Allergen Genes Research Articles

First Report of Alternaria alternata Causing Alternaria Leaf Spot of Black Nightshade in China.

Black nightshade (Solanum nigrum L.) belongs to Solanaceae family, which is distributed all over China and has become one of the malignant weeds in cotton field in Xinjiang (Zhang et al., 2020; Zhang et al., 2021). During July 2016 to August 2023, leaf spot was observed on black nightshade in Hami and Shihezi city, Xinjiang, China. The spots appeared as brown circular or irregular, with some small black dots surrounded with yellow halos. Twenty five black nightshade plants were collected from a cotton field (100 m2) for pathogen isolation in Shihezi, July 2023. Leaf samples (2×2 mm) from the boundary between diseased tissue and healthy tissue, sterilized in 70% ethanol for 45 s, dipped in 2% NaClO for 30 s, rinsed three times with sterile distilled water, placed on potato dextrose agar (PDA) after dried and cultivated at 25°C in the dark for 4 days. Twenty-five fungal isolates were obtained and purified by using the single-spore isolation method, and all isolates were found to be similar in morphology, appeared as dark brown and produced dark brown pigmentation on PDA. Conidiophores were black brown, 8 to 58 × 10 to 20 μm, with 1 to 5 transverse septa and 0 to 3 longitudinal septa, the results similar to those of Alternaria alternata isolated from buckwheat (Li et al., 2021). To confirm this identification, total genomic DNA of the isolates were extracted by CTAB method (Doyle et al., 1987). The internal transcribed spacer (rDNA-ITS), Alternaria major allergen gene (Alt α1) and glyceraldehyde-3-phosphate dehydrogenase gene (GAPDH) of the A. alternata 26 were amplified using the primers ITS1/ITS4 (Glass et al., 1995), Altα1-F/Altα1-R (Li et al., 2024) and gpd-F/gpd-R (5'-CAACGGCTTCGGTCGCAT- TG-3' / 5'-GCCAAGCAGTTGGTTGTG-3'), respectively. The rDNA-ITS, Altα1 and GAPDH gene sequences were deposited in GenBank (KX904867, PP263361, PP263360), and showed 100% identity (rDNA-ITS: 572 out of 609 bp; Alt α1: 514 out of 516 bp; GAPDH: 619 out of 619 bp) to A. alternata (KU179665; MW522975; MK451977), respectively. The multi-gene phylogenetic tree showed that the representative isolate was grouped with A. alternata and identified as A. alternata. Black nightshade was used for pathogenicity assay. Fifteen plants at 3 to 4-leaf stage were selected and sprayed with the conidial suspension (1.0×106 spores/mL) of A. alternata 26 after wound the underside of leaves (2 mm length) with inoculating needle. Five plants were sprayed with sterilized water as control. After inoculation, placed the plants in a plastic box and covered with plastic wrap to keep 80% relative humidity for 3 days at 25℃ in the greenhouse, then removed the plastic box. Symptoms of leaf spots appeared within 12 - 15 days that were similar to the symptoms observed in the field, and the fungal pathogen that was re-isolated from symptomatic leaves was identical to original pathogen on the basis of morphological and molecular analysis to fulfill Koch's postulates. This experiment was repeated three times, and the disease incidence was above 80% after inoculation at each time. To our knowledge, this is the first report of A. alternata causing leaf spot of black nightshade in China. This report will help us to monitoring distribution of the disease and providing theoretical basis for disease prevention and control.

Recycled lithium battery nanomaterials as a sustainable nanofertilizer: Reduced peanut allergenicity and improved seed quality

The rapidly increasing amount of end-of-life lithium iron phosphate (LiFePO4) batteries has raised significant environmental concerns. This study offers a strategy for a paradigm shift by transforming this growing waste into a valuable resource by recycling discarded LiFePO4 batteries and safely integrating the materials into sustainable agriculture. We used five types of LiFePO4 (10, 50 mg kg−1) applied to soil planted with peanuts in a full-culture experiment. Our results show that addition of <50 mg kg−1 of recycled nano-LiFePO4 (rn-LiFePO4) has a multifaceted positive impact on peanut because of sustainable release of nutrients and nano-specific effects, not only enhancing photosynthesis and root growth but also increasing yield by 22 %–34 % while simultaneously elevating seed nutritional quality. Moreover, a remarkable reduction (up to 99.78 % at 10 mg kg−1 rn-LiFePO4) in the expression of allergen genes was evident following exposure to LiFePO4, which showed a significant negative correlation with Fe content in the seeds. The decreased peanut allergen gene expression was mediated by a downregulation of metabolites associated with protein digestion and absorption. Furthermore, rhizosphere soil immune system enhancement may indirectly enhance immune responses to peanut allergy. This study suggests the significant potential of nanoscale LiFePO4 recycled from Li battery, including enhancing crop yield quality and mitigating peanut allergy concerns while simultaneously addressing a growing waste stream of concern.

A DNA-free and genotype-independent CRISPR/Cas9 system in soybean

Abstract Here, we report a smart genome editing system for soybean (Glycine max) using the in planta bombardment-ribonucleoprotein (iPB-RNP) method without introducing foreign DNA or requiring traditional tissue culture processes such as embryogenesis and organogenesis. Shoot apical meristem (SAM) of embryonic axes was used as the target tissue for genome editing because the SAM in soybean mature seeds has stem cells and specific cell layers that develop germ cells during the reproductive growth stage. In the iPB-RNP method, the RNP complex of the CRISPR/Cas9 system was directly delivered into SAM stem cells via particle bombardment, and genome-edited plants were generated from these SAMs. Soybean allergenic gene Gly m Bd 30K was targeted in this study. Many E0 (the first generation of genome-edited) plants in this experiment harbored mutant alleles at the targeted locus. Editing frequency of inducing mutations transmissible to the E1 generation was approximately 0.4 to 4.6 % of all E0 plants utilized in various soybean varieties. Furthermore, simultaneous mutagenesis by iPB-RNP method was also successfully performed at other loci. Our results offer a practical approach for both plant regeneration and DNA-free genome editing achieved by delivering RNP into the SAM of dicotyledonous plants.

First report of Alternaria alternata causing leaf spot on Polygonatum kingianum in China.

Polygonatum kingianum is a Chinese herbal medicine that belongs to the genus Polygonatum of the family Liliaceae. In June 2023, Polygonatum kingianum Coll. et Hemsl. in nurseries in Qujing, Yunnan Province, China, showed irregular brown spots on the leaves, whole leaf necrosis, and plant death in serious cases, with an incidence of 10-20% (Fig. S1). To identify the pathogens of P. kingianum, six diseased samples were collected from nurseries with 0.6 acre. These diseased sample leaves were soaked in 0.1% HgCl2 for 1 min and 75% ethanol for 2 min and then rinsed thrice with sterile water. Treated leaves were cut into small pieces (5×5 mm) and cultured on potato dextrose agar (PDA) for five days at 28°C. Total thirteen fungal strains were isolated from PDA medium. The nuclear ribosomal internal transcribed spacer of ribosomal DNA (ITS rDNA) region of these 13 strains was amplified by polymerase chain reaction (PCR) using universal primers ITSI/ITS4 (White et al. 1990). Sequencing and BLAST of the ITS region on NCBI showed that 11 out of 13 fungal strains belonged to the genus Alternaria, with an identity ≥99%. We selected one of the Alternaria strains, HJ-A1, for further study. The HJ-A1 colony appeared grayish brown white-to-gray with a flocculent texture on the front side and a dark gray underside on the PDA medium (Fig. S1). The conidiophores appeared brown, either single or branched, and produced numerous short conidial chains. The conidia were obclavate to obpyriform or ellipsoid in shape and contained 1-4 transverse septa and 0-2 oblique septa. The conidial diameter was 27.30µm in length and 12.27µm in width. (Fig. S1). To further determine the species of HJA1, the genomic DNA of HJ-A1 was extracted using the Lysis Buffer for PCR (AG, Hunan, China). Four Alternaria genomic DNA regions including the ITS, translation elongation factor 1-α gene (TEF1-α), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and Alternaria major allergen gene (Alt a1) were amplified by PCR using the primers as previously reported (Woudenberg et al. 2013, Hong et al. 2005). Sequence analysis revealed that the ITS (484bp) of HJ-A1 (NCBI No. PP082633), TEF1-α (267bp) of HJ-A1 (NCBI No. PP419893), GAPDH (582bp) of HJ-A1 (NCBI No. PP419892), and Alt a1 (522bp) of HJ-A1 (NCBI No. PP228046) shared the highest identity with A. alternata respectively (99≥%). A maximum likelihood phylogenetic tree was constructed with the combined sequence data sets of ITS, GAPDH, TEF, and Alt a1 using MEGA 7. The results showed that HJ-A1 strain clustered with A. alternate (Fig. S2). The pathogenicity of HJ-A1 was tested according to Koch's postulates by inoculating HJ-A1 conidia suspension (2×105 conidia/mL) into leaves of 1-year-old P. kingianum, with sterile water as a control. Each treatment group included 3 plants with 3 replicates. The tested plants were planted in a phytotron at 28℃ and 90% humidity. Three days after inoculation, symptoms similar to those under natural conditions were observed in the HJ-A1-inoculated plants, whereas no symptoms were observed in the control plants (Fig. S1). The same fungal strains were re-isolated from inoculated leaves and identified by morphologically and sequence of ITS. Previous studies showed that Alternaria alternata funji cause many plant diseases, such as fig fruit rot (Latinović N et al. 2014)，daylily leaf spot (Huang D et al. 2022), fruit blight on sesame (Cheng H et al. 2021)，leaf spot of Cynanchum atratum Bunge (Sun H et al. 2021) and so on. To our knowledge, this is the first report of A. alternata causing P. kingianum leaf spot in China. The discovery of this pathogen will help to guide the protection and control of P. kingianum disease.

The genome-wide response of Dermatophagoides pteronyssinus to cystatin A, a peptidase inhibitor from human skin, sheds light on its digestive physiology and allergenicity.

The digestive physiology of house dust mites (HDMs) is particularly relevant for their allergenicity since many of their allergens participate in digestion and are excreted into faecal pellets, a main source of exposure for allergic subjects. To gain insight into the mite dietary digestion, the genome of the HDM Dermatophagoides pteronyssinus was screened for genes encoding peptidases (n = 320), glycosylases (n = 77), lipases and esterases (n = 320), peptidase inhibitors (n = 65) and allergen-related proteins (n = 52). Basal gene expression and transcriptional responses of mites to dietary cystatin A, a cysteine endopeptidase inhibitor with previously shown antinutritional effect on mites, were analysed by RNAseq. The ingestion of cystatin A resulted in significant regulation of different cysteine endopeptidase and glycosylase genes. One Der p 1-like and two cathepsin B-like cysteine endopeptidase genes of high basal expression were induced, which suggests their prominent role in proteolytic digestion together with major allergen Der p 1. A number of genes putatively participating in the interaction of mites with their microbiota and acquired by horizontal gene transfer were repressed, including genes encoding the peptidase Der p 38, two 1,3-beta-glucanases, a lysozyme and a GH19 chitinase. Finally, the disruption of mite digestion resulted in the regulation of up to 17 allergen and isoallergen genes. Altogether, our results shed light on the putative role of specific genes in digestion and illustrate the connection between the digestive physiology of HDM and allergy.

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.

First Report of Alternaria alternata Causing Postharvest Fruit Rot of Indian Jujube (Ziziphus mauritiana) in China.

Ziziphus mauritiana Lam., commonly known as Indian jujube or ber, is a popular fruit crops grown in tropical and sub-tropical regions of China. It is commonly stored at 4℃, relative humidity of about 90%, combined with waxing or sealing with film bag. In January 2023, a postharvest fruit rot was observed on Indian jujube in three markets located in Nanchang city of Jiangxi province, China, with a disease incidence of 4 to 10%. Initially, brown spots appeared on the surface or base of thefruit, which gradually expanded into irregular brown lesions. Gray-white hyphae developed in the center of the lesions, and ultimately thefruitrotted. To isolate the pathogen, small pieces (5 × 5 mm) of ten infected fruits were surface-sterilized in 75% ethanol for 15 s and then 1% sodium hypochlorite for 30 s, rinsed three times in sterile water, plated onto potato dextrose agar (PDA), and incubated at 25°C for 3 days. Eight strains with similar morphological characteristics were isolated, and one representative isolate (JXAA-1) was used for morphological and molecular characterization. The colonies on PDA were initially olive green with white margins, and later turned dark olive or black with profuse sporulation. Conidia were borne singly or in a chain, brown, with 1 to 5 transverse septa and 0 to 3 longitudinal septa, obclavate to obpyriform, and measured 12.9 to 33.7 × 7.5 to 12.9 μm (n = 30). On the basis of morphological characteristics, the isolates were tentatively identified as Alternaria spp. (Simmons 2007). To confirm the identification, genomic DNA was extracted from the isolate JXAA-1 with the Fungi Genomic DNA Extraction Kit (Solarbio Biotech, China). The 18S nrDNA (SSU), 28S nrDNA (LSU), internal transcribed spacer of the rDNA (ITS), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), elongation factor 1-alpha (TEF1), Alternaria major allergen gene (Alt a 1), endopolygalacturonase (EndoPG) and an anonymous gene regions (OPA 10-2) were amplified and sequenced using primers NS1/NS4, LR7/LR0R, ITS5/ITS4, gpd1/gpd2, EF1-728F/EF1-986R, Alt-for/Alt-rev, PG3/PG2b, OPA10-2L/OPA10-2R, respectively (Woudenberg et al. 2015). The obtained DNA sequences (SSU: PP190241; LSU: PP190242; ITS: PP189927; GAPDH: PP196557; TEF1: PP196558; Alt a 1: PP196559; EndoPG: PP196560; and OPA 10-2: PP196561) showed 100% homology with those of A. alternata (GenBank accession nos. MT000349 [1020/1020 bp]; KP940477 [1312/1312 bp]; MK972909 [583/583 bp]; MN615421 [593/593 bp]; MN046379 [280/280 bp]; MN304714 [490/490 bp]; MN698284 [458/458 bp] and MH975214 [701/701 bp]). A maximum likelihood phylogenetic tree was constructed by combining all sequenced loci in IQTREE web servers. The isolate JXAA-1 clustered with Alternaria alternata (CBS 121336). The fungus associated with postharvest fruit rot on Z. mauritiana was thus identified as A. alternata. To evaluate the pathogenicity, six surface sterilized fruits were wounded by a sterile scalpel and inoculated with a 10 μl drop of spore suspension (1 × 105 conidia/ml) of isolate JXAA-1. Another six fruits were inoculated with sterilized ddH2O as control and the experiment was repeated three times. All fruits were incubated at 25°C and 80% relative humidity. After 5 days, all the wounded fruit inoculated with A. alternata showed similar symptoms to those observed previously, while the control fruits remained healthy. A. alternata was consistently reisolated from infected fruit and confirmed by morphological and molecular data, fulfilling Koch's postulates. A.alternata has previously been reported causing leaf spot and fruit rot on Chinese jujube (Ziziphus jujuba) in China (Bai et al. 2015; Li et al. 2021). But to our knowledge, this is the first report of A. alternata causing postharvest fruit rot on Indian jujube (Z. mauritiana) in China. Therefore, managers should pay more attention to postharvest fruit rot of jujube caused by A. alternata, the foam bag is put on after the membrane bag is sealed, the broken or infected fruit is picked out in time to reduce the spread of pathogenic fungus.

First Report of Alternaria gossypina Causing Stem Base Rot on Passilora edulis in Guangxi, China.

Passion fruit (Passilora edulis), known as the "king of fruit juices", is popular in southern China (Yuan et al. 2019). Stem base rot is a devastating disease of passion fruit commonly caused by several Fusarium spp. (Zakaria, 2022). In July 2022, typical symptoms of stem base rot were observed in a poorly managed "Qinmi No. 9" Golden passion fruit orchard in Jingxi (23°13'10"N, 106°5'23"E). The disease incidence had reached 40% (n=200) in the survey. Symptoms included ulceration and mutilation at the stem base, making the plants prone to breakage when pulled, wilting and drooping leaves above ground, and severe cases leading to the entire plant withering and dying. Fourteen plants with obvious symptoms were collected. Thin sections of plant tissue were cut from junction of sickness and health of stem, sterilized with ethanol and sodium hypochlorite, and placed on PDA medium at 28°C. Sixty fungal strains were obtained, 90% of which was identified as Fusarium based on morphology. 80% of Fusarium were F. oxysporum species complex (FOSC), but pathogenicity experiment showed all FOSC were weakly pathogenic. However, two severely pathogenic fungi with similar morphology but distinct from Fusarium were identified from the same plant. The representative strain C11 was selected for further study. C11 demonstrated a rapid growth rate, reaching a 90 mm diameter colony on PDA cultured at 25°C for 7 days. The colony displaced a round, flat shape with an overall light brown front, and cottony gray or light brown mycelium, while the reverse side was dark brown. Conidia production was observed as typically occurred in multiple chains after 14 days culture on OA medium, with round, oval or straight rod-like brown conidia ranging in size from 5.74-23.42×14.67-67.22, featuring 1-8 transverse septa and 0-3 mediastinum (Figure S1). For molecular identification, the internal transcribed spacer (ITS, OR616614), translation elongation factor 1-alpha (TEF, OR633298), alternaria major allergen (Alt a1, OR633294) gene, glyceraldehyde 3-phosphate dehydrogenase (GAPDH, OR633295), RNA polymerase subunit II gene (RPB2, OR633297), 18S Small subunit rDNA (SSU, OR616608) , 28S Large Subunit rDNA (LSU, OR616615), the KOG1058 gene regions (OR633296) and an approximately segment of the anonymous noncoding region (OPA10-2, OR633299) were amplified from C11 (Liu et al. 1999, Li et al. 2023, Andrew et al. 2009), and deposited in GenBank with accession number shown in the brackets. Phylogenetic trees were constructed in MEGA11 after splicing by BioEdit (Figure S3). Combining morphology and molecular analyses, C11 was identified as Alternaria gossypina (Woudenberg et al. 2015). To test the pathogenicity, the base of the seedling stem (20cm in height) of 50 healthy "Tainong No. 1" variety of purple passion fruit, which was more susceptible to stem-base rot, was puncture wound with needles, inoculated with 6 mm diameter colonies of fungi, and then wrapped in wet cotton (Ángel et al. 2018). Ten healthy seedlings inoculated with PDA were used as controls. These plants were cultured in an artificial greenhouse at 30±5℃with 80±5% humidity. After 15 days, the plants inoculated with C11 exhibited symptoms similar to those in the field, whereas the controls remained healthy. A. gossypina was reisolated from the diseased plant stems, with the morphology and GAPDH sequence consistent with the inoculated (Figure S1, S2). This is the first report of passion fruit stem rot caused by A. gossypina. This finding will aid in the prevention and control of stem rot in passion fruit.

First Report of Alternaria linariae Causing Postharvest Fruit Rot of Pepino (Solanum muricatum) in China.

Pepino (Solanum muricatum L.) is a popular solanaceous crop that is native to South America and is commercially grown in many countries including China for its attractive, sweet and flavorful fruits. In September 2023, a postharvest fruit rot was observed at an incidence of 7% to 10% on pepino at supermarket in Nanchang, Jiangxi, China (28.69°N, 115.81°E). Symptoms on fruits initially appeared as small black spots that later enlarged and became necrotic. To isolate the pathogen, symptomatic tissues were surface-sterilized using 75% ethanol for 15 s, then 1% sodium hypochlorite for 30 s, rinsed three times in sterile water, air dried, finally placed on potato dextrose agar (PDA) plates and incubated at 25℃ for 4 days. Ten strains (about 83% isolationfrequencyfromsymptomaticpepinofruits) with similar morphological characteristics were isolated. The colonies on PDA were initially white, gradually turning gray and eventually becoming black, and had abundant aerial mycelia. Conidia were fusiform to linetype, dark brown, measuring 50 to 100 × 10 to 28 μm (n = 30) with 5 to 10 transverse septa and 0 to 3 longitudinal septa. Based on the morphological characteristics, the pathogen was identified as Alternaria sp. (Ma et al. 2021). To further confirm species, two representative isolates (JXAL-1 and JXAL-2) were selected for molecular identification. The internal transcribed spacer (ITS) region, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), elongation factor 1-alpha (TEF1), Alternaria major allergen gene (Alt a 1) and DNA-directed RNA polymerase II core subunit (RPB2) were amplified and sequenced by using primers ITS5/ITS4, gpd1/gpd2, Alt-for/Alt-rev, EF1-728F/EF1-986R and fRPB2-5F2/fRPB2-7cR (Woudenberg et al. 2013; Woudenberg et al. 2014), respectively. These sequences were deposited into GenBank with accession number PP231808-PP231809 (ITS), PP238480-PP238481 (GAPDH), PP238482-PP238483 (Alt a 1), PP238484-PP238485 (TEF1) and PP238486-PP238487 (RPB2). A BLASTn homology search for these nucleotides showed 100% identity to ITS (KJ718182, 525 nt/525 nt), GAPDH (KJ718026, 579 nt/579 nt), Alt a 1 (KJ718694, 472 nt/472 nt), TEF1 (KJ718530, 334 nt/334 nt) and RPB2 (KJ718355, 772 nt/772 nt) sequences of Alternaria linariae CBS 107.61. The maximum likelihood analyses were performed for the combined ITS, GAPDH, Alt a 1, TEF1 and RPB2 using the IQtree web server (Trifinopoulos et al. 2016). In the phylogenetic tree, the isolates and isolates of A. linariae clustered together with 100% bootstrap support. Therefore, the fungus was identified as A. linariae. To evaluate pathogenicity, five healthy fruits of pepino were surface-sterilized with 75% ethanol, then wounded and a 5 mm diameter agar with isolate JXAL-1 was put on the wound. Another five fruits was inoculated with sterile agar plugs as control. All treated fruits were incubated at 25 ℃ with 80% humidity , and repeated twice. Five days later, all the wounded fruits inoculated with A. linariae showed similar symptoms and A. linariae was reisolated, while the control fruits remained healthy and no pathogen was isolated, fulfilling Koch's postulates. A. linariae is known as an important pathogen causing early blight of tomato and potato(Adhikari et al. 2020). To our knowledge, this is the first report of A. linariae causing postharvest fruit rot on S. muricatum in China, which expands the natural host range of A. linariae and will be helpful to develop efficient management strategies on pepino.

Alternaria tomentosae (Pleosporaceae, Dothideomycetes); novel endophytic species from Citrus grandis cv. ‘Tomentosa’ fruits in China

ABSTRACT Alternaria species show a wide range of life modes, viz. pathogens in plants, animals and human, saprobes and rarely endophytes. In the present study, three Alternaria isolates were obtained from healthy fruits of Citrus grandis cv. ‘Tomentosa’ in an industrial planting area in Guigang City, Guangxi Province, China. The nuclear internal transcribed spacer of the rDNA region (ITS), 28S large subunit rDNA (LSU), 18S small subunit rDNA (SSU), glyceraldehyde-3-phosphate dehydrogenase (gapdh), translation elongation factor 1 alpha (tef1-α), RNA polymerase second largest subunit (rpb2) and Alternaria major allergen (alt-a1) gene regions were sequenced and employed to construct phylogenetic trees. Based on the morphology and combined multi-genes phylogeny, our isolates were identified as a novel species, Alternaria tomentosae.

First Report of Leaf Blight of Hedychium coronarium Caused by Alternaria alternata in China.

Hedychium coronarium is an economically significant crop that is widely cultivated for its ornamental, aromatic, and medicinal value (Abbas et al. 2021). From 2020 to 2023, a leaf blight was observed in about 85% of H. coronarium growing in a production field (approximately 500 m2) at Southwest University, Chongqing, China (29° 150'-29° 41' N, 105° 17'-105° 44' E). Symptoms included dark brown necrotic tissue with a clear yellow border. When the disease became severe, affected leaves became dry and abscised. Symptomatic pieces (2 to 5 mm2) between necrotic and healthy tissues were collected from 20 leaf samples, then were immersed in 70% ethanol for 10 s, 0.1% mercury bichloride for 3 min, rinsed in sterile water three times, and placed onto potato dextrose agar (PDA). Four Alternaria isolates were obtained by transferring hyphal tips to new plates. All isolates had identical morphological traits. Cultures on PDA were initially white mycelium on the rim with a light brown center. At around the fourth day, the colony margin changed into light gray and the central part turned sooty black. Conidiophores were branched. Conidia were dark brown, ovoid or ellipsoid in shape, 3.4 to 13.2 μm × 4.1 to 23.5 μm (n = 50) with zero to four transverse and longitudinal septa. For molecular identification, DNA was extracted using the PlantGen DNA Kit CW0553A (Cwbio, Taizhou, China) for PCR amplification of internal transcribed spacer (ITS) region, and 28S large subunit ribosomal RNA (LSU), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), Alternaria major allergen (Alt a 1) and actin (ACT) genes (Choi et al. 2022; Xie et al. 2022; Zhang et al. 2021). BLASTn searches showed that ITS, LSU, GAPDH, Alt a 1 and ACT of four isolates had 100% homology with the corresponding sequences of A. alternata strains MZ578214, KP940477, MK903028, MN655781 and MF564199, respectively. Representative sequences of one strain (AH1) with accession numbers of OK639009, OK639186, OK664976, OK664977 and OK664978 for ITS, LSU, GAPDH, Alt a 1 and ACT regions were deposited in GenBank. The maximum-likelihood tree generated by MEGA 5.10 demonstrated that the pathogenic isolate AH1 obtained from H. coronarium leaf was grouped in the same clade with A. alternata strain CBS121348, which was supported by 100% bootstrap values. To fulfill Koch's postulates, conidia were collected from a 7-day-old culture, suspended in sterile distilled water, and adjusted to 1 × 106 conidia/mL. Leaves on 6-month-old H. coronarium were surface disinfected with 1% sodium hypochlorite solution for 1 min, rinsed twice in water, and then inoculated with AH1 using a sprayer, while leaves treated with sterile water served as negative controls.The experiment was conducted four times, and each repeat contained 10 plants. Pathogenicity testswereperformedinthe greenhouseat25°C with a 12 h photoperiod. Partial yellow lesions were observed 3 days of post-inoculation. As the disease progressed, the tawny color gradually spread across the leaf and the tip became dark brown within 7 days. The necrosis expanded and some small leaves were completely affected within 2 weeks. The pathogen was re-isolated from the lesions and re-identified through morphological traits and sequence analysis. A. alternata have been reported to cause leaf diseases in a variety of cereal crops, vegetables, and fruits across China (Sun et al. 2021; Zheng et al. 2015), which cause significant crop loss. To our knowledge, this is the first report of A. alternata causing leaf blight of H. coronarium in the world. More surveys are needed to explore the epidemiology and management strategies for disease caused by A. alternata in Southwest China.

Two novel species and a new host record of Alternaria (Pleosporales, Pleosporaceae) from sunflower (Compositae) in Myanmar.

Sunflower (Helianthusannuus L.) is a widely cultivated, fast-growing crop known for its seeds and oil, with substantial ecological and economic importance globally. However, it faces challenges from leaf diseases caused by Alternaria species, which threaten its yield. Three small-spored Alternaria species were isolated from leaf spot and blight symptoms on sunflower in Myanmar. All the species were determined based on morphological characterization and a multi-locus phylogenetic assessment of seven genes, including the internal transcribed spacer of rDNA region (ITS), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), RNA polymerase second largest subunit (RPB2), translation elongation factor 1-α (TEF1), Alternaria major allergen gene (Alt a 1), endopolygalacturonase gene (EndoPG), and an anonymous gene region (OPA10-2). The results introduced two new Alternaria species, A.myanmarensis sp. nov. and A.yamethinensis sp. nov., and a known species of A.burnsii, firstly reported from sunflower.

Identification and pathogenicity of Alternaria species causing leaf blotch and fruit spot of apple in California

In late summer 2020, symptoms of leaf blotch and fruit spot were observed in two different commercial apple orchards (cultivars ‘Pink Lady’ and ‘Modi’) in San Joaquin County, California, USA. Ninety Alternaria isolates were obtained from symptomatic leaves and fruits collected from the orchards. Based on morphological characteristics of the colonies, sporulation patterns, and conidia, the isolates were preliminarily separated into three morphogroups, tentatively identified as A. alternata, A. tenuissima and A. arborescens. Multi-locus phylogenetic analyses, using nucleotide sequences of plasma membrane ATPase, calmodulin, and Alternaria major allergen genes, showed that the isolates initially identified as A. tenuissima clustered with strains of A. alternata, following the current taxonomical arrangement of the genus. Pathogenicity tests on detached wounded apple leaves and fruits, using representative isolates of the three morphogroups, fulfilled Koch’s postulates. This is the first report of A. alternata and A. arborescens as causal agents of leaf blotch and fruit spot of apple in California.

Untargeted metabolomic analyses support the main phylogenetic groups of the common plant-associated Alternaria fungi isolated from grapevine (Vitis vinifera)

Alternaria, a cosmopolitan fungal genus is a dominant member of the grapevine (Vitis vinifera) microbiome. Several Alternaria species are known to produce a variety of secondary metabolites, which are particularly relevant to plant protection and food safety in field crops. According to previous findings, the majority of Alternaria species inhabiting grapevine belong to Alternaria sect. Alternaria. However, the phylogenetic diversity and secondary metabolite production of the distinct Alternaria species has remained unclear. In this study, our aim was to examine the genetic and metabolic diversity of endophytic Alternaria isolates associated with the above-ground tissues of the grapevine. Altogether, 270 Alternaria isolates were collected from asymptomatic leaves and grape clusters of different grapevine varieties in the Eger wine region of Hungary. After analyses of the nuclear ribosomal DNA internal transcribed spacer (ITS) and RNA polymerase second largest subunit (rpb2) sequences, 170 isolates were chosen for further analyses. Sequences of the Alternaria major allergen gene (Alt a 1), endopolygalacturonase (endoPG), OPA10-2, and KOG1058 were also included in the phylogenetic analyses. Identification of secondary metabolites and metabolite profiling of the isolates were performed using high-performance liquid chromatography (HPLC)–high-resolution tandem mass spectrometry (HR-MS/MS). The multilocus phylogeny results revealed two distinct groups in grapevine, namely A. alternata and the A. arborescens species complex (AASC). Eight main metabolites were identified in all collected Alternaria isolates, regardless of their affiliation to the species and lineages. Multivariate analyses of untargeted metabolites found no clear separations; however, a partial least squares-discriminant analysis model was able to successfully discriminate between the metabolic datasets from isolates belonging to the AASC and A. alternata. By conducting univariate analysis based on the discriminant ability of the metabolites, we also identified several features exhibiting large and significant variation between A. alternata and the AASC. The separation of these groups may suggest functional differences, which may also play a role in the functioning of the plant microbiome.

First Report of Leaf Spot Caused by Alternaria alternata on Cyclocodon lancifolius in China.

Cyclocodon lancifolius Bunge in the family Campanulaceae, and commonly known as Hong Guo Ginseng, is found in the Indonesia, Philippines, Vietnam, Japan, and China. The leaves and roots of C. lancifolius are widely used as tonics by ethnic minorities in Guizhou and Hunan Provinces in China. In addition, the fruit is edible, and it is a new resource for both medicine and food. In June 2022, symptoms of leaf spot (Fig 1 A and B.) were observed on C. lancifolius plants in the medicinal plant greenhouse of Guizhou University of Traditional Chinese Medicine (106°61'E, 26°39'N), Guizhou Province. The incidence of leaf spot on C. lancifolius was approximately 40 to 70% of all leaves in canopy. Early symptoms on leaves were small circular or irregular brown spots. As the disease progressed the lesions gradually expanded, and multiple lesions coalesced to form large irregular brown spots. Eventually the seedlings died and leaves of mature plants wilted. In order to isolate the pathogen, ten leaf pieces (5×5 mm) were cut from the junction of the diseased and the healthy tissues, surface sterilized with 75% ethanol for 30 s, 0.1% mercuric chloride (HgCl2) solution for 60 s, rinsed in sterile water three times, finally dried and placed on potato dextrose agar (PDA) and cultured in the dark at 27°C for 4 days. Five purified fungal isolates were obtained by single spore isolation. The colonies were olivaceous to dark olive with white margins and abundant aerial mycelia. On potato carrot agar (PCA) medium, these fungi produced septate conidiophores. Conidia were obclavate or ellipsoid, brown, with one to four transverse septa and one to two longitudinal septa. Spores measured 7.64 to 14.20 × 3.32 to 6.38 µm (n=50). These morphological characteristics are consistent with Alternaria alternata (S. P. Wiltshire. 1933). To further confirm the identification, four genomic DNA regions including the ribosomal DNA internal transcribed spacer (ITS), translation elongation factor 1-a gene (TEF), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), RNA polymerase II second largest subunit (RPB2), and Alternaria major allergen gene (Alt a1) were amplified and sequenced with primers ITS1/ITS4 (White et al. 1990), TEF1-728F/TEF1-986R (Carbone and Kohn 1999), gpd1/gpd2 (Berbee et al. 1999), RPB2-5F/RPB2-7cR (Liu et al. 1999), and Alt-for/Alt-rev (Hong et al. 2005), respectively. Sequences were deposited in GenBank with accession Nos. ITS: OQ128111, OQ690707, and OQ690708; TEF: OQ200380, OQ700996, and OQ700998; GAPDH: OQ200378, OQ700993, and OQ700995; RPB2:OQ200379, OQ701002, and OQ701004; Alt: OQ675614, OQ700999, and OQ701001. In a BLAST search, the sequences were 99-100% identical with corresponding sequences of A. alternata. A maximum likelihood phylogenetic tree was constructed with the combined sequence data sets of ITS, TEF, GAPDH, RPB2, and Alt a1 using MEGA 11. The isolate DHY0, DHY1, and DHY3 clustered with A. alternata (J. H. C. Woudenberg et al. 2015) (Fig. 2). To fulfill Koch's postulates, leaves on three healthy 3-month-old potted C. lancifolius seedlings were wounded with sterile needles and inoculated with 5 mm diameter mycelium, which was covered moist by sterile cotton for 24 h. Sterile water was used as the control. After inoculation, the plants were incubated at 27°C, 85% relative humidity, and a 12 h photoperiod. The experiment was repeated three times. Fifteen days after inoculation, all the leaves showed leaf spot symptoms that were similar to those observed in the greenhouse, while control leaves were asymptomatic (Fig. 1). A. alternata was successfully re-isolated from the symptomatic leaves and identified by morphology and the molecular methods described above. This pathogen has been reported to cause a leaf disease in a wide range of vegetables (Zhang et al. 2021), flowers (Zhang et al. 2022), and medicinal plants (Xing et al. 2020). To the best of our knowledge, this is the first report of A. alternata causing leaf spot on C. lancifolius in China. The accurate identification of this pathogen will provide a basis for the prevention and control of C. lancifolius leaf spot disease in the future.

First Report of Leaf Black Spot Caused by Alternaria alternata on American Persimmons in China.

American persimmons (Diospyros virginiana L.) are native to the United States. After being introduced into China, they were used as a rootstock for expanding persimmon varieties and planted in local areas due to their strong cold resistance and diverse leaf colors. In 2022, 12 plants had similar symptoms to black spot disease on the leaves of 18 American persimmons introduced in the National Field Genebank for Persimmon, Yangling, Shaanxi, China (34°17'42.80″ N, 108°04'08.21″ E). Among them, severity was highest in the 'VM10' variety (almost 100%), 'VM10' is the main cultivar in Shaanxi and Henan regions of China, and the incidence of disease in the two regions ranged between 30 and 60% in 2022. Early symptoms were irregular black-brown spots, which gradually combined into large irregular lesions with a dark brown border. The leaves began to curl, crack, scorch, and abscissed. When relative humidity was high, leaves also had signs of black sporulation and became chlorotic. To isolate the causal agent, 10 symptomatic leaves were collected from 5 diseased plants in the National Field Genebank for Persimmon. The infected leaves were cut into 20 small pieces of 5 × 5 mm from the junction of the diseased and healthy tissues and surface disinfected in 75% alcohol for 15 sec, washed with sterile water and 2% NaClO for 90 sec, rinsed three times with sterile water, dried with sterile absorbent paper, and plated on potato dextrose agar (PDA) medium. After 3 days, 12 strains of fungi were isolated from the tissue by transferring the hyphal tips of the mycelium. Among them, 10 strains had similar morphological characteristics. Fungal colonies developed on the PDA medium were initially white, then gradually changed to gray-brown with neat edges and flocculent hyphae. Conidia (n=50) light brown or medium brown, obovate or pear-shaped, and 8.27 to 15.31×17.51 to 24.31 µm, with 1 to 4 transverse septa and 0 to 2 longitudinal septa. The isolates were morphologically similar to Alternaria alternata (Simmons et al. 2007). For molecular identification, the E.Z.N.A.® Fungal DNA kit (Omega Bio-Tek) was used to extract genomic DNA from 7-day-old mycelium grown on PDA medium. The internal transcribed spacers (ITS) region, translation elongation factor 1-alpha (TEF1-α), Alternaria major allergen (Alt a1) gene, and partial RNA polymerase second largest subunit (RPB2) were amplified using ITS1/4 (Glass et al. 1995), EF1-728F/EF1-968R (Carbone and Kohn 1999), and Alt-4for /Alt-4rev (Hong et al., 2005) and RPB2-5F/RPB2-7CR (Liu et al. 1999) respectively. The sequences of a representative isolate MZS1 were deposited in GeneBank with accession numbers OP198643 for ITS, OP286949 for Tef1-α, OP286948 for Alt a1, and OP951084 for RPB2, and were 100% identical to strains of A. alternata (MN615420 for ITS, MN615423 for EF1-α, MW848792 for Alt a1 and MN615422 for RPB2). A maximum-likelihood (ML) phylogenetic tree was constructed based on the concatenated sequences of ITS, TEF1-α, Alt a1, and RPB2gene, which clustered with the A. alternata strains YZU191238 with high bootstrap support (99%). To fulfill Koch's postulates, three-month-old American persimmon 'VM10' seedlings were tested for pathogenicity. Three seedlings were sprayed with 1 × 106 spores/ml suspension in a spray pot, and the three seedlings were treated with sterilized water as a noninoculated control. All seedlings were cultured in a 25°C incubator. The experiment was performed three times under the same conditions. One week after inoculation, typical symptoms appeared on the leaves, which were similar to those observed on the leaves of the original infected persimmon trees. In the control treatment, the leaves did not show symptoms. To the best of our knowledge, this is the first report of A. alternata causing American persimmon black spots disease in China. This report will contribute to the identification of disease symptoms in the field and provide a basis for the occurrence, distribution, and control of A. alternata on American persimmon leaves.

Alternaria arborescens and A. italica Causing Leaf Blotch on Celtis julianae in China.

Celtis julianae Schneid. is widely planted as a versatile tree species with ecological and economic significance. In September 2022, a leaf blotch disease of C. julianae was observed in Nanjing, Jiangsu, China, with an infection incidence of 63%. The disease led to severe early defoliation, significantly affecting the ornamental and ecological value of the host tree. The accurate identification of pathogens is imperative to conducting further research and advancing disease control. Koch's postulates confirmed that the fungal isolates (B1-B9) were pathogenic to C. julianae. The morphology of the characteristics of the pathogen matched those of Alternaria spp. The internal transcribed spacer region (ITS), large subunit (LSU) and small subunit (SSU) regions of rRNA, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), Alternaria major allergen gene (Alt a 1), RNA polymerase second largest subunit (RPB2), and portions of translation elongation factor 1-alpha (TEF1-α) genes were sequenced. Based on multi-locus phylogenetic analyses and morphology, the pathogenic fungi were identified as Alternaria arborescens and A. italica. The findings provided useful information for disease management and enhanced the understanding of Alternaria species diversity in China. This is the first report of A. arborescens and A. italica causing leaf blotch of C. julianae in China and worldwide.

Prospects for developing allergen-depleted food crops.

In addition to the challenge of meeting global demand for food production, there are increasing concerns about food safety and the need to protect consumer health from the negative effects of foodborne allergies.Certain bio-molecules (usually proteins) present in food can act as allergens that trigger unusual immunological reactions, with potentially life-threatening consequences. The relentless working lifestyles of the modern era often incorporate poor eating habits that include readymade prepackaged and processed foods, which contain additives such as peanuts, tree nuts, wheat, and soy-based products, rather than traditional home cooking. Of the predominant allergenic foods (soybean, wheat, fish, peanut, shellfish, tree nuts, eggs, and milk), peanuts (Arachis hypogaea) are the best characterized source of allergens, followed by tree nuts (Juglans regia, Prunus amygdalus, Corylus avellana, Carya illinoinensis, Anacardium occidentale, Pistacia vera, Bertholletia excels), wheat (Triticum aestivum), soybeans (Glycine max), and kidney beans (Phaseolus vulgaris). The prevalence of food allergies has risen significantly in recent years including chance of accidental exposure to such foods. In contrast, the standards of detection, diagnosis, and cure have not kept pace and unfortunately are often suboptimal. In this review, we mainly focus on the prevalence of allergies associated with peanut, tree nuts, wheat, soybean, and kidney bean, highlighting their physiological properties and functions as well as considering research directions for tailoring allergen gene expression. In particular, we discuss how recent advances in molecular breeding, genetic engineering, and genome editing can be used to develop potential low allergen food crops that protect consumer health.

Four New Species of Small-Spored Alternaria Isolated from Solanum tuberosum and S. lycopersicum in China.

Small-spored Alternaria species have been frequently isolated from diseased leaves of Solanum plants. To clarify the diversity of small-spored Alternaria species, a total of 118 strains were obtained from leaf samples of S. tuberosum and S. lycopersicum in six provinces of China during 2022-2023. Based on morphological characterization and multi-locus phylogenetic analysis of the internal transcribed spacer of the rDNA region (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 gene (EndoPG) and an anonymous gene region (OPA10-2), seven species were determined, including four novel species and three known species (A. alternata, A. gossypina and A. arborescens). The novel species were described and illustrated as A. longxiensis sp. nov., A. lijiangensis sp. nov., A. lycopersici sp. nov. and A. solanicola sp. nov.. In addition, the pathogenicity of the seven species was evaluated on potato leaves. The species exhibited various aggressiveness, which could help in disease management.

First Report of Brown Spot Caused by Alternaria alternata on Potato (Solanum tuberosom L.) in Korea.

In June 2020, brown spot symptoms were observed in a commercial potato field located in Yeocheon, Gyeonggi Province, Korea. The symptoms were similar to those associated with early blight. Brown lesions on leaves were circular and expanded rapidly under high humidity and warm temperatures ranging 12°C at night to 30°C during daytime. Over 60% of potato (Solanum tuberosum L. cv. Superior) leaves showed the symptoms. For fungal isolation, infected leaf tissues (5 × 5 mm) from 14 infected samples were immersed in 70% ethanol for 1 min, rinsed three times in sterilized water, dried, placed on water agar amended with 100 ppm of streptomycin, and then incubated in the dark at 25°C. Hyphae emerging from the tissues were subcultured on V8-Juice agar (8% of V8-Juice, 1.5% agar, pH 7), and the obtaining cultures were subjected to single-spore isolation, resulting in 14 isolates (SYP-934~947). Three representative isolates, SYP-934 to SYP-936, were deposited in the Korean Agriculture Culture Collection (Accession Nos. KACC 410058 to KACC 410060). Conidia (n = 100) produced on the colony were brown, ellipsoid to ovoid with walls ornamented, 1 to 6 transverse and 0-3 vertical septa, and length × width of 20-45 × 7 to 24 μm (n = 100). Their morphological characteristics were consistent with Alternaria alternata (Simmons, 2007; van der Waals et al., 2011; Woundenberg et al. 2015). Sequences of the following loci in the 14 isolates were determined as described in Woundenberg et al. (2013 and 2014: the internal transcribed spacer (primer pairs VG9/ITS4, GenBank accession nos. OP581413-25), glyceraldehyde-3-phosphate dehydrogenase (gpd1/gpd2, OP588286-99), RNA polymerase second largest subunit (RPB2-5F2/fRPB2-7cR, OP588314-27), translation elongation factor 1-alpha (EF1-728F/EF1-986R, OP588300-13), Alternaria major allergen gene (Alt-For/Alt-Rev, OP588328-41), endopolygalacturonase (PG3/PG2b, OP588342-55), and an unknown gene region (OPA10-2R/OPA10-2L, OP588356-68). A neighbor-joining phylogenetic analysis based on the concatenated gene sequences, which was performed using the MEGA X program (Kumar et al., 2018), placed the 14 isolates in the clade containing A. alternata isolates. To test pathogenicity, one-month-old potato (S. tuberosum cv. Superior) plants grown in a 25°C growth chamber were sprayed with conidial suspensions (1×106 conidia/mL) prepared from 14-day-old cultures of three isolates (KACC 410058 to KACC 410060). Sterile distilled water was used as the control treatment. The inoculated pots were placed in a plastic box to maintain high humidity and incubated in the dark at 25°C for 2 days. The plants were transferred to a growth chamber (16h light with over 70% humidity at 25°C). Symptoms were first observed after 3 days post inoculation (dpi) with all three isolates, and severe brown spot symptoms were observed after 7 dpi. No symptom was observed in the control treatment. The pathogenicity assay was repeated at triplicate. Reisolated cultures from lesions were confirmed to be A. alternata based on their sequence at the rpb2 locus, thus fulfilling Koch's postulates. Alternaria alternata has been reported to cause brown spot and leaf blight on potato leaves in Israel (Dorby et al., 1984) and South Africa (van der Waals., et al. 2011). To our knowledge, this study is the first report of A. alternata causing brown spot disease in Korea.