农业微生物制剂的活性菌分离与鉴定

微生物制剂是以自然界中分离的有益菌为主体开发的产品,在健康、环保及农业生产上应用广泛。某农业微生物制剂在海南的热带农业生产中能显著增产和增强作物抗性,为了了解该微生物制剂的作用机制,本研究采用平板划线法对活性菌进行分离和纯化,对分离菌株进行生长、形态及显微观察,对16S rDNA进行序列PCR扩增、测序及生物信息学分析,鉴定活性菌的种属。结果表明,分离得到2个形态不同的细菌菌株,通过形态观察及16S rDNA序列的分子鉴定,发现这2个菌株与枯草芽孢杆菌(Bacillus subtilis)具有99.9%的同源性;2个菌株的16S rDNA序列的比较结果发现它们有7个碱基的差别,说明这2个菌株可能是不同的生理小种。

具有抑菌活性的海洋细菌的分离与鉴定

分离并筛选具有抑菌活性的海洋细菌对于开发和利用海洋微生物具有重要意义。该研究从 6份海泥样品中共分离到78株海洋细菌 ,以 6种细菌作为敏感指示菌 ,采用覆盖技术对分离菌株进行拮抗试验 ,17株海洋细菌具有抑菌活性。对其中 2株具有较强抑菌活性的海洋细菌进行革兰氏染色、耐盐试验、运动性观察、过氧化氢酶测定、明胶液化试验、硫化氢产生试验、石蕊牛奶试验、糖类发酵、硝酸盐还原等特性分析 ,依据《伯杰氏细菌鉴定手册》进行分类鉴定 ,它们分别应归属为气单孢菌属(Aeromonassp.)和假单孢菌属 (Pseudomonassp.)。

Relationship between Biological Characteristics of Beauveria bassiana (Bals.) Vuill and Pathogenicity to Bombyx mori L.

[Objective] This study was to investigate the relationship between biological characteristics of Beauveria bassiana （Bals.） Vuill and pathogenicity to Bombyx rnori L, with the aim to provide scientific basis for the control of white muscardine in Bombyx mori L. [Method] The strains isolated and purified from the 6 Beauveria bassiana biocontrol agents from all over the country and the 3 white muscardine silkworms collected from Guangxi provincial silkworm rearing areas were identified by the morphological observation and molecular biology technology. The pathogenicity of B. bassaina to silkworms was determined, and the biological characteristics such as growth diameter, sporulation and the extracellular protease activity of the different B. bassiana strains were compared. [Result] The isolated 9 strains were all B. bassaina （Bals.） Vuillemin, and all strains had high pathogenicity to silkworm, but with different pathogenicities. The growth diameter, sporulation and extracellular protease activity of different B. bassiana strains were also different, and showed correlation with the patheogenicity to silkworms. [Conclusion] B. bassiana spores production amount and exocellular protease activity had significant positive correlation with their pathogenicity to silkworm.

Isolation and characterization of bioactive fungi from shark Carcharodon carcharias' gill with biopharmaceutical prospects

Until recently, little was known about the fungi found in shark gills and their biomedicinal potential. In this article, we described the isolation, bioactivity, diversity, and secondary metabolites of bioactive fungi from the gill of a shark （Carcharodon carcharias）. A total of 115 isolates were obtained and grown in 12 culture media. Fifty-eight of these isolates demonstrated significant activity in four antimicrobial, pesticidal, and cytotoxic bioassay models. Four randomly selected bioactive isolates inhibited human cancer cell proliferation during re-screening. These active isolates were segregated into 6 genera using the internal transcribed spacer-large subunit （ITS-LSU） rDNA-sequence BLAST comparison. Four genera, Penicillium, Aspergillus, Mucor, and Chaetomium were the dominant taxa. A phylogenic tree illustrated their intergenera and intragenera genetic diversity. HPLC-DAD-HRMS analysis and subsequent database searching revealed that nine representative strains produced diverse bioactive compound profiles. These results detail the broad range of bioactive fimgi found in a shark＇s gills, revealing their biopharmaceutical potential. To the best of our knowledge, this is the first study characterizing shark gill fungi and their bioactivity.

Bioflocculant Produced by Klebsiella sp. MYC and Its Appli-cation in the Treatment of Oil-field Produced Water

Seventy-nine strains of bioflocculant-producing bacteria were isolated from 3 activated sludge samples. Among them, strain MYC was found to have the highest and stable flocculating rate for both kaolin clay suspension and oil-field produced water. The bacterial strain was identified as Klebsiella sp. MYC according to its morphological and biochemical characteristics and 16SrDNA sequence. The optimal medium for bioflocculant production by this bacterial strain was composed of cane sugar 20 g L^-1, KH2PO4 2 g L^-1, K2HPO4 5 g L^- 1, （ NH4）2SO4 0.2 g L^-1, urea 0.5 g L^- 1 and yeast extract 0.5 g L^- 1, the initial pH being 5.5. When the suspension of kaolin clay was treated with0.5% of Klebsiella sp. MYC culture broth, the flocculating rate reached more than 90.0% in the presence of 500mg L^-1 CaCI2, while the flocculating rate for oil-field produced water was near 80.0% in a pH range of 7.0 - 9.0 with the separation of oil and suspended particles from the oil-field produced water under similar conditions. The environment-friendly nature of the bioflocculant and high flocculating rate of the strain make the bioflocculant produced by Klebsiella sp. MYC an attractive bioflocculant in oil-field produced water treatment.

Isolation and Identification of Cardenolide Compounds of Gomphocarpus sinaicus and Their Fungicidal Activity Against Soil Borne and Post Harvest Fungi

This study was undertaken to explore new antifungal compounds from the methanolic extract of G. sinaicus. Two cardenolide compounds were isolated and identified by GC-MS as cardenolide glycoside, 15-hydroxy-3,4,5,6-dehydrocalotropin and cardenolide genin, 3,4,5,6-dehydrocalotropagenin. The antifungal activity of these compounds was assessed. Results revealed that both compounds showed pronounced fungicidal activity against both soil borne fungi, R. solani, F. oxysporium, and postharvest fungi, R. stolonifer, P. digtatum, compared to the standard fungicides, flutolanil and copper oxychloride, respectively. The ECs0 values of the cardenolide genin were 0.703, 13.63 and 4.22, 8.403 lag/mL forR. solani, F. oxysporium andR. stolonifer, P. digtatum respectively. On the other hand, the ECs0 values of the standard fungicide, flutolanil, were 9.49 and 61.22 ~tg/mL against R. solani and F. oxysporium. While the ECso values of copper oxychloride were 279.94 and 187.13 p.g/mL against R. stolonifer and P. digtatum, respectively. The results showed that cellulase, PME, PPO of the tested fungi was more sensitive than to cardenolide genin. The strong antifungal activity of cardenolide genin reported in this study indicated that has a potential to be used as fungicides.

Bacillus Licheniformis Dhs-40, a Flocculating Active Strain and Its Flocculating Activity

Microbial flocculants have become a hot spot in recent years. A bacterial strain with high flocculating activity was isolated from seawater samples. The strain was defined as Bacillus licheniformis dhs-40 by 16S rDNA identification and Biolog test. Ultrasonication test confirmed the flocculating activity of the strain was both in fermentation supernatant and cell. According to flocculating activity curve, the ideal fermentation time for collecting flocculating active substances was two days. The flocculating activity of the strain was sensitive to pH. The strain could only preserve flocculating activity while pH varied from 7 to 11. However, it could preserve flocculating activity while temperature varied from -20℃to 100 ℃ Saccharide, protein, lipid, nucleic acids qualitative test and RNase, Proteinase K treatment confirmed the flocculating active substances were proteins. Their flocculating activities were insensitive to Proteinase K.

Endophytic Fungi Isolated from Mangrove （Rhyzopora mucronata） and Its Antibacterial Activity on Staphylococcus aureus and Escherichia coli

Disc diffusion method was used to investigate the antibacterial activity of methanol extract of five endophytic fungi isolated from mangrove （Rhizopora mucronata） on Staphylococcus aureus ATCC 9144 and Escherichia coli ATCC 8739, Fungi were isolated from leaf, stem and root of R. mucronata. Potato dextrose agar and potato dextrose broth were used for fungi purification and fermentation. Fungal metabolites were extracted with methanol （1 ： 1 v/v） for 24 h before vacuum evaporated to yield the crude extracts. Antimicrobial activity test was done according to the Kirby-Bauer test. The inhibition zone around each disc was considered to measure antibacterial activity of endophytic fungi. Only three endophytic fungi showed inhibition activity on S. aureus ATCC 9144 and two endophytic fungi showed inhibition on E. coil ATCC 8739. The results suggested that the extracts have a high potential for enteric diseases treatment caused by foodbome pathogen.

The Biological Activity of Some Bacteria as Biocides to Protect Sesame Crop （Sesamum indicum） from Fungi Diseases

Two experiments were conducted during 2006-2007 in Complete Randomized Design （CRD） to test the biological activity of Pseudomonas bacteria as biocides to protect sesame crop from some fungi and to evaluate its efficiency as plant growth promoting First experiment investigated the effects of Pseudomonas putida2 and Pseudomonas fluorescens3 on germination and seedlings growth of sesame crop against Pythium, Alternaria and Fusarium under plastic house conditions. Second experiment conducted in large pots to investigate the effects of the two bacterial isolates on some morphological, productive and physiological characters of sesame plants against same fungi under normal conditions. Results showed very low germination and slowly seedlings growth in Pythium, Fusarium and Alternaria treatments respectively, but adding bacterial vaccine of Pseudomonas putida2 and Pseudomonas fluorescens3 as a Biocide to fungi treatments, increased the germination percent and seedlings growth of sesame crop significantly, 20 days after planting compared with control treatment. In the second experiment, control treatment scored germination by 52%, while vaccines of Pseudornonas putida2 and Pseudomonas fluorescence3 treatments increased germination percentage in Fusarium, Pythium and Alternaria treatments significantly in average more than 71%. Both isolates increased significantly Leaf number per plant, leaf area per plant, height of plant, branches number per plant, total dry weight of shoot per plant and chlorophyll content compared with other and control treatments. Similar significant effect for both isolates was recorded in seeds number per pod per plant, total weight of 1,000 seeds per plant and pods number per plant and percentage of N, P, K in total dry weight of shoot per plant and oil percentage in seeds per plant compared with other and control treatments. P. putida2 increased oil percentage in seeds by 43.3, 48.0 and 45.0% respectively while with P. fluorescens3 increased to 42.7, 44.0 and 43.7% respectively compared with control treatment （27.7%）. In general Pseudomonas putida2 increased most of growth characters much higher than Pseudomonas fluorescens3, it may related to siderophores compound and genetic factors.

Isolation and Antifungal Evaluation of Rumex cyprius Murb Extracts

Ethanolic extract of dried Rumex cyprius Murb （polygonaceae） was tested against four pathogenic fungi： three dermatophytes, Microsporum canis, Trichophyton mentagrophytes and T. rubrum, and the causative agent of chalkbrood disease of bees, Ascophaera apis. The extract showed considerable activity against all these fungi. One of the main constituents ofRumex cyprius was isolated and tested for its antimycotic activity. It showed a significant activity against the test fungi and was identified as 1,3,8-trihydroxy-6-methylanthracene-9,10-dione based on its IR, UV-V and 1^H NMR spectra.