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.

Plant Growth-Promoting Ability of Mycorrhizal Fusarium Strain KB-3 Enhanced by Its IAA Producing Endohyphal Bacterium, Klebsiella aerogenes.

Fusarium oxysporum KB-3 had been reported as a mycorrhizal fungus of Bletilla striata, which can promote the seed germination and vegetative growth. Endohyphal bacteria were demonstrated in the hyphae of the KB-3 by 16S rDNA PCR amplification and SYTO-9 fluorescent nucleic acid staining. A strain Klebsiella aerogenes KE-1 was isolated and identified based on the multilocus sequence analysis. The endohyphal bacterium was successfully removed from the wild strain KB-3 (KB-3-), and GFP-labeled KE-1 was also transferred to the cured strain KB-3- (KB-3+). The production of indole-3-acetic acid (IAA) in the culturing broths of strains of KE-1, KB-3, KB-3-, and KB-3+ was examined by HPLC. Their IAA productions were estimated using Salkowski colorimetric technique. The highest concentrations of IAA were 76.9 (at 48 h after inoculation), 31.4, 9.6, and 19.4 µg/ml (at 60 h after inoculation), respectively. Similarly, the three fungal cultural broths exhibited plant promoting abilities on the tomato root and stem growth. The results indicated that the ability of mycorrhizal Fusarium strain KB-3 to promote plant growth was enhanced because its endohyphal bacterium, Klebsiella aerogenes KE-1, produced a certain amount of IAA.

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.

Diaporthe taoicola and D. siamensis, Two New Records on Citrus sinensis in China.

Two Diaporthe species isolated from fruit of Citrus sinensis in China were characterized based on morphology and multilocus phylogeny of ITS, tef1, and tub2 gene sequences. The phylogeny indicated that the two species match Diaporthe taoicola and D. siamensis. A critical examination of phenotypic characteristics confirmed the phylogenetic results. Diaporthe taoicola was morphologically characterized by producing Alpha conidia with tapering toward both ends. Meanwhile, D. siamensis produced cylindrical or ellipsoidal Alpha conidia with two oil drops. Pathogenicity tests revealed that both species were pathogenic to fruit of C. sinensis. To our knowledge, the two species were firstly reported on Citrus sinensis in China.

Morphological and molecular identification of two new Alternaria species (Ascomycota, Pleosporaceae) in section Radicina from China.

The fungal genus Alternaria was distributed widely and found in different habitats such as plant or indoor environment. During an investigation into this genus in China, two new Alternaria species, Alternaria vulgarae and A. divaricatae were respectively isolated from diseased leaves of Foeniculum vulgare and Saposhnikovia divaricata, which both belonged to Umbelliferae. Phylogenetically, they were determined as new species belonging in the section Radicina of Alternaria based on the combined four gene fragments of ITS, TEF1, GAPDH and RPB2. Morphologically, the two species were illustrated and compared with other relevant Alternaria species in section Radicina.

Identification and Characterization of Gonatobotryum apiculatum Causing Leaf Spot and Blight on Sinowilsonia henryi.

Sinowilsonia henryi is a rare and endangered plant, as well as an endemic species in China. In July 2018, leaf spot and blight disease was observed on S. henryi in Yichang, Hubei, China. A fungus isolated from disease tissues was identified as Gonatobotryum apiculatum based on morphology and sequence analyses of ITS and LSU regions. Phylogenetic analyses indicated that the species belongs to Dothioraceae (Dothideales). Morphologically, the species produced two distinct types of conidia from authentic media, both conidia were described here. Pathogenicity tests showed that the fungus is a pathogen causing leaf spots on S. henryi. This is the first report of leaf spot and blight disease on S. henryi caused by G. apiculatum in China.

Morphology and Molecular Characterization of a Fungus from the Alternaria alternata Species Complex Causing Black Spots on Pyrus sinkiangensis (Koerle pear).

A small-spored Alternaria was found from black spots of storaged Koerle pear (Pyrus sinkiangensis), one of the economically important fruit in Xinjiang province, China. The morphology is similar to A. limoniasperae but obviously different in secondary conidiophores and conidial septa. A phylogenetic analysis using sequence datasets of ITS, GAPDH, TEF1, RPB2, Alt a1, OPA10-2, and EndoPG genes revealed that it belonged to the Alternaria alternata complex group. Pathogenicity tests illustrated that the fungus was the causal pathogen of black spot on Koerle pear fruit.

Morphology and Molecular Characterization of Alternaria argyranthemi on Chrysanthemum coronarium in China.

Chrysanthemum coronarium is an economically important plant in Asia, and used medicinally, ornamentally and as a vegetable. In April 2017, leaf spot disease on C. coronarium was observed in Shiyan, Hubei, China. A single-spore isolate was obtained and identified based on morphology and sequence analysis using four regions (rDNA ITS, GAPDH, EF-1α, and RPB2). The results indicated that the fungus is Alternaria argyranthemi. The pathogenicity tests revealed that the species could cause severe leaf spot and blight disease on the host. This is the first report of leaf spot disease on C. coronarium caused by A. argyranthemi in the world, which is also a new record of Alternaria species in China.

Identification and Characterization of Alternaria iridiaustralis Causing Leaf Spot on Iris ensata in China.

In 2016, a severe leaf spot disease was found on Iris ensata Thumb. in Nanjing, China. The symptom was elliptical, fusiform, or irregularly necrotic lesion surrounded by a yellow halo, from which a small-spored Alternaria species was isolated. The fungus was identified as Alternaria iridiaustralis based on morphological characteristics. The pathogenicity tests revealed that the fungus was the causal pathogen of the disease. Phylogenic analyses using sequences of ITS, gpd, endoPG, and RPB2 genes confirmed the morphological identification. This study is the first report of A. iridiaustralis causing leaf spots on I. ensata in China.

Alternaria brassicifolii sp. nov. Isolated from Brassica rapa subsp. pekinensis in Korea.

A new species belonging to the genus Alternaria was isolated from the necrotic leaf spots of Brassica rapa subsp. pekinensis in Yuseong district, Daejeon, Korea. It is an occasional isolate, not an etiological agent, which is morphologically similar to A. broccoli-italicae, but differs in conidial size and conidiophore shape. Phylogenetic analysis using the sequence datasets of the internal transcribed spacer (ITS) region of the rDNA, glyceraldehyde-3-phosphate dehydrogenase (gpd), and plasma membrane ATPase genes showed that it is distantly related to A. broccoli-italicae and closely related to Alternaria species in the section Pseudoalternaria, which belonged to a clade basal to the section Infectoriae. Morphologically, the species is unique because it produces solitary conidia or conidial chains (two units), unlike the four members in the section Pseudoalternaria that produce conidia as short branched chains. It exhibits weak pathogenicity in the host plant. This report includes the description and illustration of A. brassicifolii as a new species.

Manipulation of KCNE2 expression modulates action potential duration and Ito and IK in rat and mouse ventricular myocytes.

In heterologous expression systems, KCNE2 has been demonstrated to interact with multiple α-subunits of voltage-dependent cation channels and modulate their functions. However, the physiological and pathological roles of KCNE2 in cardiomyocytes are poorly understood. The present study aimed to investigate the effects of bidirectional modulation of KCNE2 expression on action potential (AP) duration (APD) and voltage-dependent K(+) channels in cardiomyocytes. Adenoviral gene delivery and RNA interference were used to either increase or decrease KCNE2 expression in cultured neonatal and adult rat or neonatal mouse ventricular myocytes. Knockdown of KCNE2 prolonged APD in both neonatal and adult myocytes, whereas overexpression of KCNE2 shortened APD in neonatal but not adult myocytes. Consistent with the alterations in APD, KCNE2 knockdown decreased transient outward K(+) current (Ito) densities in neonatal and adult myocytes, whereas KCNE2 overexpression increased Ito densities in neonatal but not adult myocytes. Furthermore, KCNE2 knockdown accelerated the rates of Ito activation and inactivation, whereas KCNE2 overexpression slowed Ito gating kinetics in neonatal but not adult myocytes. Delayed rectifier K(+) current densities were remarkably affected by manipulation of KCNE2 expression in mouse but not rat cardiomyocytes. Simulation of the AP of a rat ventricular myocyte with a mathematical model showed that alterations in Ito densities and gating properties can result in similar APD alterations in KCNE2 overexpression and knockdown cells. In conclusion, endogenous KCNE2 in cardiomyocytes is important in maintaining cardiac electrical stability mainly by regulating Ito and APD. Perturbation of KCNE2 expression may predispose the heart to ventricular arrhythmia by prolonging APD.

Occurrence of Leaf Blight on Cosmos Caused by Alternaria cosmosa in Korea.

In 2011, a leaf blight disease was observed on cosmos (Cosmos bipinnatus) leaves in Nonsan, Korea. The causal pathogen was isolated and identified based on morphological and molecular approaches. Morphological characteristics of the pathogen matched well with the Alternaria cosmosa and also easily distinguishable from Alternaria zinniae reported from cosmos seeds by producing branched beak. Phylogenetically, the pathogen could not be distinguished from A. passiflorae based on the sequence analysis of a combined data set of Alt a1 and gpd genes. However, A. passiflorae was distinguished from the present species by having conidiophores with 4 to 5 conidiogenous loci. The results indicate that the present Alternaria species is A. cosmosa. Pathogenicity tests revealed that the isolate was pathogenic to the leaves of Cosmos bipinnatus. This is the first report of Alternaria blight disease caused by A. cosmosa on cosmos in Korea.

Characterization and Pathogenicity of Alternaria burnsii from Seeds of Cucurbita maxima (Cucurbitaceae) in Bangladesh.

In the course of survey of endophytic fungi from Bangladesh pumpkin seeds in 2011~2012, two strains (CNU111042 and CNU111043) with similar colony characteristics were isolated and characterized by their morphology and by molecular phylogenetic analysis of the internal transcribed spacer, glyceraldehydes-3-phosphate dehydrogenase (gpd), and Alternaria allergen a1 (Alt a1) sequences. Phylogenetic analysis of all three sequences and their combined dataset revealed that the fungus formed a subclade within the A. alternata clade, matching A. burnsi and showing differences with its other closely related Alternaria species, such as A. longipes, A. tomato, and A. tomaticola. Long ellipsoid, obclavate or ovoid beakless conidia, shorter and thinner conidial size (16~60 [90] × 6.5~14 [~16] µm) distinguish this fungus from other related species. These isolates showed more transverse septation (2~11) and less longitudinal septation (0~3) than did other related species. Moreover, the isolate did not produce any diffusible pigment on media. Therefore, our results reveal that the newly recorded fungus from a new host, Cucurbita maxima, is Alternaria burnsii Uppal, Patel & Kamat.

A Novel Alternaria Species Isolated from Peucedanum japonicum in Korea.

We isolated and examined a new Alternaria sp., which causes leaf spots on Peucedanum japonicum in Korea, by using molecular and morphological methods. Phylogenetic analysis based on a combined internal transcribed spacer region analysis and two protein-coding genes (gpd and Alt a1) demonstrated that the causal fungus was most closely related to A. cinerariae and A. sonchi, and relevant to A. brassicae. However, conidial morphology indicated that it is a novel species within the genus Alternaria, and therefore we have assigned the fungus a new name in this study.

Characterization and Pathogenicity of Alternaria vanuatuensis, a New Record from Allium Plants in Korea and China.

Alternaria from different Allium plants was characterized by multilocus sequence analysis. Based on sequences of the ß-tubulin (BT2b), the Alternaria allergen a1 (Alt a1), and the RNA polymerase II second largest subunit (RPB2) genes and phylogenetic data analysis, isolates were divided into two groups. The two groups were identical to representative isolates of A. porri (EGS48-147) and A. vanuatuensis (EGS45-018). The conidial characteristics and pathogenicity of A. vanuatuensis also well supported the molecular characteristics. This is the first record of A. vanuatuensis E. G. Simmons & C. F. Hill from Korea and China.

Penicillium daejeonium sp. nov., a new species isolated from a grape and schisandra fruit in Korea.

Two isolates of monoverticillate Penicillium species were collected from a grape and schisandra fruit in Korea. Multigene phylogenetic analyses with the nuclear ribosomal internal transcribed spacer (ITS) region and genes encoding ß-tubulin (benA) and calmodulin (cmd), as well as morphological analyses revealed that the two isolates are members of the P. sclerotiorum complex in Penicillium subgenus Aspergilloides, but different from species of the P. sclerotiorum complex. The isolates are closely related to P. cainii, P. jacksonii, and P. viticola in terms of their multigene phylogeny, but their colony and conidiophore morphologies differ from those of closely related species. The name P. daejeonium is proposed for this unclassified new species belonging to the P. sclerotiorum complex in subgenus Aspergilloides.