Simons M, van der Bij A J, Brand I, de Weger L A, Wijffelman C A, Lugtenberg B J
Leiden University, Clusius Laboratory, The Netherlands.
Mol Plant Microbe Interact. 1996 Sep;9(7):600-7. doi: 10.1094/mpmi-9-0600.
A gnotobiotic system for studying tomato rhizosphere colonization by Pseudomonas bacteria was developed. The system is based on sterile seedlings that are inoculated with one or two strains and subsequently grown in a sterile glass tube containing quartz sand. After 7 days of growth in a climate-controlled growth chamber, the number of bacteria present on the root tip was analyzed. The system was optimized with respect to root morphology, inoculation of the seedling, and isolation of root tip bacteria. With this system, rhizosphere colonization on tomato, radish, wheat, and potato was analyzed. For detailed analysis of tomato rhizosphere colonization by some representative plant growth-promoting rhizo-bacteria, the colonization of known poor, moderate, and good potato root-colonizing Pseudomonas strains and of four Rhizobium strains was determined. All strains colonized the root tips when inoculated as single strains. When inoculated in competition with the efficient root colonizer P. fluorescens strain WCS365, many strains were outcompeted. Mutants of Pseudomonas biocontrol bacteria lacking flagella or the O-antigen of lipopolysaccharide (LPS), which were isolated in previous studies and shown to be impaired in potato rhizosphere colonization in field soil systems, showed a reduced colonization ability in the gnotobiotic system also. The gnotobiotic system was used to screen a collection of 300 random P. fluorescens WCS365::Tn5 mutants for colonization-impaired mutants. Three novel mutants were found that were outcompeted by the wild-type strain in tomato root tip colonization but were not impaired in known colonization traits such as motility, amino acid auxotrophy, and presence of the O-antigenic side chain of LPS. One strain appeared to be a thiamine auxotroph, suggesting that the root does not secrete a sufficient amount of thiamine to support growth of this strain. The other two mutants had a reduced growth rate in laboratory media, suggesting that growth rate is an important colonization factor. As the system is gnotobiotic and devoid of field-soil variables, it can also be used to study the effects of selected biotic and abiotic factors on colonization.
开发了一种用于研究假单胞菌对番茄根际定殖的悉生生物系统。该系统基于无菌幼苗，用一两种菌株接种，随后在装有石英砂的无菌玻璃管中生长。在气候控制的生长室中生长7天后，分析根尖上存在的细菌数量。该系统在根形态、幼苗接种和根尖细菌分离方面进行了优化。利用该系统，分析了番茄、萝卜、小麦和马铃薯的根际定殖情况。为了详细分析一些具有代表性的植物促生根际细菌对番茄根际的定殖情况，测定了已知在马铃薯根际定殖能力差、中等和良好的假单胞菌菌株以及四种根瘤菌菌株的定殖情况。所有菌株单独接种时都能定殖根尖。当与高效根际定殖菌荧光假单胞菌菌株WCS365竞争接种时，许多菌株被淘汰。在先前研究中分离出的缺乏鞭毛或脂多糖（LPS）O抗原的假单胞菌生物防治细菌突变体，在田间土壤系统中马铃薯根际定殖能力受损，在悉生生物系统中定殖能力也降低。该悉生生物系统用于筛选300个荧光假单胞菌WCS365::Tn5随机突变体，以寻找定殖受损突变体。发现了三个新的突变体，它们在番茄根尖定殖中被野生型菌株淘汰，但在已知的定殖特性如运动性、氨基酸营养缺陷型和LPS的O抗原侧链存在方面没有受损。一个菌株似乎是硫胺素营养缺陷型，这表明根分泌的硫胺素不足以支持该菌株的生长。另外两个突变体在实验室培养基中的生长速率降低，这表明生长速率是一个重要的定殖因素。由于该系统是悉生生物系统且没有田间土壤变量，它还可用于研究选定的生物和非生物因素对定殖的影响。
