Testing the potential of seed pellets to improve the soil health in rangelands

Experimental design and seed pellets formulation

This experiment was conducted at King’s Anvil Ranch in Altar Valley, Pima County, AZ, operated by project producer Joseph W. King. 5 random sites with 12 2m*2m plots in each site were deployed in a 20 acres highly eroded site (Figure 1). Barren areas without vegetation were selected as the study sites. Twelve plots were arranged in a three-row by four-column configuration, oriented in the east-west and north-south directions, respectively, with a 2-meter gap between each plot. Trenches were dug using a garden pick perpendicular to the potential water flow direction (mostly north to south) to prevent water or wind from moving the seed pellets. On July 17, 2023, the seed pellets were added to coincide with germination during the monsoon rains and evenly spread within each plot, except for the control (CTRL) plots (Figure 1). The unseeded NONE and CTRL plots served as negative controls to evaluate the impact of seed pellets on soil health. The NONE plots had no trenches to control for any potential effects of trenches on plant establishment (Figure 2).

Regular seed pellets contained three components: seeds, nutrients, and clay, which is used to bind the pelletizing materials (Gornish et al. 2019). Seeds of Sideoats Grama (Bouteloua curtipendula) were purchased from a natureseed.com. Sideoats Grama was selected based on the recommendation of the producer. Clay was obtained from the inventory of the Gornish lab. For each plot except for NONE, CTRL, and SEED treatments, 40 grams of seeds were pelletized into 240 grams of seed pellets (the seedling rate is calculated from recommended seedling rate for broadcasting at 10 grams per m²). Two types of organic amendments, manure and compost, and three types of microbial inoculants (Tainio BioGenesis III (plant growth-promoting bacteria), Tainio Specturm plus Myco (plant growth-promoting bacteria+ arbuscular mycorrhizal fungi), and local soil from undisturbed sites) were added as treatments. Manure was obtained in situ from Kings Ranch and compost was purchased from Tnaks’ Green Stuff. Each plot received 120 grams of the designated organic amendment. Plant growth-promoting bacteria (PGPB) and arbuscular mycorrhizal fungi (AMF) have already reached commercialization in agriculture and are frequently tested in dryland restoration (de-Bashan et al. 2012). However, their unknown survival rate and duration within seed pellets and inconsistent results in the field require further experiments in rangelands (Naamala & Smith 2021). The mass of beneficial microorganisms were added based on the instruction from vendors (2.21 g/plot for Tainio BioGenesis III and 6.13g/plot for Tainio Specturm plus Myco ). Each plot of local soil treatment was be pelletized with 120 grams of topsoil collected from nearby less-disturbed sites instead of beneficial microorganisms. Clay will be added to maintain the same total mass of pellets (80 grams) among plots.

Objective 1: Monitor plant growth and establishment to assess the effect of seed pellets on plant communities

Sparse plant cover is one of the reasons why dryland soils are vulnerable to erosion. Thus, if seed pellets can increase plant cover through facilitating the establishment of seedlings, soil health can be indirectly improved by reducing the soil’s direct exposure to disturbances. Furthermore, previous researches indicate that nutrient-rich soil amendments have the side-effect of promoting invasive species (McFarland et al. 2010). The establishment of unseeded plants will also be monitored to determine if pelletizing soil amendment can avoid that side-effect.
Ben Yang recorded plant establishment by counting plant density for each plant species within each plot once per month after seeding except for months received zero precipitation (see Table 1 in research results). We expected the number of seedings will be always higher in pelletized treatments than unpelletized treatments due to the protection from seed pellets. Treatments receiving organic amendment should arrive at maximum plant density earlier than other treatments since organic amendments can supply extra resources and retain more water. We initially planned to estimate plant cover at the end of the monsoon period (October), at the end of the winter rain season (February), and the end of monsoon periods of the next year (February and October, 2023). However, seeded plants fail to survival after monsoon ceased in October (Figure 3) and did not form continuous cover.

Objective 2: Quantify forage production to estimate the short-term economic return of seed pellets

Foliage biomass and quality are directly related to the short-term return of using seed pellets in rangeland. Both quantity and quality will be measured so that the drawback of organic soil amendments (i.e., increasing biomass but decrease forage quality) can be tested.

At the peak biomass of the 2^nd year (October, 2023), we planned to clip and weigh the foliage of all seeded plants in each plot send samples to an analytical lab to measure protein, acid detergent fiber, and phosphorous to assess foliage quality. We expected pelletized treatments have higher total biomass due to the possibly higher germination rate of seeded species. Pellets with the organic amendment were expected to have higher biomass and better forage quality compared to the plain pellets because of the additional nitrogen and organic matters. Microbial inoculants were expected to increase the quantity and quality of forage further because of their abilities to enhance the efficiency of organic amendment and promote plant growth (Hayat et al. 2010). Unfortunately, no sideoats grama established in the field as of October 2023, preventing us from assessing forage production and quality as originally proposed. To address this, we conducted two batches of greenhouse experiments. In one batch, sideoats grama was densely seeded with all unwanted plant species removed to simulate an agricultural setting. In the second batch, sideoats grama was lightly seeded without removing unwanted plant species, simulating natural conditions. These experiments were designed to evaluate the effects of microbial inoculants on forage production. We did not send the greenhouse samples for forage quality analysis as initially proposed because the microbial inoculants did not significantly affect sideoat grama, and the greenhouse results may not accurately represent forage quality under field conditions.

Objective 3: Determine the effect of seed pellets on soil health through soil health indicators

Soil health is an integrating concept summarizing physical, biological, and chemical components of the soil and has more than 43 potential indicators (Moebius-Clune et al. 2016). Considering the relevance to rangeland management and the possibility to be affected by our treatments, soil stability (wet-sieving method, Eijkelkamp), available water capacity, nitrogen (NH4+ and NO3- concentration), phosphorus, organic matter, and microbial community composition will be measured. Soil stability indicates soil’s resistance to erosion, which is critical to dryland soils that are threatened by frequent wind erosion and intense rain erosion. Available water capacity measures the range of plant available water the soil can store. Since water is the most limiting resource in arid western rangelands, available water capacity is important to plant growth and forage quality. Nitrogen and phosphorus are commonly deficient in soil and limit plant growth. Nitrogen-containing organic amendment and nitrogen-fixing microorganisms in seed pellets are very likely to increase the nitrogen content in the soil. Plant-available phosphorus might also increase because of the inoculated microorganisms mineralizing the organic phosphorus or mobilizing phosphorus from minerals (Richardson & Simpson 2011). Soil organic matter plays a central role in soil health, including soil fertility, drought resistance, aggregate stability, microbial activities, and nutrient cycling (Plaza-Bonilla et al. 2015; Fierer 2017; Moebius-Clune et al. 2016). Meanwhile, soil organic matter is naturally deficient in arid or semi-arid rangelands due to the low plant material input and high erosion (Plaza et al. 2018) and is fragile to human activities and climate change (Berdugo et al. 2020). Microbial community composition can reflect the differences in microorganisms-driven functions across treatments. It can also detect if the microbial inoculants are successfully introduced or not.

Soil samples were originally planned to be collected from each plot during the peak growing season (September 2023). We intended to analyze soil physicochemical properties at the Blankinship Lab at the University of Arizona and investigate soil microbial communities using 16S and ITS amplicon sequencing. However, due to poor plant establishment in the field at Altar Valley, we were unable to carry out these measurements. Instead, we utilized soil samples from a separate soil inoculation project at Point Reyes National Seashore to assess the impact of soil inoculation on microbial communities and its potential to improve soil health (publication in preparation).

Objective 4: Determine the lifespan of two microbial inoculants in seed pellets

A challenge in commercially available microbial inoculants is the “shelf-life” (O’Callaghan 2016). Some environmentally sensitive microorganisms can only survive for a limited time after being extracted, which then leads to inconsistent field performance. Although seed pellets in our experiment are freshly made, seeds and the inoculated microorganisms will not be activated until the first precipitation. Due to the highly varied precipitation in Western U.S. rangelands, assessment of the life span will be crucial to the effectiveness of seed pellets.
A simultaneous greenhouse experiment with soil collected from the field study sites was conducted using seed pellets with Tainio BioGenesis III, Tainio Specturm plus Myco, or no microbial inoculants (C1M0, C1M1, C1M2). Seed pellets received first watering at day 0, day 7, day 14, and day 30 after they are sown (D0, W1, W2, M1, Figure 1C). Two batches of pots were seeded. The first batch was overseeded with seed pelleting containing 15 grams of seed in each pot to examine the effect of microbial inoculants on maximum productivity. All unwanted weeds were removed. The second batch was seeded with 6.67 grams of seed in each pot (approximate to the seeding rate of the field experiment) and kept all weeds to examine if microbial inoculants would benefit unwanted plants. Both batches then received watering once per week. The total amount of water each pot received was not controlled. Instead, each pot received watering for at least 10 minutes to ensure the soil in pots were saturated. We did so since microbial inoculants might ameliorate the soil water holding capacity and affect plant growth indirectly. Plant biomass for first batch was harvested twice (April and October, 2023). Plant biomass for the second batch, soil sample and root for both batches were sampled after one year (October, 2023). The greenhouse experiment was extended compared to the initial plan because sideoats grama did not flowering nor dormant in the greenhouse and thus we cannot identify a clear time point to stop the experiment. Physicochemical properties would be measured for each replicate with the same process as the field experiment. Mycorrhizal colonization rate was quantified using microscopy. We expect soil health, plant growth, and the AMF colonization rate will all decrease with the increasing time lag for watering due to the loss of microorganisms in seed pellets. This finding will be important to determine the viability of microbial inoculants. For example, if AMF colonization rate rapidly shrinks in the first three days, a more preservative vector will be necessary. This simultaneous experiment can also verify the effect of microbial inoculants by linking microbial abundance with soil health and plant growth metrics.

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