No abstract

An acid forest soil (pH 3.9 – 4.4) from an undisturbed mixed oak stand in southern Indiana was examined for the occurrence of ammonia- and nitrite- oxidizing chemoautotrophs. Populations of both nitrifiers were detected in pH 7 most-probable-number (MPN) autotrophic media, and a Nitrosospira was isolated from highest dilution ammonia oxidizer MPN tubes. Populations of nitrite oxidizers were 10 to 1000 times higher than those of ammonia oxidizers. In pH 4.0 MPN media, ammonia oxidation was slight and unsustainable on 10% transfer to fresh medium, whereas nitrite oxidation was vigorous and sustainable. In pure culture the Nitrosospira isolate (Np IO1a) was completely inhibited by nitrapyrin at 5 μg mL−1, tolerant of 1.0 and 10.0 mM chlorate, and capable of growth only at pH 6.2 and above. Fluorescent antibodies raised against Np IO1a were used to confirm the predominance of Np IO1a in all MPN series examined. These results suggest that autotrophic ammonia oxidizers may be restricted to circumneutral microsites in this acid soil, whereas autotrophic nitrite oxidizers may not be limited to such sites.

The construction of rhizobial strains which increase plant biomass under controlled conditions has been previously reported. However, there is no evidence that these newly constructed strains increase legume yield under agricultural conditions. This work tested the hypothesis that carefully manipulating expression of additional copies of nifA and dctABD in strains of Rhizobium meliloti would increase alfalfa yield in the field. The rationale for this hypothesis is based on the positive regulatory role that nifA plays in the expression of the nifregulon and the fact that a supply of dicarboxylic acids from the plant is required as a carbon and energy source for nitrogen fixation by the Rhizobium bacteroids in the nodule. These recombinant strains, as well as the wild-type strains from which they were derived, are ideal tools to examine the effects of modifying or increasing the expression of these genes on alfalfa biomass. The experimental design comprised seven recombinant strains, two wild-type strains, and an uninoculated control. Each treatment was replicated eight times and was conducted at four field sites in Wisconsin. Recombinant strain RMBPC-2, which has an additional copy of both nifA and dctABD, increased alfalfa biomass by 12.9%o compared with the yield with the wild-type strain RMBPC and 17.9%o over that in the uninoculated control plot at the site where soil nitrogen and organic matter content was lowest. These increases were statistically significant at the 5% confidence interval for each of the three harvests made during the growing season. Strain RMBPC-2 did increase alfalfa biomass at the Hancock site; however, no other significant increases or decreases in alfalfa biomass were observed with the seven other recombinant strains at that site. At three sites where this experiment was conducted, either native rhizobial populations or soil nitrogen concentrations were high. At these sites, none of the recombinant strains affected yield. We conclude that RMBPC-2 can increase alfalfa yields under field conditions of nitrogen limitation, low endogenous rhizobial competitors, and sufficient moisture.

Two physiologically and serologically distinct strains of chemoautotrophic nitrite-oxidizing bacteria were isolated as numerically predominant members of the nitrite-oxidizer population of an undisturbed forest soil with a pH range of 4.3 to 5.2. One isolate responded as a neutrophile, characteristic of the family Nitrobacteraceae, and cross-reacted strongly with fluorescent antibody to Nitrobacter strain Engel. The second isolate responded as an acidophile in pure culture, demonstrated maximal nitrite oxidation activity at pH 5.5, and had a pH tolerance range of pH 4.1 to 7.2. Nitrite oxidase in whole cells of the acidophile sustained activity to at least pH 3.5. Cell morphology of both strains typified the genus Nitrobacter in all respects when cultured at pH 7. However, under more acidic conditions the acidophile tended to elongate and at times appeared to branch. These data provide the first evidence for the existence of an acidophilic chemoautotrophic nitrifying bacterium. Isolation of the neutrophilic Nitrobacter strain reported here complements the earlier isolation of a neutrophilic Nitrosospira strain to provide further evidence of a prominent acid-intolerant population of chemoautotrophic nitrifiers in this acid forest soil.

The association of unpasteurized apple cider with Escherichia coli O157:H7 foodborne illness has led to increased interest in potential reservoirs of this pathogen in the orchard. Fourteen U.S. orchards were surveyed in autumn 1999 to determine the incidence and prevalence of E. coli O157:H7, E. coli, total aerobic microflora, and yeasts and molds. Fruit samples (n = 63) (eight apple and two pear varieties) and soil, water, and fecal samples were collected. Samples were plated on (i) tryptic soy agar for total mesophilic aerobic count, (ii) E. coli and coliform Petrifilm for total coliforms and E. coli, and (iii) yeast and mold Petrifilm. Samples positive for coliforms and E. coli were enriched and tested for E. coli O157:H7. Fruit was also tested for internalization of microflora by aseptically removing the core, stem, and calyx areas, and the individual sections were assessed for the categories of microflora listed above. E. coli was detected in soil and water and in 6% of fruit samples (three pear samples and one apple sample), generally collected from areas previously designated as high risk in this study. However, no E. coli O157:H7 was found. Coliforms were found in 74% of fruit samples and were internalized in the cores of 40% of fruit tested. Yeasts and molds were internalized in 96.7% of samples and aerobic bacteria in 89.6%. E. coli was not found to be internalized. Total aerobic counts and total coliforms were higher in dropped and damaged fruit (P < 0.05). Findings suggest that dropped or damaged fruit should not be included in fruit designated for the production of unpasteurized juice or for the fresh or fresh-cut market. In addition, orchards should be located away from potential sources of contamination, such as pastures.