Elevated Abundance of Bacteriophage Infecting Bacteria in Soil

Abstract

Here we report the first direct counts of soil bacteriophage and show that substantial populations of these viruses exist in soil (grand mean ‫؍‬ 1.5 ؋ 10 7 g ؊1 ), at least 350-fold more than the highest numbers estimated from traditional viable plaque counts. Adding pure cultures of a Serratia phage to soil showed that the direct counting methods with electron microscopy developed here underestimated the added phage populations by at least eightfold. So, assuming natural phages were similarly underestimated, virus numbers in soil averaged 1.5 ؋ 10 8 g ؊1 , which is equivalent to 4% of the total population of bacteria. This high abundance was to some extent confirmed by hybridizing colonies grown on Serratia and Pseudomonas selective media with cocktails of phage infecting these bacteria. This showed that 8.9 and 3.9%, respectively, hybridized with colonies from the two media and confirmed the presence of phage DNA sequences in the cultivable fraction of the natural population. Thus, soil phage, like their aquatic counterparts, are likely to be important in controlling bacterial populations and mediating gene transfer in soil.Direct counts made with electron and epifluorescence microscopy have shown bacteriophage to be abundant in water from marine (5, 6, 16) and freshwater (18) habitats. They occur at densities of up to 2.5 ϫ 10 8 ml Ϫ1 and are an average of 10-fold more abundant than their bacterial hosts. Fewer direct counts have been done on sediments, and results have been more variable. Numbers of bacterial viruses in sediment are higher than in water, and counts of up to 2 ϫ 10 9 ml Ϫ1 have been recorded (11). However, bacterial counts are also higher, and in some cases, bacteriophage in sediments are more abundant than bacteria (11, 13) and sometimes less abundant (10).Although it is difficult to grow bacteriophage from soil without enrichment (31), some viable counts have been reported (1,7,8,21,22,26,27). These range from 0 to 4 ϫ 10 4 g Ϫ1 and have been determined from a wide variety of host bacteria. To date, no direct counts of bacterial viruses in this environment have been reported, so in this study we developed methods for counting the total numbers of bacteriophage in soil. We chose to count soil viruses by using transmission electron microscopy (TEM), as a direct observation of phage morphology would allow us to be confident of counting bacterial viruses. We believed that epifluorescence counting would be problematic because there are so many fine particles in soil that interact nonspecifically with most of the DNA stains used for direct virus counts in water and sediment. (greater bitter cress) and bulk soil from molehills. Suspensions were made from soil and rhizosphere samples (1 g [wet weight]) by homogenization in 10 ml of water with 5-mm-diameter glass beads first by vortex mixing for 1 min and then by shaking for 10 min on an orbital shaker. All values are expressed as wet weights, and the average water content of the soil used was 56.2% (coefficient of variation, 29.3%). Before TEM cou...

Effects of Land Uses on Fecal Indicator Bacteria in the Water and Soil of a Tropical Watershed

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Stability and activity of an Enterobacter aerogenes-specific bacteriophage under simulated gastro-intestinal conditions

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Abundance and Diversity of Viruses in Six Delaware Soils

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