Alfalfa is the third largest field crop in the US, with hay value calculated over $9.9 billion last year. However, alfalfa is susceptible to fungal pathogens and drought, which are increasing threats as climate patterns become more extreme and unpredictable. The purpose of this work is to determine if the application of the plant growth promoting rhizobacteria (PGPR) Bacillus subtilis UD1022 (‘UD1022’) along with the legume symbiont Sinorhizobium meliloti will enhance alfalfa crop productivity. The PGPR UD1022 has been shown to both inhibit common fungal pathogens including Phytotphora capsica, Rhizoctonia solani and Botrytis cinerea, as well as increasing plant resistance to drought. S. meliloti is also known to increase alfalfa resistance to drought. The combination of UD1022 and the alfalfa symbiont S. meliloiti could significantly reduce the impacts of drought and increase yield in alfalfa.
In this project we are evaluating the direct effect the PGPR UD1022 has on four common fungal pathogens of alfalfa including, Phytophthora medicaginis (root rot), Colletotrichum trifolii (anthranose), Phoma medicaginis (blackstem) and Fusarium oxysporum f. sp. medicaginis (vasucar or fusarium wilt). These pathogens have a great economic impact on the production of alfalfa; applications of fungicides represent a cost in time and money in attempting to reduce the effect of the disease and crops impacted suffer loss in yield and nutritional value. In order to determine if UD1022 produced natural compounds which inhibit the growth of these fungi, the bacteria are grown together on petri plates directly or in separate compartments. UD1022 has been shown to inhibit the growth of other common plant pathogen fungi using this method. This work is in progress currently. Once we have identified the fungal strains whose growth is significantly inhibited, those strains will be tested on live alfalfa plants maintained in controlled growth chamber conditions. This test will include treatments of plant whose roots are colonized by their symbiont S. meliloti, which may enhance the potential plant protective effect of UD1022.
To evaluate the overall growth promotion effect of inoculating both UD1022 and S. meliloti onto alfalfa, we are conducting a pot study in controlled greenhouse conditions. Field soil has been collected from the University of Delaware Research Farm, sieved and stored in a cold room until the study is started. The field soil will be homogenized with vermiculite (1:1) and seedling will be grown in this soil in 3-inch pots. The treatments include no inoculation of bacteria, S. meliloti only, UD1022 only and co-inoculation of both S. meliloti and UD1022. The biomass and nodulation of the plants will be compared to measure the response of alfalfa to the application of the PGPR. This study is planned to be initiated the first week of February 2020.
Drought poses another economically detrimental problem to alfalfa production. The potential effects of the PGPR on plant resistance to drought will be evaluated in a similar manner as the growth promotion test, using a vermiculite-perlite mix instead of field soil. The plants will be grown, treatments of bacteria applied, and then water will be withheld. Early plant growth and late stage plant growth drought tests will be conducted. The biomass and root nodules will be quantified as a measure of plant health. This test is planned to be initiated the first week of April 2020.
Growth promotion by PGPR application in the field tests are projected to be started in the field mid-April.
1. Determine direct fungal inhibition of UD1022 on six fungal pathogens of alfalfa using standard laboratory assays.
2. Test alfalfa resistance to three select alfalfa fungal pathogens when inoculated with both S. meliloti and UD1022 compared to un-inoculated and S. meliloti alone.
3. Evaluate whether inoculation with UD1022 and S. meliloti increases overall drought resistance and drought recovery in a greenhouse setting.
4. Quantify alfalfa biomass, lateral root number and number of symbiotic root nodules of plants inoculated with S. meliloti, both S. meliloti and UD1022 and no inoculation in a greenhouse setting.
5. Compare the biomass and nitrogen content of alfalfa treated with S. meliloti alone or both S. meliloti and UD1022 in the field.
The purpose of this project is to enhance alfalfa crop productivity using plant beneficial microbial inoculants. The use of nitrogen (N) fertilizers in agriculture is unsustainable and costly. Leguminous plants, such as soy and alfalfa are known to mitigate these liabilities through their ability to form symbiosis with N-fixing rhizobia bacteria in their roots. Rhizobia symbionts are commonly applied to these crops, reducing nitrogen inputs and enhancing plant growth. However, alfalfa production is susceptible to many fungal pathogens and is intolerant of drought or low water conditions. There is a growing movement in the agro-chemical industry toward the use of natural microbial inoculants to promote the growth of major food production crops. These ‘biologicals’ have been shown to benefit plants through increasing biomass, suppressing bacterial and fungal pathogens, increasing nutrient uptake and reducing the impacts of drought. This project will assess the compatibility and capabilities of the University of Delaware patented plant growth promoting rhizobacteria (PGPR) Bacillus subtilis UD1022 (hereafter UD1022) applied with the alfalfa symbiont Sinorhizobium meliloti to enhance alfalfa crop production.
Alfalfa is the third largest crop grown in the United States, with alfalfa hay valued at $9.9 billion annually. In 2017, Pennsylvania had 400,000 acres in alfalfa production, rivaling that of New York and nearly 3 times more than 13 other states in the Northeast Region combined. Alfalfa is important as a modifier of nitrogen inputs, both as a cash crop and when used in rotations. Its superior ability to take up and retain nitrogen contribute to its high protein content as fodder and makes it an ideal crop for nitrogen management. UD1022 is documented to increase pea plant biomass and nitrogen content when applied with the pea symbiont. The co-inoculation of UD1022 with S. meliloti could increase alfalfa biomass and its nutritional value.
Several factors challenge the productivity of alfalfa production, chiefly, fungal pathogens and drought. The PGPR UD1022 has been shown to inhibit common fungal pathogens including Phytotphora capsica, Rhizoctonia solani and Botrytis cinerea, likely through volatile compound production. These and other common fungal pathogens significantly reduce alfalfa crop yields and require intensive use of chemical control. The use of UD1022 in addition to S. meliloti alfalfa symbiont has the potential to drastically reduce the need for chemical controls and simultaneously provide additional benefits and protections to alfalfa.
Drought is another challenge with significant negative effects on alfalfa yields. Though certain cultivars are better equipped to resist drought events, the severity and unpredictability of such events are increasing due to climate change. UD1022 has been shown to increase plant resistance to drought. In addition to increasing lateral root proliferation, which allows greater access to soil pore water, UD1022 produces extensive biofilms. These biofilms increase soil water retention and reduces evaporation, increasing water availability to plants. S. meliloti is well documented in the literature to also aid in increasing alfalfa resistance to drought cycles. The combination of UD1022 and the alfalfa symbiont could significantly reduce the impacts of drought and increase yield in alfalfa.
- UD1022 Fungal Antagonism Assays
- Direct Antagonism Assay.
UD1022 will be used to challenge four common pathogens of alfalfa, which have been obtained from Dr. Deborah Samac of the University of Minnesota. The fungal isolates which are being tested are Phytopthora medicaginis (root rot) strain A2A1, Phoma medicaginis (spring black stem rot), strains StC 306-5 and StC 306-10, Fusarium oxysporum f. sp. medicaginis (fusarium wilt) Fom 255 and Fo 3, and Colletotrichum trifolii (anthranose) strains St. Paul AN 1 and SM. Appropriate permitting has been obtained through the USDA APHIS program. Fungal isolates are cultured on Potato Dextrose Agar (PDA) and V8 agar and stored on plates. A plug of fungal culture is cored and placed on a petri plate with a 20 microliter drop of UD1022 culture. Fungal diameter will be measured and compared to controls without UD1022. Three biological replicates will be used, and the experiment will be repeated three times. Mean fungal diameters will be compared after evaluation of normalcy. A t-Test will be applied, and significance will be accepted at P less than 0.05. This experiment will determine UD1022 direct inhibition ability on the top common alfalfa fungal pathogens.
- Indirect Antagonism Assay.
This test is similar to the direct assay, except the petri plate is divided in the middle to prevent direct interaction. UD1022 produces volatile compounds which may inhibit the growth of other organisms through the atmosphere. Results from the direct and indirect assays will be used to select three fungal pathogens to be used in UD1022 and S. meliloti inoculated plant challenge assays. Candidates (reduced fungal diameter) from either assay will be considered. UD1022 may be able to confer plant protection without directly antagonizing the pathogen. UD1022 has been shown to produce an ‘induced systemic response’ (ISR) in plants. ISR ‘primes’ the plant for pathogen attack, resulting in the plant becoming more resistant to infection.
- UD1022 Plant Fungal Suppression Assay.
Alfalfa seed will be sterilized, scarified with sulfuric acid and cold stratified for 48 hours at 4 degrees Celsius. Alfalfa seeds will be germinated on water agar plates 24 hours at room temperature in the dark. Germinated seeds will be transplanted to pots containing sterile sand and fertilized and maintained with a low nitrogen (0.5 micro Molar) solution. Plants will be kept in controlled growth chambers with 16-hour photoperiod, 55 percent relative humidity at 22 degrees Celsius. Treatments of no bacteria controls, S. meliloti and S. meliloti with UD1022 will be applied as a root drench three days after transplantation and fungal isolates will be applied to plants 10 days after transplantation. One treatment will have no bacteria and mock fungal application. Twenty-five plants will be used in each treatment arranged in a completely randomized design. Progress of the infection will be monitored daily over the course of two weeks. Results will be evaluated 20 days after transplantation (10 days after infection) using a weighted indexing system based on the progressive numbers of tissues and percentage of infection on those tissues. Statistical analysis will be a test for normal distribution and comparison of mean index values using the t-Test and significance of P less than 0.05.
- UD1022 – meliloti Plant Drought Tests.
- Early Growth Stage Drought Challenge.
The first drought experiment is intended to test drought conditions occurring at early vegetative (Stage 0) growth. Alfalfa seeds will be prepared and germinated as in Objective 2.0. Alfalfa seedlings will be transferred into pots containing vermiculite:perlite mix at 2:5 ratio by volume. A completely randomized design will be carried out in a growth chamber. Treatments will have 10 plants each. Three treatments of no inoculation (mock), S. meliloti and S. meliloti with UD1022 will be applied 5 days after placement in the chamber and irrigation maintained with fertilization of low nitrogen (0.5 milli Molar). After 3 weeks, each treatment will be randomly divided in half (5 plants each). One half will be maintained with irrigation, the other half will have drought conditions applied through suspension of irrigation. Prior to drought treatment, all pots will be watered to saturation, allowed to drain, and weighed to establish a baseline for measuring relative water content (RWC). After 10 days, plants will be documented through high quality photography and shoots harvested and weighed. Shoots will be dried at 70 degrees Celsius for 72 hours. Percent relative water content for shoots will be calculated. Shoot biomass will be compared by evaluation of means and a t-Test (after normalization analysis) will be applied with significance calculated at P less than 0.05.
- Harvest Growth Stage Drought Challenge.
This experiment will be conducted exactly as in objective 3.1, except the timing of drought application will be delayed until after 8 weeks of irrigated growth to emulate a drought occurring directly prior (late bud Stage 4) to a first cutting harvest of the alfalfa crop at first flowering (Stage 5).
- UD1022 – meliloti Alfalfa growth promotion in Greenhouse.
Field soil has been collected from an agriculture field cultivated in corn for 18 years on the University of Delaware Research Farm in Newark, DE. The top 6 inches of plowed soil was collected, sieved through 4 mm mesh and stored in 4 degree C cooler. The soil will be homogenized 1:1 with vermiculite. Soil will be used in 3-inch pots for alfalfa growth. Alfalfa plants will be germinated, prepared, potted and placed in greenhouse conditions and maintained as in Objective 2.0. Twenty-five plants will be randomly assigned to four treatments of no inoculation, S. meliloti only, UD1022 only, and S. meliloti with UD1022 applied 3 days after transplantation. Plants will be grown to Stage 5 early flowering at about 9 weeks after inoculation. Plants will be harvested, roots and shoots separated, fresh weights collected, and tissues dried at 70 degrees Celsius for 72 hours. Shoot to root ratios will be calculated. Shoot material will be sent to University of Delaware Soil Testing lab for total analysis through sample digestion and ICP analysis for P, K, Ca, Mg, Mn, Fe, Cu, B, Al, Zn, Na, total nitrogen and total carbon. Five subsamples of roots will be collected from each treatment and analyzed for nodule number, architecture and lateral root number using plant image analysis software DART. Data will be analyzed by mean comparisons and t-Tests applied with significant differences were determined at P less than 0.05.
- UD1022- meliloti Alfalfa Growth Promotion in Field.
Field plots of 3 x 3 meters will be sown with alfalfa at the University of Delaware Farm. The treatments will be no inoculation, S. meliloti only, UD1022 only and S. meliloti with UD1022. Bacteria will be applied as seed coatings in lab for S. meliloti and provided by BASF for UD1022. Each treatment will have 3 replicate plots set up in a randomized complete block design. Plots will be sown on April 15th, 2020 and harvested July 1st, 2020. Random plants will be sampled from diagonal transects every 0.25 meters (6 plants). Plants will be cut 2 inches above soil surface. Biomass will be weighed fresh, then dried as previously, and subsamples sent for analytical nutrient content as in Objective 4.0. Roots will be dug, washed and nodule number quantified. Data will be analyzed by mean comparisons and t-Tests applied with significant differences were determined.
We have no results at this time to report.
Research is in process and there are no conclusions to report at this time.
Education & Outreach Activities and Participation Summary
Currently I am submitting an abstract to present a poster at the 2020 Delmarva Soil Summit, February 27 – 27, 2020. The abstract and poster are geared to present the research goals and approaches of this proposal. This poster will describe the need for more fundamental research to integrate effective soil microbiome approaches to facilitate sustainable and regenerative agriculture. If accepted, I will have the opportunity to gain a wider audience for the use of plant beneficial bacteria to address problems in forage and commodity crops and improve soil quality.
Additional outreach will include presenting results from in vitro and in vivo fungal inhibition assays and preliminary growth promotion data will be presented in poster/short talk form at the 2020 North American Alfalfa Improvement Conference (NAAIC) held in Lansing, MI in June 1 – 3, 2020.