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
COMPLETE
Funding Source
AFRI COMPETITIVE GRANT
Reporting Frequency
Annual
Accession No.
1007947
Grant No.
2016-67013-24412
Cumulative Award Amt.
$460,482.00
Proposal No.
2015-07096
Multistate No.
(N/A)
Project Start Date
Dec 1, 2015
Project End Date
Jul 31, 2020
Grant Year
2016
Program Code
[A1151]- Plant Health and Production and Plant Products: Plant Photosynthetic Efficiency and Nutrient Utilization
Project Director
Gage, D. J.
Recipient Organization
UNIV OF CONNECTICUT
438 WHITNEY RD EXTENSION UNIT 1133
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%
Knowledge Area
102 - Soil, Plant, Water, Nutrient Relationships;

Subject Of Investigation
0110 - Soil;

Field Of Science
1040 - Molecular biology; 1103 - Other microbiology; 1100 - Bacteriology;
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 07/31/20

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:1. Problem. We encountered one unexpected problem that has slowed down our greenhouse experiments. We started our experiments using a commercial potting mix that was supposed to be nitrogen free. It clearly wasn't, and our plant took months to show signs of nitrogen starvation, an essential requirement for our experiments. We then used a sand/vermiculite mix that we had used previously, and it too had excess nitrogen and was unsuitable. The vermiculite had enough nitrogen to prevent our plants from quickly becoming nitrogen limited. Thus, we had to trial a variety of new, in-house, potting mixes in order to find one with the right water-retention, water-percolation and nitrogen characteristics. We have settled on 2:1 mix of sand and peat. 1. Changes. We have added a new technique for tracking protist movement of bacteria along root growing in soil. This makes use of an In Vivo Imaging System (IVIS) and light-emitting Pseudomonas putida. Buffer, bacteria, or bacteria plus protists are inoculated onto the plant shoot right where it meets the soil. The plant is growing in a thin, glass fronted, chamber that allows root imaging. The IVIS machine can detect light from P. putida, image it, and provide quantitative date on light intensity and location. This allows us to quickly monitor the movement of our P. putida test strain under realistic conditions, giving valuable data on transport distances and rates. We also developed a microscope-based transport assay that quantitated bacterial transport along roots in soil. This work will be submitted for publication in early 2021 under the title "Bacterial transport of beneficial bacteria by soil protists". 2. Research was shut down from April-July because of Covid. This affected our ability to conduct greenhouse experiments for Aims 3 and 4 as planned. We did finish those needed for Aim3 as discussed earlier, and those results are being analyzed and will be submitted in early 2021 under the title "Enhanced soil transport of Sinorhizobium meliloti, and nodulation of alfalfa roots, following coinoculation with soil protists". What opportunities for training and professional development has the project provided?The following undergraduate students have worked on the project: •Alycia Fulton-Engineering--Development of pore scale transport assays •Shane Hussey-MCB-protist growth curves and growth yields •Kavy Uddaraju-sequencing of protist 18S rDNA genes for identification. Photography of protists and cysts. The following graduate students have worked on the project: •Charles Bridges (PhD) -- Construction of fluorescent-proteinbacterial reporter strains •Gabrielle Corso (MS/PhD) -- MCB-- protist isolation, culturing, sequencing, greenhouse experiments, differential feeding assays. •Brian Cruz (PhD)-Engineering--Development of pore scale transport assays •Chrisotopher Hawxhurst (PhD)--Engineering-- in vitro assays of transport in soil micromodels, design and 3D-printing of chambers for IVIS transport experiments •Jamie Miccuilla (PhD)-Greenhouse experiments, IVIS transport assays •Michael Stephens (PhD)-- MCB--protist sequencing, differential feeding assays How have the results been disseminated to communities of interest?Results weredisseminated to communities of interest by: 1. Presentation of results and techniques at scientific conferences. 2. Publication of peer-reviewed journal articles 3. Presentation of results and points of interest via Twitter (@DanielGage2, @JamieMicciulla,@BrahStevovo 4. Presentation at the Gage Lab website (https://gage.mcb.uconn.edu/index.html) What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Aim1. Isolate, purify, and identify 30 non-pathogenic cyst-forming protist lines(Aims A1 and A2, year 1). This Aim is 100% 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 have ordered 3 amoeba-type protists to round out our collection. This aim is now finished. Aim2. 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 100 % complete. 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 (Sinorhizobiummelilotiprimarily andPseudomonas fluorescenssecondarily). We have modified the in vitro micromodel and have developed new, quick methods for assaying the movement of fluorescent beads and bacteria. These tests have shown that we can easily quantitate movement of protists and cargo through an emulated aggregated soil.Currently we are using this 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 want to use protists that interact with the bacterial payload. This test and a related feeding-preference test, have revealed strong feeding preferences in some of the protists under study. We have also developed a new, realistic, assays for quick evaluation of bacterial transport along root systems in soil. One method uses light production by lux-containingPseudomonasand detection by an In Vivo Imaging System (IVIS). The other makes use of RFP-labeledS. melilotiand an automated microscopic image-capture and analysis pipeline. Using the methods described above, we are now focusing on 5 protists: UC1 (Colpoda), UC5 (Cercomonas) UC6 (Thaumatomonas), UC14 (Colpoda) andAcanthamoeba castellanii. Aim3. 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 100% done. Biological Objective:To determine how well the 8 protists species chosen can moveSinorhizobium melilotialong the roots of the test plantMedicago truncatulawhen 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 have identified a suitable potting mixture and fertilization/watering procedures for the experiments. Plants become nitrogen starved without the addition of added nitrogen, or without the addition of nitrogen fixingSinorhizobium. This ended up being a difficult step to complete because of residual, unanticipated, nitrogen in many of the potting mixture ingredients that we tested for this work. Greenhouse experiments are underway and are giving positive results. These experiments have shown that some protists are capable of movingS. melilotiaround root-systems and away from the site of inoculation resulting in enhanced nodulation and enhanced plant growth. Most of the work has been done withUC1(Colpoda), UC5(Cercomonas) andAcanthamoeba castellanii. Results: This work has shown that these protists are effective at moving S. meliloti down alfalfa roots. Plants coinoculated with protists and S. meliloti developed more nodules, had them further down the root system and had increased shoot weight compared to plants inoculated with the same number of S. meliloti but without protists. This work is being written up and will be submitted in the first half of 2021 under the title: "Enhanced soil transport ofSinorhizobium meliloti,and nodulation of alfalfa roots, following coinoculation with soil protists." Aim4. 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 moveSinorhizobium melilotialong the roots of the test plantMedicago truncatulawhen 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. We did not start this Aim.It was planned to be started once Aim3 (described above) had been completed, but that aim was delayed due to complications of getting greenhouse access during shutdown of university research at UConn (our 2ndno-cost extension ran from 12/1/19 to 7/30/20 and was designed to finish out the greenhouse experiments. Unfortunately, this overlapped with Covid restrictions, but we were able to finish Aim3 during the summer after the greenhouse opened for research. Aim5. 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 is100% done. Biological Objective:To determine how well the 8 protists species chosen can moveSinorhizobium melilotialong 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 with established root systems. We completed our initial potting media, plant growth, fertilization/watering and inoculation experiments and tested protists UC1 (Colpoda), UC5 (Cercomonas) UC6 (Thaumatomonas), andAcanthamoeba castellanii,for their ability to deliverS. melilotito the infection zones of established root systems. After 6 2-month long trials, each done with slightly different protocols, we failed to see any sign that protists were able to deliverS. melilotito the infection zone at root tips. We could have continued to change the protocol, but our decision to stop was based on the fact that the experiment was designed to test, in the greenhouse, something that might be of practical use in the field. Given that we could not get the process to work under ideal conditions, pursuing it further appear to be a poor choice given limited resources.

Publications

  • Type: Conference Papers and Presentations Status: Published 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
  • Type: Conference Papers and Presentations Status: Published 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
  • Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: C. J. Hawxhurst, A. L. Kadilak, C. M. Bridges, D. J. Gage and Leslie M. Shor. (2017). Improving Biocompatibility of 3D Printed Stereolithography Resins. American Inst. Chem. Eng. Annual Meeting. San Franscisco. Talk, presented by C. Hawxhurst.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: G. Corso, B. Cruz, K. OSullivan, A. Fulton, J. Miccuila, L. Shor and D. J. Gage. (June 2017) Soil Transport of Plant Growth Promoting Rhizobacteria. Boston Bacterial Meeting. Boston, MA. Poster, presented by Gabrielle Corso
  • Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: G. Corso, B. Cruz, K. OSullivan, A. Fulton, J. Miccuila, L. Shor and D. J. Gage (January 2018). Soil Protistsenhancing transport of plant growth promoting rhizobacteria. USDA NIFA Physiology of Agricultural Plants PD Meeting, San Diego, CA
  • Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Yi-Syuan Guo, J. M. Furrer, D. J. Gage, Y. Cho and L. M. Shor. Mechanisms Whereby Microbes Promote Intermediate Soil Moisture Content 2018 AIChE Annual Meeting, Pittsburgh https://aiche.confex.com/aiche/2018/meetingapp.cgi/Paper/528536
  • Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Gabrielle E. Corso, Daniel J. Gage. (2019). A Preferential Feeding Assay Designed to Quickly Discern Protist Predation Rates and Prey Selectivity, VIII European Congress of Protistology (ECOP). Rome, Italy
  • Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Christopher J. Hawxhurst, Jamie Micciulla, Daniel J. Gage and Leslie M. Shor. (2019). Scale-up of Protist-Facilitated Biotechnology for Transporting Agrochemicals and Beneficial Bacteria Along Plant Roots. AIChE Annual Meeting, Orlando, FL
  • Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: Christopher Hawxhurst, Jamie Micciulla, Kurt Ristoph, Robert Prudhomme, Daniel J. Gage, Leslie Shor (2020). Microbial-Mediated Transport of Beneficial Bacteria and Agrochemicals. ACS BIOT 2020
  • Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: amie L. Micciulla, Christopher J. Hawxhurst, Charles M. Bridges, Leslie M. Shor, Daniel J Gage (2020). Protist facilitated transport of plant growth promoting bacteria in the rhizosphere. APS Plant Health, Denver (virtual)
  • Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: Jamie Micciulla, Christopher Hawkhurst, Charles Bridges, Leslie Shor and Daniel Gage (2020). Protist facillitated transport of rhizosphere beneficial bacteria, Sinorhizobium meliloti. ISOP virtual protist meeting.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: Stephen Taerum, Blaire Steven, Daniel Gage and Lindsay Triplett (2020). Development and validation of a PNA clamp to increase protist diversity in rhizosphere microbiome research. ISOP virtual protist meeting.
  • Type: Journal Articles Status: Published Year Published: 2020 Citation: Stephen J. Taerum, Blaire Steven, Daniel J. Gage and Lindsay R. Triplett (2020). Validation of a PNA clamping method for Reducing host DNA amplification and increasing eukaryotic diversity in rhizosphere microbiome studies, Phytobiomes J. 4:291-302
  • Type: Journal Articles Status: Other Year Published: 2021 Citation: Christopher J. Hawxhurst, Jamie Micciulla, Charles Bridges, Leslie Shor and Daniel J Gage (2021). Soil Protists Distribute Plant-beneficial Microbes along Plant Roots. In progress
  • Type: Journal Articles Status: Other Year Published: 2021 Citation: Jamie Micciulla, Joseph Gage, Leslie Shor and Daniel J Gage (2021). Enhanced soil transport of symbiotic Sinorhizobium meliloti along the roots of alfalfa following coinoculation with soil protists. In progress


Progress 12/01/17 to 11/30/18

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:As described above for accomplishments in Aim5, we were unable to demonstrate that protists were capable of moving S. meliloti to the infection zones at the root tips of established plants. We could have continued to change the protocol, but our decision to stop was based on the fact that the experiment was designed to test, in the greenhouse, something that might be of practical use in the field. Given that we could not get the process to work under ideal conditions, pursuing it further appear to be a poor choice given limited resources.? What opportunities for training and professional development has the project provided? The following undergraduate students have worked on the project: Shane Hussey-MCB-protist growth curves and growth yields The following graduate students have worked on the project: Gabrielle Corso (MS) -- MCB-- protist isolation, culturing, sequencing, greenhouse experiments, differential feeding assays. Jamie Miccuilla-Greenhouse experiments, IVIS transport assays Michael Stephens (PhD)-- MCB--protist sequencing, differential feeding assays Chrisotopher Hawxhurst (PhD)--Engineering-- in vitro assays of transport in soil micromodels, design and 3D-printing of chambers for IVIS transport experiments Charles Bridges (PhD) -- Construction of fluorescent-proteinbacterial reporter strains 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 Meetings (November 2017 and 2018) as a talk. Posters were presented at the Boston Bacterial Meeting in June 2018 and at the 2018 USDA AFRI-NIFA PD Meeting in San Diego. 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 100% complete. 2. Evaluate protist-facilitated transport using the emulated soil micromodel assay (Aim A3) and down-select to the 5 best protists for greenhouse experiments (Aim A3, year 2). This Aim is100% done. 3. Measure PFT in sterile soil pot assays for 5 protists types with 15 replicates per treatment plus appropriate controls. This aim is 75% done. As described in accomplishments, we are finishing this up and expect to submit the results for publication soon (The work has been presented at meetings in 2019) 4. Measure PFT for seed-inoculated PGPR with 5 protist types and 3 concentrations of competitor bacteria with 10replicates per treatment plus appropriate controls. 3 independent trials (Aim C3, year 3). This Aim is 0% done. This will be started as soon as we have finished the work for Aim3 and submitted it for publication. 5. Measure PFT of rhizobia in established root systems with 5 protist types and with 10 replicates pretreatment plus appropriate controls. 3 independent trials. (Aim D, year 3). This Aim is 100% done.

Impacts
What was accomplished under these goals? Aim1. Isolate, purify, and identify 30 non-pathogenic cyst-forming protist lines(Aims A1 and A2, year 1). This Aim is 100% 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 have ordered 3 amoeba-type protists to round out our collection. This aim is now finished. 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 100 % complete. 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 (Sinorhizobiummelilotiprimarily andPseudomonas fluorescenssecondarily). We have modified the in vitro micromodel and have developed new, quick methods for assaying the movement of fluorescent beads and bacteria. These tests have shown that we can easily quantitate movement of protists and cargo through an emulated aggregated soil.Currently we are using this 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 want to use protists that interact with the bacterial payload. This test and a related feeding-preference test, have revealed strong feeding preferences in some of the protists under study. We have also developed a new, realistic, assays for quick evaluation of bacterial transport along root systems in soil. One method uses light production by lux-containing Pseudomonas and detection by an In Vivo Imaging System (IVIS). The other makes use of RFP-labeled S. meliloti and an automated microscopic image-capture and analysis pipeline. Using the methods described above, we are now focusing on 5protists: UC1(Colpoda), UC5(Cercomonas) UC6(Thaumatomonas), UC14(Colpoda) andAcanthamoeba castellanii. 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 75% done. Biological Objective:To determine how well the 8 protists species chosen can moveSinorhizobium melilotialong the roots of the test plantMedicago truncatulawhen 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 have identified a suitable potting mixture and fertilization/watering procedures for the experiments. Plants become nitrogen starved without the addition of added nitrogen, or without the addition of nitrogen-fixingSinorhizobium. This ended up being a difficult step to complete because of residual, unanticipated, nitrogen in many of the potting mixture ingredients that we tested for this work. Greenhouse experiments are underway and are giving positive results. These experiments have shown that some protists are capable of movingS. meliloti around root-systems and away from the site of inoculation resulting in enhanced nodulation and enhanced plant growth. Most of the work has been done withUC1(Colpoda), UC5(Cercomonas) andAcanthamoeba castellanii. 4. Measure PFT for seed-inoculated PGPR with 5 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 moveSinorhizobium melilotialong the roots of the test plantMedicago truncatulawhen 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. We have not started this Aim yet. It will be started once Aim3 (described above) has been completed. 5. Measure PFT of rhizobia in established root systems with 5 protist types and with 10 replicates per treatment plus appropriate controls. 3 independent trials.(Aim D, year 3). This Aim is100% done. Biological Objective:To determine how well the 8 protists species chosen can moveSinorhizobium melilotialong 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 with established root systems. We completed our initial potting media, plant growth, fertilization/watering and inoculation experiments and and tested protists UC1(Colpoda), UC5(Cercomonas) UC6(Thaumatomonas), andAcanthamoeba castellanii,for their ability to deliver S. meliloti to the infection zones of established root systems. After 6 2-month long trials, each done with slightly different protocols, we failed to see any sign that protists were able to deliver S. meliloti to the infection zone at root tips. We could have continued to change the protocol, but our decision to stop was based on the fact that the experiment was designed to test, in the greenhouse, something that might be of practical use in the field. Given that we could not get the process to work under ideal conditions, pursuing it further appear to be a poor choice given limited resources.

Publications

  • Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Mechanisms Whereby Microbes Promote Intermediate Soil Moisture Content Yi-Syuan Guo, J. M. Furrer, D. J. Gage, Y. Cho and L. M. Shor 2018 AIChE Annual Meeting, Pittsburgh https://aiche.confex.com/aiche/2018/meetingapp.cgi/Paper/528536
  • Type: Conference Papers and Presentations Status: Other Year Published: 2018 Citation: Soil Protistsenhancing transport of plant growth promoting rhizobacteria G. Corso, B. Cruz, K. OSullivan, A. Fulton, J. Miccuila, L. Shor and D. J. Gage USDA NIFA Physiology of Agricultural Plants Meeting, San Diego, CA
  • Type: Conference Papers and Presentations Status: Other Year Published: 2017 Citation: Soil Transport of Plant Growth Promoting Rhizobacteria G. Corso, B. Cruz, K. OSullivan, A. Fulton, J. Miccuila, L. Shor and D. J. Gage Boston Bacterial Meeting. Boston, MA
  • Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Improving Biocompatibility of 3D Printed Stereolithography Resins C. J. Hawxhurst, A. L. Kadilak, C. M. Bridges, D. J. Gage and Leslie M. Shor 2018 AIChE Annual Meeting, Minneapolis https://www.aiche.org/conferences/aiche-annual-meeting/2017/proceeding/paper/531c-improving-biocompatibility-3d-printed-stereolithography-resins


Progress 12/01/16 to 11/30/17

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:Problems We encountered one unexpected problem that has slowed down our greenhouse experiments. We started our experiments using a commercialpotting mix that was supposed to be nitrogen free. It clearly wasn't, and our plant took months to show signs of nitrogen starvation, an essential requirement for our experiments. We then used a sand/vermiculite mix that we had used previously, and it too had excess nitrogen and was unsuitable. The vermiculite had enough nitrogen to prevent our plants from quickly becoming nitrogen limited. Thus, we had to trial a variety of new, in-house, potting mixes in order to find one with the right water-retention, water-percolation and nitrogen characteristics. We have settled on 2:1 mix of sand and peat. Changes We have added a new technique for tracking protist movement of bacteria along root growing in soil. This makes use of an In Vivo Imaging System (IVIS) and light-emitting Pseudomonas putida. Buffer, bacteria, or bacteria plus protists are inoculated onto the plant shoot right where it meets the soil. The plant is growing in a thin, glass fronted, chamber that allows root imaging. The IVIS machine can detect light fromP. putida, image it, and provide quantitative date on light intensity and location. This allows us to quickly monitor the movement of ourP. putidatest strain under realistic conditions, giving valuable data on transport distances and rates. What opportunities for training and professional development has the project provided?The following undergraduate students have worked on the project: Shane Hussey-MCB-protist growth curves and growth yields Anthony Casasanta--Engineering-- in vitro assays of transport in soil micromodels The following graduate students have worked on the project: Gabrielle Corso (PhD) -- MCB-- protist isolation, culturing, sequencing, greenhouse experiments, differential feeding assays.Jamie Miccuilla-Greenhouse experiments, IVIS transport assays. Michael Stephens (PhD)-- MCB--protist sequencing, differential feeding assays. ?Chrisotopher Hawxhurst (PhD)--Engineering-- in vitro assays of transport in soil micromodels, design and 3D-printing of chambers for IVIS transport experiments. 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 2017) as a talk. A poster was presented at the Boston Bacterial Meeting in June 2017. 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 100% complete. 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 75% done. We have modified the in vitro micromodel and have developed a new IVIS based method for assaying protist transport of bacteria in soil. We have identified four protists with characteristics the seek--high growth rates and high cyst numbers, good feeding on S. meliloti and good transport of S. meliloti and P. putida. These are UC1, UC 4, UC5 and UC6. We expect the next four protist species will be selected in the next 3 months and that the aim will be completed. 3. Measure PFT in sterile soil pot assays for 8 protists types with 15 replicates per treatment plus appropriate controls. This aim is 25% done. As described in accomplishments, this aim was set back because of difficulties in developing a suitable, nitrogen-free, potting mix. This is now done and we will begin these experiments using the four protists UC1, UC4, UC5 and UC6. Highest priority to will be to test UC6, because it has proven to be the best one, in aggregate, duiring our in vitro testing trials. 4. Measure PFT for seed-inoculated PGPR with 8 protist types and 3 concentrations of competitor bacteria with 10replicates per treatment plus appropriate controls. 3 independent trials (Aim C3, year 3). This Aim is 0% done. This will likely be started in the second half if this period. It cannot be started until the greenhouse experiments in goal #3 have been done at least twice. This is because the techniques developed in goal #3 will be used in this goal. 5. Measure PFT of rhizobia in established root systems with 8 protist types and with 10 replicates pretreatment plus appropriate controls. 3 independent trials. (Aim D, year 3). This Aim is 30% done. ? ?We have begun these greenhouse experiments and have potting mix, watering/fertilizing methods and inoculation protocols working. We will be starting the first protist (UC6) addition in March, and expect to have this goal completed by the end of this reporting 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 100% 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 have ordered 3 amoeba-type protists to round out our collection. This aim is now finished. 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 75% 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. These tests have shown that we can easily quantitate movement of protists and cargo through an emulated aggregated soil.Currently we are using this 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 want to use protists that interact with the bacterial payload. This test and a related feeding-preference test, have revealed strong feeding preferences in some of the protists under study. Half of the protist species tested so far (12 of our 20 have been tested) show no preference for Sinorhizobium or the control bacteria E. coli. The others tested split evenly with half preferring Sinorhizobium and the other half preferring E. coli. We have also developed a new, realistic, assay for quick evaluation of bacterial transport along root systems in soil. This method makes use of a Pseudomonas strain that emits light and its detection using an In Vivo Imaging System (IVIS). The IVIS allows us to track colonization of roots in soil by the light-emitting Pseudomonas strain using small glass fronted chambers over the course of 3-4 days. Initial experiments have shown that some protists are much more effective at enhancing colonization of roots than are others. This new assay should provide useful, relevant, data on bacterial transport along roots by our 20 protist species and help in choosing the best 8 for further evaluation. 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 25% 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 have identified a suitable potting mixture and fertilization/watering procedures for the experiments. Plants become nitrogen starved without the addition of added nitrogen, or without the addition of nitrogen-fixing Sinorhizobium. This ended up being a difficult step to complete because of residual, unanticipated, nitrogen in many of the potting mixture ingredients that we tested for this work. 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. This will likely be started in the second half of this period. It cannot be started until the greenhouse experiments in goal #3 have been done at least twice. This is because the techniques developed in goal #3 will be used in this goal. 5. 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 is30% 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 with established root systems. We have completed our initial potting media, plant growth, fertilization/watering and inoculation experiments and can now establish M. truncatula plants, allow them to nitrogen starve and then inoculate with Sinorhizobium at a level that allows nodulation and rescue of the plants. Alternatively, we can inoculate at a level that does not allow nodulation or rescue from nitrogen starvation. We are now beginning experiments to access the if protist facilitated transport of Sinorhizobium will allow efficient rescue of the plants when inoculated with low numbers of bacteria. That is, we are testing if protists can move the bacteria from the site of inoculation to sites on the roots where nodulation can occur and rescue the plants. For these initial experiments, we will use protist UC6, a Thaumatomonas species. While not all of our protist species have been characterized in terms of feeding preferences and ability to move bacteria in vitro, the UC6 isolate is among the most promising of those we have tested so it will be the first tested in these greenhouse experiments.

Publications

  • Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: "C. J. Hawxhurst, A. L. Kadilak, C. M. Bridges, D. J. Gage and Leslie M. Shor. (2017). Improving Biocompatibility of 3D Printed Stereolithography Resins. American Inst. Chem. Eng. Annual Meeting. San Franscisco. Talk, presented by C. Hawxhurst.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: G. Corso, B. Cruz, K. OSullivan, A. Fulton, J. Miccuila, L. Shor and D. J. Gage. (June 2017) Soil Transport of Plant Growth Promoting Rhizobacteria. Boston Bacterial Meeting. Boston, MA. Poster, presented by Gabrielle Corso.


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