| Cucumber(Cucumis sativus L.)is a popular and important vegetable crop all over the world.The continuous monoculture is a common and typical planting pattern of greenhouse cucumber in North China.However,With the increasing number of continuous intensive cultivation years and the extensive application of farmyard manure and chemical fertilizer,the greenhouse soil had occurred the problems of hardening,acidification and secondary salinization,enrichment of available phosphorus(P)and potassium(K),the imbalance of soil microbiome,which resulted in the poor growth and development of plant roots,unbalanced nutrient absorption,and the decline of cucumber yield and quality.At present,the application of beneficial functional microbial inoculants is an effective approach to solve the problem of the obstacles of greenhouse cucumber production.Based on this,this study took the cucumber greenhouse with different cultivation years in Daqingfa village of Yongqing county in the superiority area of greenhouse cucumber of Hebei province,and the Bacillus megaterium developed by our research group and its products as the main research object,to study the effects and mechanism of application of Bacillus megaterium on solubilizing soil phosphorus and potassium and improving the crop availability,promoting cucumber growth,regulating the characteristics of soil microbial community in root zone.Firstly,a field investigation was conducted to explore the changes of the diversity,composition,and structure of soil microbial communities with different cultivation years under cucumber greenhouse conditions.Then,field experiments were carried out in the cucumber greenhouse with continuous cropping for 15 and 2 years,to clarify the effects of application of Bacillus megaterium on cucumber growth and soil microbial communities in the cucumber greenhouse with different cultivation years.Finally,the pot experiment was conducted to reveal the mechanisms of action of Bacillus megaterium by RNA-seq and the metabolism data.The main results are as follows:(1)The variation of soil microbial communities to different cultivation years under cucumber greenhouse conditions were investigated.Cucumber greenhouses with continuous mono-cropping 1,8,15 and 22 years were selected in the superiority area of greenhouse cucumber in Yongqing County,Hebei Province.The characteristics of soil bacterial and fungal communities were analyzed by high-throughput sequencing.Results showed that the soil chemical properties of greenhouse were significantly affected by continuous planting for 8 or more years.Soil pH significantly decreased,whereas soil EC,organic matter(OM),total nitrogen,phosphorus,potassium(TN,TP,TK)content,and nitrate N(NO3--N),available phosphorus,potassium(AP,AK)content significantly increased.It also significantly affected the characteristics of soil microbial community in the greenhouse root zone soil:the diversity of soil fungal community was significantly decreased,the relative abundance of soil bacterial phylum Actinobacteria and fungal phylum Ascomycota were decreased,but the relative abundances of Acidobacteria and Firmicutes were increased significantly.PCoA analysis showed that the structures of soil bacterial and fungal communities were shifted by the different cultivation years.RDA analysis revealed that soil OM and NO3--N were the most important factors to drive the variation of soil bacterial community,while soil pH,OM and NO3--N were the most important factors to drive the variation of soil fungal community.(2)The effects of application of Bacillus megaterium inoculant on cucumber growth and soil microbial communities in the greenhouse with cucumber continuously cultivated for 15 years were clarified.The two-year experiment showed that,compared with conventional fertilization,conventional fertilization with the application of Bacillus megaterium inoculant significantly increased the cucumber yields by 11.8%at least and increased P and K accumulation in cucumber fruits and roots by 27.5%and 17.8%at least,respectively.Meanwhile,conventional fertilization reduced by 10%～20%P and 5%～10%K from chemical fertilizer with application of Bacillus megaterium inoculant did not decrease the yields and quality of cucumber,and the bioavailability of soil P and K.Moreover,the q-PCR result showed that Bacillus megaterium could efficiently colonize the root zone soil after inoculation.Highthroughput sequencing identified that Bacillus megaterium inoculant improved the richness of soil bacterial and fungal communities significantly.The application of Bacillus megaterium inoculant did not change the compositions of the predominant phyla Proteobacteria,Acidobacteria,Actinobacteria,Firmicutes of the indigenous soil bacterial community and predominant phylum Ascomycota of the indigenous soil fungal community,but increased the relative abundances of genera Nocardioides and Streptomyces with growth promotion and antagonism in the soil bacterial community and orders Sordariales and Hypocreales with antagonism in the soil fungal community,and decreased the pathogenic bacterial genus Xanthomonas significantly.PCoA analysis revealed that the structures of soil bacterial and fungal communities were significantly affected by Bacillus megaterium within days 20 after application.RDA analysis revealed that soil pH,AK,TP,and TK were the most important factors to drive the variation of soil bacterial community while soil AK and TK were the most important factors to drive the variation of soil fungal community.(3)The effects of application of Bacillus megaterium inoculant on cucumber growth and soil microbial communities in the greenhouse with cucumber cultivated for 2 years were clarified.The two-year experiment showed that the strain of Bacillus megaterium could quickly and efficiently colonized the root zone soil after application.The application of Bacillus megaterium inoculant at the rate of 75 L/hm~2 when the cucumber seedings transplanted was the most suitable approach to increase cucumber yields by 6.71%at least,soil available P and K by 1 1.6%and 16.3%at least,and P and K accumulation in cucumber plants by 14.9%and 17.8%at least,respectively,which has obvious effect on solubilizing soil P and K,promoting P and K absorption and cucumber growth.High-throughput sequencing identified that Bacillus megaterium inoculant increased the richness of soil bacterial community.Bacillus megaterium inoculant significantly increased the relative abundances of phyla Actinobacteria,Firmicutes and genera Lysobacter with antagonism,and genus Pseudomonas and Flavobacterium with growth promotion in the soil bacterial community,while decreased the relative abundances of the pathogenic bacterial genus of Xanthomonas.The structure of soil bacterial community was shifted by the application of Bacillus megaterium inoculant,and soil pH and AK were the most important factors to drive the variation of soil bacterial community.(4)The growth promotion mechanism of Bacillus megaterium application were revealed.It was found that Bacillus megaterium could produce Indole acetic acid(IAA),Gibberellin(GA3)and Zeatin(ZR)during the growth process,which could directly promote the growth of cucumber.Further pot experiment of interaction between Bacillus megaterium and cucumber showed that inoculation of Bacillus megaterium could effectively regulate the endogenous plant hormone levels in cucumber roots,the contents of IAA and salicylic acid(SA)were significantly increased by 1.96 and 7.79 times at day 1 after inoculation,and ZR content increased by 2.93 times at day 8 after inoculation,which promoted cucumber growth and improved the disease resistance of cucumber.Moreover,different expression genes of cucumber roots after interaction between Bacillus megaterium and cucumber were analyzed through RNA-seq technology.Results showed that 7700 genes of cucumber root were differentially expressed by the induction of Bacillus megaterium,among them,3250 genes were up-regulated,and 3820 genes were down-regulated.Through KEEG pathway enrichment analysis of differentially expressed genes,it was found that Bacillus megaterium affected Metabolism,Environmental Information Processing,Genetic Information Processing and other metabolic pathways in cucumber root.It was mainly induced the up-regulate expression of Indole-3-acetic acid-amido synthetase,Xyloglucan endotransglucosylase/hydrolase,and early flowering protein genes which were related to plant growth promotion,with the maximum upregulation of 123 times;and the up-regulate expression of calmodulin,chitinase and aspartate aminotransferase genes which were related to plant disease resistance,with the maximum upregulation of 5.00 times.At the molecular level,it was preliminarily revealed that Bacillus megaterium could regulate the differential expression of genes involved in plant hormone signaling,plant-pathogen interaction,metabolism of terpenoids and polyketides and pyruvate metabolism pathways through the interaction with plants,then promote plant growth and improve disease resistance of plant. |
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