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Fungal allergy is the third most frequent cause of respiratory pathologies and the most related to a poor prognosis of asthma. The genera Alternaria and Cladosporium are the most frequently associated with allergic respiratory diseases, with Alternaria being the one with the highest prevalence of sensitization. Alternaria alternata is an outdoor fungus whose spores disseminate in warm and dry air, reaching peak levels in temperate summers. Alternaria can also be found in damp and insufficiently ventilated houses, causing what is known as sick building syndrome. Thus, exposure to fungal allergens can occur outdoors and indoors. However, not only spores but also fungal fragments contain detectable amounts of allergens and may function as aeroallergenic sources. Allergenic extracts of Alternaria hyphae and spores are still in use for the diagnosis and treatment of allergic diseases but are variable and insufficiently standardised, as they are often a random mixture of allergenic ingredients and casual impurities. Thus, diagnosis of fungal allergy has been difficult, and knowledge about new fungal allergens is stuck. The number of allergens described in Fungi remains almost constant while new allergens are being found in the Plantae and Animalia kingdoms. Given Alt a 1 is not the unique Alternaria allergen eliciting allergy symptoms, component-resolved diagnosis strategies should be applied to diagnose fungal allergy. To date, twelve A. alternata allergens are accepted in the WHO/IUIS Allergen Nomenclature Subcommittee, many of them are enzymes: Alt a 4 (disulfide isomerase), Alt a 6 (enolase), Alt a 8 (mannitol de-hydrogenase), Alt a 10 (aldehyde dehydrogenase), Alt a 13 (glutathione-S-transferase) and Alt a MnSOD (Mn superoxide dismutase), and others have structural and regulatory functions such as Alt a 5 and Alt a 12, Alt a 3, Alt a 7. The function of Alt a 1 and Alt a 9 remains unknown. Other four allergens are included in other medical databases (e.g., Allergome): Alt a NTF2, Alt a TCTP, and Alt a 70 kDa. Despite Alt a 1 being the A. alternata major allergen, other allergens, such as enolase, Alt a 6 or MnSOD, Alt a 14 have been suggested to be included in the diagnosis panel of fungal allergy.

Severe outbreaks of Alternaria leaf blotch and fruit spot were recently observed in cv. Pink Lady apples in northern Israel, especially on fruit. Such severe outbreaks have not been reported from other countries. Symptoms involved cracks and rot around the calyx and external rot of the fruit body. Up to 80 % of the fruit in some orchards were affected by the disease. Microscopic examinations, fulfillment of Koch’s postulates and molecular (genetic) analyses confirmed the causal agent as Alternaria alternata f. sp. mali . The incidence of Alternaria increased as the degree of calyx cracking increased, or if fruit were both cracked and rotted. Injecting spore suspensions into the fruit produced typical rot symptoms. Injection assays of detached fruit of eight apple cultivars showed that cvs. Pink Lady and Golden Delicious were susceptible whereas cv. Jonathan was resistant. Pink Lady and Golden Delicious produced more fruit rot as the inoculum concentration increased. Rot in all three cultivars was moderate close to the skin but more severe close to the seed locule. Aqueous extracts taken from Jonathan fruit peel inhibited germ tube elongation of A. alternata f. sp. mali in vitro. This is the first report on heavy infection of Pink Lady fruit in Israel caused by A. alternata f. sp. mali .

In this study, a novel taxol-producing endophytic fungus, strain F3, was isolated from the fruits of Taxus cuspidata and identified as Alternaria alternata according to its macroscopic and microscopic traits and sequence analysis of internal transcribed spacer (ITS). The presence of taxol was detected by thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC) and confirmed by ultra-high-performance liquid chromatography-electrospray coupled to tandem mass spectrometry (UPLC-ESI–MS/MS) and nuclear magnetic resonance (NMR). The fermentation parameters of strain F3 were then optimized for high taxol production. The maximum taxol yield of 195.4 µg L −1 by A. alternata F3 was observed in 200-mL yeast peptone dextrose (YPD) broth, at an initial pH value of 6.0, supplemented with 0.1 g L −1 sodium acetate, 0.25 g L −1 salicylic acid, and 0.00125 g L −1 silver nitrate and inoculum size 2%, and incubated at 28 °C and 150 rpm for 8 days, which was 2.12-fold compared with the initial yield of taxol. Also, fungal taxol exhibited antitumor activity towards human lung carcinoma (A549) cell line and human cervical carcinoma (Hela) cell line with IC 50 values of 3.98 µg mL −1 and 0.35 µg mL −1 . Overall, this is the first report on taxol-producing endophytic fungus isolated from the fruits of Taxus . This study offers a novel source for the production of taxol for anticancer treatment.

This study explored the association between differentially expressed miRNA Aa-milR13 in Alternaria alternata f. sp. mali strains with and without virus infection, and the regulation of fungal host pathogenicity by viruses. MiRNA sequencing was conducted on a hypovirulent strain (QY21) with compound infection of Alternaria alternata chrysovirus 1 (AaCV1) and Alternaria alternata magoulivirus 1 (AaMV1), a hypovirulent strain (QY21-C1) with single AaMV1 infection, and a virus-free strain (QY21-C2). Fourteen miRNAs with significant differential expression were identified. Aa-milR13 was significantly downregulated in virus-infected strains and validated by RT-qPCR. Structural analysis of the Aa-milR13 sequence identified that the mature Aa-milR13 is located at the 5’ end of its precursor stem-loop structure. Knockout of Aa-milR13 in QY21-C2 resulted in slower hyphal growth, darker colony color, and reduced pathogenicity, resembling virus-infected strains. Conversely, overexpression of Aa-milR13 led to accelerated hyphal growth, lighter colony color, and significantly enhanced pathogenicity.Three target genes of Aa-milR13, subtilisin-like protein (AaSLP,cinr CC77DRAFT_1100266), DUF431-domain-containing protein (DUF431, CC77DRAFT_1022347), and high-affinity nitrate transporter NrtB (NrtB, CC77DRAFT_1056077), were predicted and confirmed by RT-qPCR to be negatively correlated with Aa-milR13 expression. Bioinformatics analysis indicated that Aa-milR13 targets the CDS regions of the potential target genes through cleavage, with Watson-Crick pairing of AU, CG, wobble pairing of GU, and various pairing patterns such as AA, AC, AG, GG, UU, UC, etc. This study suggests that fungal virus infection downregulates Aa-milR13, upregulating its targets, potentially contributing to reduced fungal pathogenicity. This is the first report of small RNAs acting as an intermediate bridge in the regulation of fungi virulence by fungal viruses. The research results lay the foundation for elucidating the mechanism of small RNA-involved regulation of fungi hypovirulence by fungal viruses and provide theoretical support for the use of fungal viruses in the control of crop fungal diseases.

Summary Fumonisin B1 (FB1) and Alternaria alternate f. sp. lycopersici (AAL)‐toxin are classified as sphinganine analog mycotoxins (SAMTs), which induce programmed cell death (PCD) in plants and pose health threat to humans who consume the contaminated crop products. Herein, Fumonisin B1 Resistant41 (FBR41), a dominant mutant allele, was identified by map‐based cloning of Arabidopsis FB1‐resistant mutant fbr41, then ectopically expressed in AAL‐toxin sensitive tomato (Solanum lycopersicum) cultivar. FBR41‐overexpressing tomato plants exhibited less severe cell death phenotype upon AAL‐toxin treatment. Analysis of free sphingoid bases showed that both fbr41 and FBR41‐overexpressing tomato plants accumulated less sphinganine and phytosphingosine upon FB1 and AAL‐toxin treatment, respectively. Alternaria stem canker is a disease caused by AAL and responsible for severe economic losses in tomato production, and FBR41‐overexpressing tomato plants exhibited enhanced resistance to AAL with decreased fungal biomass and less cell death, which was accompanied by attenuated accumulation of free sphingoid bases and jasmonate (JA). Taken together, our results indicate that FBR41 is potential in inhibiting SAMT‐induced PCD and controlling Alternaria stem canker in tomato.

Alternaria alternata botybirnavirus 1 (AaBRV1) was isolated from a strain of Alternaria alternata, causing watermelon leaf blight in our previous research. The effect of AaBRV1 on the phenotype of its host fungus, however, was not determined. In the present study, a novel strain of AaBRV1 was identified in A. tenuissima strain TJ-NH-51S-4, the causal agent of cotton Alternaria leaf spot, and designated as AaBRV1-AT1. A mycovirus AaBRV1-AT1-free strain TJ-NH-51S-4-VF was obtained by protoplast regeneration, which eliminated AaBRV1-AT1 from the mycovirus AaBRV1-AT1-infected strain TJ-NH-51S-4. Colony growth rate, spore production, and virulence of strain TJ-NH-51S-4 were greater than they were in TJ-NH-51S-4-VF, while the sensitivity of strain TJ-NH-51S-4 to difenoconazole, as measured by the EC50, was lower. AaBRV1-AT1 was capable of vertical transmission via asexual spores and horizontal transmission from strain TJ-NH-51S-4 to strain XJ-BZ-5-1hyg (another strain of A. tenuissima) through hyphal contact in pairing cultures. A total of 613 differentially expressed genes (DEGs) were identified in a comparative transcriptome analysis between TJ-NH-51S-4 and TJ-NH-51S-4-VF. Relative to strain TJ-NH-51S-4-VF, the number of up-regulated and down-regulated DEGs in strain TJ-NH-51S-4 was 286 and 327, respectively. Notably, the expression level of one DEG-encoding cytochrome P450 sterol 14α-demethylase and four DEGs encoding siderophore iron transporters were significantly up-regulated. To our knowledge, this is the first documentation of hypervirulence and reduced sensitivity to difenoconazole induced by AaBRV1-AT1 infection in A. tenuissima.

In this study, we isolated and identified pathogenic fungi from the naturally occurring fruits of red grapes, studied their biological characteristics, screened fifteen essential oil components to find the best natural antibacterial agent with the strongest inhibitory effect, and then compared the incidence of postharvest diseases and storage potential of red grapes treated with two concentrations (0.5 EC50/EC50) of essential oil components (inoculated with pathogenic fungi) during storage for 12 d at room temperature. In our research, Alternaria alternata was the primary pathogenic fungus of red grapes. Specifically, red grapes became infected which caused diseases, regardless of whether they were inoculated with Alternaria alternata in an injured or uninjured state. Our findings demonstrated that the following conditions were ideal for Alternaria alternata mycelial development and spore germination: BSA medium, D-maltose, ammonium nitrate, 28 °C, pH 6, and exposure to light. For the best Alternaria alternata spore production, OA medium, mannitol, urea, 34 °C, pH 9, and dark conditions were advised. Furthermore, with an EC50 value of 36.71 μg/mL, carvacrol demonstrated the highest inhibitory impact on Alternaria alternata among the 15 components of essential oils. In the meantime, treatment with EC50 concentration of carvacrol was found to be more effective than 0.5 EC50 concentration for controlling Alternaria alternata-induced decay disease of red grapes. The fruits exhibited remarkable improvements in the activity of defense-related enzymes, preservation of the greatest hardness and total soluble solids content, reduction in membrane lipid peroxidation in the peel, and preservation of the structural integrity of peel cells. Consequently, carvacrol was able to prevent the Alternaria alternata infestation disease that affects red grapes, and its EC50 concentration produced the greatest outcomes.

Alternaria blotch disease, caused by Alternaria alternata apple pathotype (AAAP), is one of the major fungal diseases in apple. Early field observations revealed, the anther-derived homozygote Hanfu line (HFTH1) was highly susceptible to AAAP, whereas Hanfu (HF) exhibited resistance to AAAP. To understand the molecular mechanisms underlying the difference in sensitivity of HF and HFTH1 to AAAP, we performed allele-specific expression (ASE) analysis and comparative transcriptomic analysis before and after AAAP inoculation. We reported an important immune gene, namely, MdFLS2 , which displayed strong ASE in HF with much lower expression levels of HFTH1-derived alleles. Transient overexpression of the dominant allele of MdFLS2-1 from HF in GL-3 apple leaves could enhance resistance to AAAP and induce expression of genes related to salicylic acid pathway. In addition, MdFLS2-1 was identified with an insertion of an 85-bp terminal-repeat retrotransposon in miniature (TRIM) element-like sequence in the upstream region of the nonreference allele. In contrast, only one terminal direct repeat (TDR) from TRIM-like sequence was present in the upstream region of the HFTH1-derived allele MdFLS2-2. Furthermore, the results of luciferase and β-glucuronidase reporter assays demonstrated that the intact TRIM-like sequence has enhancer activity. This suggested that insertion of the TRIM-like sequence regulates the expression level of the allele of MdFLS2 , in turn, affecting the sensitivity of HF and HFTH1 to AAAP.

Citrus alternaria brown spot, caused by the tangerine pathotype of Alternaria alternata, is one of the most severe fungal diseases affecting citrus crops. Currently, there is a critical need for rapid and visual detection techniques to identify the tangerine pathotype of A. alternata. In this study, a novel detection system was developed by combining recombinase polymerase amplification (RPA) with CRISPR/Cas12a technology, targeting the ACTT3 gene specific to the tangerine pathotype of A. alternata. Through optimization of reaction time and component concentrations, the assay demonstrated a detection sensitivity of 1 pg μL−1 within 40 min at a constant temperature of 37 °C. The results can be visually interpreted using nucleic acid test strips, offering advantages in specificity, sensitivity, and speed. This system has been successfully validated for the rapid detection of the pathogen within plant tissues, including leaves and fruits, providing an efficient and practical solution for real-time field detection of the tangerine pathotype of A. alternata.

The pathogen Alternaria alternata infects a variety of plants and crops, notably poplars, and results in large financial losses. Using twelve chemical fungicides for fungicide sensitivity tests (FSTs) on A. alternata, the result showed that prochloraz (PCZ), mancozeb (MZ), and fludioxonil (FLU) have potent inhibitory effects against the pathogen through different mechanisms. To investigate how the pathogen responded to fungicide-induced stress, transcriptome and physiological investigations were carried out after treatments with three fungicides at their corresponding 50% effective concentration (EC50) doses. The MZ treatment produced a distinct genetic response; FLU treatment produced the greatest number of differentially expressed genes (DEGs), followed by PCZ. DEGs from FLU treatment were mostly engaged in ribosome biosynthesis, those from MZ treatment in lipid and carbohydrate metabolism, and those from PCZ treatment in carbohydrate metabolism, according to Gene Ontology (GO) analysis. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis revealed that FLU and PCZ treatments were associated with ribosome biogenesis, whereas MZ treatment was linked to the pyruvate metabolic pathway. Collinear trend analysis indicates that MZ exhibits a unique pattern, with FLU treatment causing the most significant overexpression of genes, followed by PCZ. The six categories of 88 elevated DEGs associated with fungal resistance include tyrosinase, ATP-binding cassette (ABC) transporters, major facilitator superfamily (MFS) transporters, antioxidant and cellular resilience genes, as well as genes involved in cell wall and membrane biosynthesis. Notably, the pathways involved in the synthesis of melanin and ergosterol exhibited the strongest response to FLU. The results of a correlation analysis between physiological indices and resistance-related genes indicated that melanin content, malondialdehyde (MDA) content, and tyrosinase activity were positively correlated with the majority of resistance-related DEGs, whereas soluble protein content, superoxide dismutase (SOD) activity, and catalase (CAT) activity were negatively correlated, which is consistent with the observed trends in the measured physiological indicators. Taken together, this study provides a theoretical basis for developing more effective fungicides and chemical control strategies against A. alternata.