Source: UNIV OF CONNECTICUT submitted to
ENHANCED TRANSPORT OF PLANT GROWTH PROMOTING RHIZOBACTERIA BY COINOCULATED SOIL PROTISTS
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
NEW
Funding Source
Reporting Frequency
Annual
Accession No.
1007947
Grant No.
2016-67013-24412
Project No.
CONW-2015-07096
Proposal No.
2015-07096
Multistate No.
(N/A)
Program Code
A1151
Project Start Date
Dec 1, 2015
Project End Date
Nov 30, 2018
Grant Year
2016
Project Director
Gage, D. J.
Recipient Organization
UNIV OF CONNECTICUT
(N/A)
STORRS,CT 06269
Performing Department
Molecular and Cellular Biology
Non Technical Summary
The goal of this proposal is to enhance plant nutrition by developing a technology that uses soil protists to increase effectiveness plant growth promoting rhizobacteria (PGPR). Sometimes PGPR work reliably, and cost effectively. Often, however, PGPR are ineffective because they do not reach their sites of action on plant roots. This can be caused by: restricted movement of PGPR through soil; inefficient water-based transport of PGPR or competition with endogenous soil bacteria.We propose that soil protists have the capacity to move PGPR and that they could increase PGPR effectiveness by allowing PGPR to keep up with growing roots. We will isolate from Medicago truncatulaand soybean rhizospheres 30 types of cyst-forming protists. These will be characterized in an in vitro micromodel to select those that best transport bacteria to legume roots. In vitro data will be used to derive agent-based models of protist transport. The top 8 PGPR-moving protists will be used in a series of increasingly realistic experiments to determine their effectiveness in moving rhizobial bacteria. Experiments will measure the effects of protists on: rhizobial transport through soil, rhizobial effectiveness with competitors and rhizobial effectiveness on established roots.Rhizobia, the most frequently used PGPR for nutrient acquisition, are inoculated on soybean, alfalfa, bean, pea, clover and other legumes to provide cheap and sustainable nitrogen. Their use could increase if inoculation was more effective. Nitrogen acquisition in nonlegumes, phosphate acquisition, iron acquisition and biocontrol are also facilitated by PGPR and may be enhanced by protist transport of PGPR.
Animal Health Component
(N/A)
Research Effort Categories
Basic
100%
Applied
(N/A)
Developmental
0%
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
1020110110045%
1020110110345%
1020110104010%
Goals / Objectives
The long term goal of this project is to test the hypothesis thatplant nutrition can be enhanced by developing an inoculation technology that uses soil protists to increase targeting of plant growth promoting rhizobacteria ot the roots of agricultural crops. Our objectives for this project are:Within 6 months of project start, isolate, purify, and identify 30 non-pathogenic cyst-forming protist lines (Aims A1 and A2, year 1).Within 15 months, evaluate protist-facilitated transport using the emulated soil micromodel assay (Aim A3) and down-select to the 8 best protists for greenhouse experiments (Aim A3, year 2).Within 21 months, measure PFT in sterile soil pot assays for 8 protists types with 15 replicates per treatment plus appropriate controls. 3 independent trials. (Aim B2, year 2).Within 2.5 years, measure PFT for seed-inoculated PGPR with 8 protist types and 3 concentrations of competitor bacteria with 10 replicates per treatment plus appropriate controls. 3 independent trials. (Aim C3, year 3).Within 3 years, measure PFT of rhizobia in established root systems with 8 protist types and with 10 replicates per treatment plus appropriate controls. 3 independent trials. (Aim D, year 3)
Project Methods
Specific Aim A: In vitro characterization of protists for use with M. truncatula and soybean. We will purify 30 different protist lines from soil samples. Identity of the protists will be determined by sequencing their 18s rDNA. The ability of each of these lines to transport beneficial bacteria (S. meliloti and strain USDA110) will be tested in microfluidic devices designed for that purpose. Transport will be quantified by measuring bacterial position, time and relative movement with and without protists. The top 8 lines will be used for the aims below.Specific Aim B: Characterization of protist facilitated transport (PFT) of rhizobia in sterile soil. We will grow plants in a nitrogen free soil mixture and test the ability of our top 8 protist lines to transport rhizobia bacteria along the roots of their host plants and induce the formation of root nodules. Prior to these tests we will identify the inoculation site in the pot that best ensures that bacteria alone cannot reach the root system of their host. 4 weeks after plants are inoculated they will be removed from their pots and the plants will undergo a standardized analysis. This will consist of determining nodule number in each of 3 three root zones: 0-10 cm, 10-20 cm and >20 cm. We will measure values for: 1) Average number of nodules per plant, 2) average number of nodules per root zone and 3) shoot dry weight. Calculation of statistical significance, 95% confidence intervals and trends analyses will be conducted using the statistical package R.Specific Aim C: Characterization of protist facilitated transport (PFT) of rhizobia bacteria in the presence of competitor rhizobia. For this aim we will essentially repeat aim B, but do so in the presence of marked competitor bacteria, that usually prevent inoculated bacteria from efficiently inducing root nodules. Seeds will be surface sterilized, germinated and planted, one plant per pot, at the center of the pots. Soil will contain competitor bacteria. At the time of planting seeds will be inoculated with test rhizobia bacteria with and without our best 8 protist lines. Location of the inoculation will be directly on the seed at the point where the root emerges. Data collections will include: Nodule number, nodule position and shoot weight will be collected and analyzed as described for Specific Aim B. Also collected will be concentrations of viable inoculum cells and competitor cells in soil samples.Specific Aim D. Characterization of protist facilitated transport (PFT) of rhizobia on roots of established plants. For this we will determine if protists can transport beneficial bacteria along root systems that are already established. Seeds will be surface sterilized, germinated and planted in a nitrogen poor soil mixture, one plant per pot, at the center of pots. The plants will be allowed to grow for 4 weeks.When plants begin to show signs of nitrogen limitation they will be inoculated with rhizobial bacterial with and without our top 8 protist lines. Four weeks after plants are inoculated they will be removed from their pots and the plants will undergo the standardized analysis to see if protists were able to transport bacteria along the roots causing nodule formation (see Specific Aim B above).

Progress 12/01/15 to 11/30/16

Outputs
Target Audience:Target audience consisted of professional scientists, graduate students and undergraduates. Efforts consisted of presentations at scientific meetings, publications and classroom description/discussion of the work. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?The following undergraduate students have worked on the project: Kelly O'Sullivan-MCB-protist isolation and culturing Paige Orlofsky)--Engineering--in vitro assays of transport in soil micromodels Anthony Casasanta)--Engineering--in vitro assays of transport in soil micromodels The followinggraduate students have worked on the project: Gabrielle Corso (MS) -- MCB--protist isolation, culturing, sequencing, greenhouse experiements, differential feeding assays Michael Stephens (PhD)-- MCB--protist sequencing Brain Cruz (PhD)--Engineering-- in vitro assays of transport in soil micromodels, seed inoculation methods in greenhouse assays Grant Bouchillon (PhD)--Engineering--in vitro assays of transport in soil micromodels Alycia Fulton(PhD)--Engineering--in vitro assays of transport in soil micromodels How have the results been disseminated to communities of interest?The results of this work were presented at the annual American Institute of Chemical Engineers Annual Meeting (November 2016) as two posters. What do you plan to do during the next reporting period to accomplish the goals?1. Isolate, purify, and identify 30 non-pathogenic cyst-forming protist lines(Aims A1 and A2, year 1). This Aim is 90% Complete. Will will purchase and culture ~5 more protists to round out out collection 2. Evaluate protist-facilitated transport using the emulated soil micromodel assay (Aim A3) and down-select to the 8 best protists for greenhouse experiments(Aim A3, year 2).This Aim is 50% done. This Aim is 50% done. We have modified the in vitro micromodel and have developed new, quick methods for assaying the movement of fluorescent beads and bacteria. Two protist types were have been tested in the devices. These tests have shown that we can easily quantitate movement of protists and cargo through an emulated aggreagated soil. We expect that the rest of this aim will be accomplished rapidly and should be done within the next 6 months. The differential feeding assays will be down as the first down selection, followed by the transport assays in the soil micromodel. 3. Measure PFT in sterile soil pot assays for 8 protists types with 15 replicates per treatment plus appropriate controls. 3 independent trials(Aim B2, year 2).This Aim is 10% done. This Aim is 10% done. This is our highest priority for the upcoming reporting period. We will begin these experiments as soon as we have identified the best potting and watering protocols. The first experiments will be commenced as soon as possible, using protists that we know will make it into the final eight/best protist group. For example our protist UC6 is a Thamatomonas isolate that is robust, grows quickly to hight numbers, travels fast and transports S. meliloti. We will use in in our first assays, even if the other 7 best protists are not yet identified. 4. Measure PFT for seed-inoculated PGPR with 8 protist types and 3 concentrations of competitor bacteria with 10 replicates per treatment plus appropriate controls. 3 independent trials(Aim C3, year 3).This Aim is 0% done. This Aim is 0% done. This will likely not be started in the next reporting period. 5. Within 3 years, measure PFT of rhizobia in established root systems with 8 protist types and with 10 replicates per treatment plus appropriate controls. 3 independent trials.(Aim D, year 3). This Aim is0% done. This Aim is0% done. These experiments are relatively easy to do and we will begin greeen house trials duing the nextreporting period.

Impacts
What was accomplished under these goals? 1. Isolate, purify, and identify 30 non-pathogenic cyst-forming protist lines(Aims A1 and A2, year 1). This Aim is 90% Complete. Biological Objective: To isolate cyst-forming soil protists. These are the organisms that will eventually be tested to see if they can provide better distribution of plant growth promoting rhizobacteria along the roots of crop plants. Following isolation, the protists will be sequenced in order to identify them. This is being done to minimize the chance that we will be working with human, animal or plant pathogens. We have isolated purified 25 strains of protists. Of these, 16 have were found to be robust and easy to grow. These 16 have beenidentified through sequencing of 18S rDNA and none were found to be known pathogens. We will be purchasing another 5 or so protists from ATCC in order to fill out some of the protist types that we did not isolate, for example cyst-forming amoebas. In addition, we have been testing various growth media in order to rapidly grow protists to high cell/cyst numbers. 2. Evaluate protist-facilitated transport using the emulated soil micromodel assay (Aim A3) and down-select to the 8 best protists for greenhouse experiments(Aim A3, year 2).This Aim is 50% done. Biological Objective:We must reduce the number of protist species with which we are working. To characterize 30 species of protists is beyond the scope of this project. We will be selecting for further work those protists that are easy to culture, robust, reach high cyst numbers, survive desiccation and transport our test bacteria (Sinorhizobium meliloti primarily and Pseudomonas fluorescens secondarily. We have modified the in vitro micromodel and have developed new, quick methods for assaying the movement of fluorescent beads and bacteria. Two protist types were have been tested in the devices. These tests have shown that we can easily quantitate movement of protists and cargo through an emulated aggregated soil. Currently we are using a new assay to help us down sample from ~20 to ~8 protists. This assay measures the feeding preferences of the protists and lets us determine if they will readily eat/transport the bacterial payload, Sinorhizobium meliloti. This is an important test, because we will want to use protists that interact with the payload. We will also be testing a second bacterium Pseudomonas fluorescens in this assay as well, because Pseudomonads are often used to inoculate seeds during planting. We expect that the rest of this aim will be accomplished rapidly and should be done within the next 3 months 3. Measure PFT in sterile soil pot assays for 8 protists types with 15 replicates per treatment plus appropriate controls. 3 independent trials(Aim B2, year 2).This Aim is 10% done. Biological Objective:To determine how well the 8 protists species chosen can move Sinorhizobium meliloti along the roots of the test plant Medicago truncatula when competitors are absent. Test plants will be inoculated by providing cysts and beneficial bacteria at the time of planting, mimicking the inoculation of seed in the field, during planting. We are in the process of conducting greenhouse experiments to identify the best potting mixtures and water procedures for this series of experiments. 4. Measure PFT for seed-inoculated PGPR with 8 protist types and 3 concentrations of competitor bacteria with 10 replicates per treatment plus appropriate controls. 3 independent trials(Aim C3, year 3).This Aim is 0% done. Biological Objective:To determine how well the 8 protists species chosen can move Sinorhizobium meliloti along the roots of the test plant Medicago truncatula when competitors are present. Test plants will be inoculated by providing cysts and beneficial bacteria at the time of planting, mimicking the inoculation of seed in the field, during planting. 5. Within 3 years, measure PFT of rhizobia in established root systems with 8 protist types and with 10 replicates per treatment plus appropriate controls. 3 independent trials.(Aim D, year 3). This Aim is0% done. Biological Objective:To determine how well the 8 protists species chosen can move Sinorhizobium meliloti along the roots when the plant is already well established in the soil. The purpose here is to see if protists can deliver beneficial bacteria to mature or established plants. If so, this could benefit the delivery of bacterial to perennial crop plants such as grape.

Publications

  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2016 Citation: Cameron A. Harrington , Andrea L. Kadilak , Charles M. Bridges , Daniel J. Gage and Leslie M. Shor. Biocompatibility of 3D Printer Material to Bacterial Cultures. November 2016. American Institute of Chemical Engineers Annual Meeting, San Francisco, CA https://www3.aiche.org/proceedings/Abstract.aspx?PaperID=479898
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2016 Citation: Alycia J. Fulton , Brian C. Cruz , Grant M. Bouchillon , Daniel J. Gage and Leslie M. Shor. Development of a Pore-Scale Transport Assay for Protist-Facilitated Transport of Plant Growth- Promoting Bacteria. November 2016. American Institute of Chemical Engineers Annual Meeting, San Francisco, CA https://aiche.confex.com/aiche/2016/webprogram/Paper458032.html