Biodegradation of the herbicide mecoprop-p with soil depth and its relationship with class III tfdA genes

Mecoprop-p [(R)-2-(4-chloro-2-methylphenoxy) propanoic acid) is widely used 37 in agriculture and poses an environmental concern because of its susceptibility to leach 38 from soil to water. We investigated the effect of soil depth on mecoprop-p 39 biodegradation and its relationship with the number and diversity of tfdA related genes, 40 which are the most widely known genes involved in degradation of the 41 phenoxyalkanoic acid group of herbicides by bacteria. Mecoprop-p half-life (DT50) was 42 approximately 12 days in soil sampled from <30 cm depth, and increased progressively 43 with soil depth, reaching over 84 days at 70-80 cm. In sub-soil there was a lag period of 44 between 23 and 34 days prior to a phase of rapid degradation. No lag phase occurred in 45 top-soil samples prior to the onset of degradation. The maximum degradation rate was 46 the same in top-soil and sub-soil samples. Although diverse tfdAα and tfdA genes were 47 present prior to mecoprop-p degradation, real time PCR revealed that degradation was 48 associated with proliferation of tfdA genes. The number of tfdA genes and the most 49 probable number of mecoprop-p degrading organisms in soil prior to mecoprop-p 50 addition were below the limit of quantification and detection respectively. Melting 51 curves from the real time PCR analysis showed that prior to mecoprop-p degradation 52 both class I and class III tfdA genes were present in top- and sub-soil samples. However 53 at all soil depths only tfdA class III genes proliferated during degradation. Denaturing 54 gradient gel electrophoresis confirmed that class III tfdA genes were associated with 55 mecoprop-p degradation. Degradation was not associated with the induction of novel 56 tfdA genes in top- or sub-soil samples, and there were no apparent differences in tfdA 57 gene diversity with soil depth prior to or following degradation

Influence of root exudates on soil microbial diversity and activity

Interactions between plant roots and soil microorganisms in the rhizosphere are critical for plant growth. However, understanding of precisely how root exudates influence the diversity and activity of rhizosphere microorganisms is limited. The main objective of this study was to investigate the effect of radiata pine (Pinus radiata) root exudates on rhizosphere soil microbial communities, with an emphasis on the role of low molecular weight organic anions. The study involved the development and validation of new methods for investigating rhizosphere processes in a purpose-built facility. This included development of an in situ sampling technique using an anion exchange membrane strip to collect a range of organic anions exuded from radiata pine roots grown in large-scale rhizotrons. These included tartarate, quinate, formate, malate, malonate, shikimate, lactate, acetate, maleate, citrate, succinate and fumarate. Soil microbial activity and diversity were determined using dehydrogenase activity and denaturing gradient gel electrophoresis. Links between organic anions in root exudates and rhizosphere soil microbial community structures were investigated by comparing wild type and genetically modified radiata pine trees which were grown in rhizotrons for 10 months. As expected, there was considerable temporal and spatial variability in the amounts and composition of organic anions collected, and there were no consistent or significant differences determined between the two tree lines. Significant differences in rhizosphere microbial communities were detected between wild type and genetically modified pine trees; however, they were inconsistent throughout the experiment. The shifts in microbial communities could have been related to changes in exudate production and composition. Based on results from the main rhizotron experiment, a microcosm study was carried out to investigate the influence of selected pine root exudate sugars (glucose, sucrose and fructose) and organic anions (quinate, lactate and maleate) on soil microbial activity and diversity. Soil microbial activity increased up to 3-fold in all of the sugar and organic anion treatments compared to the control, except for a mixture of sugars and maleate where it decreased. The corresponding impacts on soil microbial diversity were assessed using denaturing gradient gel electrophoresis and 16S rRNA phylochips. Addition of the exudate compounds had a dramatic impact on the composition and diversity of the soil microbial community. A large number of bacterial taxa (88 to 1043) responded positively to the presence of exudate compounds, although some taxa (12 to 24) responded negatively. Organic anions had a greater impact on microbial communities than sugars, which indicated that they may have important roles in rhizosphere ecology of radiata pine. In addition, a diverse range of potentially beneficial bacterial taxa were detected in soil amended with organic anions, indicating specific regulation of rhizosphere microbial communities by root exudates. This project highlighted the considerable challenges and difficulties involved in detailed investigation of in situ rhizosphere processes. Nonetheless, the findings of this study represent a significant contribution to advancing understanding of relationships between root exudates and soil microbial diversity, which will be further enhanced by refinement and application of the specific methodologies and techniques developed

Changes to the structure of Sphingomonas spp. communities associated with biodegradation of the herbicide isoproturon in soil

The phenyl-urea herbicide isoproturon is a major contaminant of surface and ground-water in agricultural catchments. Earlier work suggested that within-field spatial variation of isoproturon degradation rate resulted from interactions between catabolizing Sphingomonas spp. and pH. In the current study, changes to the structure of Sphingomonas communities during isoproturon catabolism were investigated using Sphingomonas-specific 16S rRNA gene primers. Growth-linked catabolism at high-pH (> 7.5) sites was associated with the appearance of multiple new denaturing gradient gel electrophoresis (DGGE) bands. At low-pH sites, there was no change in DGGE banding at sites in which there was cometabolism, but at sites in which there was growth-linked catabolism, degradation was associated with the appearance of a new band not present at high pH sites. Sequencing of DGGE bands indicated that a strain related to Sphingomonas mali proliferated at low pH sites, while strain Sphingomonas sp. SRS2, a catabolic strain identified in earlier work, together with several further Sphingomonas spp., proliferated at high-pH sites. The data indicate that degradation was associated with complex changes to the structure of Sphingomonas spp. communities, the precise nature of which was spatially variable

Timing is everything: Improving predictions of winter New Zealand grass grub densities and associated damage from summer and autumn larval counts

Costelytra giveni is a serious pasture pest in New Zealand and accurate estimates of population densities are important to inform control measures. This species generally has a one-year life cycle so populations should either remain stable after eggs have hatched or decline due to larval mortality. Larval counts were obtained using a simple, standard and widely used sampling method from a series of soil cores collected from ryegrass research plots in Canterbury, New Zealand between 27 January and 16 June 2021 and a significant increase in population was recorded. Measurements on 27 January, 19 March and 5 May, represented only c. 8%, 25% and 63% of the mean densities measured on 16 June, respectively. The apparent increase in larvae is attributed to failure to find small 1st and 2nd instar individuals within the soil samples. Larvae increased in size as they transitioned from 1st to 3rd instar and later instar specimens were more easily discovered. An equation to describe the observed results provided date-related correction factors to allow a more realistic prediction of C. giveni larval densities in the winter following empirical larval counts. Larval counts measured on 27 January, 19 March, and 5 May, would need to be multiplied by 13, 4 and 1.6, respectively, to accurately estimate the larval density found on 16 June. This study showed that summer-autumn sampling using the current method can significantly underestimate winter C. giveni larval densities, potentially leading to unanticipated pasture production losses. Similar results were also found in 2022. While the equation provides a guide to population estimates, the caveat is that region and environment will influence population trends in any particular year.</p

The interconnected rhizosphere: High network complexity dominates rhizosphere assemblages.

While interactions between roots and microorganisms have been intensively studied, we know little about interactions among root-associated microbes. We used random matrix theory-based network analysis of 16S rRNA genes to identify bacterial networks associated with wild oat (Avena fatua) over two seasons in greenhouse microcosms. Rhizosphere networks were substantially more complex than those in surrounding soils, indicating the rhizosphere has a greater potential for interactions and niche-sharing. Network complexity increased as plants grew, even as diversity decreased, highlighting that community organisation is not captured by univariate diversity. Covariations were predominantly positive (&gt; 80%), suggesting that extensive mutualistic interactions may occur among rhizosphere bacteria; we identified quorum-based signalling as one potential strategy. Putative keystone taxa often had low relative abundances, suggesting low-abundance taxa may significantly contribute to rhizosphere function. Network complexity, a previously undescribed property of the rhizosphere microbiome, appears to be a defining characteristic of this habitat

Shrub characteristics & herb and soil properties.csv

Using space for time substitution, we established a paired livestock exclusion-free grazing (control) system to evaluate the effects of livestock exclusion on shrubs at Haibei Research Station. Exclosure manipulation experiments were conducted in 1997, 2003, and 2011. Small mammals cannot be eliminated using these fences. There were two 9-year exclosures, six 17-year exclosures, and one 23-year exclosure, all 30 m×30 m in size. The exclosures were adjacent to an open pasture. Control plots were delineated temporarily near the exclosures by outlining the boundary with plastic wires and wood posts in the open pasture. There were four, four, and one control plots for the 9-, 17- and 23-year exclosures, respectively. Notably, the topography, vegetation, and soil types were similar between the exclosures and corresponding plots before fencing. From August to September 2020, we conducted the sampling and investigation.  In each plot, we chose five P. fruticosa shrub patches according to the canopy area frequency distribution of shrub patches (from the investigation of shrub) and randomly chose five herb patches as far away from the shrub patches as possible in the grassy matrix. One quadrat of 50 cm×50 cm was set in each shrub and herb patch. We harvested all aboveground biomass (AGB) at ground level from the quadrats. Live aboveground biomass was divided into four functional groups (grass, sedge, legume, and non-legume forb), and dead aboveground biomass was separated into standing and soil surface litter. The classified biomass was dried in an oven to a constant weight at 65 °C and weighed.  Soil was sampled at the above-mentioned chosen five shrub patches and five herb patches in each plot. For each patch, we used soil bulk density drilling (with a cutting ring volume of 100 cm3) to extract 100 cm3 soil at 0–10, 10–20, 20–30, 30–50, 50–70, and 70–100 cm. The soil samples were rapidly brought back to the laboratory and weighed. After oven-drying at 105 °C to a constant weight, soil samples were weighed again. Soil water content was calculated as the percentage of water weight in the fresh soil. At each shrub and herb patch, we also randomly selected 3–7 sampling points and collected samples at 0–5, 5–10, 10–20, 20–30, 30–50, 50–70, and 70–100 cm using a soil auger of 5 cm diameter and completely mixed soils from the same depth. Soil samples were air-dried indoors immediately after returning to the laboratory and sieved to pass a 2 mm screen with debris eliminated. After sieving, the roots were carefully removed using tweezers. Soil-water mixtures at a 1:5 ratio (w:v) were prepared to determine pH using a pH meter. Representative sub-samples were passed through a 0.25 mm sieve for soil element measurements. Soil total carbon (STC) and total nitrogen (TN) contents were measured using an elemental analyzer (Vario EL cube; Elementar, Langenselbold, Germany) at the College of Pastoral Agriculture Science and Technology, Lanzhou University, Lanzhou, China. The soil inorganic carbon (SIC) and total phosphorus (TP) contents were determined using a SKALAR carbon element analyzer (2SN100903#; Skalar Analytical B.V., Breda, The Netherlands) and an automated discrete analyzer (Smarchem450; AMS, Italy), respectively, at the State Key Laboratory of Herbage Improvement and Grassland Agro-ecosystems, College of Pastoral Agriculture Science and Technology, Lanzhou University. Soil organic carbon (SOC) content was calculated as the difference between the STC and SIC content.  The shrub investigation included two parts: shrub patch investigation and shrub investigation of herb patches. For P. fruticosa shrub patches, it is difficult to count individuals, as P. fruticosa mainly carries out vegetative propagation by creeping stems belowground. We measured each P. fruticosa patch’s (1) canopy longest axis 2a and perpendicular 2b, which we used for the ellipse area formula (πab) as the patch canopy area (shrub patch is regarded as an ellipse); (2) largest height; (3) number of branches from previous years; and (4) number of twigs, which are young hairy red brown stems coming out in the current year. For reproductive output, we counted the number of ovaries surrounded by the calyx for each patch, selected 20–30 of the ovaries in each plot to measure the internal achene (containing one seed) number, and acquired the patch’s seed number by multiplying its ovary number by the corresponding average number of achenes in the ovary. We also estimated the number of seedlings under the patch-projected area. For the larger patches that had especially high numbers of branches, twigs, seeds, or seedlings, we selected a representative part to measure and multiplied by an estimated multiple. Potentilla fruticosa patches close to each other in the 23-year exclosure were separated by the height and color of the patches. For P. fruticosa shrubs in the herb patch, we investigated in the 50 cm×50 cm quadrats set in section 2.2.1. The shrub properties were measured in a similar manner to the shrub patch.  The aboveground biomass of P. fruticosa was nondestructively estimated by the following estimation formula (Liang et al. 2013): AB=113.02P2H+29.77 where AB, P, and H are the aboveground biomass (g), canopy perimeter (m), and height (m), respectively, of the shrub patch. The perimeter of an ellipse cannot be estimated accurately; however, there are approximation formulas, of which one of the Ramanujan formulas is famous for its briefness: P≈π[3(a+b)- √ [(3a+b)(a+3b)]] We obtained climatic data from the meteorological station installed in 1980 at the Haibei Research Station.  </p

What are we missing? Optimising DNA extraction from allophanic soils

Allophanic soils are so named because of the presence of the clay allophane, and, in Aotearoa New Zealand, are predominantly found in the North Island. They make up 5% of New Zealand soils. Due to their different physical and chemical properties to other soils, these clay-rich soils are difficult to extract DNA from. This study was conducted to maximize DNA extraction from allophanic soils for microbiota assessments. Soil samples were collected from five Aotearoa regions, and DNA was extracted using skim milk and casein. Following findings from this work, further testing using different rates of casein was carried out to maximize DNA concentration, and hence, diversity. Amplicon sequencing of the bacterial (16S) and fungal (ITS) marker genes was performed on DNA extracts, and resulting amplicon sequence variants were analyzed to determine any effects the modified extraction method had on apparent microbial biodiversity. Amending extraction protocol with either skim milk or casein significantly increased the amount of DNA extracted from high-clay soils, with skim milk returning higher DNA concentrations than casein. However, with reference to diversity, casein-amended DNA extracts confirmed higher 16S and ITS diversity compared to skim milk extracts.</p