Multi-year and multi-site effects of recurrent glyphosate applications on the wheat rhizosphere microbiome.

Glyphosate (N-(phosphonomethyl)glycine) is broad-spectrum herbicide that is extensively used worldwide, but its effects on the soil microbiome are inconsistent. To provide a sound scientific basis for herbicide re-review and registration decisions, we conducted a four-year (2013-2016) study in which we consecutively applied glyphosate to a wheat (Triticum aestivum L.)-field pea (Pisum sativum L.)-canola (Brassica napus L.)-wheat crop rotation at five sites in the Canadian prairies. The glyphosate rates were 0, 1, 2, 4 and 8 kg ae ha-1, applied pre-seeding and post-harvest every year. The wheat rhizosphere was sampled in the final year of the study and analysed for microbial biomass C (MBC), the composition and diversity of the microbiome, and activities of ß-glucosidase, N-acetyl-ß-glucosiminidase, acid phosphomonoesterase and arylsulphatase. Glyphosate did not affect MBC, the composition and diversity of prokaryotes and fungi, and the activities of three of the four enzymes measured in the wheat rhizosphere. The one effect of glyphosate was a wave-like response of N-acetyl-ß-glucosaminidase activity with increasing application rates. The experimental sites had much greater effects, driven by soil pH and organic C, on the soil microbiome composition and enzyme activities than glyphosate. Soil pH was positively correlated with the relative abundance of Acidobacteriota but negatively correlated with that of Actinobacteriota and Basidiomycota. Soil organic C was positively correlated with the relative abundances of Proteobacteriota and Verrucomicrobiota, but negatively correlated with the relative abundance of Crenachaeota. The activity of acid phosphomonoesterase declined with increasing relative abundance of Acidobacteriota, but increased with that of Actinobacteriota and Basidiomycota. The activity of N-acetyl-ß-glucosaminidase also increased with increasing relative abundance of Actinobacteriota but decreased with that of Mortierellomycota. ß-glucosidase activity also decreased with increasing relative abundance of Mortierellomycota. The core fungal species observed in at least 90% of the samples were Humicola nigrescens, Gibberella tricincta and Giberella fujikuroi. Therefore, this multi-site study on the Canadian prairies revealed no significant effects of 4-year applications of glyphosate applied at different rates on most soil microbial properties despite differences in the properties among sites. However, it is important to keep evaluating glyphosate effects on the soil microbiome and its functioning because it is the most widely used herbicide worldwide.

Investigation on gene transfer from genetically modified corn (Zea mays L.) plants to soil bacteria.

Knowledge about the prevalence and diversity of antibiotic resistance genes in soil bacteria communities is required to evaluate the possibility and ecological consequences of the transfer of these genes carried by genetically modified (GM) plants to soil bacteria. The neomycin phosphotransferase gene (nptII) conferring resistance to kanamycin and neomycin is one of the antibiotic resistance genes commonly present in GM plants. In this study, we investigated kanamycin-resistant (Km(R)) and neomycin-resistant (Nm(R)) soil bacterial populations in a 3-year field trial using a commercial GM corn (Zea mays L.) carrying the nptII gene and its near isogenic line. The results showed that a portion (2.3 - 15.6 %) of cultivable soil bacteria was naturally resistant to kanamycin or neomycin. However, no significant difference in the population level of Km(R) or Nm(R) soil bacteria was observed between the GM and non-GM corn fields. The nptII gene was not detected in any of the total 3000 Km(R) or Nm(R) isolates screened by PCR. Further, total soil bacterial cells were collected through Nycodenz gradient centrifugation and bacterial community DNA was subjected to PCR. Detection limit was about 500 cells per gram of fresh soil. Our study suggests that the nptII gene was relatively rare in the soil bacterial populations and there was no evidence of gene transfer from a GM corn plant to soil bacteria based on the data from total soil bacterial communities.

Development of real time PCR assays for detection and quantification of transgene DNA of a Bacillus thuringiensis (Bt) corn hybrid in soil samples.

Real time PCR assays were developed to detect and quantify the transgene DNA of a commercially released Bacillus thuringiensis (Bt) corn (Zea mays L.) hybrid (DKC42-23), which was derived from the event MON863 and also carried a neomycin phosphotransferase gene (the nptII gene). We applied the real time PCR assays to investigate the persistence of the transgene DNA in a field trial grown with DKC42-23 over 3 years, in combination with bacterial natural transformation. The results showed that under continuous cultivation of DKC42-23, its transgene DNA was detectable in the field plots all year around. Meanwhile, when soil DNA extracts from DKC42-23 plots were used as donor in bacterial natural transformation, successful recovery of kanamycin resistant (Km(R)) transformants indicated that the nptII gene carried by DKC42-23 could be taken up and integrated into naturally competent Pseudomonas stutzeri pMR7 cells, leading to the restoration of the antibiotic resistance of P. stutzeri pMR7. However, after the cultivation of a soybean line in the same plots for the subsequent growing season, the presence of transgene DNA of DKC42-23 was reduced to undetectable levels at the end of that growing season. Therefore, existing corn-soybean crop rotation practices reduce the availability of transgene DNA in soil and thus minimize the risks that might be attributable to horizontal gene transfer. The real time PCR assays are useful for investigating the persistence of transgene DNA derived from the MON863 event in soil environments.

Weed seed viability in composted beef cattle feedlot manure.

Manure composting has gained increased acceptance by the beef cattle (Bos taurus) feedlot industry in southern Alberta, Canada. Unlike fresh manure, compost is often promoted as being "weed-free." Studies were conducted with five weed species in 1997 and thirteen in 1999 to examine the effect of feedlot manure composting on weed seed viability. Weed seeds were buried in open-air compost windrows and recovered at various times during the thermophilic phase of composting. Windrow temperature and water contents were also measured. Germinability was zero for all composted weed seeds at all sampling times in 1997. However, some seeds remained viable (positive tetrazolium test denoting respiration) on Day 70. In 1999, only one of the thirteen species retained germinability on Day 21 and only two species had respiring seeds on Day 42. Time-viability relationships during composting were defined by exponential decay models. Lethal temperatures to eliminate viability was species-dependent. In 1999, four weed species were killed in the initial 7 d of composting at a lethal temperature of 39 degrees C while temperatures of > 60 degrees C were required for two species. Regression analysis on weed seed viability versus windrow temperature resulted in significant R2 values, which showed that only 17 to 29% of the variation in viability was accounted for by temperature. The lack of definitive relationships between temperature and weed seed viability demonstrated that factors other than temperature may play a role in eliminating weed seeds during composting.