Recipient Organization
UNIV OF HAWAII
3190 MAILE WAY
HONOLULU,HI 96822
Performing Department
(N/A)
Non Technical Summary
Current problem: In the tropics and subtropics, farms are affected by increasing input costs, high pest loads, and nutrient-poor soils. The use of certain cover crops can serve as a low-cost intervention to improve soil fertility, as they can form symbiotic associations with nitrogen fixing soil bacteria. Thus, mulching these cover crops has been shown to improve soil fertility, leading to higher crop yields. Plants in the genusCrotalariahave the added benefit of producing anti-nematode alkaloids, which can significantly reduce the nematode load in the soil, further protecting crops and improving yields. These alkaloids are only produced in plants infected with certain microbes (sterile plants do not produce them), suggesting an intimate relationship between microbial infection and alkaloid production.Plant-feeding pathogenic nematodes are a major agricultural pest globally and are responsible for approximately USD $215 billion in annual losses for only the top 20 life-sustaining crops. This figure is likely an underestimate due to the cryptic nature of pathogenic nematodes and likelihood that nematode attack may be confused with fungal damage, water stress, or other physiological issues.Thus, the development of microbial inoculants for green manure crops has the potential to significantly boost plant health through improved soil fertility. Nematodes contribute to the loss of economically important crops such as coffee, pineapple, and sugarcane in Hawai'i specifically, in addition to the damage they cause to ornamentals.Goals and methods: The goal of this research is to identify and develop specific microbial strains from natural populations ofCrotalariaacross Hawai'i. Plants and their associated microbes will be collected from these populations and used to develop inoculants which will be experimentally tested in order to identify specific strains that optimize plant health, biomass, nitrogen production, and alkaloid content. Further, we will experimentally test how different phytochemical mixtures (different combinations of alkaloids) can affect nematode populations in field and lab conditions. Taken together, the results from these objectives will improve the utility of a low-cost green manure plant that can give growers a 2-for-1 deal: improved soil fertility and effective nematode control.
Animal Health Component
20%
Research Effort Categories
Basic
60%
Applied
20%
Developmental
20%
Goals / Objectives
The goal of the proposed research is to identify effective bacterial inoculants to maximize yield and alkaloid content of an important green manure crop, assess the effect of green manure phytochemical mixtures on nematode mortality, and experimentally test the respective ability of these factors to improve the efficacy of this green manure crop in field settings.Specifically, I will address the following three objectives: (1) Characterize the effects of rhizobia infection and strain diversity onCrotalariaalkaloid production in natural populations across Hawai'i (2) Experimentally test the effects of rhizobia infection and strain diversity onCrotalariaplants grown in agricultural settings and subsequent effects on nematode populations (3) Experimentally test the ability of phytochemical mixtures to control nematode populations in both field and lab settings. Taken together, the results from these objectives will improve the utility of a low-cost green manure plant that can give growers a 2-for-1 deal: improved soil fertility and effective nematode control.(Objective 1) Characterizing the effects of rhizobial infection and strain diversity onCrotalariaalkaloid production in natural populations.In Hawai'i, there are eight species ofCrotalariagrowing in natural populations:C. spectabilis,C. pallida,C. retusa,C. pumila,C. incana,C. lanceolata,C. assamica, andC. juncea. These natural populations will be used to harvest naturally occurring nodules, and plant traits will be quantified to elucidate the relationship between naturally occurring rhizobia, alkaloid production and diversity, foliar plant nitrogen, herbivore pressure, and soil nematode population densities. These observational data collected from wild populations will determine which strains will be developed for question 2.(Objective 2) Experimentally testing the effects of rhizobial infection and strain diversity onCrotalariaplants in agricultural settings and subsequent impacts on nematode populations.Using rhizobia harvested from Q1, I will identify at lease 5 rhizobia strains to be used in field and lab experiments. In addition to experimentally infectingCrotalariaplants with these candidate strains, I will also experimentally use serial dilutions to test the effect of microbial diversity on the yield, alkaloid content, and alkaloid diversity ofCrotalariaplants and their ability to control nematode populations.(Objective 3) Experimentally testing the effects of phytochemical mixtures on nematode populations.Field experiments using monocultures and mixed plots will be conducted to test whether in-field phytochemical mixtures can be more effective at controlling nematode populations. Using plants grown in the field experiment, extracts will be made from either monocultures (single species plots) or mixed plots (containing a higher diversity of alkaloids). These mixtures will be applied to nematodes in the lab and mortality will be measured.
Project Methods
(Objective 1)Methods:I will collect from naturally occurringCrotalariapopulations.For each host plant population, I will measure root nodulation for each individual plant, andpyrrolizidinealkaloid content in nodules, leaves, seeds, and flowers. Rhizoplane microbial communities will also be characterized using metabarcode (16s rRNA gene) sequencing on an Illumina MiSeq. Alkaloid content and composition will be measured using high-performance liquid chromatography (HPLC). Protein content of nodules, leaves, seeds, and flowers will be quantified using a bicinchoninic acid assay (BCA). Each individual plant harvested for nodulation will also be measured for total height and weighed for total biomass. Each individual plant surveyed will be assessed for herbivory (as a binary trait), in addition to noting any specific species engaging in foliar herbivory.Analysis:Metabolomics data will be analyzed to see if ion intensity is predicted by strain identity. Principal components analysis will be used to dissect how much variation is explained by nodulation status and strain identity. Additional analyses such as partial least squares projection to latent structures will also be utilized. Metabolomic data will be integrated into hierarchical structural equation models with the goal of quantifying trade-offs between alkaloid concentration, chemo-diversity, total plant nitrogen content, and total plant biomass. Additional structural equation models will be constructed to test the effects of isolate identity and diversity on alkaloid content and diversity, nitrogen content, plant size, nematode load, and herbivore performance.Expected Outcomes:In the short term, observational data collected at natural populations will allow us to see whichCrotalariaspecies are correlated with lower nematode loads in the soil. In the long term, a combination of metabolomic, sequencing, nutritional, and experimental data will identify specific candidate strains that are associated with high alkaloid content, high nitrogen content, robust plant growth, and low herbivory rates.These data will be extremely valuable in informing future agricultural work on green manure crops in Hawai'i and beyond.(Objective 2)Methods:For the first experimental phase of this objective, I will use two commercially available species:C. junceaandC. spectabilis. Seeds will be germinated in growth chambers and subset into the following treatment groups (n=200 seedlings per treatment group): control (uninfected), candidate strain 1, candidate strain 2, candidate strain 3, candidate strain 4, and candidate strain 5. After two weeks of plant growth, inoculation broth for each candidate strain will be used to infect seedlings through direct pipetting (10 ul per seedling) onto the base of each stem. After six weeks of growth inside, plants will be moved into greenhouses and grown for an additional ten weeks. Plants will be transplanted into experimental plots at the agricultural campus and allowed to grow for an additional eight weeks. Nematode load at these plots will be quantified before transplantation. Finally, plants will be harvested and tilled into the soil. Total biomass and plant height will be measured for each individual plant. A subset of leaves (40 mg) will be saved from each plant for alkaloid content and diversity analysis, and foliar nitrogen analysis. Nematode load will be quantified at each plot (and around each individual plant) to assess the effect of tillage.For the second experimental phase, I will use a series of serial dilutions on the same two species to test the effect of microbial diversity on plant growth, alkaloid production and diversity, foliar nitrogen, and nematode control. An inoculation slurry will be prepared using nodules harvested from previously identified candidate populations. The inoculation slurry will be serially diluted for each candidate population (five treatment groups per candidate population; 25 treatment groups total plus a control group per species). Seeds will be germinated, inoculated, and grown using the same methods as the first experimental phase. Nematode load will be quantified in the same manner as the first phase. Total biomass and plant height will be measured for each individual plant. A subset of leaves (40 mg) will be saved from each plant for alkaloid content and diversity analysis, and foliar nitrogen analysis. Nodulation will be measured for each plant to assess the effect of microbial diversity and nodule size, number, and quality.Analysis:Metabolomics and foliar nitrogen will be analyzed using the same methods as Obj 1. Structural equation models will be constructed to evaluate the direct and indirect effects of nodulation on plant yield, alkaloid content and diversity, and nematode load. Separate models will be constructed to evaluate the effects of microbial diversity on nodulation (size, number, and quality) and direct and indirect effects on previously described response variables (i.e., plant yield may be indirectly affected by microbial diversity via degree of nodulation).Expected Outcomes:This first combination of experiments will provide vital information about the identified candidate strains and their performance in agricultural settings. For example, whether we see patterns between alkaloid content and/or diversity and strain identity observed in natural populations replicated in experimental settings is highly informative for future agroecology research. Consistently high performing strains that produce plants with high alkaloid content, high foliar nitrogen, and suppressed nematode populations in both natural and experimental settings are excellent candidates for potential microbial inoculants.(Objective 3)Methods:The first phase of this objective will consist of growing plants in both monocultures and mixtures in field settings. For the second phase, plants harvested from the field will be used to produce extracts for lab nematode toxicity experiments. Upon germination, seeds in the inoculated treatment groups will be dosed with inoculant broth (using the best performing candidate strain from Objective 2). Plants will be grown for an additional 6 weeks in the greenhouse before being transplanted into field plots. Nematode loads will be quantified before planting and after tillage. Plots will be tilled after 10 weeks of growth, with 100 mg of fresh mass harvested from each plant before tillage. For the second phase of this objective, fresh plant mass harvested from the first phase will be dried using a drying oven and then homogenized. Fresh extract (made from 50 mg dried plant mass and 50 ml methanol) will be applied directly to nematodes. For the extracts made from mixed plots, 25 mg of dried plant mass will be used from each species (50 mg total).Nematodes will be harvested locally from the University of Hawai'i agricultural research station. After 24, 48 and 72 hours, nematodes will be inspected to observe mortality, or any behavioral changes.Analysis:The presence and diversity of alkaloids in extracts will be validated using HPLC. Monocrotaline will be used as an internal standard for all extracts, and total amount of alkaloids will be quantified (in mg/g of dried plant material) in addition to the diversity of alkaloids.Expected Outcomes: In the short term, phytochemical mixtures will be tested to validate the results of in-field experiments. For instance, we expect that monocultures will be less effective at controlling nematode populations both in field and lab settings compared to mixed plots, due to reduced phytochemical diversity. These results will be valuable because they highlight the importance of phytochemical diversity as a tool in integrated pest management and cover crop development. In the long term, growers may potentially benefit from new strategies of integrated pest management, such as diversifying cover crops and green manure plots rather than planting monocultures.