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First Report of Leaf Spot on Rubber Tree Caused by Lasiodiplodia pseudotheobromae in China

Wu, R. H., Zhang, Y., Li, Z. P.
Plant disease 2019 v.103 no.4 pp. 766
DNA primers, Hevea brasiliensis, Juglans regia, Lasiodiplodia, adults, agar, cinnamon, conidia, culture media, disease severity, ethanol, fungi, grapes, internal transcribed spacers, latex, leaf spot, leaves, mercuric chloride, mycelium, pathogens, polymerase chain reaction, rubber, soaking, temperature, tissues, tropics, weather, China, Thailand
Rubber tree (Hevea brasiliensis Muell. Arg.) is an important economic crop in the tropical regions of China, which used to produce natural latex and is mainly grown in Hainan, Guangdong, and Yunnan Provinces. In August 2017, leaf spots were observed on nearly 33% of the rubber trees (n = 200, clone RRIM600) in a plantation of Sanya (Hainan Province), and the crown disease severity rating was 5 (scale 1, 3, 5, 7, 9). The disease occurred in hot weather, with temperatures ranging from 26 to 35°C. The disease mainly endangers the adult leaves of rubber trees. Symptomatic leaves initially exhibited small, dark brown, round or oval spots with an obvious yellow halo, subsequently expanding into round, semicircular, or irregularly shaped gray-white lesion with scattered black spots. There was a distinct dark brown necrotic zone at the junction of the diseased and healthy tissue, surrounded by a yellow halo. Lesion expansion was not limited by the lobular veins, but the main vein. Finally, the leaves with extensive lesions gradually dried and dropped off of the plant. The diseased tissues were excised from 10 infected leaves with typical symptoms, which were taken from several individual rubber trees and surface sterilized by soaking in 70% ethanol for 10 s followed by 1 min in 0.1% HgCl₂, rinsed three times with sterile water, and then transferred to potato dextrose agar (PDA) and incubated at 28°C. Two days after incubation, fast-growing fungal colonies (70 to 75 mm in diameter) formed with dense, aerial, whitish cottony mycelium, which then turned to dark blue or black. Conidia were initially hyaline and unicellular. Mature conidia were two-celled, cinnamon to light brown, and often with longitudinal striations. Conidia measured 21.5 to 31.85 × 12.06 to 14.49 µm (average 26.63 × 13.1 µm, n = 200). Morphological characteristics were similar to descriptions for Lasiodiplodia sp., a member of the family Botryosphaeriaceae (Alves et al. 2008; Phillips et al. 2013). The internal transcribed spacer (ITS) region was amplified with the corresponding primer pairs ITS1 and ITS4 (White et al. 1990). A 503-bp fragment was amplified and sequenced (GenBank accession no. MH422961). It showed 100% identity to Lasiodiplodia pseudotheobromae (KX244815.1). To better determine the species, primers βT2a/βT2b and EF1-688F/EF1-1251R were used for PCR-based molecular identification (Alves et al. 2008; Mahmodi et al. 2014). These sequences were submitted to GenBank (accession nos.: βT2, MH454259, 431 bp; and EF-1α, MH543322, 504 bp). In the nucleotide BLAST analysis, both showed 100% identities with βT2 (KP319261.1) and EF-1α (MH102237.1) of L. pseudotheobromae. Koch’s postulates were verified on 10 nonsymptomatic leaves that were from five plants (clone 7-33-97) collected in a disease-free rubber plantation in Haikou, Hainan Province. All leaves were surface sterilized and wounded using a sterile needle. Five leaves were inoculated with L. pseudotheobromae-colonized PDA plugs, and the other five were treated with PDA plugs without mycelium as a control. The inoculation site was kept moist for 1 day, and then the mycelium and agar disks were removed. After 5 days, symptoms similar to the original leaf lesions were observed on leaves of inoculated plants, whereas the control leaves showed no symptoms. Cultures reisolated from symptomatic leaves showed the same morphological characteristics and molecular traits as those initially isolated from infected leaves in the field. This suggests that L. pseudotheobromae mainly invades from the wound. This pathogen has been reported on grapes and English walnut (Juglans regia) in China (Dissanayake et al. 2015; Li et al. 2016). It was also previously reported on rubber tree in Thailand (Trakunyingcharoen et al. 2015). To our knowledge, this is the first report of leaf spot disease on rubber tree caused by L. pseudotheobromae in China.