated in the interaction between tree peony and A. alternata. RNA-Seq libraries were constructed from leaf samples and high-throughput sequenced using the BGISEQ-500 sequencing platform. Six distinct libraries were characterized. M1, M2 and M3 were derived from leaves that had undergone mock inoculation, while I1, I2 and I3 originated from leaves that had been inoculated with the pathogen. A range of 10.22-11.80 gigabases (Gb) of clean bases were generated, comprising 68,131,232 - 78,633,602 clean bases and 56,677 - 68,996 Unigenes. A grand total of 99,721 Unigenes were acquired, boasting a mean length of 1,266 base pairs. All these 99,721 Unigenes were annotated in various databases, including NR (Non-Redundant, 61.99%), NT (Nucleotide, 45.50%), SwissProt (46.32%), KEGG (Kyoto Encyclopedia of Genes and Genomes, 49.33%), KOG (clusters of euKaryotic Orthologous Groups, 50.18%), Pfam (Protein family, 47.16%), and GO (Gene Ontology, 34.86%). In total, 66,641 (66.83%) Unigenes had matches in at least one database. By conducting a comparative transcriptome analysis of the mock- and A. alternata-infected sample libraries, we found differentially expressed genes (DEGs) that are related to phytohormone signalling, pathogen recognition, active oxygen generation, and circadian rhythm regulation. Furthermore, multiple different kinds of transcription factors were identified. The expression levels of 10 selected genes were validated employing qRT-PCR (quantitative real-time PCR) to confirm RNA-Seq data., Conclusions: A multitude of transcriptome sequences have been generated, thus offering a valuable genetic repository for further scholarly exploration on the immune mechanisms underlying the tree peony infected by A. alternata. While the expression of most DEGs increased, a few DEGs showed decreased expression., (© 2024. The Author(s).)

Alternaria is a large genus within Pleosporaceae and consists of fungi that have up to recently been considered to be 15 separate genera, including Ulocladium . The majority of Ulocladium species after incorporation into Alternaria were placed in three sections: Ulocladioides, Pseudoulocladium , and Ulocladium . In this study, phylogeny of 26 reference strains of 22 species and 20 Russian Ulocladium -like isolates was recovered. The partial actin gene ( act ), Alternaria major allergen ( alta1 ), calmodulin ( cal ), glyceraldehyde-3-phosphate dehydrogenase ( gapdh ), RNA polymerase II second largest subunit ( rpb2 ), and translation elongation factor 1-α ( tef1 ) were sequenced for Russian isolates. All these fungi were examined using multilocus phylogenetic analysis according to the genealogical concordance phylogenetic species recognition (GCPSR) principle and the coalescent-based model Poisson tree processes (PTP, mPTP) and evaluated for the presence of recombination. All strains were combined into two clades that corresponded to the Pseudoulocladium and Ulocladioides sections. The Pseudoulocladium clade included four reference strains and nine local isolates and considered to be a single species, whereas the Ulocladioides section comprises 11 species, instead of 17 names previously adopted. Nine species were abolished by joining four other species. Species A. atra and A. multiformis were combined into the single species A. atra . Five species, A. brassicae-pekinensis, A. consortialis, A. cucurbitae, A. obovoidea , and A. terricola , were united in the species A. consortialis. Alternaria heterospora and A. subcucurbitae were combined into one species, A. subcucurbitae. Alternaria aspera, A. chartarum, A. concatenata , and A. septospora were combined into a single species, A. chartarum . Also, amplification with two different primer sets was performed to define mating-type locus 1 ( MAT1 ) idiomorph. All studied isolates were heterothallic, contradicting some prior studies. Twenty Russian Ulocladium -like isolates were assigned to five species of two sections, A. atra, A. cantlous, A. chartarum, A. consortialis , and A. subcucurbitae . Species A. cantlous and A. subcucurbitae were found in Russia for the first time.

Species in Alternaria sections Infectoriae and Pseudoalternaria are commonly isolated from agricultural crops and a variety of other plant hosts. With the increasing appreciation that species from these two sections are often the dominant taxa recovered from important cereal crops, the need for improved understanding of their biodiversity and taxonomy has grown. Given that morphological characteristics and existing molecular markers are not sufficient for distinguishing among species, we expanded the genomic resources for these sections to support research in biosystematics and species diagnostics. Whole genome assemblies for 22 strains were generated, including the first genomes from section Infectoriae or Pseudoalternaria strains sampled from Canada, which significantly increases the number of publicly released genomes, particularly for section Pseudoalternaria . We performed comprehensive phylogenomic analyses of all available genomes (n = 39) and present the first robust phylogeny for these taxa. The segregation of the two sections was strongly supported by genomewide data, and multiple lineages were detected within each section. We then provide an overview of the biosystematics of these groups by analyzing two standard molecular markers from the largest sample of section Infectoriae and Pseudoalternaria strains studied to date. The patterns of relative diversity suggest that, in many cases, multiple species described based on minor morphological differences may actually represent different strains of the same species. A list of candidate loci for development into new informative molecular markers, which are diagnostic for sections and lineages, was created from analyses of phylogenetic signals from individual genes across the entire genome.

Alternaria alternata is part of a genus comprised of over 600 different species that occur all over the world and cause damage to humans, plants and thereby to the economy. Yet, even though some species are causing tremendous issues, the past years have shown that assigning newly found isolates to known species was rather inconsistent. Most identifications are usually done on the basis of spore morphology, chemotype and molecular markers. In this work we used strains isolated from the wild as well as commercial strains of the DSMZ (German collection of microorganisms and cell cultures) as a reference, to show, that the variation within the Alternaria alternata species is comparable to the variation between different species of the genus Alternaria in regards to spore morphology and chemotype. We compared the different methods of identification and discerned the concatenation of multiple molecular markers as the deciding factor for better identification. Up until this point, usually a concatenation of two or three traditional molecular markers was used. Some of those markers being stronger some weaker. We show that the concatenation of five molecular markers improves the likeliness of a correct assignment, thus a better distinction between the different Alternaria species., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

In this study, a novel mitovirus, tentatively designated as "Alternaria alternata mitovirus 2" (AaMV2), was isolated from the fungus Alternaria alternata f. sp. mali causing apple leaf blotch disease. The complete genome of AaMV2 is 3,157 nucleotides in length, with an A+U content of 68.10%. The genome has a single large open reading frame (ORF) encoding an RNA-dependent RNA polymerase (RdRp) protein with a molecular mass of 98.10 kDa. BLAST analysis revealed that AaMV2 has the highest sequence identity to Leptosphaeria biglobosa mitovirus 6, with 79.76% and 82.86% identity at the amino acid and nucleotide level, respectively. Phylogenetic analysis suggested that AaMV2 is a new member of the genus Duamitovirus within the family Mitoviridae. This is the first report of the complete genome sequence analysis of a mitovirus in A. alternata., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Austria, part of Springer Nature.)

Previous work identified a pair of specific effectors AsCEP19 and AsCEP20 in Alternaria solani as contributors to the virulence of A. solani . Here, we constructed AsCEP19 and AsCEP20 deletion mutants in A. solani strain HWC168 to further reveal the effects of these genes on the biology and pathogenicity of A. solani . Deletion of AsCEP19 and AsCEP20 did not affect vegetative growth but did affect conidial maturation, with an increase in the percentage of abnormal conidia produced. Furthermore, we determined the expression patterns of genes involved in the conidiogenesis pathway and found that the regulatory gene abaA was significantly upregulated and chsA , a positive regulator for conidiation, was significantly downregulated in the mutant strains compared to the wild-type strain. These results suggest that AsCEP19 and AsCEP20 indirectly affect the conidial development and maturation of A. solani . Pathogenicity assays revealed significantly impaired virulence of Δ AsCEP19 , Δ AsCEP20, and Δ AsCEP19 + AsCEP20 mutants on potato and tomato plants. Moreover, we performed localization assays with green fluorescent protein-tagged proteins in chili pepper leaves. We found that AsCEP19 can specifically localize to the chloroplasts of chili pepper epidermal cells, while AsCEP20 can localize to both chloroplasts and the plasma membrane. Weighted gene co-expression network analysis revealed enrichment of genes of this module in the photosynthesis pathway, with many hub genes associated with chloroplast structure and photosynthesis. These results suggest that chloroplasts are the targets for AsCEP19 and AsCEP20., Importance: Alternaria solani is an important necrotrophic pathogen causing potato early blight. Previous studies have provide preliminary evidence that specific effectors AsCEP19 and AsCEP20 contribute to virulence, but their respective functions, localization, and pathogenic mechanisms during the infection process of A. solani remain unclear. Here, we have systematically studied the specific effectors AsCEP19 and AsCEP20 for the first time, which are essential for conidial maturation. The deletion of AsCEP19 and AsCEP20 can significantly impair fungal pathogenicity. Additionally, we preliminarily revealed that AsCEP19 and AsCEP20 target the chloroplasts of host cells. Our findings further enhance our understanding of the molecular mechanisms underlying the virulence of necrotrophic pathogens., Competing Interests: The authors declare no conflict of interest.

Aims: The Gα subunit is a major component of heterotrimeric G proteins, which play a crucial role in the development and pathogenicity of several model fungi. However, its detailed function in the causal agent of pear black spot (Alternaria alternata) is unclear. Our aim was to understand the characteristics and functions of AaGA1 in A. alternata., Methods and Results: AaGA1 was cloned from A. alternata in this study, which encodes 353 amino acids and has a "G-alpha" domain. Mutant ΔAaGA1 resulted in reduced vegetative growth, conidiation, and spore germination. Especially, mutant ΔAaGA1 produced only fewer conidia on the V8A medium, and spore formation-related genes AbaA, BrlA, and WetA were significantly downregulated. More tolerance against cell wall-inhibiting agents was observed after the deletion of AaGA1. Moreover, AaGA1 deletion led to a significant reduction in melanin and toxin production. Interestingly, deletion of AaGA1 resulted in defective appressorium-like formations, complete loss of the ability to penetrate cellophane, and decreased infection on non-wound inoculated tobacco leaves. Cell wall-degrading enzyme-related genes PME, CL, Cut2, and LC were significantly downregulated in mutant ΔAaGA1 mutant, significantly reducing virulence on wound-inoculated pear fruits., Conclusions: The G protein alpha subunit AaGA1 is indispensable for fungal development, appressorium-like formations, and pathogenicity in A. alternata., (© The Author(s) 2024. Published by Oxford University Press on behalf of Applied Microbiology International.)

Aims: To identify promising fungal endophytes that are able to produce glycyrrhizin and enhance it in licorice and the mechanisms involved., Methods and Results: Fifteen fungal endophytes were isolated from Glycyrrhiza glabra L. rhizomes among which SGGF14 and SGGF21 isolates were found to produce glycyrrhizin by 4.29 and 2.58 µg g-1 dry weight in the first generation of their culture. These isolates were identified as Fusarium solani and Alternaria tenuissima, respectively, based on morphological characteristics and sequence analysis of internal transcribed spacer, TEF1, ATPase, and CAL regions. Subsequently, G. glabra plants were inoculated with these fungal isolates to examine their effect on glycyrrhizin production, plant growth parameters and the expression of key genes involved in glycyrrhizin pathway: SQS1, SQS2, bAS, CAS, LUS, CYP88D6, and CYP72A154. Endophytes were able to enhance glycyrrhizin content by 133%-171% in the plants. Natural control (NC) plants, harboring all natural endophytes, had better growth compared to SGGF14- and SGGF21-inoculated and endophyte-free (EF) plants. Expression of SQS1, SQS2, CYP88D6, and CYP72A154 was upregulated by inoculation with endophytes. LUS and CAS were downregulated after endophyte inoculation. Expression of bAS was higher in SGGF21-inoculated plants when compared with NC, EF, and SGGF14-inoculated plants., Conclusions: Two selected fungal endophytes of G. glabra can produce glycyrrhizin and enhance glycyrrhizin content in planta by modulating the expression of key genes in glycyrrhizin biosynthetic pathway., (© The Author(s) 2024. Published by Oxford University Press on behalf of Applied Microbiology International.)

Background: Taxol, derived from Taxus trees, is a valuable natural resource for the development of anticancer drugs. Endophytic fungi from Taxus trees are a promising alternative source of Taxol. However, the impact of plant-endophytic microbial interaction on the host's Taxol biosynthesis is largely unknown., Results: In the current study, the diversity of endophytic fungi in three different Taxus species was analyzed using Internal Transcribed Spacer sequencing. A total of 271 Operational Taxonomic Units (OTUs) were identified, grouping into 2 phyla, 8 classes, 16 orders, 19 families, and 19 genera. Alpha and beta diversity analysis indicated significant differences in endophytic fungal communities among the various Taxus trees. At the genus level, Alternaria and Davidiella were predominantly found in T. mairei and T. media, respectively. By utilizing a previously published dataset, a Pearson correlation analysis was conducted to predict the taxol biosynthesis-related fungal genera. Following screening, two isolates of Alternaria (L7 and M14) were obtained. Effect of inoculation with Alternaria isolates on the gene expression and metabolite accumulation of T. mairei was determined by transcriptomic and untargeted metabolomic studies. The co-inoculation assay suggests that the two Alternaria isolates may have a negative regulatory effect on taxol biosynthesis by influencing hormone signaling pathways., Conclusion: Our findings will serve as a foundation for advancing the production and utilization of Taxus and will also aid in screening endophytic fungi related to taxol production., (© 2024. The Author(s).)

Loquat ( Eriobotrya japonica ) is an economically important subtropical fruit crop in China. Field surveys conducted in different loquat orchards located in Chongqing, Sichuan, and Fujian provinces between 2017 and 2020 resulted in a collection of 56 Alternaria -like isolates from trees exhibiting symptoms of loquat leaf spot. Multigene phylogenetic analyses using seven gene regions, namely, ITS, gapdh , RPB2 , tef1 , Alt a 1 , endoPG , and OPA10-2, showed that all the isolates belonged to the genus Alternaria , and supporting morphological analysis identified them as members of species A. alternata , A. gaisen , and A. chongqingensis sp. nov. In vitro and in vivo pathogenicity tests showed all the identified species to be pathogenic and able to cause leaf spot disease on loquat. Moreover, comprehensive phylogenetic analyses employing all combinations of the above seven gene sequences revealed the capability of Alt a 1 - tef1 - endoPG to provide a well-resolved gene tree for Alternaria spp. at the species level. This study adds to the current knowledge on an unknown species ( A. chongqingensis sp. nov.) and is the first report of A. gaisen in loquat worldwide., Competing Interests: The author(s) declare no conflict of interest.

The antifungal activity of Serratia plymuthica CCGG2742, a bacterial strain isolated from grapes berries skin, against a phytopathogenic fungus isolated from blueberries was evaluated in vitro and in vivo. In order to characterize the wild fungal isolate, phylogenetic analysis using concatenated DNA sequences from the RPB2 and TEF1 genes and of the ITS region was performed, allowing the identification of the fungal isolate that was called Alternaria tenuissima CC17. Hyphae morphology, mycelium ultrastructure, conidia and reproductive structures were in agreement with the phylogenetic analysis. The antifungal activity of the S. plymuthica strain was dependent on the composition of the culture medium. The greatest inhibition of mycelial growth of A. tenuissima CC17 by S. plymuthica CCGG2742 was observed on YTS medium, which lacks of an easily assimilable carbon source. Fungal growth medium supplemented with 50 % of bacterial supernatant decreased the conidia germination of A. tenuissima CC17 up to 32 %. Preventive applications of S. plymuthica CCGG2742 to blueberries and tomato leaves at conidia:bacteria ratio of 1:100, protected in 77.8 ± 4.6 % and 98.2 ± 0.6 % to blueberries and tomato leaves from infection caused by A. tenuissima CC17, respectively. To the best of our knowledge, this is the first report on the antifungal activity of S. plymuthica against A. tenuissima, which could be used as a biological control agent of plant diseases caused by this fungal species. In addition, the results of this work could be a starting point to attribute the real importance of A. tenuissima as a pathogen of blueberries in Chile, which until now had been considered almost exclusively to A. alternata. Likewise, this research could be relevant to start developing highly effective strategies based on S. plymuthica CCGG2742 for the control of this important phytopathogenic fungus., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Alternaria brassicicola is a part of the Alternaria complex that causes leaf blight and head rot (ABHR) in brassica crops. Infested broccoli seeds can play an important role in introducing A. brassicicola in transplant houses and production fields. However, characterization of natural seed infestation and seed-to-seedling transmission of A. brassicicola in broccoli is yet to be demonstrated. In this research, we characterized Alternaria spp. isolates from commercial broccoli seedlots for their species identity, pathogenicity, and aggressiveness on broccoli and their sensitivity to a quinone-outside inhibitor (QoI) fungicide (azoxystrobin). Two hundred commercial seedlots from two broccoli cultivars, Cultivar 1 (EC; n = 100 seedlots) and Cultivar 2 (ED; n = 100 seedlots) were, evaluated for the presence of A. brassicicola under in vitro conditions using a seedling grow-out assay. Alternaria spp. was detected in 31 and 28% of the commercial seedlots of Cultivar 1 and Cultivar 2, respectively. The seed-to-seedling transmission (%) varied considerably within each positive-infested seedlot, which ranged from 1.3 to 17.3%. Subsequent molecular identification of single-spore cultures ( n = 138) was made by sequencing four housekeeping genes: actin, the major allergen (Alta1), plasma membrane ATPase, and glyceraldehyde-3-phosphate dehydrogenase (GPD), and the sequences were concatenated and compared for the phylogenetic distance with diverse Alternaria species. Ninety-six percent ( n = 133) of the isolates formed a cluster with a known A . brassicicola based on a multigene phylogeny, which were later confirmed as A. brassicicola using a species-specific PCR assay. One hundred percent of the A. brassicicola seed isolates ( n = 133) were either highly or moderately aggressive on broccoli (cultivar Emerald Crown) based on a detached leaf assay. Sensitivity of representative A. brassicicola isolates ( n = 58) to azoxystrobin was evaluated using a spore germination assay, and the EC 50 values (effective fungicide concentration [ppm] at which germination of conidia of isolates were reduced by 50% compared to control) for each isolate was determined. A. brassicicola isolates from naturally infested commercial broccoli seeds were sensitive to azoxystrobin with considerably low EC 50 values in the range of <0.0001 to 0.33 ppm; however, there were a few isolates (14%) that showed 100-fold reduced sensitivity from the most sensitive isolate (EC 50 = 0.0001 ppm). Our results confirm that commercial broccoli seedlots can be naturally contaminated with pathogenic and aggressive A. brassicicola . We also provide evidence for the potential presence of A. brassicicola isolates with reduced azoxystrobin-sensitivity in naturally infested commercial broccoli seedlots, which has never been reported before. Together, these findings may have implications in considerations for seed-health testing, seed treatments, and greenhouse scouting to limit introduction of infested seedlots in commercial broccoli fields., Competing Interests: The author(s) declare no conflict of interest.

Transcription factor Cmr1 (Colletotrichum melanin regulation 1) and its homologs in several plant fungal pathogens are the regulators of the 1,8-dihydroxynaphthalene (DHN)-melanin biosynthesis pathway and have evolved functional diversification in morphology and pathogenicity. The fungal genus Alternaria comprises the group of "black fungi" that are rich in DHN-melanin in the primary cell wall and septa of the conidia. Some Alternaria species cause many economically important plant diseases worldwide. However, the evolution and function of Cmr1 homologs in Alternaria remain poorly understood. Here, we identified a total of forty-two Cmr1 homologs from forty-two Alternaria spp. and all contained one additional diverse fungal specific transcription factor motif. Phylogenetic analysis indicated the division of these homologs into five major clades and three branches. Dated phylogeny showed the A and D clades diverged latest and earliest, respectively. Molecular evolutionary analyses revealed that three amino acid sites of Cmr1 homologs in Alternaria were the targets of positive selection. Asmr1, the homolog of Cmr1 in the potato early blight pathogen, Alternaria solani was amplified and displayed the sequence conservation at the amino acid level in different A. solani isolates. Asmr1 was further confirmed to have the transcriptional activation activity and was upregulated during the early stage of potato infection. Deletion of asmr1 led to the decreased melanin content and pathogenicity, deformed conidial morphology, and responses to cell wall and fungicide stresses in A. solani. These results suggest positive selection and functional divergence have played a role in the evolution of Cmr1 homologs in Alternaria. KEY POINTS: • Cmr1 homologs were under positive selection in Alternaria species • Asmr1 is a functional transcription factor, involved in spore development, melanin biosynthesis, pathogenicity, and responses to cell wall and fungicide stresses in A. solani • Cmr1 might be used as a potential taxonomic marker of the genus Alternaria., (© 2024. The Author(s).)

As one of the most typical pathogens in fruit postharvest diseases, Alternaria alternata (A. alternata) can produce Alternaria toxins (ATs) aggravating fruit decay and harming human health. In this study, ATs (tenuazonic acid, alternariol monomethyl ether, and alternariol) production was inhibited effectively by 200 and 8000 mg/L MF (methyl ferulate) in vitro and in vivo. 1-Octen-3-ol and 3-octanol were the potential iconic volatile organic compounds of ATs (R 2  > 0.99). MF induced oxidative stress, resulting in physiological and metabolic disorders, membrane lipid oxidation and cell damage. It decreased precursors and energy supply by disturbing amino acid metabolism, ABC transporters, citrate cycle, pentose and glucuronate interconversions to regulate ATs synthesis. MF down-regulated the genes related to ATs synthesis (PksJ, AaTAS1, and OmtI), transport (AaMFS1 and MFS), and pathogenicity to affect ATs production and virulence. This study provided a theoretical basis for the control of ATs production., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier Ltd.)

The genus Alternaria comprises many important fungal pathogens that infect a wide variety of organisms. In this report, we present the discovery of a new double-stranded RNA (dsRNA) mycovirus called Alternaria botybirnavirus 2 (ABRV2) from a phytopathogenic strain, XC21-21C, of Alternaria sp. isolated from diseased tobacco leaves in China. The ABRV2 genome consists of two dsRNA components, namely dsRNA1 and dsRNA2, with lengths of 6,162 and 5,865 base pairs (bp), respectively. Each of these genomic dsRNAs is monocistronic, encoding hypothetical proteins of 201.6 kDa (P1) and 2193.3 kDa (P2). ABRV2 P1 and P2 share 50.54% and 63.13% amino acid sequence identity with the corresponding proteins encoded by dsRNA1 of Alternaria botybirnavirus 1 (ABRV1). Analysis of its genome organization and phylogenetic analysis revealed that ABRV2 is a new member of the genus Botybirnavirus., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Austria, part of Springer Nature.)

Alternaria alternata is a prevalent postharvest pathogen that generates diverse mycotoxins, notably alternariol (AOH) and alternariol monomethyl ether (AME), which are recurrent severe contaminants. Nitrogen sources modulate fungal growth, development, and secondary metabolism, including mycotoxin production. The GATA transcription factor AreA regulates nitrogen source utilization. However, little is known about its involvement in the regulation of nitrogen utilization in A. alternata. To examine the regulatory mechanism of AaAreA on AOH and AME biosynthesis in A. alternata, we analyzed the impact of diverse nitrogen sources on the fungal growth, conidiation and mycotoxin production. The use of a secondary nitrogen source (NaNO 3 ) enhanced mycelial elongation and sporulation more than the use of a primary source (NH 4 Cl). NaNO 3 favored greater mycotoxin accumulation than did NH 4 Cl. The regulatory roles of AaAreA were further clarified through gene knockout. The absence of AaAreA led to an overall reduction in growth in minimal media containing any nitrogen source except NH 4 Cl. AaAreA positively regulates mycotoxin biosynthesis when both NH 4 Cl and NaNO 3 are used as nitrogen sources. Subcellular localization analysis revealed abundant nuclear transport when NaNO 3 was the sole nitrogen source. The regulatory pathway of AaAreA was systematically revealed through comprehensive transcriptomic analyses. The deletion of AaAreA significantly impedes the transcription of mycotoxin biosynthetic genes, including aohR, pksI and omtI. The interaction between AaAreA and aohR, a pathway-specific transcription factor gene, demonstrated that AaAreA binds to the aohR promoter sequence (5'-GGCTATGGAAA-3'), activating its transcription. The expressed AohR regulates the expression of downstream synthase genes in the cluster, ultimately impacting mycotoxin production. This study provides valuable information to further understand how AreA regulates AOH and AME biosynthesis in A. alternata, thereby enabling the effective design of control measures for mycotoxin contamination., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

Alternaria alternata is a ubiquitous soil-borne fungus capable of causing diseases in a variety of plants and occasionally in humans. While populations of A. alternata from infected plants have received significant attention, relatively little is known about its soil populations, including its population genetic structure and antifungal susceptibilities. In addition, over the last two decades, greenhouses have become increasingly important for food and ornamental plant production throughout the world, but how greenhouses might impact microbial pathogens such as A. alternata populations remains largely unknown. Different from open crop fields, greenhouses are often more intensively cultivated, with each greenhouse being a relatively small and isolated space where temperature and humidity are higher than surrounding environments. Previous studies have shown that greenhouse populations of two common molds, Aspergillus fumigatus and A. alternata, within a small community in southwestern China were variably differentiated. However, the relative contribution of physical separation among local greenhouses to the large-scale population structure remains unknown. Here, we isolated strains of A. alternata from seven greenhouses in Shijiazhuang, northeast China. Their genetic diversity and triazole susceptibilities were analyzed and compared with each other and with 242 isolates from nine greenhouses in Kunming, southwest China. Results showed that the isolation of greenhouses located <1 km from each other locally contributed similarly to the overall genetic variation as that between the two distant geographic regions. In addition, our results indicate that greenhouses could be significant sources of triazole resistance, with greenhouses often differing in their frequencies of resistant strains to different triazoles., Importance: Greenhouses have become increasingly important for food production and food security. However, our understanding of how greenhouses may contribute to genetic variations in soil microbial populations is very limited. In this study, we obtained and analyzed soil populations of the cosmopolitan fungal pathogen Alternaria alternata in seven greenhouses in Shijiazhuang, northeast China. Our analyses revealed high proportions of isolates being resistant to agricultural triazole fungicides and medical triazole drugs, including cross-resistance to both groups of triazoles. In addition, we found that greenhouse populations of A. alternata located within a few kilometers showed similar levels of genetic differentiation as those separated by over 2,000 km between northeast and southwest China. Our study suggests that greenhouse populations of this and potentially other fungal pathogens represent an important ecological niche and an emerging threat to food security and human health., Competing Interests: The authors declare no conflict of interest.

Aims: Understanding the inhibitory effects of natural organic substances on soil-borne pathogenic fungi and the relevant molecular mechanisms are highly important for future development of green prevention and control technology against soil-borne diseases. Our study elucidates the inhibitory effect of the combined application of humic acids (HAs) and chitosan on Alternariasolani and the light on the corresponding mechanism., Methods and Results: The effect on A. solani growth by HAs incorporated with chitosan was investigated by plate culture and the corresponding mechanism was revealed using transcriptomics. The colony growth of A. solani was suppressed with the highest inhibition rate 33.33% when swine manure HAs was compounded with chitosan at a ratio of 1:4. Chitosan changed the colony morphology from round to irregularly. RNA-seq in the HAs and chitosan (HC) treatment revealed 239 differentially expressed genes compared with the control. The unigenes associated with enzymes activities related to growth and biological processes closely related to mycelial growth and metabolism were downregulated. RNA-seq also revealed that chitosan altered the expression of genes related to secondary metabolism, fungal cell wall formation and polysaccharide synthesis, and metabolism. Meanwhile, weighted gene co-expression network analysis showed that, genes expression in the module positively correlated with mycelial growth was significantly reduced in the HC treatment; and the results were verified by real-time quantitative polymerase chain reaction., Conclusions: The co-inhibition effect of HAs and chitosan on A. solani is associated with downregulated genes expression correlated with mycelial growth., (© The Author(s) 2024. Published by Oxford University Press on behalf of Applied Microbiology International.)

We isolated the dark septate endophytic (DSE) fungi from roots of typical plant species in the tundra of Changbai Mountains Nature Reserve, including Rhododendron aureum , R. conferentiatum , Vaccinium uliginosum , and Dryas octopetala , and studied their colonization. We further investigated the DSE community composition and species diversity of the four tundra plant species by using morphological characteristics combined with rDNA ITS sequence analysis. The results showed that DSE formed a typical structure of "microsclerotia" in roots of the four plant species. A total of 69 strains of DSE fungi were isolated from the root samples, belonging to 10 genera, and 12 species. They were Phialocephala fortinii , Alternaria alternata , A. tenuissima , Epicocum nigrum , Canariomyces microsporus , Colletotrichum spaethianum , C. camelliae , Leptophoria sp., Cladosporium cladosporioides , Phoma sp., Cadophora sp., and Discosia italica , respectively. The DSE fungal species diversity was rich, and all these fungal species were firstly reported as DSE fungi in the alpine tundra belt of China. Among them, Phialocephala fortinii was the common and dominant species of all tundra plants. The Simpson, Pielou, and Shannon diversity indices of DSE fungi of the four plant species of tundra differed significantly. Our results showed that tundra plants have rich diversity of DSE fungi, and they can form a good symbiotic relationship, which enhance the adaptability of tundra plants to the harsh environment.

Background: Garlic leaf spot (GLS) caused by Alternaria alternata is one of the main diseases in the garlic production areas, and its management heavily relies on dicarboximide fungicides. However, the efficacy of dicarboximides against the GLS disease has decreased year on year., Results: In the present study, 10 of 148 A. alternata strains separated from Jiangsu Province were moderately resistant (MR) to a dicarboximide fungicide procymidone (Pro MR ). Positive cross-resistance was observed between Pro and iprodione (Ipro) or fludioxonil (Fld), but not between Pro and fluazinam or azoxystrobin. Mutations at AaOS1, but not Aafhk1, were confirmed to confer the Pro resistance by constructing replacement mutants, whereas mutations at both AaOS1 and Aafhk1 decreased the gene expression level of AapksI, as well as the ability to produce mycotoxin AOH (polyketide-derived alternariol) and virulence. Additionally, more genes (AaOS1 and Aafhk1) harboring the mutations experienced a larger biological fitness penalty., Conclusion: To our knowledge, this is the first report on Pro resistance selected in garlic fields, and mutations at AaOS1 of A. alternata causing a decreased ability to produce the mycotoxin AOH. © 2024 Society of Chemical Industry., (© 2024 Society of Chemical Industry.)

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

Background: Alternaria blotch disease in Himachal Pradesh, India, caused by Alternaria spp., adversely affects apple cultivars, resulting in reduced fruit size and quality accompanied by premature leaf fall., Methods and Results: Sixteen Alternaria isolates from apple growing regions underwent comprehensive analysis including morphology, pathogenicity, and molecular characterization. Variations in conidiophore and conidia dimensions, shapes, and divisions were observed among isolates. Pathogenicity assays revealed differences in incubation periods, latent phases, and disease responses. Molecular characterization via nuclear ITS rDNA and RAPD analysis indicated 99-100% homology with Alternaria alternata, Alternaria mali, and other Alternaria spp., with a close phylogenetic relationship to Chinese isolates. Differentiation of isolates based on origin, cultural characteristics, and morphology was achieved using RAPD markers., Conclusions: The study identifies diverse genotypes and morphotypes of Alternaria contributing to apple blotch disease in Himachal Pradesh. These findings highlight the complexity of the pathogenic environment and hold significant implications for disease management in apple orchards., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

Fusarium verticillioides produces fumonisins, which are mycotoxins inhibiting sphingolipid biosynthesis in humans, animals, and other eukaryotes. Fumonisins are presumed virulence factors of plant pathogens, but may also play a role in interactions between competing fungi. We observed higher resistance to added fumonisin B 1 (FB 1 ) in fumonisin-producing Fusarium verticillioides than in nonproducing F. graminearum , and likewise between isolates of Aspergillus and Alternaria differing in production of sphinganine-analog toxins. It has been reported that in F. verticillioides , ceramide synthase encoded in the fumonisin biosynthetic gene cluster is responsible for self-resistance. We reinvestigated the role of FUM17 and FUM18 by generating a double mutant strain in a fum1 background. Nearly unchanged resistance to added FB 1 was observed compared to the parental fum1 strain. A recently developed fumonisin-sensitive baker's yeast strain allowed for the testing of candidate ceramide synthases by heterologous expression. The overexpression of the yeast LAC1 gene, but not LAG1 , increased fumonisin resistance. High-level resistance was conferred by FUM18 , but not by FUM17 . Likewise, strong resistance to FB 1 was caused by overexpression of the presumed F. verticillioides "housekeeping" ceramide synthases CER1 , CER2 , and CER3 , located outside the fumonisin cluster, indicating that F. verticillioides possesses a redundant set of insensitive targets as a self-resistance mechanism.

Jojoba shrubs are wild plants cultivated in arid and semiarid lands and characterized by tolerance to drought, salinity, and high temperatures. Fungi associated with such plants may be attributed to the tolerance of host plants against biotic stress in addition to the promotion of plant growth. Previous studies showed the importance of jojoba as jojoba oil in the agricultural field; however, no prior study discussed the role of jojoba-associated fungi (JAF) in reflecting plant health and the possibility of using JAF in biocontrol. Here, the culture-independent and culture-dependent approaches were performed to study the diversity of the jojoba-associated fungi. Then, the cultivable fungi were evaluated for in-vitro antagonistic activity and in vitro plant growth promotion assays. The metagenome analysis revealed the existence of four fungal phyla: Ascomycota, Aphelidiomycota, Basidiomycota, and Mortierellomycota. The phylum Ascomycota was the most common and had the highest relative abundance in soil, root, branch, and fruit samples (59.7%, 50.7%, 49.8%, and 52.4%, respectively). Alternaria was the most abundant genus in aboveground tissues: branch (43.7%) and fruit (32.1%), while the genus Discosia had the highest abundance in the underground samples: soil (24%) and root (30.7%). For the culture-dependent method, a total of 14 fungi were isolated, identified, and screened for their chitinolytic and antagonist activity against three phytopathogenic fungi (Fusarium oxysporum, Alternaria alternata and Rhizoctonia solani) as well as their in vitro plant growth promotion (PGP) activity. Based on ITS sequence analysis, the selected potent isolates were identified as Aspergillus stellatusEJ-JFF3, Aspergillus flavus EJ-JFF4, Stilbocrea sp. EJ-JLF1, Fusarium solani EJ-JRF3, and Amesia atrobrunneaEJ-JSF4. The endophyte strain A. flavus EJ-JFF4 exhibited the highest chitinolytic activity (9 Enzyme Index) and antagonistic potential against Fusarium oxysporum, Alternaria alternata, and Rhizoctonia solani phytopathogens with inhibitory percentages of 72, 70, and 80 respectively. Also, A. flavus EJ-JFF4 had significant multiple PGP properties, including siderophore production (69.3%), phosphate solubilization (95.4 µg ml -1 ). The greatest production of Indol-3-Acetic Acid was belonged to A. atrobrunnea EJ-JSF4 (114.5 µg ml -1 ). The analysis of FUNGuild revealed the abundance of symbiotrophs over other trophic modes, and the guild of endophytes was commonly assigned in all samples. For the first time, this study uncovered fungal diversity associated with jojoba plants using a culture-independent approach and in-vitro assessed the roles of cultivable fungal strains in promoting plant growth and biocontrol. The present study indicated the significance of jojoba shrubs as a potential source of diverse fungi with high biocontrol and PGP activities., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

Herbal medicines are widely used for clinical purposes worldwide. These herbs are susceptible to phytopathogenic fungal invasion during the culturing, harvesting, storage, and processing stages. The threat of fungal and mycotoxin contamination requires the evaluation of the health risks associated with these herbal medicines. In this study, we collected 138 samples of 23 commonly used herbs from 20 regions in China, from which we isolated a total of 200 phytopathogenic fungi. Through morphological observation and ITS sequencing, 173 fungal isolates were identified and classified into 24 genera, of which the predominant genera were Fusarium (27.74%) and Alternaria (20.81%), followed by Epicoccum (11.56%), Nigrospora (7.51%), and Trichocladium (6.84%). Quantitative analysis of the abundance of both Fusarium and Alternaria in herbal medicines via RT-qPCR revealed that the most abundant fungi were found on the herb Taraxacum mongolicum , reaching 300,000 copies/μL for Fusarium and 700 copies/μL for Alternaria . The in vitro mycotoxin productivities of the isolated Fusarium and Alternaria strains were evaluated by using liquid chromatography-tandem mass spectrometry (LC-MS/MS), and it was found that the Fusarium species mainly produced the acetyl forms of deoxynivalenol, while Alternaria species mainly produced altertoxins. These findings revealed widely distributed fungal contamination in herbal medicines and thus raise concerns for the sake of the quality and safety of herbal medicines.

Postharvest fruit rot caused by pathogens is a serious problem in the pear industry. This study investigated the fungal diversity and main pathogens and identified a new pathogen in the stored 'Huangguan' pear ( Pyrus bretschneideri Rehd.), the dominant pear variety in northern China. We sampled 20 refrigeration houses from five main producing regions in Hebei Province and used Illumina sequencing technology to detect the fungal composition. Alternaria (56.3%) was the most abundant fungus, followed by Penicillium (9.2%) and Monilinia (6.2%). We also isolated and identified nine strains of Alternaria and four strains of Penicillium . Moreover, we observed a new postharvest fruit disease in 'Huangguan' pear caused by Stemphylium eturmiunum , which was confirmed by phylogenetic analysis by combining the sequences of three conserved genes, including internal transcribed spacer, gapdh , and calmodulin. This study marks the first documentation of S . eturmiunum causing fruit rot in 'Huangguan' pears, offering valuable insights for identifying and controlling this newly identified postharvest disease., Competing Interests: The author(s) declare no conflict of interest.

Blue honeysuckle is emerging as a popular edible fruit and is also used in folk medicine. However, from June to August 2021, a serious leaf-spot disease affected the yield and quality of blue honeysuckle in Harbin, Heilongjiang Province, China; the species and characteristics of the pathogens responsible for the disease were unknown. In this study, 30 fungal isolates were obtained from infected blue honeysuckle leaves and identified as Alternaria tenuissima based on morphological and molecular characteristics and phylogenetic analyses. To the best of our knowledge, this is one of the first studies to identify A. tenuissima as the causal agent of blue honeysuckle leaf spots in China. Pathogenicity tests of the isolates revealed that most isolates were moderately pathogenic. All blue honeysuckle cultivars tested were found to be susceptible to 30 A. tenuissima isolates. In addition, elder, Dahurian rose fruit, sea buckthorn, rowan, hawthorn, bird cherry, and sorb could be infected by A. tenuissima isolates, while European cranberry bush and Nanking cherry were not infected. A . tenuissima isolates were highly sensitive to prochloraz (EC 50 ≤ 0.50 μg ml -1 ) with 86.21% efficacy at 400 μg ml -1 in the field trials. Therefore, crop rotation and application of chemical fungicides are considered to control the disease-causing leaf spots in blue honeysuckle. These results provide a basis for controlling A. tenuissima in blue honeysuckle in China., Competing Interests: The author(s) declare no conflict of interest.

Black spot, caused by Alternaria brassicicola ( Ab ), poses a serious threat to crucifer production, and knowledge of how plants respond to Ab infection is essential for black spot management. In the current study, combined transcriptomic and metabolic analysis was employed to investigate the response to Ab infection in two cabbage ( Brassica oleracea var. capitata ) genotypes, Bo257 (resistant to Ab ) and Bo190 (susceptible to Ab ). A total of 1100 and 7490 differentially expressed genes were identified in Bo257 (R&#95;mock vs. R&#95; Ab ) and Bo190 (S&#95;mock vs. S&#95; Ab ), respectively. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis revealed that "metabolic pathways", "biosynthesis of secondary metabolites", and "glucosinolate biosynthesis" were the top three enriched KEGG pathways in Bo257, while "metabolic pathways", "biosynthesis of secondary metabolites", and "carbon metabolism" were the top three enriched KEGG pathways in Bo190. Further analysis showed that genes involved in extracellular reactive oxygen species (ROS) production, jasmonic acid signaling pathway, and indolic glucosinolate biosynthesis pathway were differentially expressed in response to Ab infection. Notably, when infected with Ab , genes involved in extracellular ROS production were largely unchanged in Bo257, whereas most of these genes were upregulated in Bo190. Metabolic profiling revealed 24 and 56 differentially accumulated metabolites in Bo257 and Bo190, respectively, with the majority being primary metabolites. Further analysis revealed that dramatic accumulation of succinate was observed in Bo257 and Bo190, which may provide energy for resistance responses against Ab infection via the tricarboxylic acid cycle pathway. Collectively, this study provides comprehensive insights into the Ab -cabbage interactions and helps uncover targets for breeding Ab -resistant varieties in cabbage.

As a fruit tree with great economic value, apple is widely cultivated in China. However, apple leaf spot disease causes significant damage to apple quality and economic value. In our study, we found that MdMYB6-like is a transcription factor without auto-activation activity and with three alternative spliced variants. Among them, MdMYB6-like-β responded positively to the pathogen infection. Overexpression of MdMYB6-like-β increased the lignin content of leaves and improved the pathogenic resistance of apple flesh callus. In addition, all three alternative spliced variants of MdMYB6-like could bind to the promoter of MdBGLU H . Therefore, we believe that MdMYB6-like plays an important role in the infection process of the pathogen and lays a solid foundation for breeding disease-resistant cultivars of apple in the future.

The clinical effects of Schisandra chinensis against human disease are well-documented; however, studies on its application in controlling plant pathogens are limited. Here, we investigated its inhibitory effect on the growth of Alternaria alternata, a fungus which causes significant post-harvest losses on apples, known as black spot disease. S. chinensis fruit extract exhibited strong inhibitory effects on the growth of A. alternata with an EC 50 of 1882.00 mg/L. There were 157 compounds identified in the extract by high performance liquid chromatography-mass spectrometry, where benzocaine constituted 14.19% of the extract. Antifungal experiments showed that the inhibitory activity of benzocaine on A. alternata was 43.77-fold higher than the crude extract. The application of benzocaine before and after A. alternata inoculation on apples prevented the pathogen infection and led to mycelial distortion according to scanning electron microscopy. Transcriptome analysis revealed that there were 4226 genes differentially expressed between treated and untreated A. alternata-infected apples with benzocaine. Metabolomics analysis led to the identification of 155 metabolites. Correlation analysis between the transcriptome and metabolome revealed that benzocaine may inhibit A. alternata growth via the beta-alanine metabolic pathway. Overall, S. chinensis extract and benzocaine are environmentally friendly plant-based fungicides with potential to control A. alternata., (© 2024. The Author(s).)

Direct exposure to the fungal species Alternaria alternata is a major risk factor for the development of asthma, allergic rhinitis, and inflammation. As of November 23rd 2020, the NCBI protein database showed 11,227 proteins from A. alternata genome as hypothetical proteins (HPs). Allergens are the main causative of several life-threatening diseases, especially in fungal infections. Therefore, the main aim of the study is to identify the potentially allergenic inducible proteins from the HPs in A. alternata and their associated functional assignment for the complete understanding of the complex biological systems at the molecular level. AlgPred and Structural Database of Allergenic Proteins (SDAP) were used for the prediction of potential allergens from the HPs of A. alternata. While analyzing the proteome data, 29 potential allergens were predicted by AlgPred and further screening in SDAP confirmed the allergic response of 10 proteins. Extensive bioinformatics tools including protein family classification, sequence-function relationship, protein motif discovery, pathway interactions, and intrinsic features from the amino acid sequence were used to successfully predict the probable functions of the 10 HPs. The functions of the HPs are characterized as chitin-binding, ribosomal protein P1, thaumatin, glycosyl hydrolase, and NOB1 proteins. The subcellular localization and signal peptide prediction of these 10 proteins has further provided additional information on localization and function. The allergens prediction and functional annotation of the 10 proteins may facilitate a better understanding of the allergenic mechanism of A. alternata in asthma and other diseases. The functional domain level insights and predicted structural features of the allergenic proteins help to understand the pathogenesis and host immune tolerance. The outcomes of the study would aid in the development of specific drugs to combat A. alternata infections., (© 2024. The Author(s).)

Alternaria spp. cause different diseases in potato and tomato crops. Early blight caused by Alternaria solani and brown spot caused by Alternaria alternata are most common, but the disease complex is far more diverse. We first provide an overview of the Alternaria species infecting the two host plants to alleviate some of the confusion that arises from the taxonomic rearrangements in this fungal genus. Highlighting the diversity of Alternaria fungi on both solanaceous hosts, we review studies investigating the genetic diversity and genomes, before we present recent advances from studies elucidating host-pathogen interactions and fungicide resistances., Taxonomy: Kingdom Fungi, Phylum Ascomycota, Class Dothideomycetes, Order Pleosporales, Family Pleosporaceae, Genus Alternaria., Biology and Host Range: Alternaria spp. adopt diverse lifestyles. We specifically review Alternaria spp. that cause disease in the two solanaceous crops potato (Solanum tuberosum) and tomato (Solanum lycopersicum). They are necrotrophic pathogens with no known sexual stage, despite some signatures of recombination., Disease Symptoms: Symptoms of the early blight/brown spot disease complex include foliar lesions that first present as brown spots, depending on the species with characteristic concentric rings, which eventually lead to severe defoliation and considerable yield loss., Control: Good field hygiene can keep the disease pressure low. Some potato and tomato cultivars show differences in susceptibility, but there are no fully resistant varieties known. Therefore, the main control mechanism is treatment with fungicides., (© 2024 The Authors. Molecular Plant Pathology published by British Society for Plant Pathology and John Wiley & Sons Ltd.)

Alternaria is a plant pathogen that spreads globally and is prone to causing citrus brown spot disease and metabolizing mycotoxins, thus seriously hindering the development of this economic crop industry. Herein, a "label-free" and "turn on" visual fluorescent assay for citrus Alternaria based on CRISPR-Cas12a and rolling circle amplification (RCA) was described. Using ssDNA complementary to RCA primer as a trans-cleavage substrate for CRISPR-Cas12a, the two systems of CRISPR-Cas12a and RCA-amplified G-quadruplex were skillfully integrated. By using a portable light source for excitation, the positive sample produced obvious red fluorescence, while the negative sample remained almost colorless, making them easy to differentiate with the naked eye. In addition, the specificity was demonstrated by distinguishing Alternaria from other citrus disease related pathogens. Moreover, the practicality was verified by analyzing cultured Alternaria and Alternaria in actual citrus leaf and fruit samples. Therefore, this method may contribute to the on-site diagnosis of Alternaria., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier B.V.)

Alternaria linariae is an economically important foliar pathogen that causes early blight disease in tomatoes. Understanding genetic diversity, population genetic structure, and evolutionary potential is crucial to contemplating effective disease management strategies. We leveraged genotyping-by-sequencing (GBS) technology to compare genome-wide variation in 124 isolates of Alternaria spp. ( A. alternata , A. linariae , and A. solani ) for comparative genome analysis and to test the hypotheses of genetic differentiation and linkage disequilibrium (LD) in A. linariae collected from tomatoes in western North Carolina. We performed a pangenome-aware variant calling and filtering with GBSapp and identified 53,238 variants conserved across the reference genomes of three Alternaria spp. The highest marker density was observed on chromosome 1 (7 Mb). Both discriminant analysis of principal components and Bayesian model-based STRUCTURE analysis of A. linariae isolates revealed three subpopulations with minimal admixture. The genetic differentiation coefficients (F ST ) within A. linariae subpopulations were similar and high (0.86), indicating that alleles in the subpopulations are fixed and the genetic structure is likely due to restricted recombination. Analysis of molecular variance indicated higher variation among populations (89%) than within the population (11%). We found long-range LD between pairs of loci in A. linariae , supporting the hypothesis of low recombination expected for a fungal pathogen with limited sexual reproduction. Our findings provide evidence of a high level of population genetic differentiation in A. linariae , which reinforces the importance of developing tomato varieties with broad-spectrum resistance to various isolates of A. linariae ., Competing Interests: The author(s) declare no conflict of interest.

Plants belonging to the genera Astragalus, Oxytropis, Ipomoea, Sida , and Swainsona often contain the toxin swainsonine (SW) produced by an associated fungal symbiont. Consumption of SW-containing plants causes a serious neurological disorder in livestock, which can be fatal. In this study, a fungal endophyte, Alternaria section Undifilum , was identified in Astragalus garbancillo seeds, using polymerase chain reaction (PCR) followed by direct sequencing. In seeds, the SW concentrations were about 4 times higher than in other parts of the plant. Furthermore, microscopic examination demonstrated that the fungus mycelium grows inside the petioles and stems, on the outer surface and inside the mesocarp of the fruit, in the mesotesta and endotesta layers of the seed coat, and inside the endosperm of the seeds. Our results support the notion that the SW-producing fungus is vertically transmitted in the host plant A. garbancillo .

Endophytic fungi are an important source of novel antitumor substances. Previously, we isolated an endophytic fungus, Alternaria alstroemeria, from the medicinal plant Artemisia artemisia, whose crude extracts strongly inhibited A549 tumor cells. We obtained a transformant, namely AaLaeA OE26 , which completely loses its antitumor activity due to overexpression of the global regulator AaLaeA. Re-sequencing analysis of the genome revealed that the insertion site was in the noncoding region and did not destroy any other genes. Metabolomics analysis revealed that the level of secondary antitumor metabolic substances was significantly lower in AaLaeA OE26 compared with the wild strain, in particular flavonoids were more downregulated according to the metabolomics analysis. A further comparative transcriptome analysis revealed that a gene encoding FAD-binding domain protein (Fla1) was significantly downregulated. On the other hand, overexpression of AaFla1 led to significant enhancement of antitumor activity against A549 with a sevenfold higher inhibition ratio than the wild strain. At the same time, we also found a significant increase in the accumulation of antitumor metabolites including quercetin, gitogenin, rhodioloside, liensinine, ginsenoside Rg2 and cinobufagin. Our data suggest that the global regulator AaLaeA negatively affects the production of antitumor compounds via controlling the transcription of AaFla1 in endophytic A. alstroemeria., (© 2023 Wiley-VCH GmbH.)

Sweet basil ( Ocimum basilicum ) is an important spice herb grown in Israel for local markets and export. The crop is used as a fresh culinary herb or spice, and the essential oils are used in cosmetics and food flavorings. Due to increased demand, the production area of basil has increased in Israel. Postharvest losses due to fungal disease are a major economic concern for growers. In the summer of 2019, a leaf spot was observed in postharvest shipments of sweet basil destined for Europe; in late winter of 2022, leaf spots were observed on greenhouse-grown sweet basil. Fungal isolates from infected leaves were characterized by morphology in culture as Alternaria spp. PCR amplification of the Alternaria major allergen Alt a1 , ITS, and gdp gene regions of the recovered isolates confirmed the presence of A. alternata , a common pathogen of numerous herbs and spice plants. In vitro growth tests demonstrated that 25°C was the optimum temperature for growth of the isolates. The isolates were tested for pathogenicity and found to infect a commonly grown cultivar of basil, cultivar Eli (previously cultivar Perrie). Foliar symptoms in pathogenicity tests were identical to those observed in commercial shipments and in the field, which completed Koch's postulates. Control of the nascent disease by applying fungicides to the plants may be necessary to reduce postharvest losses., Competing Interests: The author(s) declare no conflict of interest.

Phytopathogenic Alternaria species are renown for production of toxins that contribute to virulence on host plants. Typically, these toxins belong to well-known secondary metabolite chemical classes including polyketides, non-ribosomal peptides and terpenes. However, the purported host toxin brassicicolin A produced by A. brassicicola is an isocyanide, a chemical class whose genetics and encoding gene structure is largely unknown. The chemical structure of brassicicolin A shows it to have similarity to the recently characterized fumicicolins derived from the Aspergillus fumigatus isocyanide synthase CrmA. Examination of the A. brassicicola genome identified AbcrmA, a putative homolog with 64% identity to A. fumigatus CrmA. Deletion of AbcrmA resulted in loss of production of brassicicolin A. Contrary to reports that brassicicolin A is a host-specific toxin, the ΔAbcrmA mutants were equally virulent as the wildtype on Brassica hosts. However, in line with results of A. fumigatus CrmA generated metabolites, we find that brassicicolin A increased 360-fold under copper limited conditions. Also, like A. fumigatus CrmA derived metabolites, we find brassicicolin A to be a broad-spectrum antimicrobial. We speculate that CrmA-like isocyanide synthase products provide the producing fungi a fitness advantage in copper depleted environments., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: N.P.K. declares a potential conflict of interest as co-founder of the company Terra Bioforge. N.P.K. is also a Scientific Advisory Board member for Clue Genetics, Inc. The remaining authors declare no competing interests., (Copyright © 2023. Published by Elsevier Inc.)

Black rot, caused by Alternaria radicina, seriously endangers carrots throughout the growing season, affecting both leaves and fleshy roots. In this study, we sequenced and assembled the genome of the A. radicina isolate CBR2. The genome was 34.6 Mb in size and consisted of 6 scaffolds. The sequence information provided in this genome will be used as a reference for further comparative genomics analysis of Alternaria species and will contribute to disease control in carrot production., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Tomato early blight is a significant disease that causes substantial losses to tomato yield and quality. Mefentrifluconazole, an isopropanol-azole subgroup of triazole fungicides, has been registered in China for controlling various plant diseases, including tomato early blight, grape anthracnose, and apple brown spot. However, limited information is available on the mefentrifluconazole resistance risk and mechanism in plant pathogens. The sensitivity to mefentrifluconazole of 122 isolates of Alternaria alternata, one of the causal agents of tomato early blight, collected from different provinces in China, was evaluated. The results showed a unimodal curve for the sensitivity frequency, with an average EC 50 of 0.306 μg/mL. Through fungicide adaption, six resistant mutants (N4, N5, T4, T5, NG1, and NG10) were obtained from three parental isolates, with a mutation frequency of 3.28 × 10 -4 and resistance factors ranging between 19 and 147. The survival fitness of the resistant mutants, except for NG1, was significantly lower than that of their parental isolates. Positive cross-resistance was observed between mefentrifluconazole and difenoconazole or fenbuconazole, whereas no cross-resistance was found with three non-DMI fungicides. Furthermore, three distinct point mutations were detected in the AaCYP51 protein of the resistant mutants: I300S in T4 and T5; A303T in N4, NG1, and NG10; and A303V in N5. Compared to the parental isolates, the AaCYP51 gene was overexpressed in all six resistant mutants when treated with mefentrifluconazole. In summary, the resistance risk of A. alternata to mefentrifluconazole was low, and point mutations and overexpression of the AaCYP51 gene were identified as contributing factors to mefentrifluconazole resistance in A. alternata., (Copyright © 2023 Elsevier Inc. All rights reserved.)

The wild relatives of modern tomato crops are native to South America. These plants occur in habitats as different as the Andes and the Atacama Desert and are, to some degree, all susceptible to fungal pathogens of the genus Alternaria. Alternaria is a large genus. On tomatoes, several species cause early blight, leaf spots and other diseases. We collected Alternaria-like infection lesions from the leaves of eight wild tomato species from Chile and Peru. Using molecular barcoding markers, we characterized the pathogens. The infection lesions were caused predominantly by small-spored species of Alternaria of the section Alternaria, like A. alternata, but also by Stemphylium spp., Alternaria spp. from the section Ulocladioides and other related species. Morphological observations and an infection assay confirmed this. Comparative genetic diversity analyses show a larger diversity in this wild system than in studies of cultivated Solanum species. As A. alternata has been reported to be an increasing problem in cultivated tomatoes, investigating the evolutionary potential of this pathogen is not only interesting to scientists studying wild plant pathosystems. It could also inform crop protection and breeding programs to be aware of potential epidemics caused by species still confined to South America., (© 2023 The Authors. Environmental Microbiology published by Applied Microbiology International and John Wiley & Sons Ltd.)

Quinoa ( Chenopodium quinoa Willd.) is a native American crop mainly grown in the Andes of Bolivia and Peru. During the last decades, the cultivation of quinoa has expanded to more than 125 countries. Since then, several diseases of quinoa have been characterized. A leaf disease was observed on quinoa plants growing in an experimental plot in Eastern Denmark in 2018. The symptoms produced by the associated fungi consisted of small yellow blotches on the upper surface of leaves with a pale chlorotic halo surrounding the lesion. These studies used a combination of morphology, molecular diagnostics, and pathogenicity tests to identify two different Alternaria species belonging to Alternaria sections Infectoriae and Alternata as the causal agent of observed disease symptoms. To the best of our knowledge, this is the first report of Alternaria spp. as foliar pathogens of quinoa. Our findings indicate the need for additional studies to determine potential risks to quinoa production., Competing Interests: The author(s) declare no conflict of interest.

Over 380 host plant species have been known to develop leaf spots as a result of the fungus Alternaria alternata. It is an aspiring pathogen that affects a variety of hosts and causes rots, blights, and leaf spots on different plant sections. In this investigation, the lipopeptides from the B. subtilis strains T3, T4, T5, and T6 were evaluated for their antifungal activities. In the genomic DNA, iturin, surfactin, and fengycin genes were found recovered from B. subtilis bacterium by PCR amplification. From different B. subtilis strains, antifungal Lipopeptides were extracted, identified by HPLC, and quantified with values for T3 (24 g/ml), T4 (32 g/ml), T5 (28 g/ml), and T6 (18 g/ml). To test the antifungal activity, the isolated lipopeptides from the B. subtilis T3, T4, T5, and T6 strains were applied to Alternaria alternata at a concentration of 10 g/ml. Lipopeptides were found to suppress Alternaria alternata at rates of T3 (75.14%), T4 (75.93%), T5 (80.40%), and T6 (85.88%). The T6 strain outperformed the other three by having the highest antifungal activity against Alternaria alternata (85.88%)., (© 2023. The Author(s).)

The fungal genus Alternaria , which causes a variety of crop diseases, is widely distributed in the world. Alternaria leaf blight, caused by Alternaria dauci , is one of the most common and destructive diseases in carrot. The infection of A. dauci leads to dramatic decay on both foliage and taproot in severe cases, which results in significant yield losses. In this study, we sequenced and assembled the genome of A. dauci isolate CALB1, which isolated from the major carrot producing areas of China. A total of 65 contigs were assembled, and the estimated genome size was 34.9 Mb. The draft genome of A. dauci can be used for comparative genomic analysis of Alternaria species and provide genetic information for further research on plant-pathogen interactions., Competing Interests: The author(s) declare no conflict of interest.

Astragalus adsurgens (A. adsurgens), which is considered a forage in China, grows widely in Eurasia and North America. However, Alternaria gansuense (A. gansuense) (synonym: Embellisia astragali) systematically infects A. adsurgens, producing swainsonine (SW), which poisons domesticated animals. In this study, we hypothesized that the A. gansuense SW-producing fungus is morphologically and molecularly related to the locoweed endophyte, Alternaria oxytropis (A. oxytropis), which systematically grows in host plants. Therefore, pure cultures of the fungi from diseased plants or endophytic interactions were collected from fields and assayed for SW via high-performance liquid chromatography linked to mass spectroscopy (HPLC-MS). The production of SW was also detected in A. adsurgens, A. oxytropis and diseased plants by assaying for the presence of the β-ketoacyl synthase (KS) gene, which is required for SW synthesis. Diseased A. adsurgens and pure cultures of A. gansuense have SW and the healthy-looking A. adsurgens plants also contained SW, probably because they were infected with A. gansuense. Therefore, A. adsurgens-infected A. gansuense are not safe for livestock consumption., (© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Alternaria is a plant pathogen and human allergen. Alternaria alternata is one of the most abundant fungal spores in the air. The purpose of this study was to examine whether Alternaria spp. spore concentrations can be used to predict the abundance and spatio-temporal pattern of A. alternata spores in the air. This was investigated by testing the hypothesis that A. alternata dominates airborne Alternaria spp. spores and varies spatio-temporally. Secondarily, we aimed at investigating the relationship between airborne Alternaria spp. spores and the DNA profile of A. alternata spores between two proximate (~ 7 km apart) sites. These were examined by sampling Alternaria spp. spores using Burkard 7-day and cyclone samplers for the period 2016-2018 at Worcester and Lakeside campuses of the University of Worcester, UK. Daily Alternaria spp. spores from the Burkard traps were identified using optical microscopy whilst A. alternata from the cyclone samples was detected and quantified using quantitative polymerase chain reaction (qPCR). The results showed that either A. alternata or other Alternaria species spores dominate the airborne Alternaria spore concentrations, generally depending on weather conditions. Furthermore, although Alternaria spp. spore concentrations were similar for the two proximate sites, A. alternata spore concentrations significantly varied for those sites and it is highly likely that the airborne samples contained large amounts of small fragments of A. alternata. Overall, the study shows that there is a higher abundance of airborne Alternaria allergen than reported by aerobiological networks and the majority is likely to be from spore and hyphal fragments., (© 2023. The Author(s).)

Alternaria black spot disease on pecan is caused by the opportunistic pathogen Alternaria alternata and poses a serious threat to the local South African and global pecan industry. Several diagnostic molecular marker applications have been established and used in the screening of various fungal diseases worldwide. The present study investigated the potential for polymorphism within samples of A. alternata isolates obtained from eight different geographical locations in South Africa. Pecan ( Carya illinoinensis ) leaves, shoots, and nuts-in-shuck with Alternaria black spot disease were sampled, and 222 A. alternata isolates were retrieved. For rapid screening to identify Alternaria black spot pathogens, polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis of the Alternaria major allergen ( Alt a1 ) gene region was used, followed by the digestion of the amplicons with Hae III and Hinf I endonucleases. The assay resulted in five ( Hae III) and two ( Hinf I) band patterns. Unique banding patterns from the two endonucleases showed the best profile and isolates were grouped into six clusters using a UPGMA (unweighted pair group method with arithmetic averages) distance matrix (Euclidean) dendrogram method on R-Studio. The analysis confirmed that the genetic diversity of A. alternata does not depend on host tissues or the pecan cultivation region. The grouping of selected isolates was confirmed by DNA sequence analysis. The Alt a1 phylogeny corroborated no speciation within the dendrogram groups and showed 98-100% bootstrap similarity. This study reports the first documented rapid and reliable technique for routine screening identification of pathogens causing Alternaria black spot in South Africa.

Real-time PCR methods were developed to quantify the frequency of SDHC-H134R and SDHB-H277Y mutants associated with succinate dehydrogenase inhibitors (SDHI) resistance in Alternaria populations from pistachio. The linearity of the standard curves demonstrated the applicability in the quantification of the assays. The accuracy and reliability of the qPCR protocols to determine the frequency of mutants in real samples were corroborated. Orchards visibly affected by Alternaria late blight were sampled. The frequency of mutants was determined using the qPCR assays, while the frequency of resistant phenotypes was determined using a single discriminatory dose. The statistical analysis showed that the frequencies of the mutation SDHC-H134R determined with the qPCR assay were highly correlated with those estimated with the conventional method. The survey also evidenced that resistance to boscalid is still widespread in California. Results also indicated the possible contribution of other mutations to SDHI resistance. Our results confirmed the prevalence of SDHC-H134R mutants and the occurrence of mutation SDHB-H277Y at low frequencies. The real-time PCR methods developed in this study were able to detect differences in the frequencies of resistant mutants caused by the use of chemical fungicides. Finally, the effects of two fungicide programs on the frequency of mutants resistant to SDHI and quinone outside inhibitors fungicides were studied using qPCR assays. The experiments demonstrated that the use of anilinopyrimidine and demethylation inhibitors fungicides in the same program reduced the frequency of these mutations in Alternaria populations. The qPCR methods developed and used in this study can be used to track resistance levels in the pistachio orchards on a large scale., Competing Interests: The author(s) declare no conflict of interest.

In Zherong county, Fujian province, the black spot of Pseudostellaria heterophylla often breaks out in the rainy season from April to June every year. As one of the main leaf diseases of P. heterophylla, black spot seriously affects the yield and quality of the medicinal material. To identify and characterize the pathogens causing black spot, we isolated the pathogens, identified them as a species of Alternaria according to Koch's postulates, and then tested their pathogenicity and biological characteristics. The results showed that the pathogens causing P. heterophylla black spot were A. gaisen, as evidenced by the similar colony morphology, spore characteristics, sporulation phenotype, and the same clade with A. gaisen on the phylogenetic tree(the maximum likelihood support rate of 100% and the Bayesian posterior probability of 1.00) built based on the tandem sequences of ITS, tef1, gapdh, endoPG, Alta1, OPA10-2, and KOG1077. The optimum conditions for mycelial growth of the pathogen were 25 ℃, pH 5-8, and 24 h dark culture. The lethal conditions for mycelia and spores were both treatment at 50 ℃ for 10 min. We reported for the first time the A. gaisen-caused black spot of P. heterophylla. The results could provide a theoretical basis for the diagnosis and control of P. heterophylla leaf spot diseases.