Isolation,Screening and Identification of Antagonistic Actinomycetes against Ustilago scitaminea Syd

[Objectives] This study was conducted to obtain actinomycetes strains having antagonistic effect on Ustilago scitaminea Syd.[Methods] At first, actinomycetes strains were isolated from 22 soil samples in Hainan sugarcane regions. Then, antagonistic actinomycetes against U. scitaminea were screened by confrontation culture. Finally, the taxonomic status of antagonistic actinomycetes was determined using 16S rDNA.[Results] From the 22 samples, 984 actinomycetes strains were isolated. From all the isolated strains, 23 antagonistic actinomycetes strains were obtained through primary screening, and one strains with better antagonistic effect was then obtained through secondary screening, and designated FAS. 16S rDNA identification showed that strain FAS shared 99% sequence similarity with Streptomyces cealestis US24. A phylogenetic tree was built with MAGE 7.0 software, and the results showed that strain FAS had the shortest genetic distance with S. caelestis US24. Therefore, the actinomycetes FAS was determined as S. caelestis .[Conclusions] This study provides a new biocontrol method for the biological control of sugarcane smut, thereby ensuring sustainable development of sugarcane industry and sugar industry.

Dynamic changes of inducible nitric oxide synthase expression in rat's retina and its role on blood-retinal barrier injury after acute high intraocular pressure

AIM: To clarify the role of inducible nitric oxide synthase(i NOS) in blood-retinal barrier(BRB) injury after acute high intraocular pressure(IOP) in rats.METHODS: Forty-two Sprague-Dawley(SD) rats were randomized into 7 groups [control(Cont), 3, 6, 12, 24, 48, and 72 h, n=6]. Except Cont group, other groups’ retina tissue was obtained at corresponding time points after a model of acute high IOP have been established in rats. The expression of i NOS and tight junction protein zonula occludens(ZO)-1 was detected by Western blotting. Evans blue(EB;3%) was injected into the great saphenous vein to detect the leakage of EB by spectrophotometer. Nine rats were divided into Cont, 6 h, 12 h groups, the expression of i NOS was localized by immunofluorescence. In order to verify the role of i NOS in the damage to BRB, thirty-six rats were randomly divided into 4 groups [Cont, Cont+inhibitor(Inh), 6 h and 6 h+Inh, n=9]. After treatment with the i NOSspecific inhibitor 1400 W, the expression of i NOS and ZO-1 and the leakage of BRB were detected again.RESULTS: The immunofluorescence results showed that the expression of i NOS was observed in the Cont group and 6 h group, but not in the 12 h group. i NOS was mainly expressed in the retinal nerve fiber layer, ganglion cell layer and inner nuclear layer and that it did not colocalize with the retinal ganglion cell marker Neu N but was co-expressed with the vascular endothelial cell marker CD31. Western blotting showed that in the early period(3 h, 6 h) after acute high IOP, the expression of i NOS was upregulated, then the down-regulation of i NOS were tested in the follow-up timing spots. ZO-1 expression showed a continuous downregulation after 6 h. The quantitative results for EB showed that the amount of EB leakage began to increase at 3 h after acute high IOP. At 6 h, the leakage of EB was lower, but at 12 h, the leakage of EB was highest, after which it gradually recovered but remained higher than that in the Cont group. The expression of i NOS was down-regulated after 1400 W treatment. ZO-1 expression was not significantly changed in the Cont+Inh group and the 6 h group, and significantly down-regulated in the 6 h+Inh group, and the leakage of EB was significantly increased after 1400 W treatment.CONCLUSION: These results suggest that the upregulation of i NOS expression in the early stage after acute high IOP may have a protective effect on BRB injury.

Optimization of Streptomyces caelestis Culture Conditions and Preliminarily Determination of Its Antifungal Components

Streptomyces caelestis FAS is an actinomyces strain isolated with Ustilago scitaminea as an indicator. With GAUZE s medium as the basic medium, carbon source and nitrogen source in the medium and ventilation volume were optimized, and the antifungal substances produced by S. caelestis FAS were preliminarily determined through organic solvent extraction and ammonium sulfate precipitation. The results showed that the antifungal components produced by S. caelestis FAS were proteins, and the optimal culture conditions were using corn flour as carbon source and KNO 3 as nitrogen source and culturing in 250 ml triangular flask added with 70 ml of culture medium. The crude protein obtained under these culture conditions had the best antifungal effect with an inhibition zone diameter of 25 mm. This study will provide a scientific basis for further study on the antifungal mechanism and application of S. caelestis FAS.

Identification of In-intro Antagonistic Activity of HAS1 Protein against Sporisorium scitanminea

Sugarcane smut greatly influences yield and quality of sugarcane.HAS1 gene in biocontrol strain HAS antagonistic to Sporisorium scitanminea was selected for prokaryotic expression vector construction and inducible expression,and the antagonistic effect of HAS1 protein was detected in vitro.The results showed that the expression product of antifungal HAS1 gene obtained through prokaryotic expression vector construction and inducible expression had better antagonistic effect against S.scitanminea.This study provides an experimental basis for further utilization of this protein.

Study on Acute Toxicity of Purified Protein HAS1 Expression in KM Mice

[Objectives]The antifungal protein HAS1 newly obtained was evaluated for acute toxicity safety in KM mice according to relevant national regulations,so as to eliminate people s concerns about the safety of transgenic plants.[Methods]The acute toxicity of the purified protein HAS1 was observed by intragastric administration of mice,and the poisoning symptoms,poisoning degree,recovery and death were observed.[Results]No abnormal toxicity symptoms were observed in the test group,the vehicle control group and the blank control group.The main tissues and organs were not abnormal in gross anatomy.The average body weight of each group showed an increasing trend compared with before administration at 1,3,7,11 and 14 d after administration.It was found that after giving the purified protein HAS1 to KM mice at a cumulative dose of 64 mg/kg a day,no obvious toxicity was observed in the acute toxicity test,indicating that the test substance was non-toxic by oral administration.[Conclusions]This study provides a basis for further use of the protein and its coding genes.

Colonization Ability of Bacillus subtilis HAS in Sugarcane

Bacillus subtilis HAS had good control effects on sugarcane smut in the field. Colonization dynamics of B. subtilis HAS were studied through biotics marker and pot culture, to understand the bio-control mechanism of the strain and provide experimental basis for commercialization. The results showed that B.subtilis HAS had strong colonization ability, which could be colonized in sugarcane root, stem, leaf and rhizosphere for long time, but the colonization levels were different. The colonization density of B.sutili HAS remained 103 CFU/g soil in the rhizosphere and 102 CFU/g （fresh weight） in the root after inoculation of 60 d. The colonization in the rhizosphere decreased first, then increased and decreased again, and finally stabilized. The colonization in the root first increased then decreased. Meantime, colonization status in stems and leaves showed that the colonization density in stems and leaves was 10 CFU/g （fresh weight）, indicating that the colonization level was not high in stem and leaf.

Skin Anaphylaxis Test of Purified Protein HAS1 in Cavies

The antifungal protein HAS1 is a new antifungal protein isolated from the genome of Bacillus subtilis HAS,which has a good inhibitory effect on various pathogenic fungi in sugarcane.This study aimed at evaluating the immunological transfer reaction of the test sample through repeated skin contact by observing whether the cavy skin repeatedly exposed to purified protein HAS1 suffers from allergic reaction and how strong is the allergic reaction.The results showed that the test group and the vehicle control group exhibited no allergic reaction in the skin immediately and at 24,48 and 72 h and had an mean reaction score and a sensitization rate both of 0,so the results of the naked eye observation were both nonallergenic to cavy skin;and in the positive control group,the cavies were observed to be highly sensitized immediately after removing the drug,mildly sensitized at 24 h,and not sensitized at 48 and 72 h,which meant the skin allergy was alleviated with the observation time.It is suggested that the purified protein HAS1 is negative for cavy skin anaphylaxis test (nonallergenic to cavy skin),which provides an experimental basis for further utilization of the protein and its coding gene.

Study on the anti-gout effects of four herbs extracts

Objective:To explore the anti-gout effects of four traditional Chinese medicine extracts of Eucommia,Sargentodoxa cuneata,Caulis spatholobi and Phellinus igniarius.Methods:Monosodium Urate(MSU)was used to establish a mouse gouty arritis model.With coloricine as the positive control group,the effects of four traditional Chinese medicine extracts of Ecuador,Sargentodoxa,Caulis spatholobi and Phellinus igniarius on rice were studied;The hot plate method was used to observe the analytical effects of the four groups of traditional Chinese medicine extracts;the enzyme-linked immunoassay(ELISA)was used to detect residual interference-1 beta(IL-1 beta);the four traditional Chinese medicine extracts were simultaneously determined on the white blood cells,and monocytes in the blood of mice as well as the influence of neutrophils and histopathological changes of toes.Results:Compared with the normal group,the joint diameter ratio,blood leukocytes,monocytes,neutrophils,IL-1βat different time points in the model group were significantly increased(P<0.01),and the pain percentage was significantly reduced(P<0.05).Compared with the model group,the joint diameter ratio of the colchicine group,the Eucommia group,the Sargentodoxa group,the Caulis spatholobi group and the Phellinus sibiricum group was significantly reduced(P<0.05),and the pain percentage was significantly increased(P(0.05).The four groups of traditional Chinese medicine extracts significantly decreased the white blood cells,monocytes,and neutrophils in the blood of mice(P<0.05),and the IL-1βexpression levels of the Eucommia ulmoides group,)the Sargentodoxa group and the Phellinus igniarius group significantly decreased(P<0.05).Eucommia ulmoides and Caulis spatholobi are more effective in reducing pathological damage of toes in mice.Conclusion:The water extract of Caulis spatholobi has obvious anti-gout effect,and the most significant effect on analgesia and swelling.Eucommia ulmoides and Sargentodoxa cuneata have more obvious anti-inflammatory effects and have certain analgesic and swelling effects.Phellinus Phellinus igniarius has obvious inhibitory effect on IL-1β,but it does not show good anti-gout potential.

Study on Wide Antifungal Property of Bacillus subtilis HAS

Bacillus subtilis HAS is a biocontrol strain with better inhibitory effect on Sporisorium scitaminea Sydow. In order to further understand its action range,with fungal pathogens in sugarcane production as targets,confrontation tests and tests of inhibitory effect of secondary metabolites on growth of pathogens were carried out. The results showed that B. subtilis HAS not only had better inhibitory effect on S. Scitaminea,but also inhibited the growth of various fungal pathogens in sugarcane production. The inhibitory effect was the best on Ceratocystis adiposum,Thielaviopsis paradoxa,Fusarium moniliforme and Colletotrichum fuleatum,which was reflected by the radius of inhibitory zone over 10 mm. The inhibitory rates of the secondary metabolites were about 70%. It indicates that B. subtilis HAS has wide antifungal property,providing reference for further application of this strain.