The impact of agricultural practices on microbial spatiotemporal dynamics and contributions to soil health

Bulk Soil and Plant Sampling
In August 2021 and August 2022, soil and plant samples were gathered from agricultural lands spanning North Dakota and Minnesota. In the first year, samples were taken from a total of 61 farms, including 31 conventional and 30 organic ones. Similarly, in the second year, samples were collected from 57 of the same farms, with 30 being conventional and 27 organics. USDA (United States Department of Agriculture) defines organic farming as a method that utilizes cultural, biological, and mechanical techniques to maintain on-farm resources, preserve the environment, and protect the variety of living organisms enhancing the ecosystem. (https://www.ams.usda.gov/sites/default/files/media/Organic%20Practices%20Factsheet.pdf). Conventional farms apply resources like fertilizer, water for irrigation, amendments, and pesticides evenly, without considering the natural variations in soil and crop conditions between and within fields. This leads to an uneven distribution of resources, with some areas receiving too much and others too little (Corwin & Scudiero, 2019). To prevent potential edge effects, the sampling was conducted at 25 meters from the edges. To ensure that the samples represented the entire farm, the sampling locations were chosen randomly. 5 soil cores (15 cm deep) were collected from the area around randomly chosen 5 plants resulting in a total of 25 soil cores from each of the 61 farms using a 1” diameter soil step probe. In 2021, 5 fully matured corn plants were uprooted from each farm using a shovel, and the same process was repeated in 2022 for the crop that was present at that time. The soil cores from each farm were collected in one-gallon Ziploc© bags which were then stored on ice packs in coolers during transportation to the laboratory. Once in the lab, plant particles and macro-organisms were removed from the soil and the soil cores were broken up and homogenized by sieving with a 2 mm sieve, and then divided into two 15 ml conical tubes. One of the tubes and the remaining soil in Ziplock bags were stored at -20°C for soil analysis, and the other was stored at -80°C for long-term storage for molecular analysis. In the field, the roots of the plants were clipped off at the root-shoot junction using sterilized secateurs, and 5 roots were collected and stored in a Ziplock bag and placed in coolers with ice packs during transportation to the laboratory. Once in the lab, the roots were washed with tap water to remove the soil. They were then washed in 95% EtOH for a minute and disinfected with a 10% bleach solution for a minute. Roots were again washed with 95% EtOH for 30 seconds and then rinsed with sterile water for 2 minutes. Excess water was removed using paper towels out of the processed roots. The roots were then cut into approximately 1cm pieces using scalpels, with about 40% of the cut roots from each plot stored in a 15 ml conical tube at -80°C for molecular analysis. Thinner roots were selected for mycorrhizal slide preparation and were placed in 15 ml conical tubes with 70% ethyl alcohol for longer storage. To measure the weight of fresh plants, they were wrapped and taped individually before being weighed. The collected plants were placed in brown paper bags and stored on ice packs for transporting to the lab. The plants were then dried in an oven at 40°C for approximately for 5 days to measure dry biomass.

Measurement of Soil and Plant Properties
100gm of bulk soil was measured from the soil samples held in Ziplock bags and placed in sealable bags provided by soil and plant biomass analyzing facility “AgVise Laboratories” located at Grand Forks, North Dakota for the analysis of the physiochemical properties of the soil. 50 gm of dried plant biomass was grinded and sent off to " AgVise Laboratories" for plant physiochemical properties analysis. 

Mycorrhizal Colonization Assessment
Processed roots were further washed using one ml of a one M solution of (NaPO3)6, to remove any excess soil that remained. Approximately 15 root pieces were removed from each 15 ml conical tubes and kept into a 1.5 ml microcentrifuge tube. Labeling was done accordingly. (NaPO3)6 was added to each 1.5 ml microcentrifuge tubes, and the closed tubes were shaken for two hours. After two hours, the roots were removed and washed in deionized water. A 40% w/v KOH solution was added to the roots, and the mixture were incubated at 80°C for 30-40 minutes. After that, the roots were again rinsed with deionized water. Then, a 5% ink-vinegar solution was added to the roots and kept for 30-40 minutes at 80°C for staining. For the ink-vinegar stock solution, 50 ml ink (black from Parker Quink) was mixed with 950 ml of household vinegar (5% acetic acid). After the staining, the roots were rinsed with tap water and a drop of vinegar (Vierheilig et al., 1998). The stained roots were aligned parallel to the long axis of a microscopy slide. On each slide, there were five rows of roots- each having two to three pieces. Roots were fixed into place using 50% glycerol and a cover slip. The edges of the cover slips were securely sealed with coats of clear nail polish. The arbuscular mycorrhizal colonization was observed under a compound microscope at a magnification of 40X. The spot on the root surface where the center of the horizontal and vertical crosshairs entered through the side of the root was taken as the point of intersection. All intersections between the roots and the vertical crosshair were taken into consideration. Several categories were made to count the intersections: ‘arbuscules’, ‘vesicles’, ‘arbuscules and vesicles’, ‘only hyphae’, and ‘none’ i.e., there is no fungal material present in the root. To assess each intersection, the plane of focus was moved completely through the root. If the vertical crosshair cut one or more arbuscules or vesicles or hyphae, the appropriate category was incremented by one. For the arbuscular and vesicular colonization, the sum of the corresponding category was divided by the total number of intersections studied. The hyphal colonization was estimated as the ratio of non-negative intersections. One hundred intersections were calculated per sample to get the representative numbers (McGonigle et al., 1990b).

Microbiome analysis

The soil and root samples were frozen at -20°C until DNA extraction. To extract the DNA, 200 mg of soil was used for each bulk soil sample. For each root sample, 350 mg of root pieces were used. We utilized the Qiagen DNeasy Power Soil Pro kit and Qiagen DNeasy Plant Pro kit (Qiagen Inc., Valencia, CA) to extract the DNA, following the manufacturer’s instructions. We assessed the quality, quantity, and yield of the DNA using a NanoDrop ND-1000 spectrophotometer. After quantification, each DNA sample was normalized using nuclease-free water to a final volume of 50 µl at a concentration of 5 ng/µl. Standardized DNA samples have been sent to the University of Minnesota Genomics Center for amplicon sequencing for further microbiome analysis.