Progress 09/15/23 to 09/14/24
Outputs
Target Audience:Our research progress is working to provide information to the scientific community working on plant-microbe interactions, nutrient use efficiency, and adaption to abiotic stress mechanisms. We are also committed to providing useful data to respective local soybean associations and producers, with considerations in providing additional data points to producers on a national scale for informed production practice decision making. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?At NCSU, the project provided training opportunities for three master students (Alena Jones, Benjamin Rose, and Garud Shah), two Ph.D. students (Danielle Cooney and Mariela Perez Sepulveda), one postdoctoral associate (Dr. Arjun Kafle), and two undergraduate students (Carolina Brown and Erin Clark). At the University of Missouri, the project provided training opportunities for one master student (Salina Tripathi), two Ph.D. students (Bruna Montes Luz, Prakriti Chand), a postdoctoral associate (Dr. Dhruv Aditya Srivastava), and two undergraduate students (Grace Johnson and Nick Wood). All graduate trainees have made significant progress towards graduation, and have significantly improved their skills in experimental design, various laboratory techniques (e.g. DNA extraction, stable isotope experiments, data and statistical analysis). Together with the student, the postdoctoral associates designed the experiment, and conducted all analyses including the transcriptome and network analysis. The undergraduate students helped with the experiment, and also conducted independent experiments that support earlier made observations that AM fungi are able to translocate K from a hyphal compartment to the host, even under elevated phosphate and nitrogen treatments. In addition, the postdoc Dhruv Srivastava participated in a grant writing workshop, and has improved his teaching and mentoring skills. He also wrote and won a grant from the University of Missouri Postdoc association. The postdoctoral scientists at both institutions mentored the various undergraduate students. Various presentations were delivered by all mentees. Particularly, Grace Johnson presented part of her work as her poster during the Show Me Research Week at the University of Missouri and won the second prize for her presentation of around 100 undergraduate students that presented their work. How have the results been disseminated to communities of interest?The results have been disseminated to communities of interest by research presentations to different audiences, and contributions to scientific journals (see also products). What do you plan to do during the next reporting period to accomplish the goals?Objective 1: Performing experiments to test the impact of both phosphorus and potassium availability on rubidium transport in mycorrhizalMedicago truncatula. Objective 2: RNA sequencing and analysis of soybean colonized or not by an AM fungus and under varying potassium availabilities to identify the underlying molecular mechanisms responsible for the mycorrhiza-mediated potassium transport. Objective 3: Travel to co-PI University for a wrap-up meeting and present findings to national meetings Objective 4: Finalizing the remaining publications and Final Reporting
Impacts
What was accomplished under these goals?
1- We used two-compartment systems and rubidium (Rb+) to track potassium transport (K+) in AM soybean plants under increasing sodium chloride (NaCl) treatments and under limiting and sufficient K+ conditions. Additionally, we investigated the impact of external K+ availability on soybean root development in plants exposed to salinity. Two treatments containing either limited (LK, 0.05 mM K+) or sufficient (SK, 3.75 mM K+) K+ levels were used, as well as four Na+ conditions with 0, 50, 100, and 200 mM Na+ in the form of NaCl. Both K+ and Na+ treatments were mixed to make a total of eight different treatment media, abbreviated below as LK+0/50/100/200Na or SK+0/50/100/200Na. The impact of NaCl and K+ treatments on soybean root development was tested by placing germinated seedlings on large plates for 10 days under a light shelf. Primary root length, as well as number and length of lateral roots were recorded. Although the primary root length was not affected by the NaCl treatments, possibly to maintain the acquisition of other nutrients, a strong reduction in the number and length of lateral roots was observed. We also report that there was absolutely no impact of K+ availability on root development under any NaCl treatment in young seedlings. This indicates a limited effect of external K+ availability on biomass production for this soybean cultivar to cope with NaCl exposure, at least at early developmental stages. To assess the impact of salinity and K+ supply on AM symbiosis and biomass production in soybean, plants were inoculated with R. irregularis DAOM 197198 and compared to non-treated plants through placement in two-compartment systems under various NaCl and K+ regimes. Root colonization and plant dry weight were determined after eight weeks of co-culture overall but four weeks of NaCl treatments. Plant shoots were also digested to determine Rb+, K+, and Na+ concentrations by ICP-OES or ICP-MS, and K+:Na+ ratios were calculated. We observed that although a slow decrease in shoot biomass can be observed with increasing salinity, no differences occurred between AM and non-inoculated plants in both LK and SK conditions. On the other hand, the root biomass was similar between AM and NM plants unexposed to NaCl, but multiple differences appeared at higher salinity with more biomass produced in NM plants compared to AM ones. However, the external K+ availability did not have a great influence on root weight either. Altogether, these data indicate that the symbiotic status of this soybean cultivar and the external K+ availability had very limited impacts on the plant biomass production under moderate and high salinity conditions. Our results may suggest that the soybean cultivar we used was somehow tolerant to K+ deprivation, at least in our experimental conditions. Interestingly, the AM colonization was negatively and strongly impacted by NaCl exposure: it gradually dropped from about 50 % in unexposed plants and those under moderate salinity (i.e. 50 mM of NaCl) to less than 20 % on average at high salinity (i.e. 100/200 mM of NaCl). Additionally, shoot P concentrations in NM and AM plants matched AM root colonization's response to increasing NaCl treatments. This could be due to the decrease of the number and length of lateral roots we observed in our short-term experiment, making less colonization sites available for the fungus, and/or to the fungal inability to properly grow or form symbiosis. Overall, our results demonstrate that salinity does influence soybean root development, biomass, and AM colonization, but also show a more moderate impact of K+ availability on these factors. Additionally, we observed that colonized plants accumulated less Na+ compared to NM plants in both LK and SK conditions. This observation was also made in high salinity conditions where a strong reduction of root colonization was recorded. It indicates that even if the fungus was not able to transport P to colonized plants, it has the capacity to prevent Na+ ion accumulation in surrounding roots, probably by a sequestration mechanism in hyphae. 2- Last year, we explored the K+ vs. carbon relationship in AM symbiosis in the model legume plant Medicago truncatula, using a split-root approach where two root halves of single plant are spatially separated by an impermeable plastic sheet. This year, RNA-seq on roots in these various conditions was performed and data are currently analyzed. Additionally, we investigated the gene regulatory networks that are correlated to different phenotypic traits of plants under drought stress conditions, such as biomass, potassium levels in plant tissues, water loss per above ground biomass, and colonization of the roots with arbuscules.When the plants were four weeks old, the plants were inoculated with the AM fungus DAOM 197198 (5 weeks). Two weeks later (7 weeks), all plants (AM and nonmycorrhizal plants) were inoculated with Ensifer meliloti. At week 12, all plants were fertilized with sufficient P and N levels (P and N levels in the soil were 0.25 mM P and 2 mM N, respectively), but with low or high potassium levels (K levels in the soil were 0.08 mM K or 1.6 mM K, respectively). At week 14, all plants were exposed to different drought stress levels (well-watered, mild drought stress, or severe water stress), and at week 16, all plants were harvested. We studied the expression of phosphate, ammonium and potassium transporters in AM and NM roots dependent on K fertilization and drought stress treatments. PT1, PT3, PT13, and PT11 were expressed in both, AM and NM roots, and drought stress caused a significant reduction of their expression. In contrast, PT4, a well characterized mycorrhiza-specific transporter, was highly expressed in AM roots under well-watered and severe water stress conditions. We identified mycorrhiza-specific ammonium transporters that were particularly expressed in roots under well-watered conditions. AMT2;4, AMT2;3, AMT2;5 and AMT1 are expressed in AM roots under well-watered and severe drought stress conditions, but the expression is significantly reduced under drought stress. AMT2;3 has been described as essential for arbuscular branching in PT4 mutants, whereas AMT2;2, AMT2;4 and AMT2;5 are not required. However, AMT2;4 had a higher affinity for ammonium than AMT2;3 in yeast complementation assays, and it has been proposed that the receptor activity of AMT2;3 is more critical than its transport activity. We also identified the mycorrhiza-specific potassium transporters AKT1_1, and HAK20, that are highly expressed in AM roots under well-watered conditions. HAK17, another high affinity K transporter is expressed in NM and AM roots, but its expression is significantly higher in AM than in NM roots. The expression of all these transporters is significantly reduced under severe drought stress conditions. In addition, we identified AKT2/3 that is expressed in NM and AM roots under drought stress conditions. AKT2/3 encodes a putative potassium outward rectifying channel, and these channels are typically activated by membrane depolarization caused for example by the inhibition of H+-ATPase activities and induced net H+ influx by drought stress. We also examined the expression of genes that are directly involved in the nutrient and resource exchange between both partners in the AM symbiosis. As expected, these genes showed high expression levels in AM roots under well-watered conditions, but the expression is significantly reduced under drought stress conditions. Consistent with our observation that K deprivation induces a higher colonization of the AM roots with arbuscules, several of these genes show a slightly higher expression in the AM roots when drought stress is combined with K deficiency. PT8, a mycorrhiza specific phosphate transporter, and STR1, STR2, and FatM, which are all involved in the transport of lipids to the AM fungus show a higher expression in K deficient AM roots under drought stress.
Publications
- Type:
Book Chapters
Status:
Published
Year Published:
2023
Citation:
Das D, Tripathi S, Mutyala P, Srivastava DA, B�cking H. Development and resource exchange processes in root symbioses of legumes, Book: Symbiosis in Nature, publisher IntechOpen
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2023
Citation:
Johnson G, Tripathi S, Srivastava DA, B�cking H. 2023. Role of arbuscular mycorrhizal interactions in the potassium uptake of plants. Show Me Research Week, University of Missouri, April 18-20, 2023
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2024
Citation:
Impact of the arbuscular mycorrhizal symbiosis on the physiological and molecular responses of legumes to potassium deprivation. A1402 PD Meeting, St. Louis, MO, August 22 and 23, 2024
- Type:
Journal Articles
Status:
Submitted
Year Published:
2024
Citation:
Jones A.P., Kafle A., Rose B.D., Dutta S., Vann R. B�cking H., Garcia K. The external potassium availability determines the effect of arbuscular mycorrhizal fungi on the salinity tolerance of soybeans.
- Type:
Journal Articles
Status:
Published
Year Published:
2024
Citation:
Rose B.D., Dellinger M.A., Larmour C., Polishook M.I., Higuita-Aguirre M.I., Dutta S., Cook R.L., Zimmermann S.D., Garcia K. (2024) The ectomycorrhizal fungus Paxillus ammoniavirescens influences the effects of salinity on loblolly pine in response to potassium availability. Environmental Microbiology. 26(3): e16597.
- Type:
Journal Articles
Status:
Published
Year Published:
2023
Citation:
Houdinet G., Guerrero-Gal�n C., Rose B.D., Garcia K., Zimmermann S.D. (2023) Secrets of the fungus-specific potassium channel TOK family. Trends in Microbiology. 31(5): 511-520.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2024
Citation:
Garcia K. Deciphering mycorrhiza-mediated plant potassium nutrition. August 2024. 12th International Conference On Mycorrhiza (ICOM12). Manchester, UK
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2024
Citation:
Rose B.D., Dellinger M.A., Larmour C.P., Polishook M.I., Higuita Aguirre M.I., Dutta S., Cook R.L., Zimmermann S.D., Garcia K. The ectomycorrhizal fungus Paxillus ammoniavirescens influences the effects of salinity on loblolly pine in response to potassium availability. August 2024. 12th International Conference On Mycorrhiza (ICOM12). Manchester, UK
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2024
Citation:
Garcia K. Deciphering mycorrhiza-mediated plant potassium nutrition. March 2024. Forest Resources and Environmental Conservation Seminar Series. Virginia Tech
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2024
Citation:
Rose B.D., Dellinger M.A., Larmour C.P., Polishook M.I., Higuita Aguirre M.I., Dutta S., Cook R.L., Zimmermann S.D., Garcia K. The ectomycorrhizal fungus Paxillus ammoniavirescens influences the effects of salinity on loblolly pine in response to potassium availability. April 2024. 2024 Middle Atlantic States Mycology Conference (MASMC). Duke University
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2024
Citation:
Perez Sepulveda M., Mullen M., Garcia K., Heitman J. L., Gatiboni L. The Impact of salinity on soybean growth and symbiotic bacteria strains. October 2023. ASA, CSSA, SSSA International Annual Meeting. St. Louis, MO
|
Progress 09/15/22 to 09/14/23
Outputs
Target Audience:Our research progress is working to provide information to the scientific community working on plant-microbe interactions, nutrient use efficiency, and adaption to abiotic stress mechanisms. We are also committed to providing useful data to respective local soybean associations and producers, with considerations in providing additional data points to producers on a national scale for informed production practice decision making. Due to her current administrative position, Co-PI Dr. Heike Buecking is not as actively involved in field days and stakeholder conferences than she used to be, but in meetings with stakeholder groups (for example Missouri Soybean Merchandizing Council), the benefit of microbial communities for soil health and crop nutrition are regularly discussed. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?The project provides training opportunities for two M.S. student (Salina Tripathi at the University of Missouri, and Alena Jones at NCSU), one Ph.D. student (Danielle Cooney at NCSU), two postdoctoral associates (Dr. Dhruv Srivastava at the University of Missouri, and Dr. Arjun Kafle at NCSU), and several undergraduate students (Nick Wood at the University of Missouri, and Carolina Brown and Erin Clark at NCSU). All students and postdocs from NCSU are activelly involved on the project, particularly through monthly meetings with the entire funded group. How have the results been disseminated to communities of interest?The results have been disseminated to communities of interest by research presentations to different audiences, and contributions to scientific journals (see also products). What do you plan to do during the next reporting period to accomplish the goals?NCSU: Additional experiments are currently running to investigate the impact of potassium availability on nodule formation in the same two soybean cultivars. University of Missouri: We hypothesize that the water status of the plant or an exposure of the plant to high salt concentrations will affect fungal potassium transport. To measure this effect, we will use two compartment systems with one Medicago root compartment and one fungal compartment. For these experiments, we will choose the fungal partner that in the previous experiments showed the higher potassium transfer activity. Similar to the experiments described above, we will measure the rubidium transport from the fungal compartment to the plants, when the plant or the fungal partner will be exposed to different levels of salinity (through addition of different levels of 0, 50, 100, and 200 mM NaCl) or to different water availabilities. Non-drought or drought conditions will be established by weighing the pots daily, followed by water additions, accordingly. These experiments will be conducted with nodulated or non-nodulated plants.
Impacts
What was accomplished under these goals?
Objective C1. We have used Rb+ as a proxy to test whether potassium is being directly transported by AM fungi to soybean plants under salinization in limited-potassium (LK) and sufficient-potassium (SK). Soybean cultivar (MG 5) S52RS86 was used, it is an excluder which takes out the Cl- ions. To test this, a two-compartment system is used which is composed of a plastic box (12 cm x 8 cm x 8cm : L x W x H) and a Magenta GA-7 Plant Culture box inside the plastic box. The outer box is the root compartment (RC), and the inner box is the fungal compartment (FC) for the hyphae from the RC to extend to. Plants were either inoculated with the fungus (AM) or were not inoculated (NM). The soybeans were also under four levels of salinity (0, 50, 100, & 200 mM). Each treatment contained six replicates. In the RC, plants were watered every 3 days with Long Ashton nutrient solution with limited potassium (0.05 mM, LK) or sufficient potassium (3.75 mM, SK) for a duration of eight weeks. A 30 mL solution of RbCl (3.25 mM) and KCl (0.5 mM) was added to the FCs every 3 days before harvest. Root and shoot samples were collected for the following analyses: biomass (FW & DW), colonization, and digestion. Currently, soybean shoots have been grinded and are in progress for digestion, and obtaining concentrations of Rb+, K+, Na+, P, Ca2+, Mg2+, and Fe that will be determined by ICP-OES. Overall, the results are stable throughout all treatments in the shoot DW biomass. There are not many significant differences between NM/AM in LK/HK in the varying NaCl conditions. In NaCl level 200 mM, there is a significant difference between NM and AM of the SK. NM had a higher biomass than AM. The root DW biomass kind of shows a decrease in weight of both NM/AM in LK/SK as NaCl increases. Within each NaCl treatment, NM has higher biomass than AM. There is a general decrease of colonization of both soybeans in LK and SK as NaCl treatment goes. Plants in LK had a higher colonization in 0, 50, & 100 mM NaCl treatment. It may be possible that Rhizophagus irregularis DAOM can support the plant in limited potassium. Yet, plants in the 200 mM NaCl shows that the plants in sufficient potassium had a higher colonization rate than LK. This may indicate that there is a threshold of salinity that the fungus can function properly with more potassium supply that's available. Plants in the 50 mM NaCl had a significant difference with plants in LK having more colonization. The same experiment is currently being done but with soybean cultivar MG 6. All methods and analyses are the same as the experiment with soybean MG 5. Additionally, a plate study was done in parallel to see what the direct effects of salinity on soybean root architecture are either in limited or sufficient potassium. Soybean seeds (MG 5 & MG 6) were germinated, after 3-5 days five seeds were placed on germination paper in a large-sized plate (23 x 23 cm). The media consisted of pure agar, Long Ashton nutrient solution (LK=0.05 mM, SK=3.75 mM), and NaCl (0, 50, 100, & 200 mM). Each treatment has 3 replicates (15 total seeds) each. Aluminum foil was placed over the plates to cover up the roots to act as the dark. Plates were placed on a light shelf for 10 days. On the harvest day, shoot and root samples were collected for biomass (FW & DW). From physical observation, in LK, as NaCl increases, soybean growth decreases. Currently, root length, number of lateral roots, and number of root hairs are being measured by ImageJ. Objective C2. To explore the K+ vs. C relationship in AM symbiosis in the model legume plant Medicago truncatula, we used a split-root approach where two root halves of single plant are spatially separated by a impermeable plastic sheet. Therefore, the root system of each plant was divided into two separate parts and placed in independent compartments filled with Turface. Fungal compartments (FC) which were placed in each box and supplemented or not with K+ that were only accessible to the AM fungal fungus, but not roots, through perforated holes. Plants colonized with the AM fungus (one or two root halves) had proportionally higher shoot dry weight compared to the NM plants. Similarly, non-colonized root halves displayed lower dry weight than those colonized with the AM fungus. Comparing the root weight between root halves within same conditions, almost all root halves had similar root dry weight except in one condition where one root half was NM whereas the other root half was colonized, and the corresponding FC of the colonized root half was supplemented with SK solution. AM fungal colonization rate in the roots that were inoculated with the AM fungus was around 30% to 40% and the colonization rate was not significantly different between the root halves treatments. Plants allocated less 13C in NM root halves than AM root halves. More specifically, plants allocated a similar amount of 13C to root halves of split root systems when both root halves were either non-colonized or both root halves were colonized that had similar K in the FC of the corresponding root halves. As our expectation, host plants allocated more 13C to the colonized roots notably when the fungus had access to FC which were supplemented with sufficient K. Our results clearly demonstrated that there is a clear relation between plant C allocation to the AM symbiotic roots when the fungus had access to extra K availability in mycorrhizosphere thus the fungus is capable of providing K benefits to the host plants. Plants colonized with AM fungus either to one root half or both root halves had higher K concentration in the shoot tissues compared to plants that were not colonized to both root halves. Compared to colonized plants, shoot K concentration was relatively higher, but not significantly, when both root halves were colonized with the fungus irrespective of K supply in the HC. The K concentration between root halves from each split root system of the same condition remained similar when they were not colonized or both roots were colonized with the fungus. However, the root half that was colonized with the fungus acquired a higher K concentration than the other corresponding not-colonized root half, particularly this observation was more notable when the fungus had access to sufficient K in the FC, suggesting that AM fungus was able to acquire K from the FC and transfer in the interphase of symbiotic interactions. Additionally, shoot P concentration in the colonized plants irrespective of one root or both root halves was higher than non-colonized both root halves which further conformed that AM fungi facilitate to acquire more P in the plant tissues during symbiotic interactions. In a similar trend, overall AM colonized root halves had more P than the non-colonized roots. The root P concentration in each root half of the split root system remained same when both root halves were colonized with the fungus. In conjunction with the root K concentration, the colonized root half contained more P than its associated non-colonized roots of the split root systems thus indicating K and P are conjointly transported to the host during AM symbiosis. Objective C3. This objective is currently in progress. Plants have been collected for the RNAseq experiment, and should be submitted soon for sequencing. Objective C4. This part of the project is now completed, and we are working on the corresponding publication.
Publications
- Type:
Journal Articles
Status:
Accepted
Year Published:
2022
Citation:
Kafle A., Garcia K. (2022). Cesium could be used to track potassium transport in mycorrhizal Medicago truncatula. Plant Signaling & Behavior. 17(1): 2134676.
- Type:
Journal Articles
Status:
Accepted
Year Published:
2023
Citation:
Garcia K., Cloghessy K., Cooney D.R., Shelley B., Chakraborty S., Kafle A., Sonawala U., Collier R., Jayaraman D., Pilot G., An� J-M. (2023) The putative transporter MtUMAMIT14 participates in nodule formation in Medicago truncatula. Scientific Reports. 13: 804.
- Type:
Journal Articles
Status:
Accepted
Year Published:
2023
Citation:
Rose B.D., Frank H.E.R., Garcia K. (2023) Development of split-root assays for loblolly pine (Pinus taeda L.) seedlings to study ectomycorrhizal symbioses. MethodsX. 10: 102046.
- Type:
Journal Articles
Status:
Accepted
Year Published:
2023
Citation:
Amenc L., Becquer A., Trives-Segura C., Zimmermann S.D., Garcia K., Plassard C. (2023) Overexpression of the HcPT1.1 transporter in Hebeloma cylindrosporum alters the phosphorus accumulation of Pinus pinaster and the distribution of HcPT2 in ectomycorrhizae. Frontiers in Plant Science. 14: 1135483.
|
Progress 09/15/21 to 09/14/22
Outputs
Target Audience:Our research progress is working to provide information to the scientific community working on plant-microbe interactions, nutrient use efficiency, and adaption to abiotic stress mechanisms. We are also committed to providing useful data to respective local soybean associations and producers, with considerations in providing additional data points to producers on a national scale for informed production practice decision making. Due to her current administrative position, Co-PI Dr. Heike Buecking is not as actively involved in field days and stakeholder conferences than she used to be, but in meetings with stakeholder groups (for example Missouri Soybean Merchandizing Council), the benefit of microbial communities for soil health and crop nutrition are regularly discussed. Changes/Problems:The progress on the project was affected by the move of the Co-PI of the project Heike Buecking to the University of Missouri. The move made it necessary to transfer the grant from South Dakota State University to the University of Missouri, establish a new lab, and hire personnel. Due to problems with the visa, the start date of the postdoc had to be delayed. The new laboratory at the University of Missouri is now established, and we expect that we can continue the project as planned. What opportunities for training and professional development has the project provided?The project provides training opportunities for three M.S. student (Salina Tripathi at the University of Missouri, and Alena Jones at NCSU), one Ph.D. student (Danielle Cooney at NCSU), three postdoctoral associates (Dr. Kevin Cope and Dr. Dhruv Srivastava at the University of Missouri, and Dr. Arjun Kafle at NCSU), and four undergraduate students (Nick Wood at the University of Missouri, and Nyssa Ndey-Bongo, Jacob Layton, and Anne Bonds at NCSU). Dr. Kevin Cope was financially supported by another USDA funded project, but contributed through his studies to additional results that were relevant for this project. The M.S. student and the second postdoctoral scientist started only very recently to work on this project. The late start of this personnel on this project is related to the move of the lab of Dr. Heike Bücking from South Dakota State University to the University of Missouri (previously South Dakota State University), that made the transfer of the grant necessary, and additional delays that were caused by hiring an international postdoctoral scientist to this project. All students and postdocs from NCSU were involved on the project since the beginning, except for the M.S. student who was hired recently. How have the results been disseminated to communities of interest?The results have been disseminated to communities of interest by research presentations to different audiences, and contributions to scientific journals (see also products). What do you plan to do during the next reporting period to accomplish the goals?NCSU: Additional efforts are needed on the soybean part of the project. The recently hired M.S. student is now taking part of this aspect. She is developing experiments to evaluate (1) the potassium transport in three soybean cultivars, (2) the impact of potassium availability on salt stress tolerance in these three cultivars, and (3) the transcriptional responses in mycorrhizal vs. non-mycorrhizal soybean plants under sufficient and limited potassium conditions. Additional experiments are also planned to investigate the impact of potassium availability on nodule formation in the same three soybean cultivars. University of Missouri: After the experiments for Objective 2 have successfully been completed, we will shift our focus to experiments in which we will study how environmental factors such as drought or salinity affects fungal potassium transport (Objective 1). We hypothesize that the water status of the plant or an exposure of the plant to high salt concentrations will affect fungal potassium transport. To measure this effect, we will use two compartment systems with one Medicago root compartment and one fungal compartment. For these experiments, we will choose the fungal partner that in the previous experiments showed the higher potassium transfer activity. Similar to the experiments described above, we will measure the rubidium transport from the fungal compartment to the plants, when the plant or the fungal partner will be exposed to different levels of salinity (through addition of different levels of 0, 50, 100, and 200 mM NaCl) or to different water availabilities. Non-drought or drought conditions will be established by weighing the pots daily, followed by water additions, accordingly. These experiments will be conducted with nodulated or non-nodulated plants. The progress on the project was affected by the move of the Co-PI of the project Heike Buecking to the University of Missouri. The move made it necessary to transfer the grant from South Dakota State University to the University of Missouri, establish a new lab, and hire personnel. Due to problems with the visa, the start date of the postdoc had to be delayed. The new laboratory at the University of Missouri is now established, and we expect that we can continue the project as planned.
Impacts
What was accomplished under these goals?
The plant microbiome is a largely unexplored resource of beneficial microorganisms with diverse properties and a hidden potential to manipulate plant growth and success in stressful environments. Here, we aim to understand how environmental factors (water limitation and salt exposure), nutrient availability, and carbon transport from the host to the AM fungus affect the symbiotic transport of K. We will also study whether fungal K transport plays a role for the biological nitrogen fixation of legumes. In addition, we propose to identify and characterize the molecular mechanisms used by non-mycorrhizal and mycorrhizal M. truncatula and soybean plants in response to K deficiency. Finally, we will directly translate our findings to the field, and will test the applicability of our results for field grown soybean production systems. Objective C1. To test whether AM fungi can transport K+ to Medicago truncatula, we used rubidium (Rb+) as a proxy for K+. AM plants had significantly higher shoot and root dry weight than NM plants in both LK and SK conditions. Notably, despite the similarity of shoot dry weights of AM inoculated plants between LK and SK conditions, the root dry weights were significantly lower in SK. In both LK and SK conditions, root AM colonization remained around 45%. In both LK and SK treatment, AM plants had significantly higher Rb+ concentrations compared to NM plants. NM plants grown in LK had significantly less K+ concentrations in the shoot tissues compared to plants grown in SK, thus validating the sufficient and limited K+ conditions used in our study. In both LK and SK, AM plants had significantly higher shoot K concentrations which clearly suggested that AM fungi can improve K+ nutrition in the host plants. There was no difference in shoot and root K+ concentration of the plants grown in LK compared to SK. Also, AM plants contained significantly higher shoot P concentration than NM plants providing further evidence of P benefit to the plants. The concentrations of Rb+ in the shoot demonstrated significant, strong positive correlation to shoot K+ concentrations indicating that Rb+ could be used as a proxy for K+ transport in AM symbiosis study. Furthermore, a significant, positive correlation occurred between shoot Rb+ concentrations and the percentage of AM colonized roots suggesting that the fungus is able to transport Rb+ to the colonized plants. This latter observation was reinforced by the fact that no significant correlation was observed between shoot Rb+ concentrations and shoot biomass in NM or AM plants. Objective C2. To explore the K+ vs. C relationship in AM symbiosis in Medicago truncatula, we used a split-root approach. The root system of each plant was divided into two separate parts and placed in independent compartments filled with Turface. Fungal compartments (FC) where placed in each box and supplemented or not with K+ that was only accessible to the AM fungal fungus. Plants inoculated with the AM fungus (one or two compartments) had higher shoot dry weight compared to the NM plants. Similarly, non-colonized root halves displayed lower dry weight than those colonized with the AM fungus. Comparing the root weight between root halves within same conditions, almost all root halves had similar root dry weight except in one condition where one root half was NM whereas the other root half was colonized and the corresponding FC was supplemented with HK solution. AM fungal colonization rate in the roots that were inoculated with the AM fungus was around 30% to 40% and the colonization rate was not significantly different between treatments. Additionally, we have recently started several experiments that are currently ongoing: In Experiment 1, we currently test how the availability of potassium for different arbuscular mycorrhizal fungi affects plant biomass, nutrient uptake, nitrogen fixation and carbon allocation. For this experiment, we surface sterilized Medicago seeds and pre-germinated them on moist germination paper. To accelerate lateral root formation, the tip of the primary root of the germinated seedlings was excised, and the plants were grown in a hydroponic system in a sterilized nutrient solution. After the plants had developed enough roots, the plants were transferred into sterile, custom-made 3-D printed four compartment pots. When the plants are fully colonized, we will add rubidium as a potassium replacement to one or the other fungal compartment, and will measure Rb+ transport to the plant root halves and the shoot, root and shoot biomass and nutrient contents, and the carbon allocation to each root half. The carbon allocation will be measured by placing the compartment systems into a closed container, in which 13C-labeled CO2 will be released. We recently also set up the plants for a second experiment, that will allow us to evaluate which role potassium nutrition by the fungal partner plays in carbon allocation and root nodulation. For this experiment, we will use three compartment systems with one nodulated root half and one AM root half, and one fungal compartment. After the fungus has successfully crossed over the divider into the fungal compartment, we will add only water or enrich the fungal compartment with the following tracers: 15N labelled ammonium, 33P labelled orthophosphate, and Rb as potassium tracer. Objective C3. This objective is currently in progress. A new graduate student has been hired to specifically complete the RNA-seq experiment in soybean. Currently, inoculated and non-inoculated soybean seedlings watered with limited or sufficient K+ solutions are growing, and will be collected and analyzed in the coming month. Objective C4. During the 2020 & 2021 calendar year, field-based soybean trials were conducted in North Carolina across the following environmentally diverse, and potassium limited locations. The field plot design was a randomized complete split-plot. Where evaluation was composed of unique treatment combinations of the following specifications. To evaluate the arbuscular mycorrhizal fungi, a commercially available inoculum, MycoApply© was applied or left blank on three different soybean varieties representing three maturity groups (IV, V, VI), and tested under conditions of applied additional potassium (0-0-60 KCl) or leaving soil conditions in a state of lower soil available potassium. Overall, it seems that under field conditions, we have not been able to quantify many significant differences between the addition of AM fungi compared to the control under both sufficient and low K conditions within a specific maturity group. We do observe a few differences, but most differences are a response that is driven by maturity group. This may potentially indicate that the fungi are not as essential under sufficient levels of soil K, but may be assisting in plant fitness under low soil K conditions. However, some interesting significant results were obtained for aspects of soybean seed quality relating to end products. This may allow for continued interest in the role those arbuscular mycorrhizal fungi could provide. These field results are allowing for a good initial indication for what parameters will be of continued interest to evaluate in additional to also determining what field-based conditions may hold the most potential for improving management decisions. Especially as producers look to educational and extension systems for unbiased data to help incorporate microorganisms and utilize crop management systems that are conducive to productive microorganism environments.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2022
Citation:
Cope KR, Kafle A, Yakha JK, Pfeffer PE, Strahan GD, Garcia K, Subramanian S, B�cking H. 2022. Physiological and transcriptomic response of Medicago truncatula to colonization by high-or low-benefit arbuscular mycorrhizal fungi. Mycorrhiza 32, 281-303
- Type:
Journal Articles
Status:
Published
Year Published:
2022
Citation:
Hackman J.J.*, Rose B.D.*, Frank H.E.R., Vilgalys R., Cook R.L., Garcia K. NPK fertilizer use in loblolly pine plantations: who are we really feeding? Forest Ecology and Management. 520: 120393.
- Type:
Journal Articles
Status:
Published
Year Published:
2022
Citation:
Kafle A., Cooney D.R., Shah G., Garcia K. Mycorrhiza-mediated potassium transport in Medicago truncatula can be evaluated by using rubidium as a proxy. Plant Science. 322: 111364
- Type:
Journal Articles
Status:
Published
Year Published:
2022
Citation:
Kafle A., Frank H.E.R., Rose B.D., Garcia K. (2022) Split down the middle: Studying arbuscular mycorrhizal and ectomycorrhizal symbioses using split-root assays. Journal of Experimental Botany. 73(5): 1288-1300.
- Type:
Journal Articles
Status:
Published
Year Published:
2021
Citation:
Frank H.E.R., Garcia K. (2021) Benefits provided by four ectomycorrhizal fungi to Pinus taeda under different external potassium availabilities. Mycorrhiza. 31(6): 755-766.
- Type:
Journal Articles
Status:
Published
Year Published:
2021
Citation:
Usman M., Ho-Pl�garo T., Frank H.E.R., Calvo-Polanco M., Gaillard I., Garcia K., Zimmermann S.D. (2021) Mycorrhizal symbiosis for better adaptation of trees to abiotic stress caused by climate change in temperate and boreal forests. Frontiers in Forests and Global Change. 4: 742392.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2022
Citation:
Tripathi S, Mutyala P, Bauder S, Berti M, B�cking H. 2022. The effects of management strategies on arbuscular mycorrhizal fungi for sustainable alfalfa production.International Conference on Mycorrhizae 11, Beijing, China, July 31 to August 05, 2022
- Type:
Journal Articles
Status:
Published
Year Published:
2022
Citation:
Cope KR, Yakha Y, Subramanian S, B�cking H. 2022. Breaking a bottleneck to biological nitrogen fixation in soybean: Potential plant reward mechanism for beneficial rhizobia. 25th North American Symbiotic Nitrogen Fixation Conference, June 05 to June 08, 2022
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2022
Citation:
B�cking H. 2022. Fair trade in beneficial plant microbe interactions. University of Marburg, January 13, 2022
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2021
Citation:
Cooney D.R.#, Kafle A.#, Crozier C., Gatiboni L., Wei, S., Vann R.A., Garcia K. The use of arbuscular mycorrhizal fungi to improve potassium acquisition in soybean in North Carolina. November 2021, ASA-CSSA-SSSA. Salt Lake City, UT
|
Progress 09/15/20 to 09/14/21
Outputs
Target Audience:Our research progress is working to provide information to the scientific community working on plant-microbe interactions, but also to respective local soybean associations and producers, with considerations in providing additional data points to producers on a national scale. Changes/Problems:Co-PI Bücking moved her lab to the University of Missouri. Consequently, the field work presented in this project is being done in North Carolina and Missouri, instead of North Carolina and South Dakota. What opportunities for training and professional development has the project provided?Conference attendance and presentation (posters and talks). One videoclip has been produced. How have the results been disseminated to communities of interest?Conference attendance and presentation (posters and talks). One videoclip has been produced. What do you plan to do during the next reporting period to accomplish the goals?Similar laboratory works presented on Medicago truncatula is being done in soybean Williams-82, as well as cultivars use din NC field. Also, since co-PD Bücking moved to the University of Missouri, a similar field work will start soon at that location.
Impacts
What was accomplished under these goals?
Objective C1. To demonstrate the K nutrient benefits of AM fungi to the host plant from the K limited soil. To test the impact of AM fungi on K-sufficient and -limiting conditions, Medicago truncatula seedlings were inoculated with the AM fungus Rhizophagus irregularis isolate 09 (Myco) and watered with low (-K) or high (+K) K solutions. Plants were placed in two-compartment systems containing one plant root compartment in which both plants and fungal inoculum were placed at the beginning of the experiment, and one fungal compartment that can be reached by the fungus only later on. Rubidium was placed in the fungal compartment only, allowing its transport to the host plant only when the fungus was present. Shoot and root biomasses were significantly higher in colonized plants compared to the non-mycorrhizal (NM) plants. Around 45% of the roots of inoculated plants were colonized under both K regimes. K, Rb, and P concentrations in the shoot tissue were significantly higher in mycorrhizal plants. In our experiment, we used Rb as analogous tracer for K and we observed that shoot Rb concentrations were consistently and significantly higher in mycorrhizal than in non-mycorrhizal plants. Additionally, it followed a similar pattern than shoot K concentrations. This pattern suggests that the fungus takes up and transfers K and Rb to the host plant have through a similar mechanism. Moreover, the tissue sodium (Na) concentration in mycorrhizal plants was lower than in non-mycorrhizal plants, suggesting that AM fungi prevent the accumulation of Na in host plant tissues. Objective C2. To demonstrate how the benefit of AM fungi for K nutrient is affected by soil salinity. Medicago truncatula (Jemalong A17) was used to understand the effects of external sodium addition on AM-mediated K nutrition. All plants were treated with low K solution only. Additionally, to prevent the osmotic shock of sodium chloride (NaCl) solution to the plant, we increased NaCl level incrementally starting from day 19. Final NaCl concentrations (0 mM, 50 mM, 100 mM, and 200 mM) were reached ten days later supplied every 3 days until harvest. Fungal compartments were provided with 3.25mM of RbNO3 and 0.5 mM of KNO3 on day 34 and until harvest. Plant growth was not affected by NaCl addition, probably due to the short period of time at full concentration. Interestingly, the AM root colonization was in decreasing order as increased the concentrations of salt solution. The dried shoot samples have been provided to the elemental analysis facility to quantify K, Rb, and Na concentrations. The results obtained from the facility will provide us with insightful information on the effects of salt stress on AM-mediated plant K nutrition. Similar works are currently in progress for soybean. Objective C3. Identify the plant genes that control the potassium uptake in mycorrhizal and non-mycorrhizal M. truncatula and soybean plants using gene expression networks and reverse genetics. Medicago truncatula mutants from 3 genes coding for putative potassium transporters expressed during mycorrhizal symbiosis under potassium deprivation are being analyzed. RNA-sequencing experiments are conducted on soybean Williams-82 in order to identify plant genes involved in plant potassium acquisition upon colonization with the mycorrhizal fungus Rhizophagus irregularis. Objective C4 - Examine the impact of AM communities on potassium uptake of soybeans under field conditions. Row crop production in field conditions can face a diverse host of challenges related to providing a sufficiently abundant crop for food, fuel, and fiber. One-way producers help crops mitigate environmental stress is through adequate nutrition supplied in the soil, but sometimes this can lead to over abundance of particular elements moving into water sources. Thus, the importance of understanding and researching methods to address these challenges and still provide viable solutions, such potential solution is better understanding the role and addition of arbuscular mycorrhizal fungi in a field-based setting. During the 2020 calendar year field-based soybean trials were conducted in North Carolina across the following environmentally diverse, and potassium limited locations. Piedmont Research Center, Sandhills Research Center, and Upper Coastal Plain Research Center. The field plot design was a randomized complete split-plot. Where evaluation was composed of unique treatment combinations of the following specifications. To evaluate the arbuscular mycorrhizal fungi, a commercially available inoculum, MycoApply © was applied or left blank on three different soybean varieties representing three maturity groups (IV, V, VI), and tested under conditions of applied additional potassium (0-0-60 KCl) or leaving soil conditions in a state of lower soil available potassium. Throughout the growing season of the field trial, data was collected at two timings for shoot biomass, tissue nutrient content, and chlorophyll content. To conclude out the season, producer representative data of yield, seed protein, and seed oil were collected. The following results of data were collected in 2020. Overall, it seems that under field conditions, we have not been able to quantify many significant differences between the addition of AM fungi compared to the control under both sufficient and low K conditions within a specific maturity group. We do observe a few differences, but most differences are a response that is driven by maturity group. However, this result is not of primary interest for understanding response of AM under sufficient and low levels of soil K. This may potentially indicate that the fungi are not as essential under sufficient levels of soil K, but may be assisting in plant fitness under low soil K conditions. However, some interesting significant results were obtained for aspects of soybean seed quality relating to end products. This may allow for continued interest in the role those arbuscular mycorrhizal fungi could provide. Additional data is necessary for a more robust and complete conclusion as to the potential and implication of this work. As there could also be influences from natural AM populations that are better adapted comparatively to the additions provided through our inoculant.
Publications
|
|