Progress 02/01/17 to 01/31/19
Outputs
Target Audience:The target audience for the current reporting period included fruit industry personnel (growers, packers, commercial entities), research scientists and assistants, and academic researchers. Efforts in the current reporting included publication of research results in a peer reviewed journal and local presentations. Experiential learning opportunities were provided to a recently graduated research assistant and other laboratory personnel. Changes/Problems:The PD accepted a faculty position at a university research center which began midway through the final year of the postdoctoral grant. This job opportunity for the PD supports the assertion that this postdoctoral program is beneficial for new scientists to gain experience in research and reporting that enables recipients to be competitive for transitioning to long-term leadership research appointments. What opportunities for training and professional development has the project provided?Professional development activities for the postdoctoral project director (PD) included presenting research funded by this opportunity at both industry and academic conferences, mentoring from supervisor, grant-writing training, providing mentoring and training to students and coworkers, and online training for biochemical analysis software. Presentations regarding research funded by this opportunity gave the PD further experience in public speaking, and included two industry conferences, one academic conference, and an additional in-house research station presentation. Mentoring from supervisor has included feedback on written reports and presentations, as well as valuable discussion of proposed projects and experimentation. The mentor has also supported and assisted PD in applying for a grant which could have provided support for the faculty position which the PD began in July of 2018. The PD also took a grant-writing training (Grantmanship Fundamentals) recommended by the supervisor, which was offered through the ARS web-based training system AgLearn. Mentoring and training for coworkers provided by the PD included gas chromatograph mass spectrometry instrumental maintenance and daily operation, liquid chromatograph mass spectrometry instrumental maintenance and daily operation, root sample solvent-based extraction for use in biochemical analysis, and utilizing two types of software to analyze raw data files to obtain quantitative information regarding metabolites present in samples. The PD also sought out online training for utilizing software for both biochemical as well as microbiome (Next-Gen Sequencing) analysis. How have the results been disseminated to communities of interest?Research funded by this opportunity has been presented at both industry and scientific conferences, including a training workshop provided by the PD. As a goal of this funding opportunity is to provide new scientists with further training, these presentations can also be viewed as professional development. Industry conferences attended were the International Fruit Tree Association (IFTA) conference in Wenatchee, WA and the Washington State Horticultural Society Association (WSHA) meeting in Pasco, WA. The PD received a scholarship to attend the IFTA conference, for which the PD engaged in service activities, including conference photography, speaker introduction, and providing assistance for registering conference attendees. For the WSHA meeting, the PD presented a summary of current research results connected with this project, which allows for both dissemination of information as well providing the PD with valuable public speaking experience. Both industry meetings also helped the PD to gain an understanding of useful industry trends in apple rootstock cultivar use, on-farm research, and related orchard training systems. The scientific conference attended was the American Society for Horticultural Science (ASHS) meeting, where the PD delivered a workshop presentation titled, "Exploring the rhizosphere via metabolic profiling". The presentation delivered a comparison of techniques for assessing root exudates, including those developed in connection with this project, to other scientists engaging in rhizosphere research. This conference was a valuable opportunity to synthesize a workshop training seminar, connect with fellow scientists, as well as gain an understanding of other approaches for horticultural research by attending research presentations provided by other scientists. Two peer-reviewed journal articles connected with this research have been published, per the following citations: Leisso, R., Rudell, D., Mazzola, M. 2017. Metabolic composition of apple rootstock rhizodeposits differs in a genotype-specific manner and affects growth of subsequent plantings. Soil Biology and Biochemistry. doi:10.1016/j.soilbio.2017.06.011 Leisso, R., Rudell, D., Mazzola, M. 2018. Targeted metabolic profiling indicates apple rootstock genotype-specific differences in primary and secondary metabolite production and validation quantitative contribution from vegetative growth. Frontiers in Plant Science 9: 1-15. https://doi.org/10.3389/fpls.2018.01336 Additional manuscripts are being drafted to address scion impacts on the soil microbiome and rhizodeposits. What do you plan to do during the next reporting period to accomplish the goals?This is the Final Report, per the need to convert the post-doctoral grant to a standard grant as the post-doctoral scholar has transitioned to a permanent position as an assistant professor. Remaining funds will support research related to original project goals.
Impacts
What was accomplished under these goals?
Long-term goals. The project's contribution to the long-term goals of reducing the impacts of apple replant disease and increasing fundamental understanding of the apple root rhizosphere include: determining that environmental factors, as well as scion genotype and size, impact apple root exudates rootstock genotype can impact the rhizosphere soil microbial community composition, which has implication for functional activities of these microbes and their effects on tree growth and health; effects of the scion on the rhizosphere soil microbial community are not detectable root exudates differ among scion and rootstock genotypes among several chemical classes, including phenolic compounds, sugars, sugar alcohols, organic acids, amino acids, and triterpenoids in addition to biochemical compounds identified by comparison to an authentic standard, numerous unidentified compounds released into the rhizosphere also differ according to both scion and rootstock genotype. Practical implications include: Foundational information that could inform the ability to develop precision agriculture-based solutions for soil-borne diseases, specifically considering tailoring rootstock decisions to site-specific conditions including soil type, soil biology, general climactic trends regarding temperature and precipitation; a subtext to this vision is further defining rootstock cultivar characteristics according to their optimal growth and health conditions Utilizing information regarding rootstock metabolite contrasts among genotypes to inform apple rootstock breeding programs either for disease tolerance or for supporting a beneficial microbiome The knowledge that endophytes persist in apple trees opens further questions regarding their influence on tree nutrient sequestration, growth, and transmission mechanisms, especially from a nursery production perspective Objective 1. Research accomplishments connected to contrasting the effects of the apple scion on the root exudate metabolic profile on rootstocks of differing tolerance to ARD, and determining specific metabolites that could be involved in providing scion/rootstock tolerance to ARD included both further method development and research results: When microbes were specifically excluded from experimental systems, more compounds that could feed the rhizosphere microbiome than inhibit pathogens differed between rootstock cultivars. Rootstock cultivars assessed were 'M26' and 'G935'. Libraries of metabolites confirmed by authentic standards were expanded for both apple root exudates and root tissue. Two greenhouse experiments assessed the impact of rootstock cultivar-specific (G41, G935, M9Nic29, and M26) exudates on a spatially separated orchard soil; results indicated that 1) M9Nic29 rootstock exudates select for a distinct fungal community (as assessed by (terminal restriction fragment length polymorphism analysis; T-RFLP) relative to other rootstocks and that 2) for all rootstock genotypes examined, fungal communities were influenced by rootstock exudates relative to control communities (no rootstock) over time. Phenolic metabolites including 4-hydroxybenzoic acid, benzoic acid, chlorogenic acid, phloridzin, phloroglucinol, and rutin were tested for their ability to inhibit growth of apple replant disease pathogens Pythium ultimum var. ultimum, Phytophthora cactorum, and Rhizoctonia solani AG-5. Results indicate that phloridzin, benzoic acid, and 4-hydroxybenzoic acid can all inhibit these pathogens in a concentration-dependent manner. Inhibition of Pythium ultimum by rutin was minimal and chlorogenic acid and phloroglucinol moderate at the concentrations tested. Results indicate that apple scions can impact biochemical compounds released by tree roots within first season of growth according to scion cultivar for bud-grafted apple trees. Cultivar-based differences were more profound in metabolites that would inhibit pathogen growth than in metabolites that would promote growth in the microbiome. Scions assessed were Honeycrisp, Granny Smith, and G41, grafted onto rootstock cultivar G41. In summary, with respect to Objective 1, this research project confirmed that apple root exudates differ in a cultivar-dependent manner in metabolic composition which could impact their tolerance to root pathogens. In addition, apple scion can influence the root exudate biochemical profile within the first year of growth after grafting but impacts of the exudate biochemical profile on the rhizosphere microbial community were undetectable in the current experimental system. Specific phenolic metabolites differing in quantity among rootstock cultivars were tested for their impact on pathogen growth. Some of the assayed metabolites inhibited in vitro growth of root pathogens, but inhibitory activity was only observed at concentrations in excess of that predicted to be encountered in the biological system. This information has utility both for rootstock breeding as well as enhancing fundamental knowledge regarding apple root rhizosphere physiology. Objective 2. Experimentation was performed to identify apple root biochemical compounds produced (quantitatively or qualitatively) in a rootstock genotype (cultivar) specific manner, and to understand impacts that resulting changes in soil biochemistry and microbial community have on the next generation of apple trees. Results included: When cultivated in the same orchard soil, plant biomass, disease severity, and rhizosphere microbiome composition of apple seedlings differed depending upon genotype of the rootstock cultivated in this orchard soil. Metabolic composition of root exudates differed among apple rootstock genotypes. The genotypes assessed in the present study were G41, G935, M9Nic29, and M26. Results suggest that G41 and G935 are the most similar in terms of root exudate metabolite profiles. Rhizosphere microbial community amplicon sequencing results have been received from one scion x rootstock interaction experiment and are partially analyzed; DNA from a second experiment have been submitted to a sequencing service. Root types influence the phenolic compound biochemical profile and differ between rootstock genotypes according to their tolerance to apple replant disease. Extension roots and fibrous roots have different phenolic compound profiles which also differ according to rootstock cultivar. Cultivars tested were M26 and G41. In axenic conditions, a greater number of differences were observed between rootstock genotypes M26 and G935 in classes of compounds that could "feed" microbial communities than those that could inhibit microbial growth. This is in contrast to greenhouse collected exudates, where a greater number of metabolites with the potential to inhibit microbial growth were observed to differ between genotypes. Assessment of metabolites produced by rootstocks in axenic conditions indicated phloridzin and sorbitol can be among the more abundant metabolites produced by roots; testing the impacts of these metabolites on the soil microbial community indicates that sorbitol has a significant effect on both the bacterial and fungal community structure, while phloridzin influenced only the fungal community structure and to a lesser extent than did sorbitol. In summary, with respect to Objective 2, results indicate that rootstock cultivar specific changes to orchard soil biochemistry and microbial community composition can impact growth of the next generation of trees as assessed by a plant bioassay. Furthermore, based on amplicon sequencing of rhizosphere microbial communities according to scion and rootstock graft combinations, microbial community differences may exist among scion/rootstock combinations, but are not extreme, and the biological impacts of these subtle differences are uncertain.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2018
Citation:
Leisso, R., Rudell, D., Mazzola, M. 2018. Targeted metabolic profiling indicates apple rootstock genotype-specific differences in primary and secondary metabolite production and validation quantitative contribution from vegetative growth. Frontiers in Plant Science 9: 1-15. https://doi.org/10.3389/fpls.2018.01336
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2017
Citation:
Leisso, R. and Mazzola, M. 2017. Exploring the rhizosphere via metabolic profiling. 21 Sept 2017, ASHS Annual Meeting 2017, Waikoloa, HI.
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Progress 02/01/17 to 01/31/18
Outputs
Target Audience:The target audiences reached by the project included the postdoctoral scientist project director (PD), the apple fruit industry personnel, horticulture scientists, technical staff, and students. The PD benefits from this funding opportunity, as part of its purpose is to provide recent PhD graduates (postdoctoral scientists) with research funding, mentoring from an experienced scientist, training opportunities, and opportunities to further understand the grantmanship process. The apple fruit industry was reached through this project through presentations at two horticultural conferences that took place in Washington State: the International Fruit Tree Association meeting (IFTA) and the Washington State Tree Fruit Assocation meeting (WSTFA). The scientific community was reached through a workshop presentation at the American Society for Horticultural Science meeting and a peer reviewed journal article published in Soil Biology and Biogeochemistry. Mentoring and training for students and technical staff were provided by the PD, and included gas chromatograph mass spectrometry instrumental maintenance and daily operation, liquid chromatograph mass spectrometry instrumental maintenance and daily operation, root sample solvent-based extraction for use in biochemical analysis, and utilizing two types of software to analyze raw data files to obtain quantitative information regarding metabolites present in samples. Changes/Problems:An upcoming change which will be addressed through discussion with the program directors is that the PD has accepted a faculty position at a university research location to begin midway through the final year of the postdoctoral grant. This job opportunity for the PD supports the assertion that this postdoctoral program is beneficial for new scientists to gain experience in research and reporting that enables recipients to be competitive for transitioning to long-term leadership research appointments. What opportunities for training and professional development has the project provided?Professional development activities for the postdoctoral project director (PD) during the first reporting period included presenting research funded by this opportunity at both industry and academic conferences, mentoring from supervisor, grant-writing training, providing mentoring and training to students and coworkers, and online training for biochemical analysis software. Presentations regarding research funded by this opportunity gave the PD further experience in public speaking, and included two industry conferences, one academic conference, and an additional in-house research station presentation. Mentoring from supervisor has included feedback on written reports and presentations, as well as valuable discussion of proposed projects and experimentation. The mentor has also supported and assisted PD in applying for a grant which could provide support for the faculty position which the PD will begin in July of 2018. The PD also took a grant-writing training (Grantmanship Fundamentals) recommended by the supervisor, which was offered through the ARS web-based training system AgLearn. Mentoring and training for coworkers provided by the PD included gas chromatograph mass spectrometry instrumental maintenance and daily operation, liquid chromatograph mass spectrometry instrumental maintenance and daily operation, root sample solvent-based extraction for use in biochemical analysis, and utilizing two types of software to analyze raw data files to obtain quantitative information regarding metabolites present in samples. The PD also sought out online training for utilizing software for both biochemical as well as microbiome (Next-Gen Sequencing) analysis. How have the results been disseminated to communities of interest?Research funded by this opportunity has been presented at both industry and scientific conferences, including a training workshop provided by the PD. As a goal of this funding opportunity is to provide new scientists with further training, these presentations can also be viewed as professional development. Industry conferences attended were the International Fruit Tree Association (IFTA) conference in Wenatchee, WA and the Washington State Tree Fruit Association (WSTFA) meeting in Pasco, WA. The PD received a scholarship to attend the IFTA conference, for which the PD engaged in service activities, including conference photography, speaker introduction, and providing assistance for registering conference attendees. For the WSHA meeting, the PD presented a summary of current research results connected with this project, which allows for both dissemination of information as well providing the PD with valuable public speaking experience. Both industry meetings also helped the PD to gain an understanding of useful industry trends in apple rootstock cultivar use, on-farm research, and related orchard training systems. The scientific conference attended was the American Society for Horticultural Science (ASHS) meeting, where the PD delivered a workshop presentation titled, "Exploring the rhizosphere via metabolic profiling". The presentation delivered a comparison of techniques for assessing root exudates, including those developed in connection with this project, to other scientists engaging in rhizosphere research. This conference was a valuable opportunity to synthesize a workshop training seminar, connect with fellow scientists, as well as gain an understanding of other approaches for horticultural research by attending research presentations provided by other scientists. Additionally, a peer-reviewed journal article connected with this research was published in the first reporting period, per the following citation: Leisso, R., Rudell, D., Mazzola, M. 2017. Metabolic composition of apple rootstock rhizodeposits differs in a genotype-specific manner and affects growth of subsequent plantings. Soil Biology and Biochemistry. doi:10.1016/j.soilbio.2017.06.011 What do you plan to do during the next reporting period to accomplish the goals?In the next reporting period, research aspects of the project will focus on assessing the impact of the scion on the root exudate metabolic profile, analyzing scion x rootstock rhizosphere microbial community amplicon sequencing results, and summarizing and reporting on the project through peer-reviewed journals. Per the project postdoctoral training plan, the PD will also attend the Metabolomics 2018 conference in June of 2018.
Impacts
What was accomplished under these goals?
Objective 1. Research accomplishments connected to contrasting the effects of the apple scion on the root exudate metabolic profile on rootstocks of differing tolerance to ARD, and determining specific metabolites that could be involved in providing scion/rootstock tolerance to ARD included both further method development and research results: • Methods were further developed for collecting and analyzing apple root exudates: an automatically irrigated greenhouse setup allowed for collection of percolated exudates. • A validation procedure for root exudates from trees grown in a non-sterile greenhouse environment was developed using micropropagated trees generated under sterile conditions. • Libraries of metabolites confirmed by authentic standards were expanded for both apple root exudates and root tissue. • The quantity of root exudates released into the rhizosphere generally corresponded to apple rootstock vigor. • Two greenhouse experiments assessed the impact of rootstock cultivar-specific (G41, G935, M9Nic29, and M26) exudates on a spatially separated orchard soil; results from an initial experiment indicated that 1) M9Nic29 rootstock exudates select for a distinct fungal community (as assessed by (terminal restriction fragment length polymorphism analysis; T-RFLP) relative to other rootstocks and that 2) for all rootstock genotypes examined, fungal communities were influenced by rootstock exudates relative to control communities (no rootstock) over time. • Phenolic metabolites including 4-hydroxybenzoic acid, benzoic acid, chlorogenic acid, phloridzin, phloroglucinol, and rutin were tested for their ability to inhibit growth of the apple replant disease pathogens Pythium ultimum var. ultimum, Phytophthora cactorum, and Rhizoctonia solani AG-5. Results indicate that phloridzin, benzoic acid, and 4-hydroxybenzoic acid can all inhibit these pathogens in a concentration-dependent manner. Inhibition of Pythium ultimum by rutin was minimal and chlorogenic acid and phloroglucinol moderate at the concentrations tested. In summary, with respect to Objective 1, this research project confirmed that apple root exudates differ in a cultivar-dependent manner in metabolic composition which could impact their tolerance to root pathogens. Specific phenolic metabolites differing in quantity among rootstock cultivars were tested for their impact on pathogen growth. Some of the assayed metabolites inhibited in vitro growth of root pathogens, but inhibitory activity was only observed at concentrations in excess of that predicted to be encountered in the biological system. This information has utility both for rootstock breeding as well as enhancing fundamental knowledge regarding apple root rhizosphere physiology. Objective 2. Experimentation was performed to identify apple root biochemical compounds produced (quantitatively or qualitatively) in a rootstock genotype (cultivar) specific manner, and to understand impacts that resulting changes in soil biochemistry and microbial community have on the next generation of apple trees. Results included: • When cultivated in the same orchard soil, plant biomass, disease severity, and rhizosphere microbiome composition of apple seedlings differed depending upon genotype of the rootstock cultivated in this orchard soil. • Metabolic composition of root exudates differed among apple rootstock genotypes. The genotypes assessed in the present study were G41, G935, M9Nic29, and M26. Results suggest that G41 and G935 are the most similar in terms of root exudate metabolite profiles. • pH of both soil and water infiltrated through the tree rhizosphere were altered by the presence of a rootstock but did not differ significantly among rootstocks. • Rhizosphere microbial community amplicon sequencing results have been received from one scion x rootstock interaction experiment and are partially analyzed; DNA from a second experiment have been submitted to a sequencing service. In summary, with respect to Objective 2, results indicate that rootstock cultivar specific changes to orchard soil biochemistry and microbial community composition can impact growth of the next generation of trees as assessed by a plant bioassay. Furthermore, based on amplicon sequencing of rhizosphere microbial communities according to scion and rootstock graft combinations, microbial community differences may exist among scion/rootstock combinations, but are not extreme, and the biological impacts of these subtle differences are uncertain.
Publications
- Type:
Journal Articles
Status:
Accepted
Year Published:
2017
Citation:
Leisso, R., Rudell, D., Mazzola, M. 2017. Metabolic composition of apple rootstock rhizodeposits differs in a genotype-specific manner and affects growth of subsequent plantings. Soil Biology and Biochemistry. doi:10.1016/j.soilbio.2017.06.011
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