| wheat sharp eyespot caused by Rhizoctonia cerealis is one of the important factors affecting the high and stable yield of wheat.At present,the prevention of wheat sharp eyespot is mainly based on chemical and agricultural control measures.Chemical control with fast results,good prevention and control,but the long-term use of chemical pesticides is prone to pathogenic bacteria resistance and pollution of the environment.Biological control is gaining more and more attention in plant disease control because of its advantages such as being friendly to the environment and not easy to develop drug resistance.In this study,the rhizosphere bacteria of wheat were isolated by dilution isolation method,a wheat rhizobacteria strain NY3 with strong antagonistic effect on Rhizoctonia cerealis was obtained by screening.Preliminary discussion on the mechanism of biological control and disease control effect of NY3,the fermentation medium and fermentation conditions of NY3were optimized by using single-factor test and response surface analysis.The main results are as follows:1.89 bacterial strains were isolated from wheat inter-root soil using dilution isolation method,five strains of inter-rhizosphere bacteria NY1,NY2,NY3,NY4,and NY5 with good antagonistic effect were obtained by standoff culture screening using Rhizoctonia cerealis bacteria as target pathogens.Among them,wheat rhizosphere bacteria NY3 showed the best inhibition of Rhizoctonia cerealis with 86.72%inhibition of mycelial growth.The NY3 strain was identified as Bacillus velezensis by morphological characteristics,physiological and biochemical properties,16S r DNA and gyr B sequence analysis.The effect of NY3 on the mycelial morphology of Rhizoctonia cerealis bacteria was observed by microscopy,and it was found that NY3 could increase its branching and appear twisted and broken.The effect of NY3 fermentation filtrate on the nucleus formation and germination of Rhizoctonia cerealis bacteria was measured,and the results showed that NY3 fermentation filtrate could inhibit its formation and germination.2.The antagonistic effect of rhizosphere bacteria NY3 on the 10 plant pathogenic fungi tested was investigated by plate standoff culture.The results showed that the inhibition spectrum of NY3 was wider,the distribution of its mycelial growth inhibition rate against the pathogenic fungi of the test plants ranged from 72.44%to 88.89%.NY3 showed the best inhibition effect on Rhizoctonia solani with 88.89%inhibition of mycelial growth.3.The fermentation conditions of NY3 were optimized using a single-factor test combined with response surface analysis,and the optimized optimal conditions were tryptone 15.0 g,yeast extract 5.0 g,Na Cl 10 g,inoculum 3%,incubation temperature 26℃,incubation time41 h,p H 7,and speed 202 rpm.The fermentation filtrate obtained by incubation under this condition had the best inhibition effect on Rhizoctonia cerealis with 88.96%inhibition of mycelial growth.4.Hydrolytic enzyme production and siderophore capacity of wheat rhizosphere bacteria NY3 were tested using cellulase,protease and siderophore assay media.It was found that NY3 secreted fibrinase and protease but not siderophore.The crude lipopeptide compound of NY3 was extracted using the hydrochloric acid precipitation method,and the crude extract was found to have strong antibacterial activity,with 10 mg/m L of lipopeptide crude extract inhibiting the mycelial growth of Rhizoctonia cerealis by 61.54%.The thermal and UV stability of the lipopeptide analogues produced by NY3 was investigated,the thermal stability results showed that the lipopeptides produced by NY3 had good thermal stability,the inhibition rate of mycelial growth of Rhizoctonia cereali was above 50%at the treatment temperature of 25-90℃,when the temperature was increased to 100°C,the inhibition rate still reached 47.73%.The UV stability results showed that the antibacterial activity of NY3lipopeptides crude extracts showed a decreasing trend with increasing irradiation time after UV irradiation treatment,the inhibition activity was not significantly different from that of the treatment without UV irradiation when the treatment time was 30 min and below.The inhibition activity was significantly lower than the control when the UV irradiation time was above 30 min,However,when the UV irradiation time was 120 min,the mycelial growth inhibition rate could still reach 43.94%.It can be seen that NY3 lipopeptide crude extracts are tolerant to temperature and UV light.Detection of lipopeptide-producing genes in wheat rhizosphere bacteria NY3 using five lipopeptide gene primers for iturin,fengycin,surfactin,bacilysin,and mycosubtilin.The results turned that the lanes of iturin,surfactin and bacilysin all showed a single band and the product fragment was consistent with the size of the target fragment.It is inferred that the genes for the synthesis of lipopeptides such as iturin,surfactin and bacilysin may exist in the genome of rhizosphere bacteria NY3.It is therefore speculated that this strain may metabolize and produce the above three types of lipopeptide compounds.5.The effects of NY3 root irrigation treatment on peroxidase(POD),superoxide dismutase(SOD)and malondialdehyde(MDA)contents of wheat leaves were examined at the wheat seedling stage.The results showed that NY3 root irrigation treatment significantly increased superoxide dismutase(SOD)and peroxidase(POD)activities and decreased malondialdehyde(MDA)content in wheat seedlings.6.Antibiotic induction of strain NY3 using rifampicin to obtain strain NY3~r with stable resistance to 200μg/m L rifampicin.NY3~r inoculation by seed dressing and root irrigation,the levels of labeled antagonistic bacteria in the soil were measured every 5 d,and the extinction dynamics of strain NY3~r in wheat inter-root soil over 30 d was obtained.The rhizosphere soil bacteria of wheat treated with wheat seed dressing and root irrigation were still able to reach 1.47×10~7 cfu/g and 6.97×10~6 cfu/g after 30 d.7.The effectiveness of the bacterium NY3 on the control of wheat sharp eyespot was measured in pot trials.The results showed that the efficacy of NY3 bacterial solution(1×10~9cfu/m L)seed dressing and root irrigation treatments against wheat sharp eyespot was 71.76%and 77.92%,respectively,which was lower than that of sennithromycin treatment,but the difference between them was not significant.In the field trial,the efficacy of NY3(1×10~9cfu/m L)seed dressing against wheat sharp eyespot was 46.73%,which was not significantly different from that of sennithromycin(51.26%). |
