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 leaf blight caused by Stemphylium lycopersici on Salvia splendens in China.

Salvia splendens is a popular ornamental plant in China with extensive potentials, including value in traditional Chinese medicine and in environmental restoration function (Li et al. 2008). In September 2019, leaf blight disease was observed on road side plants of S. splendens in Bayi park, Nanchang city, Jiangxi province, China. The typical symptoms appeared as irregular necrotic spots or leaf blight, accompanied by extensive scorch necrosis or ultimately defoliation. Small segments cut from diseased leaves were surface sterilized in a 2% sodium hypochlorite solution for 2 min and rinsed three times with sterile distilled water. Then, the samples were placed on potato dextrose agar (PDA) plates incubated at 25°C in darkness. Pure cultures were obtained by the hyphal tip method. Morphologically, all 11 colonies were identical to each other on PDA. Two strains, YZU 191468 and YZU 191481, were selected for further study and deposited in the Fungal Herbarium of Yangtze University (YZU), Jingzhou, Hubei, China. The 7-day-old colonies were circular, 53 to 56 mm in diameter, and consisted of white mycelium with a buff margin, and were cinnamon colored in the center of the reverse side. To examine conidial morphology, the mycelium was transferred onto potato carrot agar (PCA) and incubated at 23°C with a period of 8 h light/16 h dark for 7 days. Conidia were normally solitary or two in a chain, ellipsoid or long ellipsoid, beakless, 10 to 23×30 to 60 µm in size (n=50). Based on morphology, the isolates were consistent with Stemphylium lycopersici (Yamamoto 1960). To confirm the identification, genomic DNA was extracted from both isolates and used to amplify the internal transcribed spacer rDNA region (ITS), glyceraldehydes-3-phosphate dehydrogenase (GAPDH) and calmodulin (CAL) genes with primer pairs ITS5/ITS4, gpd1/gpd2, and CALDF1/CALDR2, respectively (Woudenberg et al. 2017). Sequences were deposited in GenBank with accession numbers OP564983 and OP564984 (ITS), OP892529 and OP892530 (GAPDH), OP584970 and OP584971 (CAL). A neighbor-joining tree was constructed with Mega 7.0 based on the combined dataset with 1,000 bootstrap replicates. The resulting phylogenetic tree showed that the strains from S. splendens clustered with S. lycopersici (CBS 122639 and CBS 124980) supported with 100% bootstrap values. The molecular analyses confirmed that the species causing leaf blight symptoms was S. lycopersici. To test pathogenicity, healthy leaves of S. splendens were surface sterilized and inoculated by mycelium blocks (6 mm in diameter) and spore suspension (1×106 spore/mL) of representative strains YZU 191468 and YZU 191481, respectively. Controls were inoculated with blocks of PDA and sterile water. Each strain was inoculated on three leaves of a plant. One clean plant was used as control. The test was replicated three times. After inoculation, the plants were covered with plastic bags and incubated in a greenhouse (25℃, 80 % relative humidity, 8 h light/16 h dark). After 5 days, the inoculated leaves exhibited dark brown spots with white mycelium, followed by withering of necrotic tissues. There were no symptoms observed on the controls. The fungal isolates inoculated leaves had the same morphological characteristics as the strains used for inoculation. S. lycopersici has been found on eggplant and Zinnia elegans in China (He et al. 2019; Yang et al. 2017). To the best of our knowledge, this is the first report of S. lycopersici causing leaf blight on S. splendens in China. This finding offers a new reference for the management and control of S. splendens leaf diseases in China.

Alternaria species in section Alternaria associated with Iris plants in China.

Plants of the Iris genus have been widely cultivated because of their medicinal, ornamental, and economic values. It commonly suffers from Alternaria leaf spot or blight disease leading to considerable losses for their commercial values. During an investigation of 14 provinces or municipalities of China from 2014 to 2022, a total of 122 Alternaria strains in section Alternaria were obtained from diseased leaves of Iris spp.. Among them, 12 representative strains were selected and identified based on morphological characterization and multi-locus phylogenetic analysis, which encompassed the internal transcribed spacer of 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), an anonymous gene region (OPA10-2), and endopolygalacturonase gene (EndoPG). The strains comprised two known species of A. alternata and A. iridicola, and two new species of A. setosae and A. tectorum, which were described and illustrated here. Their pathogenicity evaluated on Iris setosa indicated that all the strains could induce typical Alternaria leaf spot or blight symptoms. The results showed that the virulence was variable among those four species, from which A. tectorum sp. nov. was the most virulent one, followed by A. setosae sp. nov., A. iridicola and A. alternata.

New Species of Large-Spored Alternaria in Section Porri Associated with Compositae Plants in China.

Alternaria is a ubiquitous fungal genus including saprobic, endophytic, and pathogenic species associated with a wide variety of substrates. It has been separated into 29 sections and seven monotypic lineages based on molecular and morphological data. Alternaria sect. Porri is the largest section, containing the majority of large-spored Alternaria species, most of which are important plant pathogens. Since 2015, of the investigations for large-spored Alternaria species in China, 13 species were found associated with Compositae plants based on morphological comparisons and phylogenetic analyses. There were eight known species and five new species (A. anhuiensis sp. nov., A. coreopsidis sp. nov., A. nanningensis sp. nov., A. neimengguensis sp. nov., and A. sulphureus sp. nov.) distributed in the four sections of Helianthiinficientes, Porri, Sonchi, and Teretispora, and one monotypic lineage (A. argyranthemi). The multi-locus sequence analyses encompassing the internal transcribed spacer region of rDNA (ITS), glyceraldehydes-3-phosphate dehydrogenase (GAPDH), Alternaria major allergen gene (Alt a 1), translation elongation factor 1-alpha (TEF1), and RNA polymerase second largest subunit (RPB2), revealed that the new species fell into sect. Porri. Morphologically, the new species were illustrated and compared with other relevant large-spored Alternaria species in the study. Furthermore, A. calendulae, A. leucanthemi, and A. tagetica were firstly detected in Brachyactis ciliate, Carthamus tinctorius, and Calendula officinalis in China, respectively.

Evaluation of Pathogenic Fusarium spp. Associated with Soybean Seed (Glycine max) in Hubei Province, China.

Soybean (Glycine max L.) seeds showing serious symptoms from rotted pods were collected from fields during the harvesting period (July to August 2020) in Taihu Farm, Jingzhou City, Hubei Province, China. Fusarium strains were frequently encountered during fungal isolation. According to the morphology and prepathogenicity tests, six strains showing variable effects on the seeds were selected for identification based on morphology and multilocus phylogenetic analysis of the internal transcribed spacer (ITS) region of the ribosomal DNA, translation elongation factor (EF-1α), calmodulin (CAM), ß-tubulin (TUB), and partial RNA polymerase second largest subunit (RPB2), and to evaluate the pathogenic abilities on seed, root, and pod. The results indicated that the strains contained two species (Fusarium fujikuroi and F. proliferatum) in the Fusarium fujikuroi species complex (FFSC) and two species (F. luffae and F. sulawense) from the Fusarium incarnatum-equiseti species complex (FIESC). The two species of FFSC were more aggressive than those of FIESC on soybean seed, root, and pod. Among the strains, F. proliferatum YZU 201408 exhibited the most pathogenicity on all tests, with 72.2 to 90% disease severity.