First Report of Fruit Scab Caused by Alternaria alternata on Actinidia chinensis in Korea.

Kiwi (Actinidia chinesis) is an economically important fruit in Korea, with 1,300 ha cultivated and a production of approximately 25,000 tons per year (Kim and Koh, 2018; Kim and Choi, 2023). In late June 2020, fruit scab symptoms were observed on A. chinensis var. rufopulpa in an orchard in Suncheon, Korea. The incidence of scab symptoms among 20-year-old trees was over 75%, primarily superficial, but rendered the fruit less marketable. In the initial stages of the disease, small, light-brown, circular, and oval spots were formed. As the superficial spots expanded, they became cracked scabs measuring 1 to 7 cm with light edges at the later stages. To isolate the causal pathogen, two lesions were cut from two sections of symptomatic tissue, from each of seven fruits from seven trees. Lesions were surface-sterilized with 70% ethanol for 1 min and washed three times with sterilized distilled water (SDW). The sterilized pieces were placed on potato dextrose agar (PDA) and incubated in the dark at 25°C for one week. After subculturing on PDA, single-spore isolation produced 14 isolates: SYP-410 to 423). All 14 colonies appeared greyish-green and cottony on PDA after 7 d. Conidia were pale brown, ellipsoid to obclavate, with ornamented walls, 1 to 6 transverse and 0 to 3 vertical septa, and length × width of 21.5 to 53.4 × 7.3 to 19.2 µm (avg. 33.0 × 12.0 µm, n = 100). Their morphological characteristics were consistent with Alternaria spp. (van der Waals et al. 2011; Woudenberg et al. 2015). We randomly selected three isolates from the morphologically similar cultures and named them SYP-412 to 414 for further investigation. The ITS (GenBank accession nos.: OR901850 to 52), gapdh (OR924309 to 11), tef1 (OR924312 to 14), rpb2 (OR924315 to 17), Alt a1 (OR924318 to 20), endoPG (OR924321 to 23), and OPA10-2 (OR924324 to 26) sequences from SYP-412 to 414 had a 100% (515 bp/515 bp), 100% (578/578), 100% (240/240), 100% (724/724), 95.55% (451/472), 99.33% (445/448), and 100% (634/634) identity with that of type strain A. alternata CBS 918.96 (AF347032, AY278809, KC584693, KC584435, AY563302, KP124026, and KP124633), respectively. Results from the maximum likelihood phylogenetic analysis, based on the seven concatenated gene sequences, placed the representative isolates in a clade with A. alternata. Pathogenicity of SYP-412 was tested using 12 surface-sterilized two-month-old kiwifruits on a 20-year-old trees. Six kiwifruits were spray-inoculated with 5 mL of a conidial suspension (1 × 106 conidia/ml) generated after culturing in PDA medium for 7 d, with or without wounding. Another six control fruits were inoculated with SDW with and without wounding. The inoculated kiwifruits were enclosed in plastic bags to maintain high humidity for one day. Scab symptoms were observed in both wounded and unwounded fruits six weeks after inoculation, but not in the control. The pathogenicity test was performed on a total of three separate trees twice. To satisfy Koch's postulates, A. alternata was re-isolated from all the symptomatic tissues and confirmed by analyzing the ITS and rpb2 genes. Although scab disease caused by A. tenuissima (now A. alternata) has been previously reported in kiwifruit of A. chinensis var. rufopulpa in China (Woudenberg et al. 2015; Ma et al., 2019), this is the first report of its occurrence on kiwifruit in Korea and will help in future detection and control.

Rats exposed to Alternaria toxins in vivo exhibit altered liver activity highlighted by disruptions in riboflavin and acylcarnitine metabolism.

Natural toxins produced by Alternaria fungi include the mycotoxins alternariol, tenuazonic acid and altertoxins I and II. Several of these toxins have shown high toxicity even at low levels including genotoxic, mutagenic, and estrogenic effects. However, the metabolic effects of toxin exposure from Alternaria are understudied, especially in the liver as a key target. To gain insight into the impact of Alternaria toxin exposure on the liver metabolome, rats (n = 21) were exposed to either (1) a complex culture extract with defined toxin profiles from Alternaria alternata (50 mg/kg body weight), (2) the isolated, highly genotoxic altertoxin-II (ATX-II) (0.7 mg/kg of body weight) or (3) a solvent control. The complex mixture contained a spectrum of Alternaria toxins including a controlled dose of ATX-II, matching the concentration of the isolated ATX-II. Liver samples were collected after 24 h and analyzed via liquid chromatography-high-resolution mass spectrometry (LC-HRMS). Authentic reference standards (> 100) were used to identify endogenous metabolites and exogenous compounds from the administered exposures in tandem with SWATH-acquired MS/MS data which was used for non-targeted analysis/screening. Screening for metabolites produced by Alternaria revealed several compounds solely isolated in the liver of rats exposed to the complex culture, confirming results from a previously performed targeted biomonitoring study. This included the altersetin and altercrasin A that were tentatively identified. An untargeted metabolomics analysis found upregulation of acylcarnitines in rats receiving the complex Alternaria extract as well as downregulation of riboflavin in rats exposed to both ATX-II and the complex mixture. Taken together, this work provides a mechanistic view of Alternari toxin exposure and new suspect screening insights into hardly characterized Alternaria toxins.

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.

Biocontrol potential and growth-promoting effect of endophytic fungus Talaromyces muroii SD1-4 against potato leaf spot disease caused by Alternaria alternata.

BACKGROUND: Alternaria alternata is the primary pathogen of potato leaf spot disease, resulting in significant potato yield losses globally. Endophytic microorganism-based biological control, especially using microorganisms from host plants, has emerged as a promising and eco-friendly approach for managing plant diseases. Therefore, this study aimed to isolate, identify and characterize the endophytic fungi from healthy potato leaves which had great antifungal activity to the potato leaf spot pathogen of A. alternata in vitro and in vivo. RESULTS: An endophytic fungal strain SD1-4 was isolated from healthy potato leaves and was identified as Talaromyces muroii through morphological and sequencing analysis. The strain SD1-4 exhibited potent antifungal activity against the potato leaf spot pathogen A. alternata Lill, with a hyphal inhibition rate of 69.19%. Microscopic and scanning electron microscope observations revealed that the strain SD1-4 grew parallel to, coiled around, shrunk and deformed the mycelia of A. alternata Lill. Additionally, the enzyme activities of chitinase and ß-1, 3-glucanase significantly increased in the hyphae of A. alternata Lill when co-cultured with the strain SD1-4, indicating severe impairment of the cell wall function of A. alternata Lill. Furthermore, the mycelial growth and conidial germination of A. alternata Lill were significantly suppressed by the aseptic filtrate of the strain SD1-4, with inhibition rates of 79.00% and 80.67%, respectively. Decrease of leaf spot disease index from 78.36 to 37.03 was also observed in potato plants treated with the strain SD1-4, along with the significantly increased plant growth characters including plant height, root length, fresh weight, dry weight, chlorophyll content and photosynthetic rate of potato seedlings. CONCLUSION: The endophyte fungus of T. muroii SD1-4 isolated from healthy potato leaves in the present study showed high biocontrol potential against potato leaf spot disease caused by A. alternata via direct parasitism or antifungal metabolites, and had positive roles in promoting potato plant growth.

Alternaria alternata Pathogen from Cuscuta japonica Could Serve as a Potential Bioherbicide.

Dodder (Cuscuta spp.) is a dangerous parasitic plant that causes serious damage to crop production and is challenging to eliminate. Herbicide application is a common strategy to control dodder in the field, but it is costly, ineffective, and further results in hazardous outcomes. Therefore, our study aims to identify the potential pathogens in naturally occurring dodder infections which may provide efficient biocontrol options. In this regard, the pathogens were isolated from the infected plants, their pathogenicity was validated through inoculation, and the optimal culture conditions for their growth were identified by determining the pathogenicity difference. The pathogenicity range was determined in vitro using the leaves of common horticultural plants and crops. Furthermore, a small range of horticultural plants parasitized by Cuscuta reflexa in the field were inoculated with the pathogen to determine their biosafety and biocontrol potential, and the pathogens were identified by morphological and molecular characterization. We found 7 strains that were isolated after pathogen enrichment culture. Among them, Cbp6 and Cbp7 showed the highest pathogenicity against C. reflexa. After testing the inoculation of more than 50 species of plants, only 9 species showed varying degrees of lesions on leaves, which proved the high biosafety for common plants. Field spraying of these pathogens showed a good control effect on C. reflexa after 21 days; the disease severityreached 66.0%, while its host plant did not display obvious symptoms. In conclusion, the pathogens Cbp6 and Cbp7 were identified as Alternaria alternata, and the results of this study provide a theoretical basis for the biological control of dodder.

First Report on Mycotoxin Contamination of Hops (Humulus lupulus L.).

The presence of mycotoxins and other toxic metabolites in hops (Humulus lupulus L.) was assessed for the first time. In total, 62 hop samples were sampled in craft breweries, and analyzed by a multi-toxin LS-MS/MS method. The study collected samples from craft breweries in all of the Croatian counties and statistically compared the results. Based on previous reports on Alternaria spp. and Fusarium spp. contamination of hops, the study confirmed the contamination of hops with these toxins. Alternaria toxins, particularly tenuazonic acid, were found in all tested samples, while Fusarium toxins, including deoxynivalenol, were present in 98% of samples. However, no Aspergillus or Penicillium metabolites were detected, indicating proper storage conditions. In addition to the Alternaria and Fusarium toxins, abscisic acid, a drought stress indicator in hops, was also detected, as well as several unspecific metabolites. The findings suggest the need for monitoring, risk assessment, and potential regulation of Alternaria and Fusarium toxins in hops to ensure the safety of hop usage in the brewing and pharmaceutical industries. Also, four local wild varieties were tested, with similar results to the commercial varieties for toxin contamination, but the statistically significant regional differences in toxin occurrence highlight the importance and need for targeted monitoring.

Validation of a UPLC-MS/MS Method for Multi-Matrix Biomonitoring of Alternaria Toxins in Humans.

Mycotoxins, natural toxins produced by fungi, contaminate nearly 80% of global food crops. Alternaria mycotoxins, including alternariol (AOH), alternariol monomethylether (AME), and tenuazonic acid (TeA), present a health concern due to their prevalence in various plants and fruits. Exposure to these toxins exceeds the threshold of toxicological concern in some European populations, especially infants and toddlers. Despite this, regulatory standards for Alternaria toxins remain absent. The lack of toxicokinetic parameters, reference levels, and sensitive detection methods complicates risk assessment and highlights the necessity for advanced biomonitoring (HBM) techniques. This study addresses these challenges by developing and validating ultra-high performance liquid chromatography method coupled with tandem mass spectrometry to quantify AOH, AME, TeA, and their conjugates in multiple biological matrices. The validated method demonstrates robust linearity, precision, recovery (94-111%), and sensitivity across urine (LOD < 0.053 ng/mL), capillary blood (LOD < 0.029 ng/mL), and feces (LOD < 0.424 ng/g), with significantly lower LOD for TeA compared to existing methodologies. The application of minimally invasive microsampling techniques for the blood collection enhances the potential for large-scale HBM studies. These advancements represent a step toward comprehensive HBM and exposure risk assessments for Alternaria toxins, facilitating the generation of data for regulatory authorities.

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.

Detection of vinblastine and related bioactive compounds from culture extracts of endophytic fungi of Catharanthus roseus.

This study investigates the synthesis of vinblastine by endophytic fungi isolated from leaf of C. roseus. A total of 10 endophytic fungi were selected for secretion of vinca alkaloids based on the initial screening by biochemical tests and thin-layer chromatography (TLC). Out of these ten, only four fungal extracts showed positive results for presence of vinblastine at same retention time (10 min.) compared to reference compound on HPLC analysis. The detected concentration of vinblastine was maximum (17 µg/ml) in isolate no. CRL 22 followed by CRL 52, CRL 17 and CRL 28. To validate the presence of vinblastine, ultra-high-performance liquid chromatography coupled with high-resolution accurate mass spectrometry (HRMS) was employed. This analysis confirmed the presence of anhydrovinblastine, a precursor of vinblastine through the detection of molecular ions at m/z 793.4185 in extract of CRL 17. In addition to anhydrovinblastine, the intermediate compounds essential to the biosynthetic pathway of vinblastine were also detected in the extract of CRL 17. These host-origin compounds strongly suggest the presence of a biosynthetic pathway within the endophytic fungus. Based on morphological observation and sequence analysis of the ITS region of rDNA, endophytic fungi were identified as Alternaria alternata (CRL 17), Curvularia lunata (CRL 28), Aspergillus terrus (CRL 52), and Aspergillus clavatonanicus (CRL 22).

Combined analysis of transcriptomics with metabolomics provides insights into the resistance mechanism in winter jujube using L-Methionine.

Black rots lead to great economic losses in winter jujube industry. The objective of this research was to delve into the underlying mechanisms of enhanced resistance of winter jujube fruit to black rot by L-Methionine (Met) treatment. The findings revealed that the application of Met significantly curtailed lesion diameter and decay incidence in winter jujube fruit. The peroxidase (POD) activity in the Met-treated jujubes was 3.06-fold that in the control jujubes after 4 d of treatment. By day 8, the activities of phenylalanine ammonia-lyase (PAL), chitinase (CHI) and ß-1,3-glucanase (GLU) in the Met-treated jujubes had surged to their zenith, being 1.39, 1.22, and 1.52 times in the control group, respectively. At the end of storage, the flavonoid and total phenol content remained 1.58 and 1.06 times than that of the control group. Based on metabolomics and transcriptomics analysis, Met treatment upregulated 6 key differentially expressed metabolites (DEMs) (succinic acid, trans-ferulic acid, salicylic acid, delphinium pigments, (S)-abscisic acid, and hesperidin-7-neohesperidin), 12 key differentially expressed genes (DEGs) (PAL, CYP73A, COMT, 4CL, CAD, POD, UGT72E, ANS, CHS, IAA, TCH4 and PR1), which were involved in phenylpropanoid biosynthesis pathway, flavonoid biosynthesis pathway and plant hormone signal transduction pathway. Further analysis revealed that the most of the enzymes, DEMs and DEGs in this study were associated with both antioxidant and disease resistance. Consequently, Met treatment enhanced disease resistance of winter jujube fruit by elevating antioxidant capacity and triggering defense response. This study might provide theoretical support for utilizing Met in the management and prevention of post-harvest black rot in winter jujube.

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.

Development and validation of a simultaneous quantitative analytical method for two Alternaria toxins and their metabolites in plasma and urine using ultra-high-performance liquid chromatography-tandem mass spectrometry.

Much more attention has been paid to the contamination of Alternaria toxins because of food contamination and the threat to human health. In this study, an ultra-high-performance liquid chromatography-tandem mass spectrometry method was developed for the simultaneous detection of the prototypical alternariol, alternariol monomethylether, and the metabolites 4-oxhydryl alternariol, and alternariol monomethylether 3-sulfate ammonium salt of Alternaria toxins. The positive samples were used as matrix samples to optimize the different experimental conditions. 0.01% formic acid solution and acetonitrile were used as the mobile phase, and analytes were scanned in negative electron spray ionization under multiple reaction monitoring, and quantitative determination by isotope internal standard method. Application of this method to samples of human plasma and urine showed the detection of the above analytes. The results showed that the recoveries were from 80.40% to 116.4%, intra-day accuracy was between 0.6% and 8.0%, and inter-day accuracy was between 1.1% and 12.1%. The limit of detection of the four analytes ranged from 0.02 to 0.6 µg/L in urine, and 0.02 to 0.5 µg/L in plasma, respectively. Thus, the developed method was rapid and accurate for the simultaneous detection of analytes and provided a theoretical basis for the risk assessment of Alternaria toxins for human exposure.

Outbreak of Alternaria Black Spot of Pomegranate (Punica granatum L.) in Italy as a Consequence of Unusual Climatic Conditions.

Alternaria black spot of pomegranate (Punica granatum) was reported for the first time in Italy. In spring 2023, an outbreak of this disease was noticed in commercial pomegranate 'Wonderful' orchards of the municipality of Misterbianco (Sicily), following an unusually rainy period. A total of 30 randomly selected Alternaria isolates recovered from typical necrotic spots of leaves and fruits were characterized. Based on the colony morphology on solid agar media (PDA and MEA), isolates were separated into three distinct morphotypes (1, 2, and 3). The first two morphotypes comprised only isolates from fruits, while morphotype 3 comprised only isolates from leaves. Multigene phylogenetic analysis of four DNA regions, including internal transcribed spacer (ITS), translation elongation factor 1-α (EF-1α), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and a SCAR marker (OPA10-2), identified the isolates of morphotypes 1 and 2 as Alternaria alternata and morphotype 3 isolates as A. arborescens. In pathogenicity tests on unwounded leaves and fruit, the isolates of all three morphotypes produced symptoms on the leaves of three pomegranate cultivars, 'Acco', 'Wonderful', and 'Etna'. The symptoms on 'Acco' leaves were the least severe. Conversely, the fruits of 'Acco' were the most susceptible. The isolates of morphotypes 2 and 3 were not pathogenic on the fruits of 'Wonderful' and 'Etna'. This is the first report of Alternaria black spot in Italy and of A. arborescens associated with Alternaria black spot of pomegranate worldwide.

Response Mechanism of Extracellular Polymeric Substances Synthesized by Alternaria sp. on Drought Stress in Alfalfa (Medicago sativa L.).

This study investigates how extracellular polymeric substances (EPS) synthesized by dark septate endophytic (DSE) improve alfalfa's drought resistance. Drought stress was simulated in hydroponic culture, and roots were treated with different EPS concentrations to determine their effects on drought tolerance and applicable concentrations. Hydroponic solutions with 0.25 and 0.50% EPS concentrations alleviated leaf wilting and increased total plant fresh weight by 35.8 and 57.7%, respectively. SEM shows that EPS attached to the roots and may have served to protect the root system. EPS treatment significantly depressed the MDA contents of the roots, stems, and leaves. Roots responded to drought stress by increasing soluble sugar contents and antioxidant enzyme activities, while mitigating stem and leaf stress by synthesizing lipid compounds, amino acids, and organic acid metabolites. Five metabolites in the stem have been reported to be associated with plant stress tolerance and growth, namely 3-O-methyl 5-O-(2-methyl propyl) (4S)-2,6-dimethyl-4-(2-nitrophenyl)-3,4-dihydropyridine-3,5-dicarboxylate, malic acid, PA (20:1(11Z)/15:0), N-methyl-4,6,7-trihydroxy-1,2,3,4-tetrahydroisoquinoline, and 2-(S-glutathionyl) acetyl glutathione. In summary, EPS treatment induced oxidative stress and altered plant metabolism, and this in turn increased plant antioxidant capacity. The results provide a theoretical basis for the application of EPS in commercial products that increase plant resistance and ecological restoration.

Essential oils inhibiting Alternaria alternata and Colletotrichum gloeosporioides: a review.

Essential oils (EOs) have been investigated for their effectiveness against fungal fruit pathogens. The present review article summarises the EOs that inhibit Alternaria alternata and Colletotrichum gloeosporioides in the pre- and post-harvest stages of fruits. Thirty-nine scientific papers focusing on the extraction conditions and the antifungal activity of EOs were selected. The retrieved studies came mainly from China and Brazil. Hydrodistillation has been identified as the most used extractive method. The yields and chemical profiles were variable among the species. The in vitro studies were larger than the in vivo studies. The application of EOs reduced the incidence of fungal diseases in tomatoes (Lycopersicon esculentum), papaya (Carica papaya) and mango (Mangifera indica). EOs resulted as a potential ecological alternative for treating fungal diseases in fruits requiring further investigation.

Chromoblastomycosis caused by Alternaria infectoria, concurrent with myiasis, in a recipient of a kidney transplant: a compelling case report.

Neglected tropical diseases (NTDs) pose a significant threat to the health of millions of people worldwide, particularly in impoverished populations in tropical and subtropical regions. The World Health Organization (WHO) considers certain fungal infections, such as chromoblastomycosis, as NTDs. Chromoblastomycosis is a chronic fungal infection affecting the skin and subcutaneous tissue, primarily found in tropical and subtropical regions of Latin America, Africa, and Asia. This case report presents a 46-year-old female patient with chromoblastomycosis who had a history of renal transplantation and was receiving immunosuppressive therapy. The patient exhibited dark, verrucous, and ulcerative lesions on the legs, and the diagnosis was confirmed through the microscopic examination of skin scrapings by observing medlar bodies. Two sequential fungal tissue cultures and ITS sequencing verified the presence of Alternaria infectoria, not formerly described in chromoblastomycosis. Moreover, observation of fly larvae in the lesions verified the diagnosis of myiasis. Treatment with voriconazole and terbinafine resulted in complete resolution of the lesions after 5 months. This case emphasizes the importance of considering chromoblastomycosis in individuals with occupational exposure in tropical areas, as well as the challenges associated with its diagnosis, coinfections, and treatment.

Morphological and phylogenetic analyses reveal two new Alternaria species (Pleosporales, Pleosporaceae) in Alternaria section from Cucurbitaceae plants in China.

Alternaria species are commonly found as saprophytes, endophytes and plant pathogens. During a survey of small-spored Alternaria in China, two new species were discovered from Cucurbitaceae plants collected in Hubei and Sichuan provinces. This study identified two new species of Alternaria using seven genes (ITS, GAPDH, TEF1, RPB2, Alt a 1, EndoPG, and OPA10-2) for phylogenetic analyses and morphological characteristics. The two new species A.jingzhouensis and A.momordicae were described and illustrated. Alternariajingzhouensis sp. nov., associated with Citrulluslanatus, is characterized by producing muriform, ellipsoidal, flask-shaped, rostrate, and beaked conidia. It differs from A.koreana, A.ovoidea, and A.baoshanensis by bearing conidia in a simple conidiogenous locus with occasionally longer beaks in a chain, and from A.momordicae sp. nov. by having shorter beaks. Alternariamomordicae sp. nov. from Momordicacharantia was distinct from A.koreana, A.ovoidea, and A.baoshanensis by producing muriform, long ellipsoid or ovoid to obclavate, sometimes inverted club-shaped conidia on a single conidiogenous locus with a wider body and longer beak in a chain, and distinct from A.jingzhouensis sp. nov. by a longer beak conidia. These two species were clearly distinguished from other species in the section Alternaria based on DNA based phylogeny and morphological characteristics. The morphological features were discussed and compared to relevant species in the present paper.

Network analyses predict major regulators of resistance to early blight disease complex in tomato.

BACKGROUND: Early blight and brown leaf spot are often cited as the most problematic pathogens of tomato in many agricultural regions. Their causal agents are Alternaria spp., a genus of Ascomycota containing numerous necrotrophic pathogens. Breeding programs have yielded quantitatively resistant commercial cultivars, but fungicide application remains necessary to mitigate the yield losses. A major hindrance to resistance breeding is the complexity of the genetic determinants of resistance and susceptibility. In the absence of sufficiently resistant germplasm, we sequenced the transcriptomes of Heinz 1706 tomatoes treated with strongly virulent and weakly virulent isolates of Alternaria spp. 3 h post infection. We expanded existing functional gene annotations in tomato and using network statistics, we analyzed the transcriptional modules associated with defense and susceptibility. RESULTS: The induced responses are very distinct. The weakly virulent isolate induced a defense response of calcium-signaling, hormone responses, and transcription factors. These defense-associated processes were found in a single transcriptional module alongside secondary metabolite biosynthesis genes, and other defense responses. Co-expression and gene regulatory networks independently predicted several D clade ethylene response factors to be early regulators of the defense transcriptional module, as well as other transcription factors both known and novel in pathogen defense, including several JA-associated genes. In contrast, the strongly virulent isolate elicited a much weaker response, and a separate transcriptional module bereft of hormone signaling. CONCLUSIONS: Our findings have predicted major defense regulators and several targets for downstream functional analyses. Combined with our improved gene functional annotation, they suggest that defense is achieved through induction of Alternaria-specific immune pathways, and susceptibility is mediated by modulating hormone responses. The implication of multiple specific clade D ethylene response factors and upregulation of JA-associated genes suggests that host defense in this pathosystem involves ethylene response factors to modulate jasmonic acid signaling.

Baseline sensitivity and toxicity mechanisms of prochloraz to Alternaria alternata strains associated with maize leaf blight in Heilongjiang province in China.

Alternaria species are fungal pathogens that can infect maize, causing leaf blight disease and significant economic losses. This study aimed to determine the baseline sensitivity to prochloraz of A. alternata isolates obtained from diseased maize leaves collected from Heilongjiang province by assessing the half-maximal effective concentration (EC50) values. The EC50 values of prochloraz ranged from 0.0550 µg/mL to 2.3258 µg/mL, with an average of 0.9995 ± 0.5192 µg/mL. At EC50 (1.2495 µg/mL) and 2EC50 (2.4990 µg/mL), prochloraz increased the number of mycelial offshoots, disrupted the cell membrane integrity of conidia and mycelia, and resulted in a reduced ergosterol content in the mycelia. Prochloraz significantly affected the mycelial cell membrane permeability and increased the malondialdehyde (MDA) content and superoxide dismutase (SOD) activity. No cross-resistance was detected between prochloraz and other fungicides. These data demonstrate that prochloraz is a promising fungicide for managing maize leaf blight caused by A. alternata and provide novel insights into understanding the mechanism of prochloraz toxicity against A. alternata isolates.

First Report of Polygonatum kingianum Leaf Spot Caused by Alternaria alternata in Kunming, China.

Polygonatum kingianum Coll. et Hemsl., a Polygonatum species in the Asparagaceae family, plays an important role in Chinese herbal medicine (Zhao et al. 2018). P. kingianum is widely planted in the Southwestern China. In September 2023, we observed a leaf spot of P. kingianum with disease incidence of 100%, and disease index reached 60 in commercial plantings in Kunming, Yunnan province, China (24.3610°N, 102.3740°E). In the initial stage of infection, symptoms manifested as a small circular brown spot. As the spots gradually expanded, they formed oval to irregular shaped lesions with grayish-white or dark-brown borders. Progressively the entire leaf withered and died. For identification of the causal agent of the leaf spot, leaf sections (5×5 mm2) were cut from the margin of the lesion and soaked in 75% ethanol for 10 s, 1% sodium hypochlorite for 3 min, washed with sterile distilled water, dried on sterilized tissue paper and placed on potato dextrose agar (PDA). The Petri dishes were then incubated at 28℃ for 3 days with a 12-h photoperiod. A predominant fungus was isolated from 95% of the samples. Three monosporic isolates were screened using a single-spore isolation method. After 4 days of incubation the colonies were white, after 7 days turned yellow-white. Conidia were black-brown, oblong or fusiform, with 3-7 transverse septa and 0-3 longitudinal septa, with dimensions of 19.5 to 49.5 × 8.7 to 17.6 µm (n = 30). Total genomic DNA of these three isolates was extracted from mycelia by the cetyltrimethylammonium bromide (CTAB) protocol. The nucleotide sequences of the elongation factor 1-alpha (EF1α), nuclear ribosomal internal transcribed spacer (ITS), 28S nuclear ribosomal large subunit rRNA gene (LSU), 18S nuclear ribosomal small subunit rRNA gene (SSU), and the second largest subunit of nuclear DNA-directed RNA polymerase II (RPB2) gene regions were amplified using the primer pairs EF1-728F/EF1-986R (Carbone and Kohn 1999), ITS1/ITS4 (White et al. 1990), LR0R/LR5 (Schoch et al. 2012), NS1/NS4 (Schoch et al. 2012), and fRPB2-5F/fRPB2-7Cr (Liu et al. 1999), respectively. Amplicons were cloned in a pMDTM19-T vector (code no. 6013, Takara, Kusatsu, Japan) and bidirectionally sequenced. All three isolates had identical nucleotide sequences. Sequences from one isolate (PkF03) were deposited in GenBank. BLASTn analyses showed that sequences of EF1α (GenBank accession no. PP695240), ITS (PP694046), LSU (PP683406), SSU (PP683407), and RPB2 (PP695241) of isolate PkF03 were 99.6 (KP125134), 100 (KP124358), 100 (KP124510), 99.9 (KP124980), and 100% (KP124826), respectively, identical with Alternaria alternata (Fr.) Keissl. strain CBS 118815. Based on the nucleotide sequences of EF1α, ITS, LSU, SSU, and RPB2, a maximum likelihood phylogenetic tree was constructed using MEGAX with Tamura-Nei model. Isolate PkF03 was grouped in the same clade as A. alternata. According to the morphology and sequence analyses isolate PkF03 was identified as A. alternata (Woudenberg et al. 2013). To determine pathogenicity of isolate PkF03, a spore suspension (106 spores/mL) was sprayed on 1-year-old healthy leaves of P. kingianum. The control leaves were sprayed with sterile water. All plants were incubated at 28℃, 70% relative humidity, and a 12-h photoperiod. The pathogenicity tests were repeated three times with six plants in each treatment. Fifteen days post-inoculation, the inoculated leaves showed brown-yellow lesions, whereas the control leaves remained symptomless. A. alternata was reisolated from infected leaves. To our knowledge, this is the first report of A. alternata causing leaf spot on P. kingianum in Kunming, China. The results provide a scientific basis for prevention and control of the disease.