Progress 04/01/24 to 03/31/25
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 cotton 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?This project has involved a postdoctoral researcher and 3 graduate students at NC State University. Personal involved have been trained to various physiological, microbiological, molecular, and isotopic approaches through the course of the project. Also, they were given the possibility to present their work in various local meetings. In addition to technical training, the researchers have been actively involved in data analysis, manuscript preparation, and interdisciplinary collaborations, enhancing their scientific communication and critical thinking skills. How have the results been disseminated to communities of interest?Multiple manuscripts detailing our findings are currently in preparation, focusing on different aspects of AMF-mediated drought tolerance and potassium uptake in cotton. These manuscripts will be submitted to high-impact peer-reviewed journals to ensure broad dissemination within the scientific community. Additionally, a book chapter discussing the importance of soil microbes in plant potassium acquisition has been accepted for publication, providing an in-depth synthesis of our work and its implications for sustainable agriculture. What do you plan to do during the next reporting period to accomplish the goals?First, cotton plants of the ST4990 and PHY443 genotypes are being cultivated in association with arbuscular mycorrhizal fungi (AMF) to assess key physiological traits linked to drought tolerance under both sufficient potassium (SK) and limited potassium (LK) conditions. Specifically, we aim to investigate whether AMF symbiosis enhances foliar potassium levels, thereby reducing the leaf turgor loss point, improving osmotic adjustment, and accelerating stomatal responses. To further evaluate the role of AMF in drought resilience, colonized and non-colonized plants will be subjected to drought stress followed by a recovery phase, allowing us to determine the extent to which AMF contributes to improved drought resistance. Second, we are exploring the carbon transfer dynamics between the plant and AMF in the most promising cotton variety by utilizing 13CO2 labeling and rubidium (Rb) as a potassium analog tracer. Between ST4990 and PHY443 cultivars, PHY443 will be chosen for this experiment as it exhibited a higher leaf count compared to cultivar ST4990. We will use the split-root technique, which is an experimental design that allows the root system of a single seedling to be divided into two separate compartments, each exposed to different conditions. First, the seeds will be germinated in Turface medium in the growth chamber (12:12 photoperiod, no night interruption, 29/24 temp) for two weeks before being transferred to a hydroponic system. During transfer, the primary root will be cut to promote lateral root growth and divide the roots into two sections. The seedlings will grow in the hydroponic system for two additional weeks in a standard Long Ashton solution before being transferred once again to double compartments filled with soil substrate (sand, vermiculite, and perlite). Each compartment will be inoculated or not with AMF. Additionally, high or low concentrations of K will be introduced to the fungal hyphae in adjacent compartments within the main compartment. Access to these adjacent compartments by the plant roots is restricted by a dense nylon mesh, but fungal hyphae are able to move freely between both units. Once colonization of AMF in the plant roots has been established, we will move forward with carbon labeling in order to detect if preferential allocation of carbon from the cotton plants to the AMF with K+ access has occurred. This experiment is currently in progress.
Impacts
What was accomplished under these goals?
Assessment of drought resistance traits in commercial cultivars of upland cotton We have previously reported assessment of multiple hydraulic traits associated with drought tolerance for the six cotton genotypes. In 2024, another set of experiments was performed to assess plant responses to drought as this stress develops in a more gradual way, similarly to what happens in the field. This set of experiments was also performed allowing plants to dehydrate to similar levels, such that both drought avoidance and drought tolerance could be differentiated. Drought avoidance refers to the ability of plants to delay or minimize tissue dehydration during perios of drought. The genotypes DP2038 and PHY443 dehydrated slower than all other genotypes, experiencing the least negative water potentials after 16 days of drought and taking the longest times to reach a dehydration level resulting in 50% leaf embolism. These genotypes have the higher dehydration avoidance of all genotypes. The genotype DP2127 is the dehydrated faster than all other genotypes, experiencing the most negative potentials after 16 days of drought and taking the shortest time to reach a dehydration level resulting in 50% leaf embolism. This genotype is then considered to have the lowest dehydration avoidance of all genotypes. The genotypes DP2125, PHY360, and ST4490 have moderate dehydration avoidance. Data of all genotypes show that plants with greater canopy size (total leaf mass per canopy) and maximum daytime transpiration under well-water conditions are associated with a shorter time to experience 50% leaf embolism during drought. Drought avoidance refers to the ability of plants to maintain performance and avoid tissue damage as they dehydrate. Drought tolerance was assessed as canopy mortality when genotypes were dehydrated to similar water potentials (on average) and by comparing the slope of the canopy mortality and water potential relationship. When experiencing similar water potentials during drought, genotypes experience similar canopy mortality. A strong positive relationship between means minimum leaf water potential during drought and canopy mortality observed using averages of the six genotypes demonstrates that the small (non-statistically significant) differences in dehydration during drought across genotypes resulted in the observed (non-statistically significant) differences in canopy mortality across genotypes. To confirm a potential similarity of drought tolerance across genotypes, we assessed the slope of the slope of the canopy mortality and water potential relationship for each genotype. The slopes are relatively similar across genotypes, but might be the lowest for PHY360, indicating greater canopy mortality at higher water potentials (i.e. lower tolerance). Differences in slope across genotypes need to be further confirmed by statistical analyses. Still, confirmation of the lower drought tolerance of PHY360 when compared to all other genotypes would not be surprising given that this genotype has the lowest leaf embolism resistance. The ability of genotypes to recover from drought was assessed by evaluating total leaf area and the percent daytime transpiration of entire canopies after seven days of recovery. A closer look of all plants demonstrates that the ability of plants to recover was strongly correlated with the canopy mortality experienced during drought, such that plants that experience lower tissue damage and mortality during drought (either via drought avoidance or tolerance) have a better ability to recover from droughts. Altogether, our results for cotton demonstrate that the ability to avoid dehydration (drought avoidance) is critical to minimize tissue damage during drought, which ultimately impact the ability of plants to recover from drought. The genotypes DP2038 and PHY443 have the greatest drought avoidance of all genotypes. They also displayed greater drought tolerance (together with DP2115, DP2127, and ST4990) than PHY360. For this experiment, PHY443 also ranked amongst the ones with greatest recovery ability, although this is likely because of their greater water potential and lower leaf mortality during drought. Assessing the impact of AMF-mediated potassium nutrition on cotton tolerance to drought Previous drought experiments evaluated eight cotton genotypes, identifying PHY443 and ST4990 as the most susceptible to drought stress. Subsequent experiments revealed that these same cultivars exhibited the highest levels of arbuscular mycorrhizal fungi (AMF) colonization. For this study, cotton genotypes PHY443 and ST4990 were cultivated in a growth chamber. The chamber was maintained under a 12-hour photoperiod with a photosynthetic photon flux density (PPFD) of 600 µmol m²s?¹ and a carbon dioxide (CO?) concentration of 420 ppm. The experiment consisted of four treatment groups for each cultivar: (1) AMF with low potassium (AMLK), (2) AMF with high potassium (AMHK), (3) non-AMF with low potassium (NMLK), and (4) non-AMF with high potassium (NMHK). Each treatment included six replicates, resulting in a total of 24 experimental plots per cultivar. Throughout the experiment, the ST4990 cultivar exhibited a lower number of leaves compared to the PHY443 cultivar. However, ST4990 demonstrated a greater degree of root colonization by AMF than PHY443. Despite higher nominal values, no significant differences were observed in root colonization percentages among treatments. The lowest colonization rate was recorded for PHY443 in the AMLK treatment (27%), whereas the highest colonization rate (51%) was observed for ST4990 in the AMLK treatment. Root fresh mass did not differ significantly for PHY443. For ST4990, no significant differences were detected between NMHK and AMHK treatments, nor between NMLK and AMLK treatments. However, AMHK significantly differed from NMLK. Shoot fresh and dry weight did not show significant differences among treatments or cultivars. Although no significant differences were observed in total shoot fresh and dry weight, total fresh leaf mass varied within the ST4990 cultivar, with NMHK exhibiting the highest fresh leaf mass, while AMLK presented the lowest. Water potential did not differ between HK and LK treatments for PHY443. However, significant differences were detected between NMHK and AMHK, with NMHK exhibiting a less negative water potential than AMHK. In contrast, for the ST4990 cultivar, a significant difference was observed between NMHK and NMLK, where NMHK maintained a less negative water potential compared to NMLK. SPAD values exhibited a decreasing trend from Day 9 to Day 51 in both cultivars. In PHY443, significant differences were detected between LK treatments from Day 9 to Day 23, after which the treatments no longer differed. Additionally, NMLK and NMHK treatments did not differ throughout the experiment, nor did AMLK and AMHK. However, NMHK and AMHK significantly differed on all measurement days, with NMHK consistently exhibiting the highest SPAD values. In ST4990, LK treatments generally did not differ, except on Day 16. HK treatments showed no differences on Days 9, 23, and 43 but exhibited significant differences on other measurement days. The results show that AMF colonization was more pronounced in ST4990 compared to PHY443, especially under LK conditions. Despite a less favorable appearance, ST4990 displayed a clear metabolic cost associated with the colonization. Although no significant differences were observed, a higher colonization rate was foreseen in plants experiencing greater stress. Overall, these findings elucidate critical interactions between potassium availability and AMF association, which warrant further investigation through refined and precise measurements in future experiments.
Publications
- Type:
Book Chapters
Status:
Accepted
Year Published:
2025
Citation:
Cooney D.R., Rose B.D., Jones A.P., Frank H.E.R., Nanda Kafle G., Kafle A., Garcia K. (2025) Mutualistic relationships between roots and soil microbes facilitate plant potassium acquisition. In: L�ttge, U., C�novas, F.M., Risue�o Almeida, M.C., Leuschner, C., Pretzsch, H. (eds) Progress in Botany Vol. 86. Progress in Botany, vol 86. Springer, Cham. (In Press)
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Progress 04/01/23 to 03/31/24
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 cotton associations and producers, with considerations in providing additional data points to producers on a national scale for informed production practice decision making. Changes/Problems:Project started with one postdoctoral researcher. Unfortunately, personal reasons forced the postdoctoral researcher to go back to her country and stop working on the project. Since then, a new Ph.D. student has been hired and is now fully active on the project. However, it caused multiple months of gap between these two individuals and resulted in multiple trainings that delayed significantly the development of some experiments in the project. What opportunities for training and professional development has the project provided?This project has involved a postdoctoral researcher and more recently a Ph.D. student at NC State University. Personal involved have been trained to various physiological, microbiological, molecular, and isotopic approaches through the course of the project. Also, they were given the possibility to present their work in various local meetings. How have the results been disseminated to communities of interest?The drought screening of the six cotton genotypes allowed us to have a more comprehensive understanding of the mechanisms resulting in leaf mortality in the species. For instance, we found an interesting association between leaf mortality and dehydration level and between leaf mortality and the level of xylem embolism (failure in the water transport). These results are currently being organized into a scientific manuscript, which will be published as an open access journal, allowing us to disseminate our results. We also expect the involved students to present these results in conferences, symposiums, and field days. What do you plan to do during the next reporting period to accomplish the goals?First, cotton plants of the genotypes ST4990 and PHY443 will be cultivated with AMF to be checked physiological traits related to drought tolerance upon symbiosis and at SK and LK conditions. For instance, we want to test whether AMF can increase the foliar levels of K, and thus lower the leaf turgor loss point, improve the plant's ability to osmotically adjust, and increase the stomatal speed. Colonized and non-colonized plants will be then exposed to drought and recovery to assess whether AMF can improve drought resistance in plants. Second, we are also going to evaluate the plant carbon transfer from the fungus to the most promising variety using 13CO2 and Rb as K tracer. We will also determine if this transfer is affected by drought and whether this association and K increase will result in higher drought tolerance. Thirdly, we will identify regulators of K nutrition in colonized or non-colonized plants of the most promising cotton variety using RNA-Seq and co-expression network studies comparable to the one we performed in early studies (Garcia et al., 2017). Additionally, the same cotton variety will be used to describe changes in the fungal microbiota of roots upon K deprivation and/or drought condition.
Impacts
What was accomplished under these goals?
Assessing germination and root development of multiple cotton cultivars We assessed the impact of low and high potassium (K) conditions 8 different cotton cultivars already in trials in the state of NC (DP2038, DP2115, DP2127, ST4990, ST4550, ST5091, PHY360, and PHY443). Among these cultivars, PHY443 resulted in a higher germination rate and ST4550 was the less germinating cultivar. The root architecture was also studied using the root scanning software WinRhizo after three weeks of growth. Results from the root architecture revealed that the roots from PHY443 in the absence of K had less lateral root formation and growth was concentrated into primary roots. In contrast, roots grown in the presence of K resulted in a significant amount of branching/lateral root formation. A similar response was also observed in the DP 2038 cultivar. Evaluating drought tolerance of multiple cotton cultivars Among the eight cultivars tested above, we selected the six genotypes that germinated the most (ST4990, PHY443, PHY360, DP2038, DP2127, DP2115) to screen for low drought resistance. Drought resistance was defined based on the rate of leaf mortality after a dry-down experiment in greenhouse. All the plants were cultivated from seeds in 6-L plastic pots containing a mixture of 50% Sun Gro Propagation Growing Mix and 50% river bottom sand. Daily irrigation was performed to field capacity using nutrient solution. Plants were grown in the North Carolina State University Phytotron chambers, and conditions were set to 18-h photoperiod with photosynthetic photon flux density (PPFD) of 600 µmol m-2 s-1, and day: night temperature cycles of 28:22°C for 30 days after germination. Thereafter, the plants were moved to a glasshouse at the Method Road Greenhouse Complex and cultivated for more than 17 days before starting the dry-down experiment. Dry-down experiment A dry-down experiment was initiated 50 d after emergence. Genotypes were split into well-watered and drought treatments, saturated with water the evening before, allowed to drain freely, and the pot surface was covered with plastic bags the following morning to eliminate water loss by evaporation. Plants were allowed exposed to drought by withholding irrigation. After four days of drought, plants were assessed for leaf water potential and percent canopy damage. Leaf water potential was measured at midday using a fully expanded leaf. For that, leaves were collected, bagged with damp paper towels for 10 min to ensure whole leaf equilibration, and measured using a Scholander pressure chamber. Percent canopy damage was assessed at the end of the experiment as the percentage of dead leaves over total leaf number). Total leaf number included leaves that abscised during the drought. The level of xylem embolism at the end of the drought was also estimated for each leaf water potential measurement. For that we performed optical vulnerability curves to determine the leaf embolism resistance of the six cotton genotypes. The embolism levels associated with each leaf water potential measurement were obtained from the optical vulnerability curves. At the end, the relationship between canopy mortality and leaf water potential and between canopy mortality and embolism level were tested. Leaf embolism resistance Additional plants were cultivated in the same conditions as the other group of plants and the vulnerability curves of leaves were constructed using the optical vulnerability method. Plants were brought to the laboratory, removed from pots, and had their roots carefully washed. Plants were held overnight with their roots underwater with aeration systems to ensure that observations started on fully hydrated individuals. The next morning, leaves were used to construct vulnerability curves for each species. Results and Prospects Plants of all genotypes decline their water potential as the drought progressed. After four days of drought, all genotypes had reached approximately -1.8 ± 0.3 MPa. No difference in the minimum leaf water potential was observed across genotypes at the end of the drought. Despite experiencing similar dehydration levels, plants of the eight genotypes displayed contrasting canopy mortality rates (i.e. percentage of canopy leaves that were dead). Three genotypes exhibited the highest canopy mortality rates: ST4990, PHY443, and PHY360, which exhibited 64, 51, and 65% leaf mortality, respectively. The other three genotypes exhibited on average 28 ± 9% mortality. Even though leaf water potential averages across species did not differ at the end of the experiment, canopy mortality rate closely correlated with leaf water potential, at the individual level (R2 = 0.75, P <0.01). This correlation demonstrates that individuals dehydrating more have more chances of experiencing damage. Canopy mortality rate also correlated with leaf embolism (R2 = 0.75, P <0.01). This demonstrates that the mechanism driving leaf mortality in dehydrated leaves is the hydraulic dysfunction of the xylem caused by embolism. Individuals of ST4990, PHY443, and PHY360 were mostly associated with the low water potential, high xylem embolism, and high canopy mortality spectrum of these two correlations. So far, out data points ST4990, PHY443, and PHY360 as the most drought susceptible genotypes. Further studies on how AMF can improve K nutrition and therefore drought resistance will be performed using them. Evaluating the impact of AM symbiosis on potassium nutrition in cotton Preliminary experiments on all 8 cultivars showed that only two responded the most to AM inoculation: PHY443 and ST4990, which are also among the most drought susceptible genotypes we tested (see above). Therefore, we evaluatee the response of these two cultivars under low and high K conditions and inoculated or not with AM fungi. The sterilized cotton seeds were germinated until 2 leaf stage, and the plants were watered with deionized water when needed. Pots with two-compartment system were used. They are constituted of one root compartment when plants were placed and inoculated or not, and one fungal compartment that can only be reached by the fungal hyphae due to the presence of a double layer of 50um mesh (Kafle et al., 2022; Kafle & Garcia, 2022). Both compartments were filled with the potting substrate (Turface or 70% sand / 30% vermiculite), and germinated cotton plants were carefully transferred into the root compartment. Plants in the mycorrhizal treatment were inoculated with 500 spores per pot. The trays were then placed in the growth chamber. Plants were watered with 200 ml of sufficient- (SK) or limiting-K (LK) solutions every third day directly in the tray. For the fungal compartments, 30 ml of 1/10th dilution of SK or LK were added once a week to prevent the pile up of nutrients. One week before harvest, rubidium was added to the fungal compartment only to track K movement upon symbiosis. This experiment revealed that root colonization was higher in SK compared to LK with PHY443, and lower in SK compared to LK with ST4990. Also, the root fresh biomass of PHY443 was in AMF-inoculated plants as compared to non-inoculated plants, and higher in NM plants at LK compared to SK. No difference was noted in AM plants between K treatments. The root dry biomass was higher under LK in the AMF-inoculated plants compared to non-inoculated plants. Also, AM plants at LK displayed a significantly higher biomass than AM plants at SK, and no difference between any NM plants was observed. Regarding the shoot fresh biomass, no significant differences were observed for PHY443 cultivar. ST4490 genotype is still under investigation. Data for other parameters like shoot nutrient concentrations, particularly K, sodium, and rubidium from this experiment are still being processed.
Publications
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