The ecology of Bacillus thuringiensis on the Phylloplane: colonization from soil, plasmid transfer, and interaction with larvae of Pieris brassicae

Seedlings of clover (Triflorium hybridum) were colonized by Bacillus thuringiensis when spores and seeds were co-inoculated into soil. Both a strain isolated in the vegetative form from the phylloplane of clover, 2810-S-4, and a laboratory strain, HD-1, were able to colonize clover to a density of about 1000 CFU/g leaf when seeds were sown in sterile soil and to a density of about 300 CFU/g leaf in nonsterile soil. A strain lacking the characteristic insecticidal crystal proteins produced a similar level of colonization over a 5-week period as the wild type strain, indicating that crystal production was not a mitigating factor during colonization. A small plasmid, pBC16, was transferred between strains of B. thuringiensis when donor and recipient strains were sprayed in vegetative form onto leaves of clover and pak choi (Brassica campestris var. chinensis). The rate of transfer was about 0.1 transconjugants/recipient and was dependent on the plant species. The levels of B. thuringiensis that naturally colonized leaves of pak choi produced negligible levels of mortality in third instar larvae of Pieris brassicae feeding on the plants. Considerable multiplication occurred in the excreted frass but not in the guts of living insects. Spores in the frass could be a source of recolonization from the soil and be transferred to other plants. These findings illustrate a possible cycle, not dependent on insect pathology, by which B. thuringiensis diversifies and maintains itself in nature.

Isotopic composition of tropospheric and soil N2O from successive depths of agricultural plots with contrasting crops and nitrogen amendments

Agricultural soils are the dominant contributor to increases in atmospheric nitrous oxide (N2O). Few studies have investigated the natural N and O isotopic composition of soil N2O. We collected soil gas samples using horizontal sampling tubes installed at successive depths under five contrasting agricultural crops (e.g., unamended alfalfa, fertilized cereal), and tropospheric air samples. Mean d 15N and d 18O values of soil N2O ranged from -28.0 to +8.9‰, and from +29.0 to +53.6‰. The mean d 15N and d 18O values of tropospheric N2O were +4.6 ± 0.7‰ and +48.3 ± 0.2‰, respectively. In general, d values were lowest at depth, they were negatively correlated to soil [N2O], and d 15N was positively correlated to d 18O for every treatment on all sampling dates. N2O from the different agricultural treatments had distinct d 15N and d 18O values that varied among sampling dates. Fertilized treatments had soil N2O with low d values, but the unamended alfalfa yielded N2O with the lowest d values. Diffusion was not the predominant process controlling N2O concentration profiles. Based on isotopic and concentration data, it appears that soil N2O was consumed, as it moved from deeper to shallower soil layers. To better assess the main process(es) controlling N2O within a soil profile, we propose a conceptual model that integrates data on net N2O production or consumption and isotopic data. The direct local impact of agricultural N2O on the isotopic composition of tropospheric N2O was recorded by a shift toward lower d values of locally measured tropospheric N2O on a day with very high soil N2O emissions.

The Genetics of Postglacial Colonization and Peripherality in Northwestern Populations of the Spring Peeper, Pseudacris crucifer

Glaciation over the Pleistocene induced dramatic range fluctuations for species across North America such that postglacial recolonization by southern refugial lineages has characterized the genetic structure of northern North American species. Based on the leading edge model of postglacial range expansion, dispersal and rapid population growth in these northern taxa is expected to produce vast areas of genetic homogeneity. Previous work on the widely distributed spring peeper (Pseudacris crucifer) revealed six distinct mitochondrial lineages that diverged between 3-11 mya, expanding and contracting with glacial cycles. Beginning 16,000 yBP, receding glaciers permitted Eastern lineage refugia residing in the southern Appalachians to migrate northward into the St. Lawrence Valley then westward through most of central Canada. Peripheral populations at the northwestern range limit of P. crucifer in central Manitoba are likely descended from this westward expanding Eastern lineage. According to the central-marginal hypothesis, founder effects from colonization as well as limited gene flow is expected to reveal genetic differentiation and lower genetic diversity in peripheral populations. The goal of my study is to further our understanding of peripheral range dynamics in peripheral Manitoba populations of P. crucifer by determining their genetic affinity and diversity relative to more central populations in Ontario and Minnesota. In this study I amplified and aligned cytochrome b sequences from sample sites across central Manitoba to reconstruct a Bayesian phylogeny for P. crucifer; additionally, microsatellite loci were genotyped to estimate genetic diversity. Results from this study affirmed Eastern lineage descent for peripheral Manitoba sites by aligning with Ontario. Initial colonization by the Interior lineage between glacial retreat and the appearance of arid vicariance events may explain the apparent introgression of non-Eastern lineages in Manitoba. However, genetic diversity measured in expected heterozygosity (H¬e) was not found to be significantly different in Manitoba genotypes. Greater isolation by distance and inbreeding relative to Ontario and Minnesota is likely the primary driver of genetic variation in these sites. Further sampling is necessary to generate a more complete genetic population structure for P. crucifer.