| Crop residue is a kind of abundant,widely distributed and renewable biomass resources in the world.However,the unreasonable use of crop residue not only wastes the straw resources,but also brings serious problems to the environment and human safety.Incorporation of crop residue to field is a simple and effective method to be applied for the current stage,however,directly incorporation without any treatments could bring a series of problems,such as affecting sowing and seedling germination and leading to crop disease.It was proposed to be a effective solution by the use of lignocelluloses-degrading microorganisms to accelerate the degradation of crop residue,for those are mainly Trichoderma and Bacillus.In this study,26 strains which can produce cellulases were preliminary screened from 30 Bacillus strains stored in laboratory,through transparent circle formation in CMC plate.Two Bacillus candidate strains which showed high enzyme activity and biocontrol potential were then screened through extracellular enzyme activity measurement and antimicrobial activity against Rhizoctorzia solani.Furthermore,the influence of weight-loss rate of wheat residue and degradation rate of cellulose,hemicelluloses and lignin by the two Bacillus strains were tested under the liquid fermentation conditions,and Bacillus pumilus GBSW19 which can effective degrade crop residue were finally obtained.In addition,the degrading effects of B.pumilus GBSW19 and its mixed strains on rice residue were also investigated in pot experiments.The results indicated that the application of B.pumilus GBSW19 solely or compound strains both degrade rice residue efficiently,and the degradation rate of rice residue at 15 d reached 58.5%and 77.6%,respectively.This result suggested that B.pumilus GBSW19 and its mixed strains have potential for practical application.However,further studies are required to investigate its authentic degrading effects on crop residue in field condition.The efficient crop residue-decomposing strain Bacillus pumilus GBSW19 was then applied in a rice-wheat rotation field during the period from 2013 to 2014.Crop residues were smashed and then incorporated into field directly or pre-sprayed with GBSW19 and control strains.The results indicated that in winter wheat field,the degradation rate of previous crop residue(rice)was relative slow and the degradation rate at 30 d was only 39.7%,whereas in summer rice field,the degradation rate of previous crop residue(wheat)significantly increased and the degradation rate at 32 d was 68.6%.In both cases,the degradation of crop residue treated with GBSW19 significantly excelled other treatments.We also investigated the influence of straw incorporation to field on the growth of rice and wheat.There were no significant differences between the emergence rate and the white roots amount in all treatments,but plant height and root length of rice during the later stage of growth significantly increased when applied with GBSW19,eventually resulting thousand kernel weight increased by 10.7%and grain yield increased by 11.1%compared with straw returning directly.In addition,the application of GBSW19 changed the acidic soil to neutral pH and significantly improved the soil organic matter,total nitrogen and available nitrogen content in 0-20 cm soil compared with other treatments during the whole investigating period.In conclusion,appropriate pretreatment before straw returning did not affect normal growth of next season crops.Moreover,the application of Bacillus pumilus GBSW19 not only accelerated the degradation of crop residue,but also adjusted soil pH,increased soil fertility and crop yields.Therefore,the Bacillus pumilus GBSW19 strain has great value in future application.In order to further study the characterstics of lignocelluloses degrading enzymes in GBSW19,we predicted the potential genes involved in cellulose and hemeicellulose degradation based on the analysis of a genome-sequenced Bacillus pumilus strain SAFR-032.Nine cellulases and hemicellulases coding genes were successfully cloned from B.pumilus GBSW19 and prokaryotic expression system was adopted for proteins expression.Among these,a ?-mannanase encoding gene Bpman5 with a length of 1134 bp shared a low amino acid similarities to the reported mannose(<50%).Bpman5 contains a signal peptide at residues 1-28 and a catalytic domain of glycosyl hydrolase family GH 5.It showed the classical TIM(?/?)8-barrel architecture and 7 of 8 active sites residue in GH5 mannanases were found in Bpman5.Moreover,electrophoretic purified mannase Bpman5 was obtained through affinity purification.The basic characteristics of Bpman5 showed that the temperature and pH optima were around 65 ? and 6.5,respectively.Meanwhile it is stable in the range from pH 5-11 at 60 ?.In addition,the purified enzyme can be activated by several ions and organic solvents and is resistant to detergents.These results indicated that Bpman5 is a thermo-,pH-and detergents-stable enzyme,thus has great application potential in the furture.Furthermore,the Bpman5 is only active towards P-mannan substrates like galactomannan(LBG)and glucomannan(konjac),but showed no activity against a-mannan,which confirms it is a typical ?-mannanase.The hydrolysis products of LBG by Bpman5 from B.pumilus GBSW19 were analyzed by thin layer chromatography(TLC).The main hydrolysis products were mannobiose(M2),mannotriose(M3),mannopentose(M5)and mannohexaose(M6),with minor mannose(M1).M3 was detected during the hydrolysis of M4,but equal amount of M1 was not accumulated,indicating that the formation of M3 during M4 hydrolysis should result from transglycosylation.Due to the hydrolysis product of LBG by Bpman5 were manno-olifosaccharides with lower DP,it provides us a useful experiment material to study the biological activity of small molecular mannan oligosaccharides in the next step.Oligosaccharide is a kind of biotic and non-protein elicitor.Many researches reported that oligosaccharide elicitors can induce plant systemic resistance and/or promote plant growth.It is a new biological pesticide and can be used as resistance inducing agent.To date,the studies of oligosaccharides were mainly on gluco-oligosaccharides,chitosan oligosaccharides,chitooligosaccharide,laminarin oligosaccharides and xyloglucan oligosaccharides.There is rare study of manno-oligosaccharides on plants.In this study,manno-oligosaccharides(MOS)were prepared from locust bean gum(LBG)by hydrolysis using purified ?-mannase Bpman5 we described above.MOS with a DP of 2-6 and minor mannose were then obtained through "centrifugation-microfiltration-ultrafiltration" steps.The hydrolysis condition for MOS production was also optimized,and the optimum condition was 10 mg/ml LBG incubated with 10 U/mg Bpman5 for 24 h.We found that these small MOS could significantly improve plant resistance to Xanthomonas oryzae and Phytophthora nicotianae through spatial separation treatment.Further research indicated that MOS treatments may result in Ca2+ influx of plant guard cell and accumulation of ROS and NO.These signal modules may further participate in plant defense responses,such as inducing stomata closure and hypersensitivity,activating transcriptional level of defense related genes involved in SA and JA signal pathway,and ultimately improve plant resistance.Besides these,MOS treatments can also promote root length,plant height and fresh weight of rice.Therefore,MOS we reported here not only can be used as plant resistance inducing agent,but also can be used as growth promoting factor which exhibit great application prospects in agricultural and related field. |
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