Screening and Identification of Cotton Verticillium Wilt Antagonistic Bacteria Strain 7-30

[Objective]The aim of this study was to screen the antagonistic spore-forming bacteria of Verticillium dahliae and identify its physiological and biochemical characteristics.[Method]Taking the cotton verticillium wilt pathogen Verticillium dahliae V-190 as the test microorganism,the antagonistic spore-forming bacteria were screened.Through the preliminary screening and secondary screening,an antagonistic bacteria strain 7-30 with stronger antibacterial activity was obtained,and its morphological characteristics,physiological and biochemical characteristics were also identified.[Result]84 antagonistic bacteria strains were isolated from soil in various places by the preliminary screening.Especially,18 strains with better antagonistic ability were screened again,so an antagonistic bacteria strain 7-30 with the diameter of inhibition zone 18.9 mm and stronger antibacterial activity was obtained.According to its morphological characteristics,physiological and biochemical characteristics,the antagonistic bacteria strain 7-30 was identified as Bacillus subtilis primarily.[Conclusion]The strain 7-30 was obtained as the antagonistic spore-forming bacteria of Verticillium dahliae.

Screening of Soil Antagonistic Bacteria for Watermelon Fusarium Wilt

[Objective] The aim of this study was to screen bacteria strains with stable antagonistic effect against watermelon fusarium wilt from soil and investigate the biological control of watermelon fusarium wilt by applying the antagonistic bacteria strains into soil.[Method] Actinomycete strains,fluorescent bacteria strains and bacillus strains were isolated from soil samples by the dilution-plate method,then its resistance was screened respectively by the improved confront culture method after colonies were purified.Finally,bacteria strains with better antagonistic effect were identified.[Result] 29 bacteria strains with stable antagonistic effect against watermelon fusarium wilt were screened from 39 soil samples,which contained 15 fluorescent bacteria strains,5 bacillus strains and 9 actinomycete strains.Furthermore,three antagonistic bacteria strains of FM2,FM3 and FM4 with the strongest antagonism were identified primarily.[Conclusion] According to cultural characteristics,morphological observation,biochemical and physiological tests,FM2 belongs to bacillus subtilis,while FM3 and FM4 belong to micrococcus.

Screening and Identification of Antagonistic Bacteria against Soil-borne Diseases of Tomato

[Objective] This study was conducted to screen resources for biological control of soil-borne diseases of tomato. [Method] Antagonistic bacteria against Ral- stonia solanacearum and Fusarium oxysporum were isolated and purified from soil samples by dilution plating and confronting incubation on PDA plates, then the antibacterial spectrum and metabolic secretions of the bacterial isolates were measured, and their species were identified by establishing phylogenetic trees with Neighbor-Joining method. [Result] From the 60 soil samples, 10 of 59 antagonistic bacteria isolates against R. solanacearum showed inhibition zone diameter 〉 25 mm, and 4 of 30 strains against F. oxysporum exhibited inhibition rate 〉 30%. The bacteriostatic substances and antibacterial spectrum analysis on above 14 isolates revealed that four strains N23-2, N58-2, NF59-3 and NF61-1 had good antago- nism against pathogenic bacteria and fungi; 12 strains produced cellulose, 11 strains produced proteases, and 6 strains produced siderophores. The molecular identification experiments indicated that four strains were members of Paenibacillus, three strains of Streptomyces, three strains of Pseudomonas strains, and four strains of Bacillus strains.

Screening of Antagonistic Bacteria against Phytophthora infestans and Its Inhibition Effect

[ Objective ] The paper was to screen bacterial strain with significant antagonistic effect against Phytophthora infestans, so as to provide basis for further development and utilization of antagonistic bacteria to inhibit P. infestans and control potato late bright. [ Method] Plate dual culture and filter paper method were used to determine the inhibition effect of strains in vivo, fermentation broth and bacterial liquid of 61 strains against P. infestans and the resistance-induction effect of SR13-2 strain. [ Result] The inhibition rate of 24 strains among 61 tested strains against mycelial growth of P. infestans was greater than 60%, and the inhibi- tion effect of HT-6 strain was the strongest with the inhibition rate of 89.92%. However, fermentation broth of all tested strains had no significant inhibition effect against P. infestans, while the inhibition effect of bacterial liquid of most strains was significantly higher than strain in vivo; the inhibition effect of $34-1 strain was the strongest with inhibition rate of 91.50%. The bacterial liquid of SR13-2 strain was found to have significant resistance-induction effect with protective rate of 60%. [ Conclusion] The inhibition effect of strains in vivo and fermentation broth of antagonistic strains S34-1 and SR13-2 had no relationship with each other, while bacterial liquid had great application potential in controlling potato late bright.

Screening of Antagonistic Actinomyces against Soft Rot of Amorphophallus konjac and Its Antibacterial Activity

[Objective] Antagonistic actinomyces of Amorphophallus konjac soft rot were isolated from the soil,the antibacterial activity of the fermentation was detected,and its active components were separated.[Method] The antagonistic actinomycetes were screened using plate confrontation culture method.The SJK18 antagonistic strain with strong activity was conducted single-factor tests using different carbon sources,nitrogen sources,temperatures and pH value as the culture condition,and the antibacterial activities of fermentation liquid was determined,respectively.The fermentation liquid of antagonistic strain was extracted with alcohol and acetic ester,and their antibacterial activities were determined.[Result] A total of 112 strains of actinomycetes were obtained,and 11 strains had antibacterial activity.The antibacterial activity of SJK18 strain was the strongest in the single-factor test,when the corn powder was adopted as the carbon sources,KNO3 was the nitrogen source,the temperature was 30 ℃ and the pH was 6.0,the appropriate pH value for the activity was 5.5-10.0.The diameters of inhibition zone of alcohol extract and raffinate of ethyl acetate were 17.0 and 20.0 mm,respectively,while the extraction of ethyl acetate had no antibacterial activity.[Conclusion] A strain of actinomyces with strong antibacterial activity against soft rot of A.konjac was obtained.

Screening and identification of an antagonistic bacterium strain LHB02 and the optimization of its fermentation conditions

A strain LHB02 with strong antibacterial activity against some aquatic pathogens was screened from the coastal marine sediment in Fujian province, China. LHB02 was identified as Bacillus subtilis, based on its 16S rRNA sequence, together with the morphological, physiological and biochemical characteristics. The antagonistic activity of strain LHB02 and its optimal fermentation conditions were also investigated. The results showed that LHB02 had strong antagonistic activity against 3 species of vibrios： Vibrio harveyi,Vibrio alginolyticus and Vibrio anguillarum, and the optimum fermentation conditions for the strain LHB02 were as follows： KB culture medium （peptone 20 g, glycerol 10 mL, K2HPO4 1.5 g, MgSO 4 .7H2O 1.5 g, H2O 1 000 mL）; temperature, 28 ℃; pH ,7.0; culture time, 36 h; and inoculation amount, 1.5% （v/v）.

Screening and Identification of Antagonistic Strain against Phytophthora sojae

[ Objective] The paper was to screen the antagonistic strain against Phytophthora sojae with biocontrol potential, and provide basis for searching control measures and designing new control strategies against P. sojae. [ Method] The rhizosphere soil of soybean was collected from three different places in Heilongjiang Province, and various soil microorganisms were isolated. Dual culture method was used to screen the microorganism with antagonistic effect against P. sojae. On this basis, the growth inhibition rate of the microorganism with stronger antagonistic effect against P. sojae was determined, and its control effect against P. sojae was also measured. [ Result] A strain of bacterium with relatively good antagonistic effect was isolated from soil, and named as strain B048. Dual test showed that the growth inhibition rate of antagonistic bacterium 11048 against P. sojac reached 97.5%. Antagonistic endurance tests showed that the width of inhibition zone was still 20.0 mm after dual culture with P. sojac for21 d. In potting experiment, the control effect of B048 against P. sojae was 100%. The antagonistic bacterium was primarily identified to be Bacillus pumilus through morphology and 16S rDNA sequence analysis. [Condusion] The antagonistic bacterium B048 had good prospect to be developed as the biocontrol bacterium against P. sojae.

Determination of Growth-promoting and Antagonistic Action of Endophytic Bacteria Strains Itb57 and Itb295 of Tobacco

[ Objective ] The paper was to study the growth-promoting and antagonistic action of endophytic bacteria strains Itb57 and Itb295 of tobacco to explore their functions in biological control. [ Method] The growth-promoting effects of bacterial suspension ~ff endophytic bacteria Itb57 and Itb295 on tobacco seedling un- der different treatment modes were studied using potting method in greenhouse. The antagonistic action of bacterial suspension of endophytic bacteria Itb57 and Itb295 on Phytophthora nicotianae, Alternaria alternata and Botrytis cinerea were measured by duel culture method. [ Result] Bacterial suspensions of enduphytic bacteria Itb57 and Itb295 had certain growth-promoting effects on tobacco seedling, which could significantly increase the fresh weight and dry weight in aerial part; the growth-promoting effect of soaking ＋ spraying and irrigating treatment was the best. Itb57 strain had good antagonistic action against P. nicotianae. A. alterna- ta and B. cinerea, while Itb295 strain only had good antifungal effect against P. nicotianae. [ Conclusion] The results provided basis for the study and application of tobacco endophytic bacteria strains Itb57 and Itb295 in biocontrol of tobacco diseases.

Antagonistical Mode of Pichia membranefaciens to Rhizopus stolonifer in Wounds of Peach Fruit

膜醭毕赤酵母 (PichiamembranefaciensHansen)是本实验室从果实上分离获得的一种能有效防治桃果实采后软腐病的拮抗菌。本文将P .membranefaciens与葡枝根霉 (Rhizopusstolonifer)在桃果实伤口部位共培养 2 4h后 ,用扫描电子显微镜观测了它们的拮抗作用。结果表明 ,在有病原菌的地方聚集了大量的酵母拮抗菌 ,而且拮抗菌紧密地吸附在病原菌的菌丝体上。结合以前的研究结果可以推断 ,P .membranefaciens主要通过与病原菌进行营养和空间的竞争 ,紧密地吸附在病原菌菌丝体上分泌能降解病原菌细胞壁的水解酶 (如几丁质酶和 β_1,3_葡聚糖酶 ) ,并可能诱导寄主产生抗性 ,从而抑制桃软腐病的发生。

Screening,identification and antagonistic effect of antagonistic bacteria JTFM1001 against aflatoxin contamination in corn

Aflatoxin is a strong carcinogenic and toxic fungal toxin produced by Aspergillus flavus and other Aspergillus species,and can seriously threaten the health of consumers,thus becoming a global concern.Corn,as an important oil and economic crop,is highly susceptible contaminated by aflatoxin.In this study,antagonistic bacteria with strong inhibitory effect on aflatoxin were screened to provide support for the treatment aflatoxin contamination control in corn.Ten strains which have strong antagonistic effects against A.flavus were isolated from healthy corn from different corn producing areas in China.Among them,the antagonistic bacteria JTFM1001 through corn kernels in vivo and field experiment,the inhibition effect of aflatoxin contamination reached above 70%and 55%,respectively.And the strain was identified as Bacillus subtilis based on its morphological,physiological and biochemical characteristics and phylogenetic analysis of 16S rDNA.In addition,our data showed that it can colonize in the rhizosphere and survive for a long time,forming the dominant flora,with broad application prospect.Finally,we were surprised to find that the antibacterial metabolites secreted by the antagonistic bacteria was one of the mechanisms of its inhibition of A.flavus and aflatoxin.This will provide us with new ideas and perspectives on the effective prevention and control of aflatoxin contamination in corn and corn oil.

Screening antagonistic microbes for seedling blight of rice and study on antagonistic mechanisms

It is shown by the result of the dual-cultured experiment that the inhibitory rate of DZW-47 was 60.42%, and the inhibitory rates of R.solani by actinomyces ZLR-2 and ZLR-11 were 43.75% and 43.05%, lower than that of DZW-47. The inhibitory mycelia growth mechanism of different strains on R.solani was quite different, with DZW-3 mainly on the aspect of hyperparasitism, DZW-21 on the synergism of hyperparasitism and metabolite, DZW-47 on the synergism of nutrient competition and secondary metabolite, ZLR-2 and ZLR-11 on producing secondary metabolite. Controlling efficiency of seedling bed accorded basically with that of the broth. The controlling efficiency of DZW-47, ZLR-2, ZLR-11, DZW-21 and DZW-3 were 97.20%, 95.7%, 94.6%, 93.6% and 89.20%, respectively.

Genetic Diversity of Antagonistic Endophytic Bacteria Isolated from Sophora alopecuroide

[ Objective ] The paper was to study and analyze the genetic diversity of 48 endophytic bacteria isolated from Sophora alopecuroide with strong antagonis- tic effect against Fusarium oxysporum and Verticillium dahliae, and carry out sequence measurement and phylogenetic analysis on 10 representative strains. [ Meth- od] Using total DNA ERIC-PCR and 16S rRNA sequencing analysis method, a total of 48 endophytic bacteria were carried out genetic diversity and phylogenetic analysis. [Result] ERIC-PCR fingerprint map showed that 48 endophytic bacteria were divided into 6 ERIC groups and 2 strains with independent groups at Wat- son distance of 0.31. Representative strains were selected from each group to determine 16S rRNA gene for phylogenetic analysis, and the results showed that these strains belonged to Bacillus atrophaeus, Bacillus subtilis, Bacillus cereus, Bacillus megaterium, Bacillus pumilus and Serratia marcescens, respectively. [ Conclu- sion ] The genetic diversity of 48 strains of bacteria was obvious. Determination of 48 strains of bacteria on diversity and phylogenetic status could lay foundation for study on mechanism of F. oxysporum and V. dahliae, which could also provide new strain sources for biological control of diseases in cotton production.

Antagonistic Effect of Russula virescens(Schaeff.) Fr.against Bortrytis cinerea

[Objective] The paper was to study the antagonistic effect of Russula virescens （Schaeff.） Ft. against Bortrytis cinerea. [Method] Using the confrontation culture method, the antagonistic effect of mycelium, fermentation broth and mycelial extract of R. virescens against B. cinerea was studied. [Result] The antagonistic ef- fect of R. vivesscens against B. cinerea was mainly manifested as hyperparasitism of mycelium and inhibition effect of metabolites. The inhibition rate of confrontation growth was between 46.9% and 52.3%, and hyperparasitism phenomenon was obvi- ous. The fermentation broth of R. virescens cultured by PDB medium had the strongest growth inhibition effect, and the growth inhibition rate against B. cinerea was the highest of 61.59%. The thermal stability of fermentation broth was good, the mycelial extract of R. virescens had no inhibition effect against the growth of B. cinerea. [Conclusion] The study provided theoretical basis for subsequent differentiation and drug sensitivity of B. cinerea.

Screening and Identification of Antagonistic Bacterium ZG-10 against Spot Blotch Disease in Pakchoi

[ Objective] The purpose was to screen bacterium with antagonistic effect against pathogen of spot blotch disease in pakchoi in vegetable field. [Method] More than 200 strains of bacteria which could produce spore were isolated from soil in different places. Through screening and rescreening, the bacteria with higher antibacterial activity were conducted observation about thallus shapes and colony characters, a series of physiological and biochemical tests were performed. [Result] Rescreening results indicated that the strains including ZG-10, ZG-19, ZG-59, ZG-72 and ZG-31 had significant antibacterial activity, which had very high research value and good application prospect for biocontrol on spot blotch disease in pakchoi; the strain ZG-10 was identified to be Bacillus subtilis. [ Conclusion] The strain ZG-10 had biocontrol potential and good development prospect. This research laid certain basis for subsequent research and strated a new way for the application of antagonistic strain and proteinum polypeptide in agdculture.

Study on AFLP Technical Protocol for Antagonistic Strains of Streptomyces

[Objective] The aim of this study was to investigate the preparation method and amplification system of antagonistic streptomyces DNA templates based on AFLP assays, and also provide a basis for the application of AFLP technology in the analysis of streptomyces or even actinomyces. [Method] The DNAs were extracted by the modified CTAB method and amplified by the Pst Ⅰ/Mse Ⅰ AFLP kit and its reaction system. The amplified products were analyzed by the denatured polyacrylamide gel electrophoresis. [Result] The genomic DNAs of ten antagonistic strains of Streptomyces were extracted and tested. The result of 0.8% agarose gel electrophoresis showed that the major DNA bands were clear without degradation and RNA residue, with the fragment sizes ranging from 37.64 to 40.86 Kb. By ultraviolet spectrophotometry, the OD260/OD280 values varying from 1.625 to 1.833 were obtained. Furthermore, the agarose gel electrophoresis of DNA products digested by Pst Ⅰ/Mse Ⅰ presented the dispersed fluorescent long band, which indicated that the enzymatic hydrolysis was fully carried out. The amplified bands of DNA templates by the screened three pairs of primers were clear with rich polymorphism. [Conclusion] The preparation method and amplification system of DNA template established in this study can be used in the AFLP analysis of Streptomyces.

Composition of Wheat Rhizosphere Antagonistic Bacteria and Wheat Sharp Eyespot as Affected by Rice Straw Mulching

To assess the effect of rice straw mulching on changes of antagonistic bacteria and the incidence of wheat sharp eyespot, a multi-year field study was performed to compare unmulched plots and the plots mulched with rice straw for two or three years. Bacterial and fungal populations were evaluated in the cultures prepared from the wheat rhizosphere and bulk soils. Rice straw mulching increased the number of pseudomonas colony forming units in wheat rhizosphere and bulk soils. The proportion of total bacteria that were fluorescent pseudomonads was higher in mulched than in unmulched soil. Bacterial isolates antagonistic to Rhizoctonia cerealis were identified using an inhibition zone test. A series of these isolates were typed by partial sequencing of the 16S rRNA gene. Pseudomonads had higher antagonistic activity against R. cerealis than other species, and more than 80% of rhizosphere fluorescent pseudomonads were antagonistic to R. eerealis. The disease indices were lower in the mulched plots than in the unmulched control. These results suggest that rice straw mulching in a rice-wheat rotation increases the number of fluorescent pseudomonads. Additionally, these fluorescent pseudomonads may contribute to the control of wheat sharp eyespot.

Antagonistic Potential against Pathogenic Microorganisms and Hydrogen Peroxide Production of Indigenous Lactobacilli Isolated from Vagina of Chinese Pregnant Women

Objective To investigate the indigenous lactobacilli from the vagina of pregnant women and to screen the isolates with antagonistic potential against pathogenic microorganisms. Methods The strains were isolated from pregnant women＇s vagina and identified using the API50CH system. The ability of the isolates to produce hydrogen peroxide was analyzed semi-quantitatively using the TMB-HRP-MRS agar. The antagonistic effects of the isolates on pathogenic microorganisms were determined with a double layer agar plate. Results One hundred and three lactobacilli strains were isolated from 60 samples of vaginal secretion from healthy pregnant women. Among them, 78 strains could produce hydrogen peroxide, in which 68%, 80%, 80%, and 88% had antagonistic effects against Candida albicans CMCC98001, Staphylococcus aureus CMCC26003, Escherichia coli CMCC44113, and Pseudomonas aeruginosa CMCC10110, respectively. Conclusion The recovery of hydrogen peroxide-producing lactobacilli decreases with the increasing pregnant age and time. The most commonly isolated species from vagina of Chinese pregnant women are Lactobacillus acidophilus and Lactobacillus crispatus. Most of L. acidophilus and L. crispatus produce a high H2O2 level.

Fermentation optimization to improve production of antagonistic metabolites by Bacillus subtilis strain BS501a

To improve antagonistic metabolites production of Bacillus subtilis strain BS501a, physical parameters of fermentation and metal inorganic salts in medium, namely initial pH value, culture temperature, fermentation time, concentrations of CaC12, FeSO4, ZnSO4, MnSO4 and MgSO4, were optimized using one-factor-at-a-time and orthogonal tests. The results show that the optimal physical parameters of fermentation are an initial pH of 7.0, a culture temperature of 30 ~C, and a fermentation time of 48 h. The optimal concentrations of metal inorganic salts in basal medium are 10.2 mmol/L CaCl2, 0.4 mmol/L FeSO4, 3.5 mmol/L ZnSO4, 0.6 mmol/L MnSO4 and 2.0 mmol/L MgSO4. Among the metal inorganic salts, MgSO4 and MnSO4 play important roles in the improvement of the antagonistic metabolites production of B. subtilis strain BS501a; especially, MgSO4 contributes a highly significant effect. The average diameter of inhibition zone of the BS501a filtered fermentation supernatant （FFS） cultured in the optimal fermentation conditions against Magnaporthe grisea DWBJ329 reaches 71.4 mm, and there is 2.4-fold increase in antifungal activity as compared with 21.2 mm under the pre-optimized conditions.

Diversity of Gram negative bacteria antagonistic against major pathogens of rice from rice seed in the tropic environment

With the use of a seed washing technique, more than 4000 Gram negative bacteria were isolated by two improved isolation methods from 446 batches of 1 kg rice seed samples obtained from 22 provinces in the Philippines. They were initially characterized on the basis of colony morphology and results of biochemical and pathogenicity tests. Six hundred and fifty two strains were further identified by Biolog, from which 133 were selected for fatty acid methyl ester (FAME) analysis together with 80 standard reference!strains. Sixteen species or types of Pseudomonas and 17 genera of non pseudomonads were identified, more than one third of which have not been recorded in rice. The most predominant species observed were P. putida and P. fulva. About 17% of the strains of Pseudomonas and 2% of the non pseudomonads were antagonistic to one or more fungal or bacterial pathogens of rice. Rice seed is an important source of biological control agents.

Mechanism of antagonistic effects of Andrographis paniculata methanolic extract against Staphylococcus aureus

Objective: To investigate the effects of Andrographis paniculata(Burm.f.) Wall. Ex Nees(A. paniculata) on expressions and activities of catalase, superoxide dismutase and alkylhydroperoxide reductase C in Staphylococcus aureus(S. aureus) with respect to its survival in vitro. Methods: Antioxidative property of methanolic leaves extract of A. paniculata(0.06 mg/m L). Minimum inhibitory concentration(MIC) was determined by its ability to reduce hydrogen peroxide(H_2O_2) toxicity against S. aureus ATCC 25923(3.8×10~8 cfu/m L). Effects of the extract on expressions of kat A(encoding catalase), sod A and sod M [encoding superoxide dismutases(SODs)], and ahp C [encoding alkylhydroperoxide reductase C(AhpC)] in S. aureuswere determined by RT-q PCR and corresponding enzyme activity assays were performed. Nitroblue tetrazolium reduction(NBT) assay was performed to determine effects of the extract on intracellular and extracellular levels of O_2-in S. aureus. Results: Cells challenged with 7.5 mmol/L H_2O_2 showed 0% survival in 30 min whereas 25% survived after treatment with the extract and H_2O_2. Cells that were treated with the extract alone had 43% survival in the same exposure period. Expressions of sod A and sod M genes in extract-treated cells were lowered 0.8-fold and 0.7-fold, respectively with decrease in total SOD activity of 26.8 U compared to untreated cells, 32.4 U(P<0.05). In contrast, extract-treated S. aureus cells showed 3.3-fold increase in kat A expression with corresponding increase in catalase activity of 1.828 U compared to untreated cells which was 1.248 U,(P<0.05). More profoundly, ahp C expression was increased 61-fold in extract-treated cells,(P<0.05) with corresponding increase in Ahp C activity of 0.018 U compared to untreated cells, 0.012U,(P<0.05). Extract-treated cells had significantly lower intra-and extracellular O_2-levels with absorbance readings(A_(575nm)) of 0.340 and 0.524 compared to untreated cells which were 0.516 and 0.928(P<0.05), respectively. Conclusions: Taken together these results suggest that the low MIC of A. paniculatamethanolic leaves extract(0.06 mg/m L) reduce H_2O_2 toxicity and more importantly, was in itself effectively inhibitory against S. aureus. Further, our observations suggest that a probable mode of its inhibitory mechanism against S. aureus is by reducing total SOD activity through downregulation of sod A and sod M expressions.