First Report of Alternaria blumeae Causing Leaf Blight on Tomato in Myanmar

Abstract

Tomato (Solanum lycopersicum) originating from western South America is widely cultivated as a commercial crop around the world. It is rich in a series of nutrition such as vitamin C, phytochemical lycopene, and so on. In February 2019, leaf blight symptoms were observed on tomato plants in a commercial growing area in Wegyi village, Pyinmana township, Naypyitaw, Myanmar. Diseased leaves showed blight symptoms with a dark brown margin and diffused chlorosis on the entire leaf blade, particularly on older leaves. Ten symptomatic leaves from different plants were randomly collected to isolate the causal agents. Infected leaves were cut into small sections, placed on moist filter papers in plastic dishes, and incubated at 25°C. Large-spored Alternaria that developed from the diseased tissues were single-spore isolated. All isolates (11) were identical to each other in morphology and deposited in the Fungi Herbarium of Yangtze University (YZU), Jingzhou, Hubei, China. Two isolates, YZU 191010 and YZU 191011, were used for further study. After being grown on potato dextrose agar (PDA) at 25°C in darkness for 7 days, colonies were white-velvety, reverse side amber brown, cinnamon-buff margin, and 36 to 38 mm in diameter. The two isolates were transferred onto potato carrot agar (PCA) and incubated at 22°C under a photoperiod of 8 h light/16 h dark for 7 days to stimulate sporulation. Conidiophores were long, erect, 70 to 100 × 3 to 6 µm in size. Conidia were solitary, long-ovoid, ellipsoid in body with six to nine transverse septa, and 65 to 120 × 16 to 36 µm in size. Conidial beaks were filamentous, 70 to 110 × 2 to 5 µm in size. Based on morphology, the fungus was identical to Alternaria blumeae described by Simmons (2007). Genomic DNA was extracted, and five gene regions including the internal transcribed spacer rDNA region (ITS), translation-elongation factor 1 alpha (TEF1), glyceraldehydes-3-phosphate dehydrogenase (GAPDH), Alternaria major allergen gene (ALT), and RNA polymerase second largest subunit (RPB2) were amplified with specific primers ITS4/ITS5, EF1-728F/EF1-986R, GPD1/GPD2, Alt-for/Alt-rev, and RPB2-5F/RPB2-7cR, respectively (Woudenberg et al. 2014). The resulting sequences of YZU 191010 and YZU 191011 were deposited in GenBank with accession numbers MN612547 and MN612548 (ITS), MN612551 and MN612552 (GAPDH), MN612545 and MN612546 (ALT), MN612553 and MN612554 (TEF1), and MN612549 and MN612550 (RPB2). Related gene sequences were retrieved from GenBank. Phylogenetic trees of the five combined genes were constructed based on Bayesian methods and maximum likelihood with an evolutionary model of GTR+I+G. The results showed that the two strains were clustered with A. blumeae (CBS 117364 and CBS 117215), supported by a Bayesian posterior probability value of 1.0 and a RAxML bootstrap value of 100%. Besides, the five gene sequences of the present fungus were 100% identical to the sequences of type strain A. blumeae (CBS 117364). Pathogenicity tests were conducted on a local tomato cultivar collected from Wegyi village. Seeds were sown in a tray to obtain the seedlings. Three-week-old seedlings were transplanted into pots for further growth. Then, 5 weeks later, the inoculation tests were performed with two isolates using two methods: mycelium plugs (2 mm diameter) from 3-day-old colonies on PDA and spore suspension (1 × 10⁵ spore/ml) prepared from 7-day-old-colonies on PCA. Aseptic PDA plugs and sterile distilled water were used as negative controls. The inoculated plants were then maintained in a greenhouse at 25°C with high humidity and 12-h light period. Dark small spots were observed after 3 days on both treatments, and they enlarged gradually. After 7 days, the spots reached to 18 to 25 × 26 to 32 mm and 7 to 18 × 8 to 25 mm in size, respectively. The symptoms were similar to the original symptoms that occurred in the field. Control leaves were symptomless. The tests were repeated three times. Koch’s postulates were fulfilled by reisolating the pathogen from inoculated leaves and identified as A. blumeae by ALT gene sequence. A. blumeae was first reported on Blumea aurita (Compositae) in Thailand (Simmons 2007) and recently was discovered on wilted tissues of Solanum tuberosum (Solanaceae) in China (Liu et al. 2019). To the best of our knowledge, this is the first report of A. blumeae causing leaf blight on S. lycopersicum (Solanaceae) in Myanmar.