Final report for LNC13-355
This project was completed in 2017 and all of our major performance targets were obtained, if not exceeded as they were described in the proposal. A publication from Lani Meyer’s MS Thesis was published in Scientia Horticulturae. A second publication has been in developed from Ravin Poudel’s PhD Dissertation and will be submitted in 2018 which describes the microbial ecology portion of the project. Furthermore, we were successful at acquiring a NIFA-SCRI CAP grant in collaboration with NC State and numerous other institutions that was built on data generated from this project. Similarly, David Loewen is currently preparing to defend his MS Thesis in May 2018 and although the majority of his work is funded by NIFA-SCRI, it was built on knowledge and infrastructure that was developed with this project. We were very successful at communicating the results of our work to scientific and grower audiences in six states (KS, MO, IA, MN, OK, HI) in 2017 in addition to more than 8 field days, twilight tours, and other hands-on educational opportunities at the Olathe Horticulture Center. Education efforts also included the preparation of a tomato grafting extension publication that will be published in 2018 through KSRE. All of these venues were very successful at making changes in knowledge and behavior of growers. Since 2013, this project has contributed directly towards technology adoption and more than 17 growers in the NCR-SARE region to utilized over 18,500 grafted plants in their high tunnels since this time.
The objectives of this project are (1) to identify rootstocks that improve productivity and reduce disease losses in the Great Plains through a series of on-farm and university research trials, (2) to determine optimum grafting/healing conditions that reduce risk of crop failure and increases the successful implementation of grafting for small-scale growers, (3) evaluate the effects of rootstocks on rhizosphere microbial communities; and (4) disseminate grafted propagation methods as well as the results of our research through an integrated extension program. We will perform a series of replicated field studies including at least two on-farm trials per year that evaluate tomato rootstocks for their performance in regards to soilborne disease, crop yield, and marketability. Specific outputs include (1) Extension publications, field tours, web materials, videos, and hands-on workshops; (2) Research journal publications, presentations, and teaching materials. Short-term outcomes include (1) Increased grower knowledge for grafting implementation; (2) Increased scientific community understanding of rhizobiome interactions. Mid- and Long-term outcomes include: (1) Changes in grower behavior related to propagation of grafted plants and rootstock selection; (2) Increased local availability of grafted plants; (3) Further scientific exploration of grafting and rhizobiome interactions, in support of fuller understanding of mechanisms of disease resistance and increased vigor. This proposal builds on a project that is supported by the Ceres Trust Organic Research Initiative.
This research is not specifically hypothesis driven. However, he objectives of this project are (1) to identify rootstocks that improve productivity and reduce disease losses in the Great Plains through a series of on-farm and university research trials, (2) to determine optimum grafting/healing conditions that reduce risk of crop failure and increases the successful implementation of grafting for small-scale growers, (3) evaluate the effects of rootstocks on rhizosphere microbial communities; and (4) disseminate grafted propagation methods as well as the results of our research through an integrated extension program.
1) Identify rootstocks that improve productivity and reduce disease losses under Great Plains conditions through a series of on-farm and university research station trials: Multiple rootstocks (>5) will be trialed at several on-farm and research station trials to determine the viability of grafting in the Great Plains. These trials will be arranged in a randomized complete block design (RCBD) and will include four replications, with multiple plants per treatment per rep. The trials will be conducted utilizing heirloom (‘Cherokee Purple’) and hybrid (‘BHN 589’) scions. These cultivars are highly utilized in Kansas and throughout the United States, particularly for high tunnel production. A trial utilizing ‘Cherokee Purple’ scion will be conducted at the KSU Student Organic Farm in Manhattan, KS (open-field). Five rootstock treatments will be compared with nongrafted controls to determine effects on crop performance (yield and plant growth). Each plot will contain six to eight plants (determined by location) and will be blocked to reduce bias conditions in the field study. A similar trial will be performed at the John C. Pair Horticultural Research and Extension Center near Wichita, KS, and this trial will utilize ‘BHN 589’ for nongrafted controls and for scions of grafted plants. Additionally, trials at the Olathe Research and Extension Center and at the two on-farm locations (Clark Farm (high tunnel) and Gieringer’s Orchard (high tunnel)) will test three additional rootstocks in addition to the ones being trialed at the other two on-farm locations (both in high tunnels-Gibbs Rd. Farm and Common Harvest, which are supported by CERES project). All trials will be monitored for incidence of any major soilborne diseases through visual assessments and microscopy and culturing methods, as needed. Fruit yield and plant growth data (plant height and shoot biomass) will be collected. All data will be analyzed using the MIXED procedure (SAS 9.1; Cary, NC).
2) Determine optimum grafting/healing conditions that reduce risk of crop failure and increases the successful implementation of grafting for small-scale growers; Various greenhouse experiments will be conducted to determine the effects of healing chamber temperature and humidity on graft survival. Additionally, novel methods for healing chamber design will be developed that cater towards production for small-acreage specialty crop growers. These include the development of chambers that reduce the risk of high temperatures by using alternative materials and various ventilation systems. These experiments will be performed at KSU-Olathe in Olathe, KS as well as the greenhouse complex at Kansas State University in Manhattan, KS. The goal of these studies will be to design a healing chamber that provides a suitable environment for graft union formation while reducing the risk of crop failure due to excessive heat. Preliminary data has shown that high humidity and/or stress in the healing chamber during days 5-8 can lead to significant growth of adventitious roots by the scion. We will determine the role of humidity at promoting adventitious roots as well as transplant fertility. Plants that are fertilized at the end of the healing process may be more prone to growing adventitious roots as the scion tissue is capable of absorbing nutrients through foliar feeding. A detailed evaluation of foliar fertilization and its role in promoting root growth will be performed. Multiple identical batches of grafted plants will be produced over time and analyzed in a RCBD whereby each of the individual batches represent blocked replicates. Four to six replicates will be utilized based on the needs of each study and will be analyzed using the MIXED procedure (SAS 9.1; Cary, NC).
3) Evaluate the effects of rootstocks and grower/location on soil and rhizosphere microbial communities;
Whole plants will be sampled from each plot in three high tunnel locations (KSU-Olathe, On-farm 1, On-farm 2) in order to determine the effect of rootstock genotype rootstocks on the ecology of the rhizobiome. A self-grafted control will be utilized to determine any effects of the grafting process on the microbial ecology of the plant. There will be a minimum of four blocks in a randomized complete block design, where a plot (experimental unit) consists of at least five plants with 18 inch spacing in row. At KSU-Olathe, there will be six replications (1 rep per high tunnel) and plots will include 8 plants each. The scion will be ‘BHN 589’ (a regionally-popular high tunnel cultivar), and this scion will be utilized at the on-farm locations in order to facilitate cross-location comparisons. Plants will be sampled twice at KSU-Olathe (peak vegetative growth and peak harvest) and once at the on-farm locations (peak harvest).
Sampling for microbial community assessment. At sampling, plant roots from each plot will be collected, sectioned into 1cm lengths and 20 pieces selected for analyses. After sonication in 0.1% Triton-X and diH2O, the sonication liquid will be collected in a 30ml syringe and passed through 0.22µl nylon membrane to collect fungi, bacteria and suspended particles. The remaining tissues will be dried at 50°C and weight recorded. A subsample of plants will be evaluated for total root biomass. The DNA extracted from tissues will be analyzed by quantitative PCR (qPCR) and relative ITS (fungi) and 16S (bacteria) copy numbers compared as described in Fierer et al (2005).
Separation of the superficial and endophytic rhizobiomes. In molecular analyses, it is uncertain whether epi- or endophytes are considered. For bacteria, the tissues are often sonicated to avoid chloroplast contamination and to maximize epiphyllous bacteria (Kadivar & Stapleton 2006). It is our experience that our choice of next-next generation sequencing tools will permit analyses of both superficial microbial communities as well as those embedded into the root matrix. As a result, we will aim to separate epi- and endophytic communities. The root tissues will be transported on ice to the laboratory, where twenty 1cm pieces will be sampled from each of the tomato plants and pooled in sterile extraction buffer (MoBio UltraClean Soil DNA). To separate superficial and endophytic communities, the tissues will be sonicated for 1min to dislodge superficial bacteria and fungi, transferred from supernatant into clean extraction buffer and the supernatant transferred into a second extraction system. The genomic DNA will be extracted from the roots and particles within the supernatant as per manufacturer’s instructions after homogenization in a FastPrep instrument (MP Biomedicals, CA). Each extract will be PCR-amplified and sequenced to determine the epi- and endophytic communities. Some overlap between the fractions will occur, but true endophytes will be more abundant in the homogenized tissues than in the sonicated supernatant.
Rhizobiome community analysis by high throughput sequencing. Our research group has adopted multiple high throughput sequencing platforms, including the Ion Torrent in the fall of 2012. We will adopt these tools to provide superior sequencing depth at reasonable cost. To extract microbial DNA, the tissues will be homogenized in FastPrep instrument (MP Biomedicals, CA), DNA extracted following the MoBio UltraClean (MoBio Laboratories, CA) instructions and eluted in 100µl of the final buffer S5. The fungal ampicons will be produced with ITS1F and ITS4 fusion primers (Jumpponen and Jones 2009): preliminary data indicate that they reliably generate amplicons adequate to provide richness/diversity estimators for fungal communities. The bacterial amplicons will be produced similarly with 9f and 338R fusion primers to estimate richness/diversity and community composition of bacterial communities.
From the derived OTU (Operational Taxonomic Unit) frequency data, we will calculate common estimators of diversity (including richness, Shannon’s diversity, Simpson’s dominance, and evenness). These data will be used to test hypotheses about diversity responses among the treatments. Hypotheses of interest include (a) Lower disease rootstocks will be associated with higher microbial diversity, (b) Lower disease rootstocks will be associated with higher frequency of beneficial microbes, and (c) Lower disease rootstocks will be associated with higher biomass of beneficial microbes. Note also that we will be able to evaluate responses on various taxonomic levels (species, genus, family) over time to optimize the value of our conclusions and to provide a taxonomic framework for the observed molecular OTU responses. We will evaluate the effects of rootstocks on bacterial and fungal community diversity and on the frequency of particular OTUs using generalized linear models in R. We will focus on OTUs classed as beneficial and pathogenic groups.
4) Extension and Outreach Program: Currently, a major barrier for growers attempting to utilize grafted plants is the ability to purchase or produce their own plants. Therefore, a primary objective of this project is to provide grower training on grafted propagation in addition to the disseminating the results of this specific project. The details of the extension plan are described in the “outreach plan” (see below).
We were successful at building a strong data set with this project that will be very valuable for growers in relation to rootstock selection. Two rootstocks that we evaluated were not suitable for this area while an additional 3-4 are showing consistent benefits across three years of data, even where little disease pressure is evident. This is extremely important information for growers and has been delivered effectively in order to institute change in knowledge and behavior.
The details of much of this work are provided in the M.S. Thesis by Lani Meyer:
Education was provided for the general public, students, university staff, and growers in a number of ways including traditional research presentations, extension presentations, field days, and hands-on workshops, in addition to one-on-one mentoring.
Educational & Outreach Activities
- Was a guest lecturer in Spring 2015 (Meyer), 2016 (Loewen), and 2017 (Rivard) HORT 350 Plant Propagation – Tomato Grafting Technique
- S.A. Masterson, M.M. Kennelly, R.R. Janke, and C.L. Rivard. 2016. Microclimate and Scion Leaf Removal to Improve the Success of Grafted Tomato Seedlings. HortTechnology 26:261-269. (1,2,6)
- Horticulture Sciences Day at Johnson County Community College (Overland Park, KS) (Meyer)
- Truman State University Agriculture and Biology Seminar Series in Kirksville, MO (Rivard)
- Grafting workshop with hands-on grating activity in Lawrence, KS (Meyer, Rivard)
- Lincoln University High Tunnel Conference in Independence, MO (Rivard)
- Phytobiomes Conference in Washington DC (Garrett, Poudel)
- American Phytopathological Society (APS) conference in Pasadena, CA (Garrett, Kennelly, Poudel)
- Lincoln University In-Service Education for Extension Educators in Jefferson City, MO (Rivard)
- Texas High Tunnel Growers Conference in College Station, TX (Rivard)
- Florida Genetics Symposium in Gainesville, FL (Poudel, Garrett)
- National Grafting Symposium in Grand Rapids, MI (Meyer, Rivard)
- D. Loewen and C.L.Rivard. Benefits and Technique for using grafted tomatoes. Missouri Tomato Growers’ School. 10 Aug, 2017. Joplin, MO.
- Data and knowledge gained from the project was included in:
- Buller, K.Oxley, and C.L. Rivard. 2017. Growing Under Cover: A Kansas Grower’s Guide. (Volume 2) Kansas Rural Center Publication (44 page manual)
- Rivard, D. Pryor, and S. Masterson. 2013. All about Tomato Grafting (5 part series). KSRE YouTube Channel. Currently >59,000 views (>13,000 views in 2017)
- All data from research trials was analyzed and presented at venues including:
- Annual ASHS Conferences from 2013-2017 (FL, LA, GA, HI).
- International Society of Horticulture Science (2014, Brisbane, Australia)
- National Grafting Symposium (Savannah, GA)
- MOSES Conference in 2014 and 2016 (La Crosse, WI)
- Great Plains Growers Conference in St. Joseph, MO (Loewen, Meyer, Rivard)
- Two grafting classes (guest lecture and lab) delivered at KSU in HORT 350 – Plant Propagation in Spring 2017 (Loewen, Rivard)
- Wisconsin Vegetable Grower’s Conference in Wisconsin Dells, WI. (Rivard)
- Urban Food Systems Symposium in Olathe, KS (Meyer, Rivard)
- OHREC public field day (Rivard, Loewen)
- OHREC commercial vegetable growers field day (Rivard, Kennelly, Loewen)
- Discussed results of project at KSRE Soil Health Bootcamp (Rivard, Kennelly)
- Tour of OHREC with Butler County Extension Master Gardeners. 9 June.
- Tour of OHREC with Sedgwick County Extension Master Gardeners. 16 August.
- Evaluating whether or not a grower should implement grafting
- Propagation of grafted plants
Utilization of grafted plants to increase productivity in high tunnel operations
- We were successful at building a strong data set with this project that will be very valuable for growers in relation to rootstock selection. Two rootstocks that we evaluated were not suitable for this area while an additional 3-4 are showing consistent benefits across three years of data, even where little disease pressure is evident. This is extremely important information for growers and has been delivered effectively in order to institute change in knowledge and behavior.
- During January 2015, we coordinated a “Tomato Grafting Clinic” at the Great Plains Growers Conference in St. Joseph, MO. Within the clinic was a self-guided tour related to grafted propagation, rootstock selection, and economic data. This included printed slides, poster presentations, all tools and supplies needed for grafting, several examples of healing chambers, and numerous videos, which looped on-screen for participants to watch. We also included plants and training where participants could graft their own plants. This was a new approach, which relied on active learning by participants.
- In December 2015, we took the “Tomato Grafting Clinic” to the Great Lakes Expo, a much larger fruit and vegetable growers conference (>3000 participants) that occurs annually in Grand Rapids, MI. The clinic was a collaboration with Ohio State University as part of a larger SCRI CAP project on grafting that we are part of and also supported the National Grafting Symposium, which was held just prior to the growers conference. While there, Lani Meyer presented the results of her research in addition to teaching farmers that visited the clinic throughout the conference. Cary Rivard also participated in the conference and helped to coordinate and deliver and in-service training that was provided to ~30 extension agents from across the country that have a particular interest in grafting. Two farmers (Josh Smith and Craig Marklinger) from the Gibbs Road Farm were in attendance as well. Although this event was not directly supported by this NCR-SARE project, much of the information delivered by the KSU team was developed through the support of this project.
- One of the Grower-collaborators on this project was invited to speak at the National Grafting Symposium in Savannah, GA, and his interaction with the project has established him as a leader in this area. After his experiences at the symposium, he has established better contacts for information about grafting nationally and has already networked with vendors to identify a source of grafted plants.
- During 2016, we were successful at securing an SCRI CAP award in collaboration with NCSU, OSU, UFL, UA, UC-Davis, and several other institutions. This new project builds on data collected during the SARE project as we are currently conducting a meta-analysis of rootstock data since 2010.
- In spring 2018, more than 9000 grafted plants were provided by KSU to at least 11 growers for use in their high tunnels.
- As a result of the research conducted on this project, we have changed the way we grow grafted plants at the research station. Furthermore, there continues to be a lack of availability for grafted plants in the region. In 2016, we began doing custom grafting for local growers. As a result, more than 14 growers in Kansas and Missouri have utilized more than 18,500 grafted plants in their high tunnels.