Progress report for GNE19-217
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 plant growth chambers. Field soil was collected from the University of Delaware Research Farm, sieved and stored in a cold room until the study was started. The field soil was homogenized with vermiculite (1:1) and seedling were 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 are being compared to measure the response of alfalfa to the application of the PGPR. This has been completed; shoot biomass and nodulation data are being evaluated and elemental analysis of plant material is in progress.
Due to time constraints caused in part by the suspension of University of Delaware research during Covid-19 lock-down, drought studies and field studies have been discontinued for this project.
- Determine direct fungal inhibition of UD1022 on four fungal pathogens of alfalfa using standard laboratory assays.
Evaluate biofilm and non-ribosomal protein B. subtilis UD1022 mutants for their role in fungal inhibition.
- Test alfalfa resistance to Phytophthora medicaginis A2A1 oomycete alfalfa pathogen when inoculated with both S. meliloti and UD1022 compared to un-inoculated and S. meliloti alone.
- Quantify alfalfa biomass and number of symbiotic root nodules of plants inoculated with S. meliloti, UD1022, both S. meliloti and UD1022 and no inoculation in a plant growth chamber. Characterize differences in elemental content of plant shoots between treatments.
Based on the statement of the problem, rationale, and justification of need stated in your proposal, describe the purpose of your project and why the research is important for sustainable agriculture in the northeast.
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.
Fungal pathogens pose a key challenge to the productivity of alfalfa production. 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.
- UD1022 Fungal Antagonism Assays
- Direct Antagonism Assay.
UD1022 is used to challenge four common pathogens of alfalfa, which were 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 was obtained through the USDA APHIS program. Fungal isolates are cultured on Potato Dextrose Agar (PDA) and V8 agar (A2A1) and stored on plates. A 7 mm plug of fungal culture is cored and placed on a petri plate with a 20 microliter drop of UD1022 culture at a distance of 2 cm. Fungal diameter was measured and compared to controls without UD1022. Three biological replicates were used, and the experiment was repeated three times. Mean fungal diameters were compared after evaluation of normalcy. A Tukey-Kramer test was applied, and significance will be accepted at P less than 0.05. This experiment determined 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 were used to select one fungal pathogen to be used in UD1022 and S. meliloti inoculated plant challenge assays. Phytophthora medicaginis A2A1 was selected for alfalfa challenge.
- UD1022 Plant Fungal Suppression Assay.
Alfalfa seed will be sterilized and cold stratified overnight at 4 degrees C. Germinated seeds were transplanted to pots containing a mix of peat:vermiculite:Turface (3:2:1). Plants are maintained in controlled growth chambers with 16-hour photoperiod, 55 percent relative humidity at 22 degrees C. Plants are thinned to 10 per pot and 24 pots are included for each treatment: treatments of no bacteria controls, S. meliloti, UD1022 and S. meliloti with UD1022 are inoculated at time of seed sowing, a ‘booster’ of UD1022 is applied 9 DAP to ‘UD1022’ treatments. 10 mL A2A1 commutated culture is drenched onto plants 10 DAP. One set (12 pots per treatment) will be infected, and one set (12 pots) will be subjected to ‘mock’ inoculation’ as ‘control’ plants. 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 Tukey-Kramer test and significance of P less than 0.05.
- 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 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 Fungal Antagonism Assays
- Direct Antagonism Assay.
Figure 1. Direct Antagonism of UD1022. Inhibition was evaluate after 7 days of growth. Colletotrichum trifolii (anthranose) strains St. Paul AN 1 and SM were moderately inhibited by UD1022. Fusarium oxysporum f. sp. medicaginis (fusarium wilt) Fom 255 and Fo 3 were not inhibited by UD1022. Phoma medicaginis (spring black stem rot), strains StC 306-5 and StC 306-10 were inhibited by 50 – 60% by UD1022. Phytopthora medicaginis (root rot) strain A2A1was inhibited by about 66% due to UD1022.
1.2 Indirect Antagonism Assay
Figure 2. Indirect Antagonism. UD1022 does not inhibit any of the fungal strains indirectly.
1.2 Addendum/Expansion of Fungal Inhibition Analysis.
Several UD1022 mutant strains ineffective in selected non-ribosomal peptides and biofilm genes were used in direct fungal challenge assays against P. medicaginis A2A1 and P. medicaginis StC306-5.
Figure 3. UD1022 NRP Mutant Inhibition of Phythophthora medicaginis A2A1.
Non-ribosomal peptides (NRP) including surfactin and plipastatin have been implicated in the antagonism of common plant fungal pathogens. UD1022 plipastatin (ppsB), surfactin (srfAC), surfactin critical gene sfp do not seem to contribute to the inhibition of A2A1 (Figure 3). 3 separate experiments with 3 replicates per treatment within each experiment.
Figure 4. UD1022 Biofilm Mutant Inhibition of P. medicaginis A2A1.
UD1022 genes responsible for biofilm and spore formation (epsTasA, spo0A and sinI) do seem to be necessary to varying degrees for fungal inhibition. Interestingly, the gene encoding the metalolactamase protein YtnP (which cleaves N-acyl homoserine lactones (AHLs)) seems to have a role in inhibition of P. medicaginis A2A1 (Figure 4). Phytophthora have not been reported to produce AHLs in the literature.
Phoma medicaginis StC306-5 results are in the process of being evaluated; there is indication that different UD1022 genes are involved in the inhibition as compared to Phytophthora medicaginis A2A1.
Understanding the genes and mechanisms of UD1022 inhibition of these important alfalfa pathogens would contribute greatly to developing methods to mitigate these economically devastating plant disease agents.
2.0 UD1022 In Vivo Fungal Suppression Assay.
Two replicates are currently in progress in the growth chamber; third replicate is being launched this week (January 15, 2021). First results are expected last week of January and subsequent evaluations will be collected through mid-February.
3.0 UD1022 – S. meliloti Alfalfa growth promotion in Greenhouse.
All replicates of this experiment have been recently collected and statistical evaluations of shoot biomass and nodulation are in progress. Shoot elemental analysis is pending.
Research is in process and there are no conclusions to report at this time.
Education & Outreach Activities and Participation Summary
Conferences and opportunities to share results of research have been severely impacted by the Covid-19 pandemic. Researchers will be actively looking for forums where the findings of this work may be communicated to the broader community.
Additional outreach will include presenting results from in vitro and in vivo fungal inhibition assays and growth promotion data may be presented in poster/short talk form at the 2022 North American Alfalfa Improvement Conference (NAAIC) held in Lansing, MI.
Pending results collection and evaluation.