Progress report for GS20-224
Nitrogen (N) is a main limiting factor for plant growth in agroecosystems. Given the unintended consequences for climate change and environmental impacts caused by highly inefficient N use in modern agroecosystems, increasing nitrogen-use efficiency in agriculture is a central focus of intensive research in agriculture. Mortierella elongata, a member of the early-diverging Mortierellomycota, is a dominant fungus among agricultural soils and functions as saprotroph and root endophyte, affecting several soil processes. Little is known about the effects of M. elongata on soil N transformation processes and the structure of the nitrifying community. Our preliminary results show that M. elongata isolates have a great ability to promote crop growth, but it remains unknown whether the promoting effects of M. elongata on crop growth are associated with its regulation of soil N dynamics and plant-available N. Our recent work shows that integrating two years of bahiagrass (Paspalum notatum) into conventional peanut (Arachis hypogea L.) and cotton (Gossypium hirsutum L.) cropping systems, known as sod-based rotation (SBR), has greater N availability compared with a conventional peanut-cotton-cotton rotation (CR). The proposed work focuses on a plant bioassay using cotton grown in soil from SBR and CR. We will combine stable isotope analysis with molecular tools to quantify the effects of M. elongata on microbially-derived N transformation processes and plant-available N dynamics, as well as on crop productivity under different crop rotations. Our objective is to better predict soil N transformations, helping growers improve economic viability with less external inputs while making agriculture more sustainable.
1) Determine the molecular role of Mortierella in regulating soil N transformations and plant N dynamics under different crop rotation systems using 15N tracers;
2) Measure the effects of Mortierella on community structure and functional genes of the soil nitrifying microorganisms under different crop rotations;
3) Quantify the consequences of objectives 1 or/and 2 on cotton productivity.
1) Mortierella induces higher plant-available N and soil N content and reduces N2O emission under sod-based rotation (SBR) compared to conventional rotation (CR);
2) Mortierella increases the abundance and functional gene activity of the nitrifying community in SBR systems in comparison to CR;
3) The SBR system inoculated with Mortierella will have the highest cotton productivity.
Soil source and bioassay setup for both objectives
We will set up a plant bioassay comparing CR and SBR soil in a growth chamber at the North Florida Research and Education Center, Quincy, FL. Soils will be collected after cotton harvest in the four-year bahiagrass-bahiagrass-peanut-cotton rotation (SBR) and the three-year peanut-cotton-cotton rotation (CR). Thus, soils will be collected from cotton plots in the single cotton phase of SBR and the two cotton phases of CR (first and second-year cotton). Cotton seedlings be transplanted into closed- system pots (20 cm × 20 cm × 50 cm) (Fig. 1B). M. elongata PMI 624 will be used as the inoculum. Labeled 15N-urea at a rate of 1% (w/w) will be used as N fertilizer to provide 28 kg N ha-1 and trace N transformations. This experiment comprises six treatments (the three cotton soils with or without inoculation with M. elongata PMI 624) sampled destructively at three sampling points, with three replicates per sampling point. This experiment will be assembled in a completely randomized block design, with a total of 54 pots.
Fig. 1 (A) Conceptual view of the central hypothesis that Mortierella increases the efficiency in soil N cycling and crop productivity underlying SBR compared to CR. (B) Mortierella-cotton paired bioassays will be performed to assess our hypotheses.
Objective1. Determine the molecular role of Mortierella in regulating soil and plant N dynamics under different crop rotation systems using 15N tracers
Bulk soil samples will be taken monthly and rhizosphere soil and root will be collected at planting, growing, and harvest season. Gas and leachate will be collected biweekly. The δ15N values (the abundance of stable 15N isotopes) in soils (bulk and rhizosphere soil), gas, microbial biomass, leachate, and root will be measured to determine how M. elongata PMI 624 affects soil N transformations and plant-available N. The isotopic analysis of N2O, N2, soil NH4+ and NO3–, and leachate will be determined by the method of Castellano-Hinojosa et al., (2020), and 15N-enrichment of root will be determined as reported by Andresen et al. (2011). Fluorometric enzyme assays using 4-Methylumbelliferone (MUB) substrates will be used to identify chitinase activity in the bulk and rhizosphere soil and to determine the molecular role of M. elongata PMI 624 on soil and plant N dynamics.
Objective 2. Measure the effects of Mortierella on community structure and functional genes of the soil nitrifying microorganisms under different crop rotations
By analyzing the DNA amplicon sequence data of collected rhizosphere and bulk soil, we can determine the effects of M. elongata PMI 624 on the variation and composition of the soil nitrifying community. After collecting soil and root, samples will be stored at -80 oC. Soil DNA will be extracted using the DNA PowerSoil kit (MoBio, Carlsbad, CA, USA) following the manufacturer’s instructions. Two-step PCR will be used to generate the amplicon library. Bacterial16S rRNA and fungal ITS gene fragments will be amplified using 341F/806R and ITS1/ITS4, respectively. All amplicons will be pooled at equimolar concentrations (20 ng μl-1), and sequenced using Illumina Miseq (v2 250bp, 6Gb sequencing capacity) (Illumina Inc., San Diego, CA, USA). qPCR will be used to quantify the expression of N-associated genes (amoA, nosZ, nirK, and nirS) in the rhizosphere and bulk soil. These results will show how M. elongata PMI 624 affects N transformation processes at the molecular level. Root metatranscriptomics (the analysis of RNA sequence to study gene expression of microbes within natural environments) will be used to measure changes in microbial diversity and N-related functional genes of root with M. elongata PMI 624 inoculation under different cropping systems. Root RNA extraction and cDNA library construction will use the method of Liao et al. (2018). cDNA pools will be sequenced on Illumina HiSeq 2000 instruments in the Duke Center for Genomic and Computational Biology.
The data of bacterial 16S rRNA and fungal ITS gene sequencing will be processed by the QIIME 1.8.0-dev pipeline. RNA sequence assembly and annotation will follow the method of Liao et al. (2018).
Objective 3. Quantify the consequences of objectives 1 or/and 2 on crop productivity
At harvest, we will measure cotton plants for biomass and yields to link results from objectives 1 or/and 2 with crop productivity. Cotton plants will be oven-dried at 65 oC for four days to obtain dry crop biomass. Lint and seed yield will be calculated by grinding a 100g subsample from each pot. Soil properties and chemistry (pH, potassium, calcium, sodium, magnesium, total phosphorus, total organic carbon, and total nitrogen) will be tested to determine the effects of M. elongata PMI 624 on variations in soil chemical properties. Bulk soil samples will be air-dried and sieved to 2 mm, and sieved soil samples will be analyzed for soil chemical properties at the Agricultural and Environmental Services lab of the University of Georgia.
We evaluated the impacts of three Mortierella isolates (PMI 77, PMI 624, and PMI 93) on plant height, plant dry biomass, and leaf area of different crops using Mortierella-inoculated plants in greenhouse bioassays (Fig. 2). Mortierella isolates had a significant impact on watermelon growth compared to non-inoculated controls. Isolates PMI 624 and PMI 93 significantly promoted at least one variable for corn and tomato relative to PMI 77 and control, and the same effect of PMI 624 and PMI 93 also occurred in the leaf area and plant dry weight of squash. Although all three Mortierella isolates positively affected the growth of bahiagrass, there was a trend of greater bahiagrass growth with inoculation of PMI 624 relative to PMI 77 and PMI 93. However, it remains unclear if the growth-promoting effects of Mortierella isolates are associated with Mortierella-induced N transformations.
Fig. 2 The impacts of Mortierella isolates on the growth of different crop species. Different lower-case letters represent significant differences across different isolates at P < 0.05 by one-way ANOVA (Zhang et al. in review).
Educational & Outreach Activities
We published a journal article entitled Mortierella elongata Increases Plant Biomass among Non-Leguminous Crop Species in Agronomy (doi:10.3390/agronomy10050754). In this publication, we found that Mortierella elongata generally promoted metrics of the plant performance among a diverse set of importantly non-leguminous crop species, including the cover crop of SBR (bahiagrass). Our recent EDIS article (The Plant-Growth-Promoting Fungus, Mortierella elongata: Its Biology, Ecological Distribution, and Activities Promoting Plant Growth) provides a brief overview of Mortierella from biological, taxonomical, ecological, and functional perspectives to help readers learn biology and potential modes of action of this fungus. Due to the Covid-19, we had a virtual sod-based rotation field day hosted by North Florida Research and Education Center in October 2020. I worked with my graduate student (Kaile Zhang, co-PI of this student grant project) and delivered a presentation regarding the impacts of the SBR system on soil health-soil C cycling (https://www.youtube.com/watch?v=6a15ROJgXZE&t=304s). This year, we led an In-service training (Soil health-Chemical and Biological health) with 22 agents and another 4 professionals (Dr. Ann Blout, Dr. Cheryl Mach, Dr. Yang Lin, and Dr. Sheeja George). This training aims to identify the chemical and biological indicators of different cropping systems across Florida (including SBR). On February 22, 2021, we had virtual training for agents in terms of introducing the importance of soil health in agroecosystems and how to take soil samples from the field. To date, we are processing soil samples and analyzing chemical and biological data. Here, we plan to generate a potential soil health indicator list using scientific-based evidence and will schedule a virtual meeting in July to report our results to the agents
To gain in-depth knowledge on the understanding of roles that soil biota play on soil nutrient cycling under diversified crop rotation systems (in respect to part of the scope in this proposed study), we collected the lectures and led a review article publication (entitled: How soil biota regulate C cycling and soil C pools in diversified crop rotations. Reference ID: 108219). The article has been accepted by Soil Biology and Biochemistry. Besides, our research manuscript studying how long-term SBR affects the nematode community and nematode-microbiome interactions is under review by Applied Soil Ecology. We are also preparing another research manuscript in terms of studying the effects of root microbiomes on cotton performance.