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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