Masters Thesis

Physiological impact of phosphite on soil bacteria

Phosphite (Pt), a traditional fungicide and novel fertilizer alternative used for P sourcing in agricultural soils, offers a potential for the conservation of phosphate (Pi), an essential and non-renewable limited resource. This potential has relied on the ability of some soil bacterial species to oxidize Pt to Pi, as a P source. However, bacterial species lacking this ability may suffer detrimental effects by increased use of Pt in the environment. We set out to explore the physiological impact of P fertilizers on bacteria in rhizosphere soil and to elucidate possible broad outcomes of Pt application to agricultural soils. Bacterial populations from destructive greenhouse samples of rhizosphere soil under various P treatments were examined over 113 days. Pt-oxidizing bacteria were enriched in Pt-treated soil (changing from 18 to 39% of culturable bacteria) but were disadvantaged in Pi-treated soil, decreasing 73.6% (changing from 30 to 7% of culturable bacteria). In contrast, non-oxidizers increased 3.1-fold in Pt-treated soil, exhibiting survival in soil samples with low Pi but high levels of Pt. Pt-oxidizers were determined to grow differently in Pi relative to Pt, in pure culture. Three Pt-oxidizers (A. wautersii, P. stutzeri, and P. putida) exhibited 4.6, 1.47, 1.56-fold shorter generation times in 2.0 mM Pi compared to 2.0 mM Pt indicating that although these bacteria can oxidize Pt, Pi as a P source results in faster growth. In Pt, A. wautersii and P. putida final cell mass decreased 43.9% and 22.1% (respectively), compared to growth in Pi, further supporting the benefit of Pi over Pt as a P source in these bacteria. In contrast, P. stutzeri showed a 15.3% increase in final cell mass in Pt compared to Pi, suggesting that Pt may be more useful as a P source for this organism. To check for detrimental effects of high levels of Pi on Pt-oxidizers which did poorly in the Pi-treated soil compared to non-oxidizers, we compared generation times and net final cell mass of three Pt-oxidizers (P. putida, S. melitoti, and Methylobacterium), to those of three non-oxidizers (P 6B, Flavobacterium, P. oleovorans subsp. lubricantis), while growing in high Pi (1.6 mM). The (g) for the Pt-oxidizers ranged from 7.74 – 13.51 hours, while the (g) for the non-oxidizer group ranged from 5.24 – 6.98 hours. Although the growth rates of the Pt-oxidizers were slower in high Pi compared to non-oxidizers, showing detriment to the oxidizers, the Pt-oxidizers reached a higher overall increase in cell mass based on a greater max increase in A values (0.60 – 0.74 A) in high Pi compared to that observed for the non-oxidizers (0.51 – 0.54 A). Both (g) and cell mass show unique growth differences linked to the bacteria’s Pt-oxidation status. Growing the same non-oxidizers in low Pi (0.2 mM), we explored an ability to tolerate Pi-scarcity as a means for non-oxidizer increases in the Pt-treated soil where only low Pi levels were present. In low Pi, non-oxidizers grew slower ((g)= 6.68 - 11.60 hrs) compared to in high Pi ((g)= 5.24 - 6.98 hrs). Net increase of cell mass was the same in two of the organisms but was limiting for Flavobacterium (low, 0.40 +/-0.010 A, high, 0.54 +/- 0.015 A). LMB stained Flavobacterium cells showed metachromatic granules after growth on LBA, elucidating storage, a possible means of non-oxidizer survival in Pt-treated soil. Two Pt-oxidizers, Methylobacterium and Acidovorax, showed metachromatic granules after growth on LBA and Pt solid media. Also, we observed at least 100 uM (5.35% of original Pt) released Pi during growth of Pt-oxidizers (Methylobacterium, S. melitoti, and P. putida) in liquid media containing Pt as the sole P source. P. putida showed Pi in media after one day, supporting Pt oxidation followed by Pi secretion by cells instead of cell death as Pi release mechanism. Minimum inhibitory concentrations of Pt were attempted for both non-oxidizers and Pt-oxidizers. pH was shown as the agent of inhibition in all but one organism. While Pt-oxidizer, P. putida, was less sensitive to lethality, P. oleovorans subsp. lubricantis, a non-oxidizer, was inhibited at 50% lower (12.5 mM MIC, 25 mM MBC) Pt concentration compared to all the other studied organisms and pH control confirmed Pt as the probable inhibitory agent. Furthermore, an immediate arrest in growth occurred upon addition of Pt (at the MIC) to mid-exponential phase cultures of P. oleovorans subsp. lubricantis growing on Pi, suggesting competition between Pt and Pi at essential binding sites for transport or metabolism. A. wautersii exhibited a diauxie effect when transferred from a Pi pre-growth to the Pt growth media in the prior growth assay showing no immediate interruption of growth by the introduction of Pt. From these studies, it is clear that both Pi and Pt treatments affect soil bacterial composition and physiology thus their application to the environment merits further investigation.

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