In Situ Monitoring of Streptothricin Production byStreptomyces rocheiF20 in Soil and Rhizosphere

Abstract

The onset of streptothricin (ST) biosynthesis in Streptomyces rochei F20 was studied by using reverse transcription-PCR (RT-PCR) to detect transcripts of ST genes during growth in liquid medium, soil, and the rhizosphere. In situ results correlated with those obtained in vitro, illustrating the growth phase-dependent manner of ST production by F20. Maximal transcription of ST resistance (sttR) and biosynthesis (sttA) genes occurred during the transition between the exponential and stationary phases of growth, when the specific growth rate () started to decline. A higher level of gene expression of sttR versus sttA was observed in all experiments. In liquid culture, maximal transcript accumulation of the sttA gene was only ca. 40% that of the sttR gene. sttA and sttR mRNAs were detected in soil containing approximately 10 6 CFU of growing cells g of soil ؊1 . sttR mRNA was detected in sterile and nonsterile rhizosphere colonized with growing mycelium of F20 at 1.2 ؋ 10 6 and 4.0 ؋ 10 5 CFU g of soil ؊1 , respectively. However, neither sttR nor sttA transcripts were detected by RT-PCR in the rhizoplane, which supported a lower population density of F20 than the rhizosphere.The involvement of antibiotics in biological control has been reported (3,14,24,27). However, antibiotic production in natural environments such as soil is difficult to demonstrate, because low levels of nutrients limit growth and production (32). Detection of antibiotic production in soil could be achieved only in the presence of nutrient sources, such as roots, seeds, or straw fragments. In addition, many antibiotics are adsorbed to clay particles and are difficult to detect by extraction (22,23). Only a few studies using conventional extraction methods have provided direct evidence supporting the crucial role of antibiotics in disease suppression by biological control agents in situ. By use of a bioassay technique, geldanamycin was detected at 88 g g of soil Ϫ1 in pea rhizosphere where Streptomyces hygroscopicus var. geldanus was used to control Rhizoctonia solani infection (24). Levels of thiostrepton production in sterile soil were reported by Wellington et al. (31) within the range of 30 to 50 ng g of soil Ϫ1 following extraction and bioassay via a specific thiostrepton-inducible promoter coupled to a resistance gene. A similar method using a highly sensitive inducible promoter driving gfp expression was used to assay oxytetracycline production by Streptomyces rimosus in soil microcosms (11). Fluorescence-activated cell sorter analysis was used to detect fluorescing cells in soil extracts. Extraction of phenazine from wheat rhizosphere inoculated with Pseudomonas fluorescens 2-79 and Pseudomonas aureofaciens 30-84 and the use of mutants with phenazine blocks unequivocally proved the significance of antibiotic production in control of take-all disease caused by Gaeumannomyces graminis var. tritici (27). Other studies have demonstrated the positive correlation between production of antibiotics using fluorescent pseudomonads and control of...

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