Identification of Biocontrol Strain HJX1 and Determination of Its Antifungal Spectrum

A biocontrol strain HJX1,which had good inhibitory effect against Fusarium oxysporum f. sp. cubense,was identified as Bacillus subtilis based on traditional morphology,16 S rRNA gene sequence and fatty acid profiles. The sequence of 16 S rRNA gene had 99% homology to B. subtilis according to the DNA fragment of 1 521 bp amplified with a pair of universal primers,P0 and P6,of bacteria. Confront culture with pathogenic fungi showed that this strain had good inhibitory effect against 10 pathogenic fungi.

Cloning and Analysis of Chitinase Gene in Bacillus subtilis HAS

[Objective] The paper was to study the possible mechanism of chitinase gene. [Method] The chitinase gene with inhibitory effect against a variety of pathogenic fungi was cloned and analyzed in this study. [Result] The genome of HAS strain contained an ORF of 834 bp. The homology of coding protein corresponding to ORF with chitosanase [Bacillus subtilis subsp. subtilis str. 168](ref|NP_390566.1) was the highest of 98%(272/277).[Conclusion]Through the analysis of amino acid sequence and secondary structure of the protease, tertiary structure prediction and analysis, HAS strain may play a pivotal role in controlling Sporisorium scitaminea Syd.

Cloning and Analysis of TasA Gene in Bacillus subtilis HAS

In order to clarify the mechanism for the inhibitory action of the Bacillus subtilis strain HAS on Sporisorium scitamineum （ Syd. ）, which can cause sugarcane smut, the full-length TasA gene which encodes a protein with broad-spectrum antimicrobial activity, was PCR-amplified from HAS, and cloned into pMD18-T vector. Sequence analysis indicated that the full-length TasA cloned from HAS consisted of 786 nucleotides, and shared 99% homology in nucleotide sequence with the TasA gene sequence published in Genbank （AJ871386.1）. It was predicted that the molecular weight of TasA protein was about 28 kD. Base transitions or transversions ~curred at positions 104, 164, 169,250, 399,623 and 627, at the 2nd, 2nd, 3rd , 1st , 3rd, 2nd and 3rd bases of TasA codons. The mutations in the seven bases may cause the missense mutations of the polypeptide chain. Compared with the amino acid sequences of the TasA protein encoded by Bacillus aubtilis subsp, subtilis str. 168, mutations in two amino acids at positions 150 and 209 of the protein encoded by the cloned TasA gene were found, and as a result, an ala- nine was replaced with a threonine.

Molecular Detection of Sugarcane Ratoon Stunting Disease in Hainan Sugarcane-growing Areas

[Objective]The paper was to clarify the occurrence of sugarcane ratoon stunting disease(RSD)in Hainan sugarcane-growing areas.[Method]In 2019,270 samples of sugarcane leaves were collected from six main sugarcane-growing areas in Hainan Province,and RSD was detected by PCR assay with specific primers.[Result]RSD was detected out in 41 out of 270 sugarcane samples,with an average detection rate of15.19%.The detection rates of RSD were different in six sugarcane-growing areas;the detection rate of RSD in Danzhou sugarcane-growing area was the highest of 22.00%;the detection rate of RSD in Lingao sugarcane-growing area was the lowest of 9.26%.RSD was detected out in 8 out of10 main sugarcane cultivars,among which Xintaitang 22 suffered the heaviest damage,with the positive detection rate of 45.83%;RSD had not been detected out in Zhongtang 1 and Zhongtang 2,while the positive detection rates of RSD in the remaining seven sugarcane cultivars were10.00%-31.25%.[Conclusion]RSD commonly occurs in Hainan sugarcane-growing areas.The research results provide a basis for scientific prevention and control of RSD and promotion and application of healthy virus-free sugarcane seedlings.

Screening and Identification of Antagonistic Strain HAS against Sporisorium scitaminea Sydow

The present study was to screen and identify a biocontrol strain with antagonistic effect against Sporisorium scitaminea Sydow. 16 S rDNA gene fragments were amplified by universal primers P0 and P6. The results showed that 16 S rDNA gene fragment with the size of 1 533 bp was obtained,and its sequence had the homology of 99% with the sequence of Bacillus subtilis. The morphological characteristics of the strain were as follows： the mycelium of strain HAS was short-rod shape,and could move; Gram staining was positive; 3% KOH solubility test was negative; flagellum was grown around the cells; spores were grown in the middle,elliptic,and cysts were slightly swelled. The strain was preliminarily identified as B. subtilis. Dual culture with pathogenic fungi showed that the strain had good inhibitory effect against various pathogenic fungi causing diseases on sugarcane and other crops. Therefore,strain HAS isolated in the study was identified to be B.subtilis,which had good control effect against S. scitaminea.

Expression of the Grifola frondosa Trehalose Synthase Gene and Improvement of Drought-Tolerance in Sugarcane （Saccharum officinarum L.）

Trehalose Is a nonreduclng dlsaccharlde of glucose that functions as a protectant In the stabilization of blologlcal structures and enhances stress tolerance to abiotic stresses in organisms. We report here the expression of a Grlfola frondosa trehalose synthase （TSase） gene for Improving drought tolerance In sugarcane （Saccharum offlclnarum L.）. The expression of the transgene was under the control of two tandem copies of the CaMV35S promoter and transferred Into sugarcane by Agrobacterium tumefaciens EHA105. The transgenlc plants accumulated high levels of trehalose, up to 8.805-12.863 mg/g fresh weight, whereas It was present at undetectable level in nontransgenlc plants. It has been reported that transgenlc plants transformed with Escherlchla coil TPS （trehalose-6-phosphatesynthase） and/or TPP （trehalose-6-phosphate phosphatase） are severely stunted and have root morphologlc alterations. Interestingly, our transgenlc sugarcane plants had no obvious morphological changes and no growth Inhibition in the field. Trehalose accumulation in 35S-35S：TSase plants resulted In In- creased drought tolerance, as shown by the drought and the drought physiological Indexes, such as the rate of bound water/free water, plasma membrane permeability, malondlaldehyde content, chlorophyll a and b contents, and activity of SOD and POD of the excised leaves. These results suggest that transgenlc plants transformed with the TSase gene can accumulate high levels of trehalose and have enhanced tolerance to drought.