INRAe, Université de Montpellier, UMR1333-DGIMI, 34095 Montpellier Cedex 05, France.
INRAe, Université de Montpellier, UMR1333-DGIMI, 34095 Montpellier Cedex 05, France.
J Microbiol Methods. 2020 May;172:105911. doi: 10.1016/j.mimet.2020.105911. Epub 2020 Mar 30.
Pseudomonas protegens is a rhizosphere pseudomonad with a high agronomical potential (entomopathogenic and beneficial to plants) and bio-catalytic activities, but no selective medium has been described for its isolation. We developed a semi-selective minimum agar medium for the specific isolation and growth of P. protegens. We searched for both (i) a carbon source allowing the growth of P. protegens but potentially inhibiting the growth of other pseudomonads and (ii) an antimicrobial agent suppressing other members of the bacterial rhizosphere community. The M9-PP-agar medium consists of M9 base agar with adipic acid as the only carbon source and Irgasan® as an anti-bacterial agent. We tested the selectivity and sensitivity of M9-PP-agar by measuring the growth of 68 bacterial strains from 36 different species on this medium. Ten of the species tested were able to grow on M9-PP-agar medium: four species from the Pseudomonadaceae (Pseudomonas aeruginosa, Pseudomonas protegens, Pseudomonas putida, Stenotrophomonas maltophilia) as well as Achromobacter xylosoxidans, Agrobacterium tumefaciens, Brevundimonas sp., Serratia liquefaciens, Serratia marcescens and Variovorax paradoxus. All colonies were white, except for those of P. protegens (12 strains), which were typically brown. We demonstrated the efficiency of the M9-PP agar medium for P. protegens isolation, by inoculating two soils with the reference strain P. protegens CHAO and then reisolating them. We also developed a fitF-PCR test targeting a regulator gene of the insecticidal P. protegens fit locus, for the rapid molecular detection of P. protegens colonies. We, therefore, developed a highly specific process for the routine isolation of new P. protegens strains from the soil environment, based on the use of a semi-selective medium and the specific color of colonies.
生防假单胞菌是一种根际假单胞菌，具有很高的农业潜力（具有昆虫病原性和对植物有益）和生物催化活性，但尚未描述其分离的选择性培养基。我们开发了一种半选择性最小琼脂培养基，用于特定分离和生长生防假单胞菌。我们同时寻找（i）一种允许生防假单胞菌生长但可能抑制其他假单胞菌生长的碳源，以及（ii）一种抑制细菌根际群落其他成员的抗菌剂。M9-PP-agar 培养基由 M9 基础琼脂和己二酸组成，己二酸是唯一的碳源，而 Irgasan®是一种抗菌剂。我们通过测量 68 株来自 36 个不同种的细菌菌株在该培养基上的生长情况，测试了 M9-PP-agar 的选择性和敏感性。在所测试的 10 个物种中，有 4 个种（铜绿假单胞菌、生防假单胞菌、恶臭假单胞菌、嗜麦芽寡养单胞菌）和无色杆菌属、根癌农杆菌、短小芽孢杆菌、液化沙雷氏菌、粘质沙雷氏菌和奇异变形菌能够在 M9-PP-agar 培养基上生长。所有菌落均为白色，除生防假单胞菌（12 株）外，其典型颜色为棕色。我们通过用参考菌株生防假单胞菌 CHAO 接种两种土壤，然后重新分离它们，证明了 M9-PP 琼脂培养基用于生防假单胞菌分离的效率。我们还开发了一种 fitF-PCR 测试，针对杀虫生防假单胞菌 fit 基因座的调节基因，用于快速分子检测生防假单胞菌菌落。因此，我们开发了一种基于使用半选择性培养基和菌落特异性颜色的新的生防假单胞菌菌株从土壤环境中常规分离的高度特异性方法。
