Alternaria solani core effector Aex59 is a new member of the Alt a 1 protein family and is recognized as a PAMP.

Early blight caused by Alternaria solani is a destructive disease in potato production. Here, through systematically screening of an effector protein pool consisting of 115 small cysteine-containing candidate Aex (Alternariaextracellular proteins) in A. solani, we identified a core effector protein named Aex59, a pathogen-associated molecular pattern (PAMP) molecule. Aex59 is uniquely present in the Ascomycota of fungi and can activate defense responses in multiple plants. Targeted gene disruption showed that Aex59 is a virulence factor and participates in spore development. Perception of Aex59 in Nicotiana benthamiana does not depend on the receptor-like kinases Brassinosteroid-associated kinase1 (BAK1) and Suppressor of BIR1-1 (SOBIR1), which are required for multiple pattern recognition receptors (PRR) pathways. Sequence analysis revealed that Aex59 is a new member of the Alt a 1 protein family and is a potential molecular marker capable of aiding in the classification of the fungi Alternaria spp.

The G-protein alpha subunit AaGA1 positively regulates vegetative growth, appressorium-like formation, and pathogenicity in Alternaria alternata.

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.

Invasive phaeohyphomycosis co-infection with Alternaria spp. and Curvularia spp. in a neutropenic host.

Phaeohyphomycoses are infections caused by dark-walled dematiaceous fungi. Alternaria and Curvularia are two genera of dematiaceous molds known to cause invasive fungal rhinosinusitis, particularly in immunocompromised patients. Co-infection with two dematiaceous fungi is rarely reported in the literature. This report describes a case of biopsy proven invasive fungal rhinosinusitis with Alternaria spp. and Curvularia spp. co-infection in a neutropenic host. The infection characteristics, microbiologic findings, and treatment are described.

JA/ethylene and NaWRKY6/3 regulated Alternaria resistance depends on ethylene response factor 1B-like in wild tobacco.

Biosynthesis of the phytoalexins scopoletin and scopolin in Nicotiana species is regulated by upstream signals including jasmonate (JA), ethylene (ET) and NaWRKY3 in response to the necrotrophic fungus Alternaria alternata, which causes brown spot disease. However, how these signals are coordinated to regulate these phytoalexins remains unknown. By analyzing RNA sequencing data and RNA interference, we identified NaERF1B-like (NaERF1B-L) as a key player in Nicotiana attenuata during A. alternata infection by regulating the transcripts of Feruloyl-CoA 6'-hydroxylase 1 (NaF6'H1), encoding a key enzyme for scopoletin biosynthesis, and NaVS1-like (NaVS1-L), a putative biosynthetic gene of the phytoalexin solavetivone. We further demonstrated that the synergistic induction of these two genes by JA and ET signaling is mediated by NaERF1B-L. Additionally, we found that the two closely related proteins NaWRKY6 and NaWRKY3 physically interact to enhance NaERF1B-L expression by directly binding and activating the NaERF1B-L promoter. Collectively, our current results demonstrate that NaERF1B-L plays a positive role in resistance to A. alternata by modulating phytoalexins biosynthesis through the integration of JA/ET and NaWRKY6/3 signaling. Our findings reveal a fine-tuned transcriptional regulatory hierarchy mediated by NaERF1B-L for brown spot disease resistance in wild tobacco.

Bioprocessing of camptothecin from Alternaria brassicicola, an endophyte of Catharanthus roseus, with a strong antiproliferative activity and inhibition to Topoisomerases.

Suppression of fungal camptothecin (CPT) biosynthesis with the preservation and successive subculturing is the challenge that impedes fungi from the industrial application, so, screening for a novel fungal isolate with a conceivable stable producing potency of CPT was the main objective of this work. Catharanthus roseus with diverse contents of bioactive metabolites could have a plethora of novel endophytes with unique metabolic properties. Among the endophytes of C. roseus, Alternaria brassicicola EFBL-NV OR131587.1 was the highest CPT producer (96.5 µg/L). The structural identity of the putative CPT was verified by HPLC, FTIR, HNMR and LC-MS/MS, with a molecular mass 349 m/z, and molecular fragmentation patterns that typically identical to the authentic one. The purified A. brassicicola CPT has a strong antiproliferative activity towards UO-31 (0.75 µM) and MCF7 (3.2 µM), with selectivity index 30.8, and 7.1, respectively, in addition to resilient activity to inhibit Topo II (IC50 value 0.26 nM) than Topo 1 (IC50 value 3.2 nM). The purified CPT combat the wound healing of UO-31 cells by ~ 52%, stops their matrix formation, cell migration and metastasis. The purified CPT arrest the cellular division of the UO-31 at the S-phase, and inducing their cellular apoptosis by ~ 20.4 folds, compared to the control cells. Upon bioprocessing with the surface response methodology, the CPT yield by A. brassicicola was improved by ~ 3.3 folds, compared to control. The metabolic potency of synthesis of CPT by A. brassicicola was attenuated with the fungal storage and subculturing, losing ~ 50% of their CPT productivity by the 6th month of storage and 6th generation. Practically, the CPT productivity of the attenuated A. brassicicola was restored by addition of 1% surface sterilized leaves of C. roseus, ensuring the eliciting of cryptic gene cluster of A. brassicicola CPT via the plant microbiome-A. brassicicola interactions. So, for the first time, a novel endophytic isolate A. brassicicola, from C. roseus, was explored to have a relatively stable CPT biosynthetic machinery, with an affordable feasibility to restore their CPT productivity using C. roseus microbiome, in addition to the unique affinity of the extracted CPT to inhibit Topoisomerase I and II.

Molecular characterization of a new botybirnavirus that infects Alternaria sp. from tobacco.

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.

First Report of Tobacco Leaf Tip Blight Caused by Alternaria gossypina in China.

Tobacco (Nicotiana tabacum L.) belongs to the family Solanaceae, an economically significant crop (Zhou et al. 2023). Twelve samples with leaf spots were collected in Keti Village, Changshun County, Zunyi City, Guizhou province, China in 2022. Twenty-five percent of the samples had dry lesions near the leaf tip which resulted leaf tip blight after development. Fungi were isolated by a previous method (Wei et al. 2022). Six Alternaria strains were obtained and preserved in the Fungal Herbarium of Yangtze University (YZU), Jingzhou, Hubei, China. Among them, one strain YZU 221477 showed distinct cultural characteristics out of five A. alternata strains, which was again determined by growing on potato dextrose agar (PDA) at 25°C for 7 days in dark to evaluate. The colonies (60 mm in diameter) were white cottony in the center surrounded by vinaceous purple. To examine the morphology, mycelia were inoculated onto potato carrot agar (PCA) at 22°C, following an 8 h light/16 h dark photoperiod (Simmons 2007). Conidia were obclavate or ovoid, normally 3-5 conidial units per chain, 20-38 × 10-16.5 µm, 3 to 5 transverse septa, beakless or a short beak (4-30 µm). The observation results were consistent with those of A. gossypina (Zhang 2003). Total genomic DNA was extracted using the CTAB method and seven gene regions including internal transcribed spacer of rDNA (ITS), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), translation elongation factor 1 alpha (TEF1), RNA polymerase second largest subunit (RPB2), Alternaria major allergen gene (Alt a 1), endopolygalacturonase (EndoPG) and an anonymous gene region (OPA10-2) were amplified with ITS5/ITS4, gpd1/gpd2, EF1-728F/EF1-986R, RPB2-5F/RPB2-7cR, Alt-for/Alt-rev, PG3/PG2b and OPA10-2L/OPA10-2R primers, respectively. All sequences were deposited in GenBank (ITS: OR710806; GAPDH: PP057862; TEF1: PP158601; RPB2: PP057863; Alt a 1: PP057865; EndoPG: PP057861; OPA10-2: PP057864). Combining with relevant sequences retrieved from the NCBI database were used for the phylogenetic analysis. Maximum Likelihood (ML) tree was constructed with RAxML v.7.2.8 employing GTRCAT model using 1000 bootstrap (BS) replicates to assess statistical support. The results indicated that the present strain grouped with A. gossypina (type strain of CBS 104.32) supported with 73% bootstrap values, also having a support of 0.83 Bayesian posterior probabilities values. Based on morphology and molecular evidence, the strain YZU 221477 is identified as Alternaria gossypina. Pathogenicity was examined to fulfill Koch's postulates. Mycelial plugs (6 mm diameter) of the present strain and A. alternata cultivated on PDA were taken from the margin and inoculated onto viable tobacco leaves (Cultivar: Yunyan 87, n=3) growing forty days, while controls were inoculated with sterile PDA. The assay was conducted three times. The plants were maintained at 25°C with humidity levels over 85% in a greenhouse. Leaves were evaluated after 7 days, necrotic spots encircled by yellow halos were on both inoculums, except controls. Pathogen re-isolation confirmed that it was the same as inoculated fungus based on morphology. A. gossypina was firstly found on cotton (Hopkins 1931), late reported to induce disease on Minneola, Nopalea, Hibiscus, Citrus, Solanum and Ageratina. To our knowledge, this is the first report of A. gossypina causing tobacco leaf tip blight in China, and it also provides a basis for controlling of tobacco leaf tip blight.

Fusarium, Scedosporium and Other Rare Mold Invasive Infections: Over Twenty-Five-Year Experience of a European Tertiary-Care Center.

Invasive mold infections (IMD) are an emerging concern due to the growing prevalence of patients at risk, encompassing but not limited to allogeneic hematopoietic stem cell transplant recipients, hematological malignancies patients, solid organ transplant recipients and intensive care unit patients. In contrast with invasive aspergillosis and mucormycosis, other hyalohyphomycoses and phaeohyphomycoses remain poorly known. We conducted a retrospective analysis of the clinical, biological, microbiological and evolutive features of 92 IMD having occurred in patients in our tertiary-care center over more than 25 years. A quarter of these infections were due to multiple molds. Molds involved were Fusarium spp. (36.2% of IMD with a single agent, 43.5% of IMD with multiple agents), followed by Scedosporium spp. (respectively 14.5% and 26.1%) and Alternaria spp. (respectively 13.0% and 8.7%). Mortality at day 84 was higher for Fusarium spp., Scedosporium spp. or multiple pathogens IMD compared with Alternaria or other pathogens (51.7% vs. 17.6%, p < 0.05). Mortality at day 84 was also influenced by host factor: higher among hematology and alloHSCT patients than in other patients (30.6% vs. 20.9% at day 42 and 50.0% vs. 27.9% at day 84, p = 0.041). Better awareness, understanding and treatments are awaited to improve patient prognosis.

Cynara cardunculus L. var. scolymus L. Landrace "Carciofo Ortano" as a Source of Bioactive Compounds.

The preservation of agricultural biodiversity and socioeconomic development are relevant both to enhance domestic production and to support innovation. In the search for new biomolecules, we have focused on the "Carciofo Ortano" landrace, growth in the northern part of the Lazio region. Artichoke cultivation generates substantial by-products, including leaves, stems, and roots, which could serve as valuable sources of biomolecules and prebiotic dietary fiber. To valorize the leaf waste of the "Carciofo Ortano" landrace, a multidisciplinary approach was applied. Chemical analysis using HPLC-DAD identified mono-O- and di-O-caffeoylquinic acids and the sesquiterpene cynaropicrin in all artichoke leaf extracts. SPME-GC/MS analyses detected aliphatic alcohols in the fresh leaf samples. Antiproliferative and cytotoxic studies on cancer (SH-SY5Y, MCF-7, MDA) and normal (MCF-10A) human cell lines revealed that leaf extracts induced a selective dose and time-dependent biological effect. While showing slight activity against environmental bacterial strains, artichoke leaf extracts exhibited significant antifungal activity against the phytopathogenic fungus Alternaria alternata. Overall, the results highlight the potential of "Carciofo Ortano" cultivation by-products as a rich source of biomolecules with versatile applications in humans, animals, and the environment.

The LOV-domain blue-light receptor LreA of the fungus Alternaria alternata binds predominantly FAD as chromophore and acts as a light and temperature sensor.

Light and temperature sensing are important features of many organisms. Light may provide energy but may also be used by non-photosynthetic organisms for orientation in the environment. Recent evidence suggests that plant and fungal phytochrome and plant phototropin serve dual functions as light and temperature sensors. Here we characterized the fungal LOV-domain blue-light receptor LreA of Alternaria alternata and show that it predominantly contains FAD as chromophore. Blue-light illumination induced ROS production followed by protein agglomeration in vitro. In vivo ROS may control LreA activity. LreA acts as a blue-light photoreceptor but also triggers temperature-shift-induced gene expression. Both responses required the conserved amino acid cysteine 421. We therefore propose that temperature mimics the photoresponse, which could be the ancient function of the chromoprotein. Temperature-dependent gene expression control with LreA was distinct from the response with phytochrome suggesting fine-tuned, photoreceptor-specific gene regulation.

NaWRKY70 is a key regulator of Nicotiana attenuata resistance to Alternaria alternata through regulation of phytohormones and phytoalexins biosynthesis.

Jasmonate (JA) and abscisic acid (ABA) are two major phytohormones involved in pathogen resistance. However, how their biosynthesis is regulated is not well understood. We silenced NaWRKY70 in wild tobacco Nicotiana attenuata and determined its role in regulating genes involved in the production of JA, ABA and the phytoalexin capsidiol in response to the fungal pathogen Alternaria alternata using techniques including electrophoretic mobility shift, chromatin immunoprecipitation, transient overexpression and virus-induced gene silencing. Silencing NaWRKY70 dramatically reduced both basal and A. alternata-induced jasmonoyl-isoleucine (JA-Ile) and ABA. Further evidence showed that NaWRKY70 directly binds to the W-boxes of the promoters of NaAOS and NaJAR4 (JA biosynthesis), NaNCED1 and NaXD1-like (ABA biosynthesis), and NaMPK4 (ABA signaling) to activate their expression, while binding but repressing the expression of NaCYP707A4-like3 (ABA degradation). Additionally, NaWRKY70 regulates capsidiol production through its key enzyme genes NaEASs and NaEAHs, and interacts with its regulator NaERF2-like to enhance their expression, whereas ABA negatively regulates capsidiol biosynthesis. Our results highlight the key role of NaWRKY70 in controlling both JA-Ile and ABA production, as well as capsidiol production, thus providing new insight into the defense mechanism of plant resistance to A. alternata.

Profile and in silico analysis of metabolite compounds of the endophytic fungus Alternaria alternata K-10 from Drymoglossum piloselloides as antioxidants and antibacterials.

Endophytic fungi are known for producing secondary metabolites with valuable biological activities, including antiviral, anticancer, antibacterial, and antioxidant properties. This study aims to evaluate an endophytic fungus from Dragon Scales leaves (Drymoglossum piloselloides) and analyze its metabolites as antioxidants and antibacterials. In this study, an endophytic fungus was isolated from the leaves of Dragon Scales (D. piloselloides) and identified using molecular analysis of the Internal Transcribed Spacer (ITS) ribosomal RNA locus. The fungus was authenticated as Alternaria alternata strain K-10. Crude extracts were obtained using n-hexane and ethyl acetate and analyzed via GC-MS Shimadzu-QP 2010 Ultra with NIST spectral library. Antibacterial activity was observed against Bacillus subtilis, Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa using the paper disc method, showing inhibition zones of 8.7-9.3 mm and 8.8-9.4 mm for ethyl acetate and n-hexane extracts, respectively. Ethyl acetate and n-hexane extracts exhibited strong antioxidant potential against 2,2-diphenyl-1-picrylhydrazil (DPPH) radical (IC50 values of 50.99 µg mL-1 and 74.44 µg mL-1, respectively). GC-MS analysis revealed 40 compounds in both extracts, some of which, including 2-ethylhexyl ester benzoic acid, benzo-b-dihydropyran-6-hydroxy-4-4-5-7-8-pentamethyl, diethyl phthalate, and octadecanoic acid, were identified through in silico analysis and found to possess antioxidant properties. These findings hold implications for potential applications of the plant and its biological constituent to be developed as lead compounds in the medical sector.

Physio-molecular responses of tomato cultivars to biotic stress: Exploring the interplay between Alternaria alternata OP881811 infection and plant defence mechanisms.

Plant fungal diseases impose a formidable challenge for global agricultural productivity, a meticulous examination of host-pathogen interactions. In this intricate study, an exhaustive investigation was conducted on infected tomatoes obtained from Egyptian fields, leading to the precise molecular identification of the fungal isolate as Alternaria alternata (OP881811), and the isolate showed high identity with Chinese isolates (ON973896 and ON790502). Subsequently, fourteen diverse tomato cultivars; Cv Ferment, Cv 103, Cv Damber, Cv 186, Cv 4094, Cv Angham, Cv N 17, Cv Gesma, Cv 010, Cv branch, cv 2020, Cv 023, Cv Gana and Cv 380 were meticulously assessed to discern their susceptibility levels upon inoculation with Alternaria alternata. Thorough scrutiny of disease symptom manifestation and the extent of tomato leaf damage ensued, enabling a comprehensive evaluation of cultivar responses. Results unveiled a spectrum of plant susceptibility, with three cultivars exhibiting heightened vulnerability (Cv Ferment, Cv 103 and Cv Damber), five cultivars displaying moderate susceptibility (Cv 186, Cv 4094, Cv Angham, Cv N 17 and Cv Gesma), and six cultivars demonstrating remarkable resilience to the pathogen (Cv 010, Cv branch, cv, 2020; Cv 023, Cv Gana and Cv 380). In order to gain a thorough understanding of the underlying physiological patterns indicative of plant resistance against A. alternata, an in-depth exploration of polyphenols, flavonoids, and antioxidant enzymes ensued. These key indicators were closely examined, offering valuable insights into the interplay between plant physiology and pathogen response. Robust correlations emerged, with higher contents of these compounds correlating with heightened susceptibility, while lower levels were indicative of enhanced plant tolerance. In tandem with the physiological assessment, a thorough investigation of four pivotal defensive genes (PR5, PPO, PR3, and POX) was undertaken, employing cutting-edge Real-Time PCR technology. Gene expression profiles displayed intriguing variations across the evaluated tomato cultivars, ultimately facilitating the classification of cultivars into distinct groups based on their levels of resistance, moderate susceptibility, or heightened sensitivity. By unravelling the intricate dynamics of plant susceptibility, physiological responses, and patterns of gene expression, this comprehensive study paves the way for targeted strategies to combat plant fungal diseases. The findings contribute valuable insights into host-pathogen interactions and empower agricultural stakeholders with the knowledge required to fortify crop resilience and safeguard global food security.

First Report of Alternaria alternata Causing Leaf blight on Epimedium sagittatum (Sieb. et Zucc.) Maxim. in China.

Epimedium sagittatum (Sieb.et Zucc.) Maxim. is an important material of traditional Chinese medicine because of the rich content of flavonoids that are used to treat osteoporosis, liver cancer, and sexual dysfunction (Liu et al. 2013). A leaf blight was observed on E. sagittatum in Zhumadian City, China (32°58'12" N, 114°37'48" E, continental monsoon climate) in June 2021. Survey indicated that about 18% of the plants were infected in a 266-ha commercial planting area. The initial symptoms were white patches with tan borders, irregular in outline, with small black particles visible on the center of the lesions. In a week or so, patches extended throughout the leaf, and then leaves withered. Thirty leaves with symptoms collected from five different sites were cut into 5×5 mm pieces, and then surface-sterilized with 75% ethanol for 15 s followed by rinsing with double distilled water (ddH2O) three times. The pieces were then disinfested with 0.1% HgCl2 solution for 30 s, and rinsed with ddH2O, then placed onto potato-dextrose agar medium (PDA) and incubated in the dark for 3 d at 28°C. Eight fungal isolates were purified; of these, only the isolate HY2-1 infected the host plant and was selected for further morphological characterization. The colonies of HY2-1 were olive green with loose aerial hyphae on PDA. Conidiophores were single or branched, producing brown conidia in short chains. Conidia were obclavate, obpyriform, or ellipsoidal, 15.9-47.3 µm × 7.6-16.6 µm (n=50) and pale brown or dark brown with a short cylindrical beak at the tip that contained 1-5 transverse septa and 0-4 longitudinal septa. Morphological characteristics of the isolate were identical with those of Alternaria species (Huang et al. 2022). For molecular identification, the internal transcribed spacers (ITS), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Weir et al. 2012), major allergen Alt a 1(Alt a 1) and translation elongation factor 1-α gene (TEF) (Lawrence et al. 2013) were amplified and sequenced using the primers ITS4/5, GDF/GDR, Alt-F/R, and EF1-728F/986R, respectively. The results of the sequencing were uploaded to GenBank as ITS (OR418487), GAPDH (OR419792), Alt a 1 (OR419794), and TEF (OR419796), respectively. Phylogenetic analyses were performed by concatenating all the sequenced loci using the Bayesian method in Phylosuite (Zhang et al.2020). The phylogenetic tree indicated that the isolate belongs to the A. alternata clade with a bootstrap value of 75%. The pathogen was identified as A. alternata based on the morphological and molecular results. To satisfy Koch's postulates, a conidial suspension (106 conidia/mL) of the HY2-1 was prepared with ddH2O to infect the healthy plants. Ninety healthy leaves on 30 plants in pots were punctured using a sterilized needle, and then inoculated by spraying the conidial suspension on the wounded leaves in a greenhouse at 25°C and 80% relative humidity. The control plants were sprayed with ddH2O. The plants showed similar symptoms to the original infected plant 15 d after inoculation. The controls showed no symptoms. A pure culture of A. alternata was isolated and identified again as previously described. Leaf blight caused by A. alternata has been reported on Taro (Liu et al. 2020), Toona ciliata (Wang et al. 2023), etc. To our knowledge, this is the first report of E. sagittatum leaf blight caused by A. alternata in China. The results will help to develop effective control strategies for leaf blight on E. sagittatum.

Occurrence of Leaf Spot on Elaeocarpus decipiens Caused by Alternaria alternata, a new disease in China.

Elaeocarpus decipiens is widely cultivated as an ornamental tree of commercial importance in southern China. During March 2018 to March 2021, leaf spot disease was observed in about 40% of E. decipiens on the campus of Jiangnan University in Wuxi, Jiangsu, China (31.48°N, 120.46°E). Leaf symptoms began as small, light brown lesions that enlarged, turned olive brown in color and then became necrotic. Ten symptomatic leaves were collected from five different trees on the Jiangnan University campus and surface sterilized with 75% ethanol for 30 seconds, followed by 1% sodium hypochlorite for 1 minute, and rinsed three times with sterile distilled water before being cultured onto potato dextrose agar and incubated in the dark at 25°C for 5 days. Five purified fungal isolates were obtained by the single spore isolation method. Emergent fungal colonies were olive-green in color with 1 to 3 mm white margins and abundant aerial hyphae. Conidia were borne in chains or singly and were obclavate or obpyriform and measured 6.5 to 17.4 × 21.3 to 32.8 µm (n=50) with one to seven transverse septa and zero to three longitudinal septa. Based on morphological characteristics, the pathogen was identified as Alternaria spp.(Simmons 2007). Three representative isolates, At1, At2 and At3, were selected for molecular identification, total genomic DNA of the fungus isolates were extracted with Plant/Fungi DNA Isolation Kit (Sigma-Aldrich, Ontario, Canada). Plasma membrane ATPase (ATP) gene, chitin synthase (CHS) gene and translation elongation factor 1-alpha (EF1) gene were amplified with primers ATPDF1/ATPDR1, CHS-79F/CHS-345R (Lawrence et al. 2013) and EF1-728F/EF1-986R (Carbone and Kohn 1999). The amplification results of the three isolate genes were consistent, and we deposited the results of the ATP (MN046377), CHS (MN046378) and EF1 (MN046379) sequences of At1 in the NCBI GeneBank. The ATPase gene from the representative isolate At1 shared 99.83% similarity to A. alternata causing leaf Spot of Codonopsis pilosula in China (OM362504, Shi et al. 2022), the CHS gene shared 100% similarity to A. alternata causing brown leaf spot on Paris polyphylla var. chinensis in China (MK391053, Fu et al. 2019), and the EF1 gene shared 100% similarity to A. alternata CBS 916.96 ex-type on Arachis hypogaea in India (KC584634). A phylogenetic tree constructed with the EF1 gene using the neighbor-joining algorithm in MEGA 11 software with 1,000 bootstrap replicates revealed that the examined isolate, At1, belongs to the fungus A. alternata. For pathogenicity tests, 10 leaves of five healthy plants were sprayed with spore suspensions (1 × 107 conidia/ml) of the 10-day-old isolates (At1, At2 and At3, respectively). As a control, five plants were sprayed with sterile distilled water. After inoculation, use the bags to moisturize for 48 hours. Pathogenicity tests were conducted three times. Fourteen days after inoculation, olive brown necrotic lesions developed on inoculated leaves while control leaves remained symptomless. The pathogen was reisolated from infected leaves and confirmed as A. alternata based on morphological characteristics and molecular markers. To date, A. alternata has been reported to cause leaf spot disease on many plants inculuding Ficus religiosa (Du et al. 2022), Tilia miqueliana (Yue et al. 2023), Ligustrum japonicum (Fang et al. 2023) and so on. To our knowledge, this is the first report of the occurrence of A. alternata causing leaf spot on E. decipiens in China. The increasing area of E. decipiens cultivation and global climate change have led to an increase in the incidence of E. decipiens diseases, which should be taken into account by forest conservationists.

First Report of Alternaria Leaf Spot Caused by Alternaria alternata on Ulmus parvifolia in Jiangsu, China.

Ulmus parvifolia Jacq. is an important tree with ornamental value, which is widely planted in Hebei and southern regions of China. In September 2022, a leaf spot symptom was observed on about approximately 20% U. parvifolia seedlings growing a tree farm (20000 m2) of Jiangsu Academy of Forestry (118°45'57.30″E, 31°51'27. 94″N). Gray to black spots appeared on leaves of seedlings. Five diseased leaves were collected from five different seedlings. The pieces were excised from the margins between healthy and diseased tissues, surface sterilized in 75% ethanol for 30 s and then in 1.5% NaClO for 90 s, rinsed three times in sterilized distilled water, plated on potato dextrose agar (PDA) and incubated at 25℃ in the darkness. Pure cultures were obtained by monosporic isolation. Six isolates with identical morphological features and the internal transcribed spacer (ITS) sequences were obtained (the isolate rate of 67%), and identified as Alternaria sp. A representative isolate, LY-1-1 was used for the further study. The colony of LY-1-1, growing on PDA was cotton-like and brown in color with gray-white aerial hyphae on their surfaces, and its reverse was dark grey. The conidia were ovate to pear-shaped, brown in color, with 1 to 4 transverse septa and 0 to 1 longitudinal septa, parietal cells extending into the beak, and measured 7.1 to 12.5×3.8 to 7.1 µm (n=35). These characteristics were consistent with the description of Alternaria sp. (Simmons 2007). The regions of ITS, large subunit ribosomal RNA (LSU), small subunit ribosomal RNA (SSU), anonymous region OPA10-2 genomic sequence (OPA10-2), Alternaria 1 major allergen (Alta1), glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and translation elongation factor 1-alpha (TEF1) genes (GenBank Accession No. OR047916, OR051904, OR047919, OR061065, OR061063, OR061064, and OR061062, respectively) were amplified (White et al. 1990; Woudenberg et al. 2015) and sequenced. These obtained sequences showed 99.86-100% similarity to the ITS (514/515 bp) of A. alternata isolate SPM-2 (OR378581), LSU (801/801 bp) of isolate B9 (OR366492), SSU (1019/1020 bp) of strain LSU0766 (MT000349), OPA10-2 (632/633 bp) of strain 19-1 (MN185000), Alta1 (470/470 bp) of strain CMML21-73 (OQ831518), GAPDH (177/177 bp) of isolate CS36-3 (KY814638), and TEF1 (240/240 bp) of isolate SY-6 (OP980553). A neighbor-joining phylogenetic tree was generated by combining all sequenced loci in MEGA7. The isolate LY-1-1 clustered in the A. alternata clade with 98% bootstrap support. Three 3-month-old U. parvifolia seedlings were wounded with a sterile needle and inoculated with 20 µL conidia suspension (1×106 spores/mL) on the left sides of leaves. Inoculation on the right side with 20 µL of sterile water was treated as a control. All inoculated plants were incubated in a greenhouse at 25℃, 80% relative humidity, and a 12-h light/dark cycle. The experiment was repeated three times. After 5 days of inoculation, typical gray to black spots were found on the left sides of all inoculated leaves, and the control did not have any leaf spot symptoms. Subsequently, the same fungus was reisolated and identified based on morphological and molecular traits, fulfilling Koch's postulates. The A. alternata has been reported to cause leaf spot on pecan (Wu et al. 2020), fruit spot on olive (Alam et al. 2019) and fruit rot on lychee (Alam et al. 2017). However, there are no other reports of A. alternata on U. parvifolia in the world. Thus, this study provides an important reference for the biology, epidemiology of A. alternata.

Fermentation of waste water from agar processing with Bacillus subtilis by metabolomic analysis.

Fungal infection has become a major threat to crop loss and affects food safety. The waste water from agar processing industries extraction has a number of active substances, which could be further transformed by microorganisms to synthesize antifungal active substances. In this study, Bacillus subtilis was used to ferment the waste water from agar processing industries extraction to analyze the antifungal activity of the fermentation broth on Alternaria alternata and Alternaria spp. Results showed that 25% of the fermentation broth was the most effective in inhibited A. alternata and Alternaria spp., with fungal inhibition rates of 99.9% and 96.1%, respectively, and a minimum inhibitory concentration (MIC) was 0.156 µg/mL. Metabolomic analysis showed that flavonoid polyphenols such as coniferyl aldehyde, glycycoumarin, glycitin, and procyanidin A1 may enhance the inhibitory activity against the two pathogenic fungal strains. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis showed that polyphenols involved in the biosynthesis pathways of isoflavonoid and phenylpropanoid were upregulated after fermentation. The laser confocal microscopy analyses and cell conductivity showed that the cytoplasm of fungi treated with fermentation broth was destroyed. This study provides a research basis for the development of new natural antifungal agents and rational use of seaweed agar waste. KEY POINTS: • Bacillus subtilis fermented waste water has antifungal activity • Bacillus subtilis could transform active substances in waste water • Waste water is a potential raw material for producing antifungal agents.

Synergistic induction of phytoalexins in Nicotiana attenuata by jasmonate and ethylene signaling mediated by NaWRKY70.

Production of the phytoalexins scopoletin and scopolin is regulated by jasmonate (JA) and ethylene signaling in Nicotiana species in response to Alternaria alternata, the necrotrophic fungal pathogen that causes brown spot disease. However, how these two signaling pathways are coordinated to control this process remains unclear. In this study, we found that the levels of these two phytoalexins and transcripts of their key enzyme gene, feruloyl-CoA 6'-hydroxylase 1 (NaF6'H1), were synergistically induced in Nicotiana attenuata by co-treatment with methyl jasmonate (MeJA) and ethephon. By combination of RNA sequencing and virus-induced gene silencing, we identified a WRKY transcription factor, NaWRKY70, which had a similar expression pattern to NaF6'H1 and was responsible for A. alternata-induced NaF6'H1 expression. Further evidence from stable transformed plants with RNA interference, knock out and overexpression of NaWRKY70 demonstrated that it is a key player in the synergistic induction of phytoalexins and plant resistance to A. alternata. Electrophoretic mobility shift, chromatin immunoprecipitation-quantitative PCR, and dual-luciferase assays revealed that NaWRKY70 can bind directly to the NaF6'H1 promoter and activate its expression. Furthermore, the key regulator of the ethylene pathway, NaEIN3-like1, can directly bind to the NaWRKY70 promoter and activate its expression. Meanwhile, NaMYC2s, important JA pathway transcription factors, also indirectly regulate the expression of NaWRKY70 and NaF6'H1 to control scopoletin and scopolin production. Our data reveal that these phytoalexins are synergistically induced by JA and ethylene signaling during A. alternata infection, which is largely mediated by NaWRKY70, thus providing new insights into the defense responses against A. alternata in Nicotiana species.

AaLaeA targets AaFla1 to mediate the production of antitumor compound in Alternaria alstroemeria.

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 AaLaeAOE26 , 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 AaLaeAOE26 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.

Multifunctionality of AsCFEM6 and AsCFEM12 effectors from the potato early blight pathogen Alternaria solani.

Plant pathogens secrete fungal-specific common in several fungal extracellular membrane (CFEM) effectors to manipulate host immunity and contribute to their virulence. Little is known about effectors and their functions in Alternaria solani, the necrotrophic fungal pathogen causing potato early blight. To identify candidate CFEM effector genes, we mined A. solani genome databases. This led to the identification of 12 genes encoding CFEM proteins (termed AsCFEM1-AsCFEM12) and 6 of them were confirmed to be putative secreted effectors. In planta expression revealed that AsCFEM6 and AsCFEM12 have elicitor function that triggers plant defense response including cell death in different botanical families. Targeted gene disruption of AsCFEM6 and AsCFEM12 resulted in a change in spore development, significant reduction of virulence on potato and eggplant susceptible cultivars, increased resistance to fungicide stress, variation in iron acquisition and utilization, and the involvement in 1,8-dihydroxynaphthalene (DHN) melanin biosynthesis pathway. Using maximum likelihood method, we found that positive selection likely caused the polymorphism within AsCFEM6 and AsCFEM12 homologs in different Alternaria spp. Site-directed mutagenesis analysis indicated that positive selection sites within their CFEM domains are required for cell death induction in Nicotiana benthamiana and are critical for response to abiotic stress in yeast. These results demonstrate that AsCFEM effectors possess additional functions beyond their roles in host plant immune response and pathogen virulence.