Identification of smut-responsive genes in sugarcane using cDNA-SRAP.

Sugarcane smut, caused by the fungus Sporisorium scitamineum, is one of the main diseases that affect sugarcane worldwide. In the present study, the cDNA-SRAP technique was used to identify genes that are likely to be involved in the response of sugarcane to S. scitamineum infection. In total, 21 bands with significant differential expression during cDNA-SRAP analysis were cloned and sequenced. Real-time qPCR confirmation demonstrated that expression of 19 of these 21 differential bands was consistent with the expression observed during cDNA-SRAP analysis, with a deduced false positive rate of 9.5%. Sequence alignment indicated that 18 of 19 differentially expressed genes showed homologies from 19% to 100% to certain genes in GenBank, including the following genes: topoisomerase (EU048780), ethylene insensitive (EU048778), and tetraspanin (EU048770). A real-time qPCR assay showed that during 0-72 h after pathogen infection, expression of the topoisomerase and the ethylene insensitive genes was upregulated, whereas expression of the tetraspanin gene was downregulated, identical to the expression patterns observed under salicylic acid treatment. Therefore, all three genes are thought to play a role during S. scitamineum challenge, but with different functions. To our knowledge, this is the first report on the application of cDNA-SRAP in differential gene expression analysis of sugarcane during a sugarcane-S. scitamineum interaction. The results obtained also contribute to a better understanding of the molecular mechanisms associated with sugarcane-S. scitamineum interactions.

Molecular variation of Sporisorium scitamineum in Mainland China revealed by internal transcribed spacers.

Sugarcane smut caused by the fungus Sporisorium scitamineum is a worldwide disease and also one of the most prevalent diseases in sugarcane production in mainland China. To study molecular variation in S. scitamineum, 23 S. scitamineum isolates from the 6 primary sugar-cane production areas in mainland, China (Guangxi, Yunnan, Guangdong, Hainan, Fujian, and Jiangxi Provinces), were assessed using internal transcribed spacer (ITS) methods. The results of ITS sequence analysis showed that the organisms can be defined at the genus level, including Ustilago and Sporisorium, and can also differentiate between closely related species. This method was not suitable for phylogenetic relationship analysis of different S. scitamineum isolates and could not provide support regarding their race ascription at the molecular level. The results of the present study will be useful for studies examining the molecular diversity of S. scitamineum and for establishing a genetic foundation for their pathogenicity differentiation and new race detection. In addition, our results can provide useful information for the pathogen selection principle in sugarcane smut resistance breeding and variety distribution.

First Report of Phoma sp. Causing Twisting and Curling of Crown Leaves of Sugarcane in Mainland of China.

Sugarcane is a major sugar and the leading energy crop worldwide and Guangxi is the largest sugarcane production area in China (2011 Sugar Annual China, www.gain.fas.usda.gov). During survey of sugarcane crops in September 2012 and June 2013, ~5 to 10% of sugarcane (cvs. FN-40 and ROC22) planted in Chongzuo and Laibing, Guangxi Zhuang Autonomous Region, P. R. China, had twisted and curling symptoms of crown leaves similar to sugarcane Pokkah boeng disease (caused by Fusarium moniliforme Sheldon). The symptoms started appearing as yellowing on midribs and leaf margins that spread further to the entire leaf, along with twisting and curling of crown leaves. The symptomatic leaf tissues (5 × 5 mm) were surface-sterilized by 0.1% HgCl2 solution for 30 s, followed by rinsing three times in sterile water, placed on potato dextrose agar (PDA), and then incubated in darkness at 28°C. After 3 days of incubation, the isolated fungal colony appeared as white villous, spherical, radial, and dense colorless mycelium from the top, while it was reddish-brown at the bottom and later became grayish. Chlamydospores were also observed with a diameter of 5 to 10 µm and were dark brown, unicellular, intercalary, and smooth. The binucleate hyphae were colorless and transparent. Pycnidia appeared on the colonies after 20 days, and were dark brown, subglobose, and 150 to 230 µm in diameter, and the conidia were ~3 to 7 × 2.5 to 6 µm, unicellular, colorless, and ovoid to oval. The fungal isolates from the symptomatic leaves were obtained and pathogenicity was evaluated. Conidial suspensions (107 CFU/ml) of the single isolate from FN-40 were micro-injected into 20 sugarcane seedlings of cultivar FN-40. Another 20 seedlings were injected with water without conidia as control. The inoculated plants were grown in a growth chamber at 28°C with a 16-h photoperiod. Twisted and curly symptoms similar to the field appeared on the inoculated leaves at 10 days after inoculation, while the control leaves remained asymptomatic. The fungus was re-isolated and identified. Genomic DNA from the cultured fungal isolate was extracted with a modified Fungal DNA Midi Kit (Omega Bio-Tek, Inc., Norcross, GA), and amplified using fungus-conserved primer sequences (ITS1: 5'-TCCGTAGGTGAACCTGCGG-3' and ITS4: 5'-TCCTCCGCTTATTGATATGC-3'). The consensus rDNA-internal transcribed spacer sequence (GenBank Accession No. KC524502) was 100% identical with 97% coverage to the ITS sequence from Phoma sp. 3. TMS-2011 (HQ631000.1) in GenBank (3). The fungus Phoma sp. was identified on the basis of morphological characteristics (2,4) and the ITS sequence of rDNA (1,3). Disease caused by Phoma sp. has been reported earlier on sugarcane from Pakistan, Hawaii, and Taiwan, causing leaf blight and curling (2,4). However, to the best of our knowledge, this is the first report of Phoma sp. causing twisting and curling of crown leaves of sugarcane in mainland of China. References: (1) M. M. Aveskamp et al. Stud. Mycol. 65:1, 2010. (2) A. Sanguino and H. Tokeski. ISSCT Proc. 17:1555, 1980. (3) P. Shrestha et al. Appl. Environ. Microbiol. 77:5490, 2011. (4) Z. N. Wang. Rep. Taiwan Sugar Res. Inst. 129:1, 1980.