| Camellia oleifera is one of the important oil crops and economic tree species in China,and the market demand is large,but it is easy to infect many diseases during its growth and development,among which anthracnose is especially serious.At present,chemical control is the main control method,but the side effect is too big.Although biological control is a good control strategy,there are some biological control agents at present,but most of them are single bacteria,which have weak competitiveness,poor stability and narrow control range under field conditions,so their efficacy is not ideal in practice.The use of artificial flora for control is becoming a trend because of its higher effectiveness,versatility,and stability in the environment.Based on the characteristics of the phyllosphere microbial community in C.oleifera,the synthetic bacterial community was constructed according to the dominant flora and the key microorganisms in the interaction network.The main cultivated C.oleifera in Hunan Province was used as the experimental material to isolate and screen the phyllosphere bacteria with the function of inhibiting bacteria and producing siderophore from C.oleifera,and to explore the control effect of the synthetic bacterial community on C.oleifera anthracnose.The main results are as follows:1.MiSeq sequencing of the bacterial community in the phyllosphere of C.oleifera was carried out.Phyllospheric bacteria of C.oleifera belong to 31 phyla,63 classes,139 orders,259 families,451 genera and 671 species.Among the three cultivars,You County had the lowest diversity and the most uniform species distribution.The species richness of Asustek was the highest and significantly different from that of You County(p<0.05).The diversity of bacterial community in You County area was the highest among the three areas,and the species distribution in Changsha area was the most uniform.Wangcheng has the highest species richness.The diversity and evenness index of bacterial community in the phyllosphere of C.oleifera from You County and Changsha were significantly different(p<0.05).Phyllosphere bacterial community was mainly composed of Proteobacteria,Firmicutes,Chloroflex,Actinobacteria,Bacteroidetes,Planctomycetes and Acidobacteria.At the genus level,the composition,relative abundance and dominant flora of the bacterial community in the phyllosphere of different C.oleifera were different.The difference of bacterial community structure in different regions was greater than that in different cultivars,and the difference of microbial community structure in different regions was significant.In the interaction network of bacterial community in the phyllosphere of C.oleifera,most of the microorganisms had positive interactions,and the symbiotic relationship was dominant,while the competitive relationship was weak.The top 5 microbial species with the greatest centrality were Exiguobacterium,Methylobacterium,Paenibacillus,Pseudonocardia and Ochrobactrum.2.A total of 1093 strains of bacteria were isolated from the leaves of C.oleifera,and 214 strains were screened by plate confrontation method against C.fructicola.Molecular identification showed that the strains were mainly from seven genera,namely Bacillus,Pseudomonas,Bacteroides,Stenotrophomonas,Microbacterium and Acinetobacter.Among them,Bacillus accounted for 60%,which was the main strain of antagonistic bacteria.Antagonistic bacteria from C.oleifera leaf were used to screen siderophore-producing bacteria,and their siderophore-producing ability was quantitatively detected.The results showed that 6 strains(4-1,6-9,6-19,5-1,C2 and 28)had the highest siderophore production ability,and the strain 6-9 had the highest siderophore production ability.3.According to the relative abundance and centrality ranking of the community,5 strains were selected from 32 strains of antagonistic bacteria against C.fructicola with siderophore production ability,and 26 synthetic bacterial communities were constructed.The study on the colonization effect of synthetic bacteria on C.oleifera leaves showed that the colonization effect of synthetic bacteria was better than that of single bacteria.On the 30th day in laboratory,the viable count of Y12/28/6-9/6-19 was 9.4893×104 CFU/mL,and the viable count of single bacterium 28 was 2.8294×104 CFU/mL,which was 3.35 times of that of single bacterium.On the 30th day,the viable count of Y12/28/6-9/6-19 was 5.4333×102 CFU/mL,and that of single bacterium 28 was 1.8314×102 CFU/mL,which was 2.96 times of that of single bacterium.In vitro control effect of the synthetic bacteria groups on the anthracnose of C.oleifera showed that the control effect of the synthetic bacteria groups was obviously higher than that of the single strain.The control effect of the synthetic bacteria groups Y12/28/6-9/6-19 and C2/Y12/6-9/28 was 100%,and the control effect of the single strain 6-9 was only 16.21%.Combined with leaf colonization test and in vitro control test,the best flora was Y12/28/6-9/6-19.4.The effects of the optimal synthetic bacteria on the defense enzyme activity and the expression of related resistance genes in C.oleifera leaves were studied.The results showed that the synthetic bacteria could significantly improve the defense enzyme activity and the expression of related resistance genes in C.oleifera leaves.The results showed that the activities of defense-related enzymes such as POD,SOD,PPO and PAL in C.oleifera leaves could be improved by inoculating Cercospora fructicola,inoculating synthetic bacterial community and spraying pathogenic bacteria for 12 h.The expression levels of resistance genes such as POD,SOD1 and PR1B1 were increased by synthetic flora and C.fruitosum.In terms of improving the activity of defense enzymes and the expression level of defense-related diseaseresistant genes,the synthetic flora is significantly higher than that of pathogenic bacteria. |
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