Construction Of Multifunction Rhizobium Engineering Strain

Rhizobium engineering strain were obtained deriving from rhizobium and bacillus withantagonist to pathogens of soybean root rot and soybean cyst nematode were screened withinsoybean nodule, after study the system of both strains protoplast preparation, regeneration andamalgamation. The characters of this engineering strain were fixing nitrogen, promotinggrowth and antagonist to pathogens. Its fermentation conditions, action mechanism on fungiand plant parasite nematode, resistance induced were studied systematically. Results showedas follows:1. 786 strains were obtained from 138 soil samples collected from more than 20provinces of China. Among them, 351 strain were bacillus and 435 strain were rhizobium. Theresults showed that there were 79 bacteria strains with antagonist to fungi of soybean root rot,in which, the effective bacillus strains were occupied 81.01%, and rhizobium only 18.99%;rhizobium L396 and bacillus Snb2 were screened out from strains with effectively neamticidetoxin to Heterodera glycines. The mortality of H. glycine treated with suspensions of L396and Snb2 were 21.6% and 66.7% respectively. Through re-screening in shaking flasks, affectof L396 and Snb2 bacterial culture filtrates on J2 of H. glycines were 69.05% and 39.13%;meanwhile, strain L396 and Snb2 had high effect against 12 pathogens; After seeking seedwith bacteria suspension, both strains promoted growth the seeding.2. According to traditional taxonomy including morphology and Physio-biochemical,the bio-control bacteria strains L396 and Snb2 were classified as Sinorhizobium sp. andBacillus sp.; The sequences of 16S rDNA gene of L396 and Snb2 were sequenced andanalyzed, the results showed that strain L396 and Snb2 belong to Sinorhizobium fredii,Bacillus subtilis, respectively.3. Take 40μg/mL Penicilium and 50℃, 120min heat treated as fusion marker of parentsstrain Snb2 and L396. The best optimal protoplast preparation, regeneration andamalgamation conditions were researched. Under optimal condition, the ratio of L396 andSnb2 protoplast forming was57.17% and 43.53%, respectively, and protoplast fusion rate was9.39×10^-4. 398 fusants were obtained after amalgamation and only 49 fusants survived transferred six times on medium with 40μg/mL Penicilium.4. RH5 was picked out based on colony morphology, size ofthalli, effect againstpathogens and difference of soluble protein among all fusants. RH5 had distinctive activeagainst pathogens and parasitic nematode among which could nodule on root of soybean. Themortality of J2 treated with Bacterial culture filtrates of RH5 was 44%.5. The fermentation conditions of RH5 were investigated by single factor and designsof orthogonal experiments, results were as follow: the optimum carbon and nitrogen resourcewas lactose and urea, respectively; the liquid medium of fermentation of RH5 constitutes bylactose 1.0%, urea 0.35%, ammonium nitrate0.35%, magnesium chloride 0.35%, PotassiumSulfate0.8%, dipotassium hydrophosphate 0.3%, ferrous sulfate 0.002%; the optimumfermentation condition included the volume of medium of shaking flasks 60mL/250 mL, theinitial pH 6-7, inoculation volume in shaking flasks 3mL.6. The effect of Bacterial culture filtrates of RH5 on spore germination and mycelialconfiguration were tested. The germination of spore were inhibited, the ratio of germation was2.30% treated with culture filtrates without dilution and sprout of germinated spore wasmalformated. Protoplast agglomerate appeared in hypha treated culture filtrates, hypha wereswelled and dissolved gradually. RH5 produced some nematicide: HCN,H2S, had ability ofamylase and oxygenation hydroxybenzene, It had some effect on total sugar content andprotein content of H. glycines.7. The seedling was promoted inoculated with RH5 suspension. The activity defenceenzymes were promoted, the increase range followed the bacteria concentration, the resultindicated the optimum inoculation was 10¹⁰cfu/mL.