First Report of Leaf Spot Disease Caused by Aspergillus tubingensis on Jatropha curcas in Yunnan, China

Jatropha curcas L. is widely distributed in China, especially in Yunnan, Sichuan, and Guizhou provinces (Liu et al. 2012). It is famous for its seeds used as a biodiesel raw material (Fairless 2007). During summer from 2009 to 2015, leaf spots were widely observed on J. curcas in Yunnan, China. Early symptoms usually developed along leaf veins. Spots were irregular, gradually increased in size and number, turned dark brown and dry, and finally the entire infected leaves turned yellow and fell. Five isolates from Mengzi County in Yunnan Province, all appearing similar, were successfully isolated using infected tissues and single-spore on potato dextrose agar (PDA). The colony on PDA was black at the center and white at the edge, 6 to 7 cm in diameter after cultured 5 days at 28°C. Each yellow conidiophore with single vesicle was not septate. The globose and yellow vesicle was covered by a layer of phialides 34 to 36 μm in diameter. The phialides were greenish and 22 to 34 μm long. The conidia that were 2.4 to 4.8 μm in diameter on each phialide were globose and dark brown, and their surface initially smooth gradually produced some thorns. The sizes of the vesicles and conidia were obtained after measuring 50 of them randomly. Identity of the fungus was further investigated by extracting DNA from one of our isolates, named BSZ-6(2), and sequence comparison of the internal transcribed spacer (ITS) gene (primers ITS1/ITS4), the β-tubulin gene (primers Bt2a/Bt2b), and the CaM gene (primers CMD5/CMD6). BLASTn analysis of the ITS gene (KJ190960), β-tubulin gene (KX768539), and CaM gene (KX768538) obtained with cognate sequences available in GenBank database revealed 99, 99, and 100% sequence identity, respectively, to Aspergillus tubingensis (Schober) Moss (JN585941 of ITS, HQ6342754 of β-tubulin, FR751418 of CaM). These morphological and molecular characteristics of the isolate BSZ-6(2) were consistent with those of A. tubingensis (Zhang et al. 2008; Samson et al. 2014). A 60-µl drop of each conidial suspension (1.0 × 106 conidia/ml) of five isolates from Mengzi County were sprayed on five healthy leaves of each 2-year-old seedling in the rainy season of 2012 and 2013; in parallel, 60 µl of sterile water was sprayed on five healthy leaves on another seedling as a control. Five replicates were set for each treatment. Five days postinoculation, symptoms similar to naturally infected leaves developed on all the inoculated leaves, but not on the control leaves. In addition, five pure cultures reisolated from the infected leaves were morphologically identical to the inoculated A. tubingensis (Samson et al. 2014), thus completing Koch’s postulates. Pathogenic tests with the five isolates conducted the same way in the dry season of 2012 and 2013 did not produce any symptoms on the leaves. Aspergillus tubingensis can also cause postharvest fruit rot of Prunus salicina Lindl., as reported by Wang et al. (2012). To our knowledge, this is first report of A. tubingensis causing leaf spot of J. curcas in Yunnan, China, as well as worldwide. © The American Phytopathological Society.