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First Report of Alternaria steviae Causing Black Leaf Spot of Stevia in China

Yan, M. F., Liu, B., Wang, Y. X., Zhu, J., Yang, P. S., Xiao, X., Jiang, J. X.
Plant disease 2018 v.102 no.12 pp. 2650
Alternaria, DNA, Stevia rebaudiana, conidia, conidiophores, culture media, ethanol, glyceraldehyde-3-phosphate dehydrogenase, greenhouses, internal transcribed spacers, leaf spot, leaves, mercuric chloride, pathogenicity, photoperiod, polymerase chain reaction, sequence analysis, tissues, China
Stevia (Stevia rebaudiana Bertoni), an herb native to South America, is now commercially cultivated in China as well as other countries for use as a natural sweetener. In June 2017, black leaf spots were observed on stevia (cv. Puxing 3) growing in a 2-ha commercial stevia field with approximately 60% of leaves infected in Gan county of Jiangxi province, China. Infected foliage displayed dark brown, elliptical or irregular, 1.0 to 1.5 cm in diameter lesions surrounded with a yellow halo. Small pieces from the margin of necrotic leaf tissue were surface sterilized in 70% ethanol for 10 s followed in 0.1% HgCl₂ for 1 min. After being washed three times in sterile distilled water and dried on sterilized filter paper, the pieces were transferred onto acidified potato dextrose agar (PDA) plates and incubated at 28°C with a 12-h light-dark cycle. Alternaria isolates were consistently recovered from pieces of symptomatic tissues on PDA plates. Colonies on PDA were gray-green and covered the entire 9-cm PDA plate after 7 days of incubation at 28°C. Conidiophores were brown, straight or slightly curved, unbranched, three to seven transverse septa, and 41.6 to 104 × 3.9 to 5.8 μm in size. Conidia were brown, ovoid to ellipsoid, with a colorless long filamentous beak. Conidia bodies measured 31.2 to 56 × 13 to 18.2 µm, with three to seven transverse and one to three longitudinal septa, and the beaks were 57.2 to 124 µm long and 1.5 to 2.5 µm wide. These cultural and morphological characteristics suggested an Alternaria species. Genomic DNA of a representative isolate (PX1) was extracted and amplified by polymerase chain reaction (PCR) using ITS1/ITS4 for the internal transcribed spacer (ITS) region, EF1-728F/EF1-986R for the tef1 region, gpd1/gpd2 for the glyceraldehyde 3-phosphate dehydrogenase (GAPDH) region, and Alt-for/Alt-rev for the Alt a 1 region (Woudenberg et al. 2014). The PCR products were sequenced (GenBank accession nos. MF471694 and MG601507 to MG601509, respectively) and shared 100% homology with the corresponding sequence of Alternaria steviae (NR136108 for ITS, KJ718596 for tef1, and KJ718756 for Alt a 1, respectively) and 99% homology with the published GAPDH (KJ718078) sequence of A. steviae. To test pathogenicity, a conidial suspension with 1 × 10⁵ conidia/ml of isolate PX1 was sprayed on six leaves of each of three healthy plants (cv. Puxing 3), whereas control plants were sprayed with sterilized water. The inoculated plants were incubated in a greenhouse at 28°C under moist conditions. Typical symptoms similar to those in the field appeared on the inoculated leaves 3 to 4 days after inoculation, whereas the control leaves remained symptomless. The original isolate was successfully reisolated from diseased leaves, fulfilling Koch’s postulates. Occurrence of the disease caused by A. steviae has previously been reported in Japan (Ishiba et al. 1982). As far as we know, this is the first report of A. steviae causing black leaf spot of stevia in China.