Establishment of a Rapid qPCR Detection Method for Burkholderia stagnalis
DONG Jin-Bao1, DENG Ying-Long1, YANG Bi-Chen1, ZHANG Ying-Long1, HUANG Hong-Ping1, ZHU You-Yong1, HE Xia-Hong1,2,*, GUO Li-Wei1,*
1 State Key Laboratory for Conservation and Utilization of Bio-Resources in Yunnan, Yunnan Agricultural University, Kunming 650201, China; 2 Provincial Key Laboratory for Conservation and Utilization of In-forest Resource, Southwest Forestry University, Kunming 650224, China
Abstract：A strain of Burkholderia stagnalis B11 was previously isolated by our research group, which exhibits an effective biocontrol effect against root rot disease of Panax notoginseng. To establish a rapid detection method based on qPCR for quantifying the colonization capacity of strain B11 in soil, a comparative genomic analysis was conducted. A specific gene, gene 1214 (681 bp), located within the MIBiG cluster BGC0001120 and annotated as homolog of burkholderic acid synthesis genes, was selected as the target. Specific primers targeting strain B11 were designed using NCBI Primer-BLAST. qPCR results showed that the designed primers exhibited good specificity. The established standard curve equation was y=-3.3689x+11.343 (R2=0.9991), with an amplification efficiency of 98.08% and a sensitivity of 0.02 pg/µL. This method was used to detect the colonization of B. stagnalis B11 in continuous cropping soil of P. notoginseng. The result showed that the abundance of B11 in the soil decreased with the increase of the bacterial suspension dilution ratio. A significant decrease occurred at a 10-fold dilution, and no significant difference compared with the control (CK) was observed at a 100-fold dilution. The colonization dynamics exhibited an initial increase followed by a subsequent decrease, with stable colonization maintained in the soil from 1 to 5 d after irrigation. This study established a qPCR method that enabled rapid quantitative detection of B. stagnalis B11, which provides a reliable technical means for evaluating the colonization capacity of this strain in the soil environment and facilitating the development of an efficient detection system.
[1] 高圣风, 刘爱勤, 桑利伟, 等. 2017. 枯草芽胞杆菌VD18R19在胡椒上的定殖动态及促生作用和对胡椒瘟病的防治效果[J]. 中国生物防治学报, 33(5): 650-657. (Gao S F, Liu A Q, Sang L W, et al.2017. Colonization dynamics and growth promotion effect of Bacillus subtilis VD18R19 in black pepper plant and its biological control effects on phytophthora rot disease[J]. Chinese Journal of Biological Control, 33(5): 650-657.) [2] 梁子英, 刘芳. 2020. 实时荧光定量PCR技术及其应用研究进展[J]. 现代农业科技,(6): 1-3, 8. (Liang Z Y, Liu F.2020. Research progress on real-time quantitative PCR technology and its application[J]. Modern Agricultural Science and Technology,(6): 1-3, 8.) [3] 刘晓萌, 苏振贺, 宣立峰, 等. 2022. 枯草芽胞杆菌HMB19198在番茄叶片上定殖能力的分子检测[J]. 中国生物防治学报, 38(2): 487-494. (Liu X M, Su Z H, Xuan L F, et al.2022. Quantitative detection of Bacillus subtilis HMB19198 on tomato leaf by real-time PCR[J]. Chinese Journal of Biological Control, 38(2): 487-494.) [4] 孙正祥, 孟祥佳, 龙欣钰, 等. 2021. 伯克霍尔德氏菌YZU-S230对西瓜枯萎病的防效及其促生作用[J]. 长江大学学报(自然科学版), 18(2): 82-88. (Sun Z X, Meng X J, Long X Y, et al.2021. Effects of Burkholderia sp. YZU-S230 on the control and growth promotion of watermelon Fusarium wilt[J]. Journal of Yangtze University (Natural Science Edition), 18(2): 82-88.) [5] 陶萌, 李世东, 张拥华. 2009. 土壤中生防菌粉红粘帚霉67-1的荧光定量PCR检测方法[J]. 中国生物防治, 25(1): 35-40. (Tao M, Li S D, Zhang Y H.2009. Development of real-time PCR quantitative assay for rapid detection of Gliocladium roseum 67-1, an effective biocontrol agent, in soil[J]. Chinese Journal of Biological Control, 25(1): 35-40.) [6] 王晓强. 2016. 植物根际促生菌Lyc2和XW10的鉴定及抑菌机理研究[D]. 博士学位论文, 山东农业大学, 导师: 李向东, pp. 17-18. (Wang X Q.2016. Identification and antimicrobial mechanism of plant growth-promoting rhizobacteria Lyc2 and XW10[D]. Thesis for Ph.D., Shandong Agricultural University, Supervisor: Li X D, pp. 17-18.) [7] 徐志周, 王明元, 杜锦鹏, 等. 2019. 一株香蕉枯萎病拮抗菌HQB-1的分离鉴定及其发酵条件优化[J]. 微生物学通报, 46(7): 1611-1618. (Xu Z Z, Wang M Y, Du J P, et al.2019. Isolation, identification and fermentation optimization of an antagonistic bacterial strain HQB-1 against banana wilt disease[J]. Microbiology China, 46(7): 1611-1618.) [8] 杨弘华, 宋燕燕, 林晓波, 等. 2016. 实时荧光定量PCR技术应用研究[J]. 山东化工, 45(22): 46-47. (Yang H H, Song Y Y, Lin X B, et al.2016. Summary of research and development of real time fluorescent PCR technology[J]. Shandong Chemical Industry, 45(22): 46-47.) [9] 杨威, 刘苏闵, 郭坚华. 2010. 细菌定殖能力与其生物防治功能相关性研究进展[J]. 中国生物防治, 26(S1): 90-94. (Yang W, Liu S M, Guo J H.2010. Relationship between bacterial colonization and biocontrol efficacy[J]. Chinese Journal of Biological Control, 26(S1): 90-94.) [10] 杨云. 2024. 基于Burkholderia arboris PN-1对三七根腐病害的生防作用及其机制研究[D]. 博士学位论文, 云南师范大学, 导师: 官会林, pp. 15-16. (Yang Y.2024. Study on the biocontrol effects and mechanisms of Burkholderia arboris PN-1 on root rot of Panax notoginseng[D]. Thesis for Ph.D., Yunnan Normal University, Supervisor: Guan H L, pp. 15-16.) [11] 张玉勋, 李光, 张光明. 2000. 拮抗细菌在大棚温室番茄叶片定殖及对灰霉病害的控制效果[J]. 植物病理学报, (1): 91. (Zhang Y X, Li G, Zhang G M.2000. Colonization of antagonistic bacteria strains on tomato leaf in green house and their effects on gray mold (Botrytis cinerea)[J]. Acta Phytopathologica Sinica, (1): 91.) [12] Abbasian F, Ghafar-Zadeh E, Magierowski S.2018. Microbiological sensing technologies: A review[J]. Bioengineering, 5(1): 20. [13] Baek K Y, Lee H H, Son G J, et al.2018. Specific and sensitive primers developed by comparative genomics to detect bacterial pathogens in grains[J]. The Plant Pathology Journal, 34(2): 104-112. [14] Bergmark L, Poulsen P H B, Al-Soud W A, et al.2012. Assessment of the specificity of Burkholderia and Pseudomonas qPCR assays for detection of these genera in soil using 454 pyrosequencing[J]. FEMS Microbiology Letters, 333(1): 77-84. [15] Burghard V, Wende S, Ulrich A.2023. Strain-specific quantitative detection of two putative biocontrol strains for suppression of ash dieback[J]. Biological Control, 187: 105376. [16] Chapela M J, Garrido-Maestu A, Cabado A G.2015. Detection of foodborne pathogens by qPCR: A practical approach for food industry applications[J]. Cogent Food & Agriculture, 1(1): 1013771. [17] Chiarini L, Bevivino A, Dalmastri C, et al.2006. Burkholderia cepacia complex species: Health hazards and biotechnological potential[J]. Trends in Microbiology, 14(6): 277-286. [18] Coenye T, Vandamme P.2003. Diversity and significance of Burkholderia species occupying diverse ecological niches[J]. Environmental Microbiology, 5(9): 719-29. [19] Han D, Chen J, Chen W, et al.2023. Bongkrekic acid and Burkholderia gladioli pathovar cocovenenans: Formidable foe and ascending threat to food safety[J]. Foods, 12(21): 3926. [20] Higuchi R, Fockler C, Dollinger G, et al.1993. Kinetic PCR analysis: Real-time monitoring of DNA amplification reactions[J]. Nature Biotechnology, 11(9): 1026-1030. [21] Kim H J, Park S H, Lee T H, et al.2006. Identification of Salmonella enterica serovar Typhimurium using specific PCR primers obtained by comparative genomics in Salmonella serovars[J]. Journal of Food Protection, 69(7): 1653-1661. [22] Lee C, Lee H H, Mannaa M, et al.2018. Genomics-based sensitive and specific novel primers for simultaneous detection of Burkholderia glumae and Burkholderia gladioli in rice seeds[J]. Plant Pathology Journal, 34(6): 490-498. [23] Li P D, Zhu Z R, Zhang Y Z, et al.2022. The phyllosphere microbiome shifts toward combating melanose pathogen[J]. Microbiome, 10(1): 56-56. [24] Liu A X, Phillips K, Jia J Y, et al.2023. Development of a qPCR detection approach for pathogenic Burkholderia cenocepacia associated with fresh vegetables[J]. Food Microbiology, 115: 104333-104333. [25] Maeda Y, Shinohara H, Kiba A, et al.2006. Phylogenetic study and multiplex PCR-based detection of Burkholderia plantarii, Burkholderia glumae and Burkholderia gladioli using gyrB and rpoD sequences[J]. International Journal of Systematic and Evolutionary Microbiology, 56(Pt 5): 1031-1038. [26] Savazzini F, Longa C M O, Pertot I, et al.2008. Real-time PCR for detection and quantification of the biocontrol agent Trichoderma atroviride strain SC1 in soil[J]. Journal of Microbiological Methods, 73(2): 185-194. [27] Su X B, Shi Y, Li R H, et al.2019. Application of qPCR assays based on haloacids transporter gene dehp2 for discrimination of Burkholderia and Paraburkholderia[J]. BMC Microbiology, 19(1): 36. [28] Tan S H L, Yuwana P, Sia M H S.2021. Partial characterization of bacteriocin-like compound (BLIS) produced by Burkholderia stagnalis strain K23/3 against Burkholderia pseudomallei[J]. Malaysian Journal of Microbiology, 17(6): 1-15. [29] Taylor M J, Bentham R H, Ross K E.2014. Limitations of using propidium monoazide with qPCR to discriminate between live and dead Legionella in biofilm samples[J]. Microbiology Insights, 7(7): 15-24. [30] Yang S D, Xiao J, Liang T, et al.2021. Response of soil biological properties and bacterial diversity to different levels of nitrogen application in sugarcane fields[J]. AMB Express, 11(1): 172-172. [31] Zhu X F, Ning W Q, Xiao W, et al.2025. Isolation and identification of Burkholderia stagnalis YJ-2 from the rhizosphere soil of Woodsia ilvensis to explore its potential as a biocontrol agent against plant fungal diseases[J]. Microorganisms, 13(6): 1289-1289.
