M. Simons et al., GNOTOBIOTIC SYSTEM FOR STUDYING RHIZOSPHERE COLONIZATION BY PLANT GROWTH-PROMOTING PSEUDOMONAS BACTERIA, Molecular plant-microbe interactions, 9(7), 1996, pp. 600-607
A gnotobiotic system for studying tomato rhizosphere colonization by P
seudomonas bacteria was developed. The system is based on sterile seed
lings that are inoculated with one or two strains and subsequently gro
wn in a sterile glass tube containing quartz sand. After 7 days of gro
wth in a climate-controlled growth chamber, the number of bacteria pre
sent on the root tip was analyzed. The system was optimized with respe
ct to root morphology, inoculation of the seedling, and isolation of r
oot tip bacteria. With this system, rhizosphere colonization on tomato
, radish, wheat, and potato was analyzed. For detailed analysis of tom
ato rhizosphere colonization by some representative plant growth-promo
ting rhizobacteria, the colonization of known poor, moderate, and good
potato root-colonizing Pseudomonas strains and of four Rhizobium stra
ins was determined. All strains colonized the root tips when inoculate
d as single strains. When inoculated in competition with the efficient
root colonizer P. fluorescens strain WCS365, many strains were outcom
peted. Mutants of Pseudomonas biocontrol bacteria lacking flagella or
the O-antigen of lipopolysaccharide (LPS), which were isolated in prev
ious studies and shown to be impaired in potato rhizosphere colonizati
on 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 colon
ization-impaired mutants. Three novel mutants were found that were out
competed by the wild-type strain in tomato root tip colonization but w
ere not impaired in known colonization traits such as motility, amino
acid auxotrophy, and presence of the O-antigenic side chain of LPS. On
e 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 labor
atory media, suggesting that growth rate is an important colonization
factor. As the system is gnotobiotic and devoid of field-soil variable
s, it can also be used to study the effects of selected biotic and abi
otic factors on colonization.