Impact of Cropping Systems, Soil Inoculum, and Plant Species Identity on Soil Bacterial Community Structure Authors: Suzanne L. Ishaq, Stephen P. Johnson, Zach J. Miller, Erik A. Lehnhoff, Sarah Olivo, Carl J. Yeoman, and Fabian D. Menalled The final publication is available at Springer via http://dx.doi.org/10.1007/s00248-016-0861-2. Ishaq, Suzanne L. , Stephen P. Johnson, Zach J. Miller, Erik A. Lehnhoff, Sarah Olivo, Carl J. Yeoman, and Fabian D. Menalled. "Impact of Cropping Systems, Soil Inoculum, and Plant Species Identity on Soil Bacterial Community Structure." Microbial Ecology 73, no. 2 (February 2017): 417-434. DOI: 10.1007/s00248-016-0861-2. Made available through Montana State University’s ScholarWorks scholarworks.montana.edu Impact of Cropping Systems, Soil Inoculum, and Plant Species Identity on Soil Bacterial Community Structure 1,2 & 2 & 3 & 4 & Suzanne L. Ishaq Stephen P. Johnson Zach J. Miller Erik A. Lehnhoff 1 1 2 Sarah Olivo & Carl J. Yeoman & Fabian D. Menalled 1 Department of Animal and Range Sciences, Montana State University, P.O. Box 172900, Bozeman, MT 59717, USA 2 Department of Land Resources and Environmental Sciences, Montana State University, P.O. Box 173120, Bozeman, MT 59717, USA 3 Western Agriculture Research Center, Montana State University, Bozeman, MT, USA 4 Department of Entomology, Plant Pathology and Weed Science, New Mexico State University, Las Cruces, NM, USA Abstract Farming practices affect the soil microbial commu- then individual farm. Living inoculum-treated soil had greater nity, which in turn impacts crop growth and crop-weed inter- species richness and was more diverse than sterile inoculum- actions. This study assessed the modification of soil bacterial treated soil (observed OTUs, Chao, inverse Simpson, community structure by organic or conventional cropping sys- Shannon, P < 0.001) and had more discriminant taxa delineat- tems, weed species identity [Amaranthus retroflexus L. (red- ing groups (linear discriminant analysis). Living inoculum soil root pigweed) or Avena fatua L. (wild oat)], and living or contained more Chloroflexi and Acidobacteria, while the sterilized inoculum. Soil from eight paired USDA-certified sterile inoculum soil had more Bacteroidetes, Firmicutes, organic and conventional farms in north-central Montana Gemmatimonadetes, and Verrucomicrobia. Organically was used as living or autoclave-sterilized inoculant into farmed inoculum-treated soil had greater species richness, steam-pasteurized potting soil, planted with Am. retroflexus more diversity (observed OTUs, Chao, Shannon, P <0.05), or Av. fatua and grown for two consecutive 8-week periods and more discriminant taxa than conventionally farmed to condition soil nutrients and biota. Subsequently, the V3-V4 inoculum-treated soil. Cyanobacteria were higher in pots regions of the microbial 16S rRNA gene were sequenced by growing Am. retroflexus, regardless of inoculum type, for Illumina MiSeq. Treatments clustered significantly, with three of the four organic farms. Results highlight the potential living or sterilized inoculum being the strongest delineating of cropping systems and species identity to modify soil factor, followed by organic or conventional cropping system, bacterial communities, subsequently modifying plant growth and crop-weed competition. Keywords 16S rRNA . Avena fatua . Amaranthus retroflexus . Conventionalfarming . Illumina MiSeq . Organic farming . Soil microbial diversity Introduction Soil microbial community characteristics are driven by a myr- iad of factors, including climate, soil physical and chemical traits, the abundance and diversity of plant species living at the site, and the influx of nutrients and minerals by water, animals, or plant detritus [1–4]. In general, an increase in microbial phylogenetic diversity leads to improved soil health, makes the system more resilient to physical or chemical disturbances, modifies plant competition, and increases plant production by facilitating access to nutrients [1, 5–9]. However, on a species- by-species basis, microorganisms can positively or negatively affect plant growth and health through various pathways conditioned soil bacterial community structure. This was done (reviewed in [10]). Additionally, the interactions between soil by comparing bacterial 16S ribosomal RNA (rRNA) gene microbiota and nutrients are complex, and increased microbial populations in potting soil that had received either a living phylogenic diversity does not always lead to increased micro- or a sterilized soil inoculum taken from organically or conven- bial functionality (reviewed in [11, 12]). Thus, more investi- tionally farmed soil and had the problematic weed species gation into the dynamics between microorganisms, soil nutri- Amaranthus retroflexus L. (redroot pigweed) or Avena fatua ents, and plants is needed to understand how these interactions L. (wild oat) growing in it. shape microbial diversity and soil quality. Although it is difficult to generalize the impact that farming/cropping systems have on soil biology, the following are common differences between conventional and organic Methods crop management in general, and applicable to dryland farms in Montana. In organic farming, mechanical practices, such as Sample Collection tillage, are commonly used to control weeds and terminate cover crops, and have been associated with a decrease in mi- Soil was collected on July 17–18, 2013, from eight paired crobial biomass [13] and diversity [14]. However, organic USDA-certified organic and conventional farms. Each farm farms often have increased weed diversity [15–17], and higher pair was located approximately 3.3 km (SD = 4.5) apart, with plant diversity has been linked with increased microbial bio- pairs located across north-central Montana (mean distance be- mass [18], respiration, and fungal abundance [2]. In conven- tween pairs 137 km, SD = 40). Pairs included Big Sandy tional farming, agriculture intensification with fertilizers and (BSa) organic (48.068895 N 110.011836 W) and convention- herbicides has a negative impact on meso- and macrobiota in al (48.066465 N 110.012038 W), Dutton (Dut) organic the short term and microbiota over time [19]. Crop rotations (47.778568 N 111.578379 W) and conventional also differ between systems, with the organic farms typically (47.772951 N 111.565377 W), Havre (Hav) organic having diverse rotations and using cover crops, green manure (48.850477 N 109.971366 W) and conventional crops, or animal manures as source of nutrients, while con- (48.826182 N 110.076707 W), and Lewistown (Lew) organic ventional farms typically grow only one or two crops and rely (47.114102 N 109.715622 W) and conventional on synthetic fertilizers as a nutrient source. Previous studies (47.118478 N 109.699570 W). Conventional farms practiced have demonstrated that diverse crop rotations increase the a no-tillage wheat-fallow rotation, and organic farms had been diversity of soil biota [14]. Finally, organic versus convention- managed with intensive tillage and diverse crop rotations. al changes in soil microbiota differ with landscape complexity Fields for soil sampling were not randomly selected due to [20]. logistical constraints, such as the pairing of farms by cropping Plant growth can have a species-specific impact on soil system, for fields to be planted with a wheat crop [except the microorganisms [21–23]. For example, a previous study organic Dutton farm planted with Triticum turanicum Jakubz. showed that plant growth increased the density of microorgan- (Khorasan wheat var. Kamut), a close wheat relative], and isms, especially pseudomonad bacteria, over unplanted soil obtaining permission from landowners. Sampled fields repre- [4]. Microbial density was most increased with clover sented a range of soil characteristics (loams, clay loams, and (Trifolium repens) and ryegrass (Lolium perenne)over silty clay loams), temperatures (mean max of 12.9–14.8 °C; bentgrass (Agrostis capillaris)orwheat(Triticum aestivum) mean min (−2.7 to −0.9 °C), and annual precipitation (265– [4]. Likewise, canola (Brassica napus) and wheat selected 388 mm) (see Table S1 [29]). for different bacterial communities, with wheat-associated soil Within each field, soil was collected to a depth of 13 cm having lower overall diversity yet more abundant Bacillus in using 6-cm-diameter cores every 10 m along a 200-m W- the rhizoplane, and Cartobacter, Rathayibacter, shaped transect, for a total of 21 subsamples/transect. Streptococcus, and other genera more abundant in canola Transects were located at least 100 m from any field edge roots [24]. and positioned on relatively uniform slopes. Subsamples A number of studies compared soil quality and microbial were pooled by field and refrigerated at 4 °C until the phylogenetic or functional diversity between ecosystems, in- experiment began. Bulk soil samples from each field were cluding different farming/cropping systems [25–28]; howev- sent for nutrient testing to Agvise Laboratories er, very little work had been done using soil from these eco- (Northwood, ND) (see Table S1 [29]). First, the soils were systems to condition other soil as an inoculum. Most studies air-dried for 2 weeks and sifted through a 1-cm2 sieve. of this nature involve the addition of a single microorganism Half the soil was autoclave-sterilized to 120 °C at 15 psi to soil [25–27], instead of using the entire soil microbiota [28]. for two 1-h periods with 24 h between sterilizations (ster- The present study sought to further investigate