Biotechnology Bulletin

Plant-Microbiome Interactions：An Eco-Evolutionary Perspective

CHENG Sai-sai, GONG Xin, XUE Wen-feng, WAN Bing-bing, LIU Man-qiang, HU Feng

2020, 36(9): 3-13. doi:10.13560/j.cnki.biotech.bull.1985.2020-0978

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Microorganisms thriving at the plant surfaces and internal tissues play critical roles in plant growth,development,adaptation,and diversification. Numerous studies have revealed the great functional capacity of plant-associated microbiome,however,it is not clear how plant-associated microbiome assembles in plant evolutionary process. In this paper,the ecological adaptation and the evolutionary mechanism of plant-microbiome interactions are summarized based on the evolutionary trajectory of plant phylogeny,plant-microbiome in crop domestication,and the microbe-microbe interaction and evolution under host constraints. Understanding the evolutionary drivers of plant-microbiome interactions may contribute to restoring the beneficial connections between plants and microbes,thus achieving the goals of high yield and high efficiency by increasing crop nutrient absorption,yield and quality. Strengthening the strategy of combining molecular biology and crop genomics will be the key to regulate plant microbiome to promote agricultural sustainability. Meanwhile,the outlook of eco-evolutionary of plant-microbiome interactions is highlighted.

Rhizospheric Chemical Signals and Soil Nutrient Transformation

LU Yu-fang, SHI Wei-ming

2020, 36(9): 14-24. doi:10.13560/j.cnki.biotech.bull.1985.2020-0703

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Though nutrient cycling in a farmland ecosystem is mainly driven by soil microbes,rhizospheric chemical signals can significantly change the composition of soil microbes and affect nutrient transformation, thus it plays a key role in promoting plant growth and resisting biotic and abiotic stresses. At present,the signal transmission between plants and mycorrhizal fungi or rhizobia in early stage has been extended to the study of its interactions with non-symbiotic microorganisms. Recently, it has been found that rhizospheric signal substances can shape the rhizosphere beneficial microbiome. This paper focuses on the recent domestic and international research progress in rhizospheric chemical signals and soil nutrient transformation. First,the concept of the chemical signals in the rhizosphere is proposed,and the types and functions of the signal substances are summarized. Then the effects of signal substances on the conversion of nitrogen,phosphorus and iron nutrients involved in the plant-plant,plant-microbe and microbe-microbe interactions are further discussed,and the deficiencies of current researches are analyzed. Finally,the future research direction of rhizospheric chemical signals is prospected,and the importance of techniques such as chemical detection,multi-omics and synthetic bacterial community in the rhizosphere chemical signaling research is emphasized.

Technical Systems of Reorganization and Construction of Crop Rhizosphere Microbiome

SUN Yu, CHANG Jing-jing, TIAN Chun-jie

2020, 36(9): 25-30. doi:10.13560/j.cnki.biotech.bull.1985.2020-0796

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The rhizosphere microbiome is of vital importance in growth and stress-resistance of crops. The improvement,reorganization and construction of rhizosphere microbiome are essential for improving the stress resistance of crops. In this study,three technical systems of reorganization and construction of rhizosphere microbiome are discussed preliminarily,including soil suspension inoculation,intercropping of crops and the application of soil memory. The characteristics and application feasibilities of the technical systems are explained from the theoretical level and practical application,thereby providing evidences for the application of rhizosphere microbiome in further sustainable development of agriculture.

Application of Rhizospheric Biocontrol Consortia and the Potential Mechanisms of Their Enhancing Efficacy on Disease-suppressive Effect

WANG Wei-xiong, SHEN Bo, JIA Hong-bai, QIAO Jun-qing, NIU Ben

2020, 36(9): 31-41. doi:10.13560/j.cnki.biotech.bull.1985.2020-0994

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The development and application of rhizospheric biocontrol microorganism-based biopesticide is an environmentally friendly strategy for the control of plant diseases. Two major barriers limiting the disease-inhibiting function of rhizosphere biocontrol microbes are inadequate host and soil colonization as well as inefficient suppression of phytopathogen growth. Currently,it has been verified from a fair amount of studies that rhizospheric biocontrol consortia are capable of improving the disease-suppressing effect of biocontrol microorganisms of rhizosphere. First,we review the current status of applying rhizosphere biocontrol consortia in managing plant diseases and the potential mechanisms of enhancing disease-suppressive efficacy such as regulating colonization,pathogen inhibition and induced systemic resistance by microbial interactions within the rhizosphere biocontrol consortia. Then,we discuss the relationships between the effect of the rhizospheric biocontrol consortia on enhancing the disease-inhibiting function and the factors of growth,biofilm formation,motility,resource competitiveness,and biosynthesis of antimicrobial compounds of the rhizosphere biocontrol microbes,respectively. Further,we prospect the directions of future study on rhizosphere biocontrol consortia and the tactics for elevating the efficiencies of their construction and application. Finally,we point out the concerned scientific topics in further deepening the study on the efficacy-enhancing mechanism of rhizospheric biocontrol consortia,and emphasize the strategy for constructing them by simplifying rhizosphere microbiome and the importance of applying rhizosphere biocontrol consortia together with a single strain of the disease-suppressive microorganisms.

Research Progressing in Signals and Molecular Mechanisms of Plant Growth-Promoting Rhizobacteria to Regulate Plant Root Development

FU Yan-song, LI Yu-cong, XU Zhi-hui, SHAO Jia-hui, LIU Yun-peng, XUAN Wei, ZHANG Rui-fu

2020, 36(9): 42-48. doi:10.13560/j.cnki.biotech.bull.1985.2020-0997

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Plant growth-promoting rhizobacteria（PGPRs）play essential roles in plant growth and health,they are the major source of microbial fertilizers,and contribute significantly to the reduction of chemical fertilizer input and green development of agriculture. PGPRs promote plant growth and improve nutrients use efficiency through two central mechanisms：activating rhizosphere nutrients and improving root development. Rhizosphere colonization of PGPRs can regulate the root system architecture（RSA）,affecting the elongation of the primary root and promoting the formation of lateral roots and root hairs. PGPRs secrete phytohormones to be involved in the root development directly,and some signal molecules to regulate RSA indirectly mediated by the plant endogenous molecular pathways. PGPRs have various effects on plant RSA,many novel signals secrete from PGPRs to affect plant RSA,and their regulating mechanisms are required to be explored,which may be used to form the ideal RSAs for different crops to increase nutrient use efficiency. Finally,these studies will enhance the contribution of PGPRs to the reduction of chemical fertilizers input and the green development of agriculture. This paper reviewed the identified phytohormones and signals secreted by PGPRs,summarized their molecular mechanisms to regulate plant RSA,and proposed the perspectives for future research.

Research Advances in Terpenoids Synthesis and Accumulation in Plants as Influenced by Arbuscular Mycorrhizal Symbiosis

XIE Wei, HAO Zhi-peng, GUO Lan-ping, ZHANG Xin, ZHANG Shu-bin, WANG You-shan, CHEN Bao-dong

2020, 36(9): 49-63. doi:10.13560/j.cnki.biotech.bull.1985.2020-0117

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Terpenoids play critical role in plants growth and coping with environmental stresses,and are the major active components in medicinal plants. Arbuscular mycorrhizal（AM）fungi are a group of soil-dwelling fungi that may form symbiotic associations with most terrestrial plants. AM symbiosis can improve plant growth and regulate plant secondary metabolisms,therefore AM inoculation is a promising strategy for promoting terpenoid-active ingredient accumulations in medicinal plants. In this paper,current research progresses in AM fungi and plant terpenoid metabolism was reviewed by analyzing the relevant literature published from 2000 to 2019. Factors influencing mycorrhizal effects on plant terpenoid metabolism,such as host plant families,terpenoid species,terpenoid accumulation organs,AM fungal species,experimental conditions,and experimental treatment types,were summarized. Moreover,we discussed the nutritional and non-nutritional mechanisms of mycorrhizal effects on plant terpenoids synthesis,transport and accumulation,which can be influenced by cultivation systems,inoculation methods,and environmental factors. Finally,we proposed the deficiencies and research perspectives in the research field.

Research Progress on Salt Tolerance and Growth-promoting Mechanism of Bacillus

HU Yu-jie, ZHU Xiu-ling, DING Yan-qin, DU Bing-hai, WANG Cheng-qiang

2020, 36(9): 64-74. doi:10.13560/j.cnki.biotech.bull.1985.2020-0746

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Soil salinization seriously restricts the development of agriculture and this is one of urgent problems to be solved in China. Bacillus has strong stress resistance and excellent salt tolerance,thus which can alleviate the damage caused by salt stress and improve the salt tolerance of plants. This paper briefly summarizes the general characteristics and application of Bacillus as well as the salt-tolerant genes of Bacillus,and reviews the salt-tolerant and growth-promoting mechanism of Bacillus on plant growth from seven aspects. In addition,this paper also discusses some issues in the research and development of Bacillus,and prospects the interactions between Bacillus as plant rhizosphere growth-promoting bacterium and plants or soil,aiming to provide theoretical reference for the improvement of saline-alkali land,the industrial application of microbial strains,and the growth promotion of crops.

Mechanisms of Salt Tolerance and Growth Promotion in Plant Induced by Plant Growth-Promoting Rhizobacteria

PAN Jing, HUANG Cui-hua, PENG Fei, YOU Quan-gang, LIU Fei-yao, XUE Xian

2020, 36(9): 75-87. doi:10.13560/j.cnki.biotech.bull.1985.2020-0511

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Soil salinization is one of the main manifestations of global land desertification. Since the constant interference of natural factors and anthropogenic activities,the land area affected by salinization is increasing year by year,and this seriously restricts agricultural development and threatens ecological security. Plant growth-promoting rhizobacteria（PGPR）,a group of bacteria that reside in the vicinity of rhizosphere or attached to roots,could not only improve the salt tolerance of plants to adapt to saline habitats,but also cooperate with host plants to remediate saline land. This review expounds both the growth promotion effects of PGPR on host plant under salt stress and the relevant mechanisms of PGPR to induce salt tolerance of host plants,and then gives an outlook for the future development trend based on the above discussion,aiming to serve the improvement of saline soil and the ecological restoration and reconstruction in saline habitat.

Research Progress on the Relationship Between Soil Microorganism and Tobacco Bacterial Wilt

LI Jun-ling, MA Xiao-han, ZHANG Yu-dan, JIA Wei, XU Zi-cheng

2020, 36(9): 88-99. doi:10.13560/j.cnki.biotech.bull.1985.2019-1268

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Tobacco bacterial wilt is a soil borne disease caused by Ralstonia solanacearum,and seriously threatens the tobacco production in China. Occurrence of tobacco bacterial wilt is closely related to the variation of soil microbe. Here,the relationship between tobacco bacterial wilt and soil microbes is systematically reviewed in the aspect of soil properties,microbial community structure and ecological network. In addition,the related mechanisms of biological control for tobacco bacterial wilt are also summarized,aiming to provide references for ecological control of tobacco bacterial wilt.

Effects of Inoculation of Rhizobacteria Containing ACC Deaminase on Soybean Growth Under Alkaline Stress

LIANG Ye, HE Chu-ting, YANG Yue, ZHANG Yu-fen, JIANG Fan

2020, 36(9): 100-108. doi:10.13560/j.cnki.biotech.bull.1985.2020-0936

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Alkaline stress leads to severe crop loss. Inoculation of ACC-deaminase-containing rhizobacteria can improve plant resistance to several abiotic stresses,however,little research has been done on whether such bacteria can improve plant resistance to alkalinity. Using the ACC deaminase-containing rhizobacteria Variovorax paradoxus 5C-2 and Glycine max（L.）Merr. Huang 13 as the experimental materials,we studied the effects of the inoculation of V. paradoxus 5C-2 on the growth of soybean under alkali stress. The results showed that 40 mmol/L NaCl treatment presented no effect on soybean growth,but at 16 d after treated with alkaline salts（pH values 8.12 and 9.04,respectively）containing 40 mmol/L Na⁺,the biomass of soybean aboveground and root,total lateral root length,chlorophyll content and photosynthetic efficiency dramatically reduced,while stomatal resistance significantly increased. After inoculation of the rhizobacteria,the dry weight of shoot and root increased. Under the condition of pH 9.04,the total lateral root length,chlorophyll content and maximum photosynthetic potential of soybean increased clearly after the inoculation,and stomatal resistance decreased significantly. These experimental results demonstrated that,under the high pH condition,inoculation of rhizospheric bacteria V. paradoxus 5C-2 may alleviate the stress effects of alkaline salts on soybean plant.

Isolation and Identification of IAA-producing Bacillus sp on Potato Rhizosphere and Its Growth-promoting Effect

LI Pei-gen, YAO Ya-qian, SONG Ji-xiang, WANG Tian-qi, ZHOU Bo, WANG Bing, LIN Rong-shan

2020, 36(9): 109-116. doi:10.13560/j.cnki.biotech.bull.1985.2019-1203

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Eleven Bacillus strains that have promising ability of producing IAA were isolated from the potato rhizosphere soil in Heilongjiang. The strain HL379 with high yield was identified by morphology,Biolog and 16S rDNA sequence analysis. The pot experiments of growing Arabidopsis thaliana and potato were carried out to study its growth promotion function. The strain was identified as Bacillus aryabhattai,and the yield of IAA was 47.8 mg/L. Growth experiments in the pot showed that potato plant height,fresh weight,dry weight,and root length increased by 13%,32%,107% and 20.8%,respectively,and potato yield increased by 38%,meaning that it had a significant promotion effect on potato growth. It reveals that HL379 has certain application potential in plant growth promotion.

Effects of Crop Rotation on Bacterial Communities in Cotton Rhizosphere Soil

WANG Hong-jie, LIU Shao-dong, LIU Rui-hua, ZHANG Si-ping, YANG Jun, PANG Chao-you

2020, 36(9): 117-124. doi:10.13560/j.cnki.biotech.bull.1985.2020-0685

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In order to analyze the effect of crop rotation on bacterial diversity and community structure in the rhizosphere soil of cotton,cotton was used as the research object,peanut and millet as rotation crop materials. IonS5^TMXL high-throughput sequencing platform was used to sequence the 16S rDNA of each treated soil sample. The results showed that the crop rotation increased the yield of cotton,by 32.19% with peanut-cotton rotation and 18.13% with millet-cotton,respectively. Though rotation was not sufficient to completely alter the bacterial community structure in the rhizosphere soil of cotton,it led to a significant increase in the relative abundances of Bacteroidetes,Proteobacteria,Planctomycetes,and Sphingomonas in the flora. Enrichment of these beneficial phylum or genera inhibited the activity of pathogens and led to a decrease in bacterial community richness. Rotation planting may affect soil bacteria to improve quality of cotton life,and then increase cotton yield.

Screening and Identification of an Antagonistic Strain Against Fusarium oxyporum f. sp. cucumerinum and Optimization of Culture Conditions

ZHANG Mei-jun, WU Qing, YIN Cui, WANG Ni, MA Xiao-qing, MA Xiao-xia, CAO Yun-e

2020, 36(9): 125-136. doi:10.13560/j.cnki.biotech.bull.1985.2020-0578

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An antagonistic strain was screened from wormcast and it inhibited the Fusarium oxyporum f. sp. cucumerinum（Foc）,and its inhibitory capability was improved via optimizing culture condition. Using the Foc as experimental pathogen and wormcast as the experimental material,an antagonistic strain named WQ-5 with strong antagonistic activity to Foc was isolated using dilution plate coating and plate confrontation method. The strain WQ-5 was identified by morphological observation,physiological and biochemical characteristics and 16S rRNA sequence analysis. The culture conditions were optimized by single factor and response surface analysis,and its control effect was verified by seed germination test and pot experiment. The strain WQ-5 was identified as Bacillus amyloliquefaciens. The optimized culture conditions,i.e. fructose 30 g/L,peptone 18.9 g/L,magnesium sulfate 3 g/L,glycerol 1 g/L,dipotassium hydrogen phosphate 1.5 g/L,pH 5.8,liquid-loading rate 38.6%,rotating speed 240 r/min,inoculation rate 5%,temperature 30℃ and cultural time 24 h,increased inhibition zone diameter by 24.8% and the inhibition rate of pathogens by 30.39%,as compared to the non-optimized conditions. The control effect experiment showed that the strain WQ-5 reduced the disease incidence of seeds by 20%,increased germination rate by 150%,significantly enhanced the root length,stem length and vigor index of seeds,decreased the disease index of plants by 48.65%,and decreased the disease index of plants by 51.67%. In conclusion,the strain WQ-5 demonstrates promising control effect on Fusarium wilt caused by Foc and presents potential research and application value.

Effects of Livestock Manure and Straw Returning to Field on Microbial Community Structure Around Maize Rhizosphere

XU Lai-peng, WAN Xian-hua, SUN Xiang-li, CAO Yan-fang, LI Hui, TIAN Ya-dong, LIU Xiao-jun, KANG Xiang-tao, WANG Yan-bin

2020, 36(9): 137-146. doi:10.13560/j.cnki.biotech.bull.1985.2020-0913

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Livestock manure and straw as organic matter may increase the abundance and diversity of soil microorganisms,improve soil grain structure and thus increase crop yield. In this study,microorganisms around maize rhizosphere were used as the research object to explore the effects of livestock manure and straw returning to field on the microbial structure. The Illumina Miseq technology was used for high-throughput sequencing of the V4-V5 variable region of 16S rDNA gene from the maize rhizobacteria of two treatments（LM group：livestock manure;SR group：straw returning to field）. The results showed that：1）there was no significant difference in Shannon index,Simpson index and Chao1 index between SR group and LM group（P >0.05）,both had high flora abundance;2）the main phyla composition was similar at the phylum level,the relative abundance of Firmicutes and Chloroflexi in SR group was higher than that in LM group（P < 0.01）,and the relative abundance of Proteobacteria in LM group was higher than that in SR group（P < 0.01）;3）the main genus composition was similar at the genus level,the relative abundance of Bacillus spp. in SR group was higher than that in LM group（P < 0.01）;4）the functional prediction of rhizosphere microbial community mainly concentrated in the metabolic pathways of substances such as carbon,amino acids,coenzymes and vitamins,terpenoids and polyketides,etc. The above results revealed that the microbial community structure of maize rhizosphere was similar under the application of livestock manure and straw returning to field,only the proportion of some bacteria was different,but the difference of effect on maize yield was not obvious（P > 0.05）,indicating that continuous straw returning to field had the same effect as livestock manure applied.

Screening,Identification and Phosphate-solubilizing Characteristics of Phosphate-solubilizing Paraburkholderia sp. from Pinus massoniana Rhizosphere Soil

LÜ Jun, PAN Hong-xiang, YU Cun

2020, 36(9): 147-156. doi:10.13560/j.cnki.biotech.bull.1985.2020-0470

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Screening a highly efficient phosphate-solubilizing bacteria from the rhizosphere soil of Pinus massoniana is to provide strain resources for artificial planting and microbial fertilizer of P. massoniana seedlings. The plate dilution method and molybdenum antimony resistance colorimetric method were used to isolate and screen phosphate-solubilizing bacteria from the rhizosphere soil of P. massoniana. The strain was identified based on the morphological,physiological and biochemical properties and the analysis of 16S rDNA gene sequence. By measuring the abilities of the strain solubilizing 4 poorly soluble phosphates,a more easily soluble phosphate was screened out,and the solubilizing characteristics of this phosphate by the strain under different conditions were explored. Finally,the effects of the strain on the growth of P. massoniana seedlings were analyzed by measuring the seedling biomass,plant phosphorus content,and available phosphorus content in the rhizosphere soil. A strain of bacterium WJ2 with strong phosphorus-solubilizing capacity was screened from the rhizosphere of P. massoniana. The amount of solubilized phosphorus reached 244.13 mg/L after the strain was cultured in inorganic phosphate liquid medium for 5 d. The strain was identified as Paraburkholderia sp. The highest phosphate solubilizing capacity of the strain in 4 phosphorus source media was Ca₃（PO₄）₂（204.42 mg/L）> AlPO₄（177.70 mg/L）> FePO₄·2H₂O（151.82 mg/L）> CaHPO₄·2H₂O（108.19 mg/L）. Pearson correlation analysis showed that there was a significantly negative correlation between the changes of phosphate solubilizing capacity and pH of WJ2 in these 4 different inorganic phosphorus sources media. The strain WJ2 utilized various carbon sources such as lactose and glucose,and effectively solubilized Ca₃（PO₄）₂ while using urea,ammonium sulfate,ammonium nitrate as nitrogen sources,and it had a high ability of solubilizing phosphate under the conditions of C/N 10∶1,concentration of NaCl 0 g/L,and initial pH 8.0. In the pot experiment,the seedling height,taproot length and fresh weight of P. massoniana seedlings after inoculating the strain WJ2 increased by 11.74%,45.73% and 46.00%,respectively,compared with the control. In addition,the soil available phosphorus and plant phosphorus content of P. massoniana inoculated with the strain WJ2 also increased significantly,the increase amounts were 18.32% and 13.82%,respectively. The strain WJ2 solubilizes a variety of insoluble inorganic phosphates,and presents a significant effect on increasing the effective phosphorus content of the soil and promoting the growth of P. massoniana seedlings. Thus,WJ2 is a promising microbial resource for the production of high-efficiency biological phosphorus fertilizer.

Isolation of Efficient Nitrogen-fixing Bacteria from the Rhizosphere of Sophora flavescens and the Growth-promoting Effect of Compound Microbial Fertilizer on Seedlings

LEI Hai-ying, ZHAO Qing-song, YANG Xiao, WANG Mao-mao, BAI Jie, SUN Yong-qi, WANG Zhi-jun

2020, 36(9): 157-166. doi:10.13560/j.cnki.biotech.bull.1985.2020-0379

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This work aims to obtain plant growth promoting rhizobacteria（PGPR）and clarify its growth-promoting characteristics,so as to promote the application of compound microbial fertilizer in matrix for Sophora flavescens seedlings. Serial dilution-plating method was used to isolate highly efficient rhizosphere nitrogen-fixing bacteria from the rhizosphere soil of three-year-old Sophora flavescens. The isolated bacteria were identified by morphological characteristics,physiological and biochemical characteristics and 16S rRNA gene sequence. Their growth-promoting ability of IAA production and phosphate-solubilizing,and nitrogenase activity were determined for screening fine strains to study their growth-promoting effects on potted S. flavescens seedlings. As a result,20 strains of S. flavescens PGPR were isolated and identified as Pseudomonas,Bacillus,Ensifer,Arthrobacter and Staphylococcus epidermidis;among them,10 strains were Pseudomonas and 4 strains were Bacillus. Bacillus strain NF2-4 had the strongest IAA production of 451 mg/L and phosphate-solubilizing capacity of 1.908 mg/L,the nitrogenase activity was 1.24 nmol/（g·h）,and thus NF2-4 and Pseudomonas sp. NF1-17 with the stronger growth promotion were used as the compound microbial fertilizer for pot experiment. The results showed that the growth promotion ability to S. flavescens seedlings was compound microbial fertilizer > microbial fertilizer NF2-4 > microbial fertilizer NF1-17 > control group. The compound microbial fertilizer demonstrated the best growth promoting effect on S. flavescens seedling.

Difference Analysis of the Community Diversity of Fungi in the Rhizosphere Soil of Zanthoxylum nitidum（Roxb.）DC in Different Regions

ZHANG Miao, CHEN Yu-feng, CHEN Long, HUANG Piao-ling, WEI Lu-ling

2020, 36(9): 167-179. doi:10.13560/j.cnki.biotech.bull.1985.2020-0667

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To understand the community diversity differences of fungi in the rhizosphere soil of Zanthoxylum nitidum（Roxb.）DC in different regions,the rhizosphere soils of Z. nitidum in 6 different regions were studied. The ITS1-2 of fungi in soil samples was sequenced on the high-throughput platform. The community diversity differences of fungi in the rhizosphere soil were analyzed by contrast,and the effect of environmental factors on fungal community was discussed combined redundancy analysis. Research showed that the community diversity of fungi in 6 regions was rich,while there were significant differences in fungal community structure and species richness among the samples. Ascomycota,unclassified,Basidiomycota,Mortierellomycota and Rozelomycota were common. The dominant community composition of each sample varied,but the dominance of Ascomycete was significant. The order of fungal community diversity from high to low was as B3> A3> A2> B2> B1> A1. Environmental factors such as soil pH,organic matter content,soil clay,soil sand,and soil powder（sand）grains all can affect the community composition and species abundance of fungi in the samples,but the influence degree of environmental factors on each sample differed.

Preparation of Drought-resistant and Salt-tolerant Bacteria and Its Effect on Germination of Licorice Seeds

ZHANG Xiao-jia, LU Ya-jun, ZHANG Wen-jin, ZHANG Yu, CUI Gao-chang, LANG Duo-yong, ZHANG Xin-hui

2020, 36(9): 180-193. doi:10.13560/j.cnki.biotech.bull.1985.2020-0120

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The objective of this work is to prepare the highly effective stress-resistant microbial agent and to study their effects on the growth,development and physiological and biochemical characteristics of licorice under drought and salt stress. Single factor and orthogonal experiments were used to optimize the fermentation medium of Bacillus pumilus. Single factor experiments were combined with the Box-Behnken test to optimize the fermentation process,and a highly effective stress-resistant microbial agent was obtained. The hydroponic germination test was used to verify its effects on licorice growth under drought and salt stress and its mechanism. Results showed that the optimized fermentation medium was as：maltose 4.0%,soy peptone 3.0%,KH₂PO₄ 0.01%,and the rest was water. The optimized fermentation process was as：fermentation time 42 h,fermentation temperature 34℃,initial pH 7.00,shaking speed 230 r/min,the inoculation amount 2.0%,and the liquid content 100 mL/250 mL. The germination and growth indexes of licorice seedlings under drought and salt stress were improved to varying degrees. The inoculated agent significantly increased the contents of antioxidants（glutathione and ascorbate）and osmotic regulators（soluble sugars and soluble proteins）. Inoculation with bacterial agents under drought and salt stress significantly increased the activities of carbon and nitrogen metabolism enzymes. The activity of nitrate reductase increased by 77% under drought stress,and the activity of sucrose invertase increased by 445.5% under salt stress. In conclusion,the B. pumilus obtained in this study may improve the drought resistance and salt tolerance of licorice by increasing the content of antioxidants,osmoregulatory substances and regulating the activities of carbon and nitrogen metabolism enzymes,thereby promote its growth.

Isolation of Specific Phage of Ralstonia solanacearum and Its Effects on Control of Soil-borne Bacterial Wilt Disease

WANG Xiao-fang, HOU Yu-gang, YANG Ke-ming, WANG Jia-ning, WEI Zhong, XU Yang-chun, SHEN Qi-rong

2020, 36(9): 194-201. doi:10.13560/j.cnki.biotech.bull.1985.2020-0816

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Soil-borne Ralstonia solanacearum is one of the most serious bacterial diseases,and phage,a virus specifically infecting bacteria,plays an important role in the control of R. solanacearum. Previous studies revealed that R. solanacearum in different regions have high polymorphism,while the phage is highly specific to host and its infection to R. solanacearum in the same region is strong. This study intends to in situ screen efficient and stable phage against R. solanacearum in a specific region,and to explore its antibacterial stability. A strain of R. solanacearum isolated from the Kirin Greenhouse in Nanjing was used as the host to screen its obligate phage from the diseased soil in the same area. A phage NJ-P3 with the strongest bacteriostatic ability was screened from it,its basic biological characteristics,stability and best preservation methods were tested indoors,and pot experiment to explore its optimal application methods. The phage NJ-P3 was a lytic podoviridae and there was a special protein in the genome that may be related to its strong lysis. Phage NJ-P3 had strong resistance,and tolerated to high temperature of 50℃,4-10 of pH,and 80 min of ultraviolet irradiation. NJ-P3 stored in SM buffer showed the highest stability in low temperature but can be stored in short time under normal temperature condition. Pot experiment demonstrated that the control effect on the soil-borne R. solanacearum was better compared to traditional soil drench（SD）method and increased by 37.5% on the average by the stem injection（SI）method for inoculating phage NJ-P3. In sum,we obtained a highly efficient and stable R. solanacearum-specific phage,and systematically explored the optimal storage conditions and application methods,which lays a theoretical foundation for the establishment of phage resource library of R. solanacearum and widespread application of phage in the prevention and control of soil-borne diseases in the future.

Isolation and Identification of Bacillus amyloliquefaciens T-6 and Its Potential of Resisting Disease and Promoting Growth

YAO Ya-qian, CHENG Na-na, LI Pei-gen, LU Yi, WANG Yan-gang, LIN Rong-shan, ZHOU Bo, WANG Bing

2020, 36(9): 202-210. doi:10.13560/j.cnki.biotech.bull.1985.2020-0847

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One Bacillus strain with antagonistic effect on peach root rot and owned a wide antibacterial range was isolated in situ from the peach orchard in Sihong,Jiangsu province. After analysis in morphology,Biolog physiology and biochemistry,16S rDNA sequence and specific PCR,the strain was identified as Bacillus amyloliquefaciens. The enzyme activity of bacteriostatic substance and growth promotion performance test revealed that the T-6 strain had the ability of producing protease,chitinase,as well as the ability of phosphorus-dissolving,phosphate-resolving and potassium-dissolving. In the pot experiment,the dry weight,total root length,and chlorophyll ratio of tomato seedlings in the T-6 treatment group increased by 31.81%,50.79%,and 35.11%,respectively,and the control effect reached 54.69%. Therefore,it is concluded that T-6 strain has great potential in biological control of soil-borne diseases such as peach root rot.

Effect of Morchella Mycelium on Soil Enzymatic Activity

WEI Jing-jing, ZHANG Hao-ran, WANG Zhi-ge, WANG Hui-chun

2020, 36(9): 211-217. doi:10.13560/j.cnki.biotech.bull.1985.2020-0090

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This work aims to study on the effect of Morchella mycelium on soil enzymatic activity. The sucrase and amylase activities of soil samples were determined by 3,5-dinitrosalicylic acid colorimetric method with single factor and orthogonal experimental design. The optimal condition of the soil sucrase activity was that the temperature was 28℃,the time was 3 d,the amount of added mycelium was 1∶5,and the soil sucrase activity increased 12 times compared with the control group. The optimal condition of soil amylase activity was as such：the amount of added mycelium was 1∶5,the temperature was 28℃,the time was 7 d,and the soil amylase activity increased 18 times compared with the control group. Morchella mycelium can improve the sucrase and amylase activities in the soil in a short period of time,which is of significance for improving soil.

Selection and Validation of Reference Genes in the Seeds of Paeonia delavayi in Quantitative Real-time PCR Analysis

PAN Mo-han, LU Tian-quan, TIAN Bo

2020, 36(9): 218-226. doi:10.13560/j.cnki.biotech.bull.1985.2019-1208

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The objective of this work is to analyze the expression stability of internal reference genes in the seeds at different developmental stages of Paeonia delavayi by real-time quantitative PCR（RT-qPCR）,and to screen the most stable reference genes in the maturation of seeds. RT-qPCR was used to analyze their expression differences of 8 housekeeping internal reference genes such as GAPC,PEPC,CYC,ACT,EF2α,TUB,ACP1 and RPL1 in P. delavayi seeds at different developmental stages. Three software（GeNorm,Norm Finder and BestKeeper）were applied to evaluate the expression stabilities of above genes in the seeds at different developmental stages of P. delavayi. The results from RT-qPCR and software demonstrated that the expression stabilities of 8 reference genes varied,the most stable ones were CYC and EF2,while the least sable one was ACT. RT-qPCR analysis of some related genes in the lipid metabolism and synthesis pathway of P. delavayi presented the same expression trend,which verified that CYC and EF2α were suitable as reference genes. In conclusion,2 internal reference genes,CYC and EF2α,are obtained to be the suitable internal reference genes for the seeds of P. delavayi.

Cloning and Expression Analysis of Oxaloacetate Hydrolase（LeOAH1）Gene from Lentinula edodes

DUAN Ying-ce, HU Zi-yi, YANG Fan, LI Jin-tao, WU Xiang-li, ZHANG Rui-ying

2020, 36(9): 227-234. doi:10.13560/j.cnki.biotech.bull.1985.2019-1246

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Oxaloacetate acetylhydrolase/Oxaloacetate hydrolase is one of the key enzymes for oxalate synthesis in Lentinula edodes. The LeOAH1 was cloned and its expression in vivo and in vitro was analyzed to lay the foundation for the functional research of LeOAH1. LeOAH1 was cloned from L. edodes L808 and bioinformatics analysis was conducted. The expression of LeOAH1 and the content of oxalic acid secreted by L. edodes under different pH conditions were respectively detected by RT-PCR and HPLC during the vegetative phase. In addition,a recombinant prokaryotic expression vector pET28a-oah1 was constructed and successfully expressed in Escherichia coli. The results showed that the length of gDNA of the LeOAH1 was 1 906 bp,the length of cDNA was 1 356 bp,encoding 451 amino acids,the protein molecular weight was about 48.9 kD,and the isoelectric point was 6.80. Bioinformatics analysis revealed that the enzyme was an unstable hydrophobic protein with four secondary structures of α-helix（40.08%）,random coil（34.59%）,extended chain（17.52%）,and β-sheet（7.10%）level structure;it was in the cytoplasm by subcellular localization. The sequence alignment results demonstrated that the enzyme had a conserved domain. The phylogenetic tree analysis suggested that the protein had the highest similarity with OAH from Japanese mushroom with 82% homology,followed by Pluteus cervinus with 64% homology. The RT-PCR and HPLC results showed that the expressions of LeOAH1 gene and the amount of oxalic acid secreted by L. edodes both increased with the increase of pH. The molecular weight of the induced protein in E. coli was in line with the predicted results. The gene characteristics and expression pattern of oxaloacetate hydrolase,as well as its relationship with oxalic acid secretion are preliminarily clarified.

Fusion Expression of Somatostatin with a Thermostable Xylanase and Characterization of the Fusion Protein

HUANG Kun-long, SU Xiao-yun, YAO Bin

2020, 36(9): 235-243. doi:10.13560/j.cnki.biotech.bull.1985.2020-0079

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The aim of this study is to obtain the fusion protein CbXyn10C-SS with antigenicity and immunogenicity,to efficiently express it,and to characterize the enzymatic properties. Somatostatin（SS）impedes feed transformation efficiency and animal growth in multiple ways. Therefore,using SS to immunize animals can avoid the inhibitory effect. However,the research of SS immunization mostly uses DNA vaccine as the carrier and highly efficient recombinant production of SS protein is seldom reported. Xylanase is widely used in feed,which can improve feed conversion rate. Till now,recombinant xylanase has been successfully expressed in microbial cells. The SS gene was fused to the 3' terminus of the thermostable xylanase gene CbXyn10C derived from the Caldicellulosiruptor bescii. The fusion protein CbXyn10C-SS presented antigenicity and immunogenicity. Being highly thermostable and efficient in catalysis,CbXyn10C-SS also demonstrated very similar biochemical characteristics and catalytic parameters to those of CbXyn10C,which is therefore suitable for the feed pelleting process. The successfully and efficiently expressed fusion protein CbXyn10C-SS has potential application value as a feed xylanase and somatostatin antigen.

Exploration of Key Functional Genes Affecting Milk Production Traits in Dairy Cattle Based on RNA-seq

CHEN Yi-dan, ZHANG Yu, YANG Jie, ZHANG Qin, JIANG Li

2020, 36(9): 244-252. doi:10.13560/j.cnki.biotech.bull.1985.2020-0255

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Exploring genetic variants affecting milk production traits is very crucial for molecular breeding in dairy cattle in China. In current study,RNA-seq technology was used to detect differential expression and genetic variation of genes in the blood samples between 2 groups of cows with high and low milk production. As a result,431 differentially expressed genes were detected between the 2 groups. GO and pathway analysis showed that these differentially expressed genes were enriched in biological processes such as metabolic processes,hormone regulation,cell migration and immune function. In addition,a total of 373 049 SNPs and 42 273 Indels were detected,of which 75 363 were novel genetic mutations. By integrating the allele frequency distribution of genetic variations with the expression level of genes in the 2 groups,12 candidate functional genes affecting milk production traits were suggested,namely ASS1,CKB,GGT1,MGAM,SDSL,HP,LTF,MMP9,UPP1,DEFB4A,PGLYRP1 and MS4A8,and in them 14 important genetic variations occurred.

Construction of srtA-Knockout Strain in Staphylococcus aureus by CRISPR/Cas9 Technology

JIANG Cheng-hui, ZENG Qiao-ying, WANG Meng, PAN Yang-yang, LIU Xu-ming, SHANG Tian-tian

2020, 36(9): 253-265. doi:10.13560/j.cnki.biotech.bull.1985.2020-0195

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This work aims to construct a srtA-deleted strain and srtA-complemented strain that is resistant to methicillin-resistant Staphylococcus aureus（MRSA）USA300 strain by CRISPR/Cas9,and to analyze its toxic effect on strain. The pCasSA-sgRNA plasmid was constructed using pCasSA vector and products of 3 pairs of srtA gRNAs. Then the pCasSA-sgRNA plasmid was modified through deletion S. aureus RN4220 strain,and transferred into USA300 strain,and the efficiency of cutting pCasSA-sgRNA plasmid was measured. The homologous sequences in the upstream（srtA-L）and downstream（srtA-R）of the srtA were amplified by PCR respectively,and their products were ligated by overlapping PCR. Then ligated products were inserted into pCasSA-sgRNA plasmid for constructing deletion pCasSA-sgRNA-srtA plasmid. The pCasSA-sgRNA-srtA plasmid was transferred into USA300 strain,and the srtA-deleted strains were obtained by screening. The growths of USA300 after the srtA deletion were compared,and their pathogenicity in mice were evaluated based on the survival rate of infected mice,bacteria burdens in organs,and histopathological changes in kidney. Meanwhile,the pLI50 plasmid was used as the vector to construct the complemented plasmid pLI50-srtA and complemented strain to verify whether the phenotypes were various in different strains. Two srtA-deleted strains were obtained after chloramphenicol screening. The deletion of srtA did not affect the growth of USA300;however,it reduced the mortality of the infected mice,as well as the bacteria burdens and pathological changes in kidney,which could be recovered by srtA complemented strains. The srtA-deleted strains and -complemented strains in the USA300 were successfully constructed in this study.

Optimization of GoldenBraid Digestion-ligation Reaction System

XU Mei-hui, ZHANG Yao-jie, TANG Ke-xuan, MIAO Zhi-qi

2020, 36(9): 266-274. doi:10.13560/j.cnki.biotech.bull.1985.2020-0161

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The objective of this work is to establish an efficient and stable GoldenBraid（GB）restriction-ligation assembly reaction system. In this study,enzyme digestion time,buffer system,restriction enzymes and ratio of substrates were optimized to improve the efficiency of effective cloning,and the optimal reaction system was determined. Results showed that the percentage of effective cloning was nearly 99% under 4 min enzyme digestion time,1 μL FastDigest Buffer（10×）buffer system,1 mmol/L ATP,0.5 μL FastDigest Esp3I or FastDigest Eco31I,and 3∶1 molar ratio of the inserts and acceptor plasmid in reaction system,which was over 4 times of that before optimization（BsmBI 8% and BsaI 23%）,and digestion-ligation efficiency was nearly 100%. The percentage of effective cloning significantly increased by optimizing reaction system even with less volume of restriction enzymes,compared to the original protocol with 1 μL restriction enzymes. This indicates that the efficiency and practicability of digestion-ligation in GB system may be improved via the optimized process.

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2020, 36(9): 267-267.

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