Performing Department
(N/A)
Non Technical Summary
SummaryClimate change is predicted to increase the variability in timing and amount of precipitationacross the globe, and strategies to mitigate the negative impacts are necessary. Components ofthe plant microbiome are capable of enhancing drought tolerance in agricultural and rangelandcommunities and could sustain ecosystem function and agricultural yields in the face of climatechange. Arbuscular mycorrhizal fungi are beneficial plant symbionts known to promotedrought tolerance. We propose to leverage the International Collection of (Vesicular-)Arbuscular Mycorrhizal Fungi (INVAM), our lab culture collection, and AM fungal sequencedata from across precipitation gradients to select AM fungal isolates that are likely to beresistant to drought and confer drought tolerance to associated host plant species.The geographic origin of AM fungal accessions in INVAM span the globe. Each of theapproximately 1000 isolates are characterized by their spore morphology and hyphalcharacteristics. In addition, our lab has additional AMF that we have isolated from Indiana,Illinois, Missouri, Kansas and Oklahoma. We will use phylogenetic and morphological traits,and geographic origin, to identify and predict which AMF species or isolates are mostproficient at conferring drought tolerance to their plant hosts. Using these data and amplicondatasets of AMF across natural moisture gradients and experimental manipulations ofprecipitation in the midwestern USA, we will identify AM fungal candidates for testing. We willthen establish greenhouse experiments to test their ability to increase drought tolerance onSorghum (Sorghum bicolor) and Big Bluestem (Andropogon gerardii).We will measure the ability of AMF to confer drought tolerance through growth,assimilation and stomatal conductance response to drought. We will then evaluate whether thesemetrics are best predicted by fungal traits, isolate origin, or information on distribution. The bestfungal isolates will be used in field tests of drought resilience in future trials. This researchadvances efforts of best utilize plant microbiomes.
Animal Health Component
60%
Research Effort Categories
Basic
40%
Applied
60%
Developmental
(N/A)
Goals / Objectives
Climate change poses challenges to agriculture and ecosystem productivity (Abbass et al. 2022), as much of North America is projected to experience a drier climate, and a likelihood of drought (Tripathi et al. 2023). Drought could already be responsible for global declines in crop yield of many commodity crops (Zhu al. 2014). One approach to increasing drought resilience in crop and rangeland productivity involves manipulation of the plant microbiome (Hu et al. 2022, Janssen & Hofmockel 2019). The plant microbiome is diverse and variation in microbiome composition affects plant resilience and productivity under drought conditions (Saleem et al. 2019; Wang et al. 2019). A challenge in managing the microbiome for drought resilience is identifying members of the plant microbiome that best confer drought tolerance. We address this challenge by focusing on the composition of Arbuscular Mycorrhizal fungi (AMF), critical components of the microbiome that have been shown to confer plant drought tolerance. AMF are obligate plant symbionts which have coevolved with plants for ~400million years (Remy et al. 1995). AMF can increase drought tolerance of many agricultural crops (Chareesri et al. 2020; Subramanian et al. 2006; Watts-Williams et al. 2022). The extraradical hyphae of AMF expand surface area used for acquiring water by plant roots (Abdalla et al. 2023). Because drought can limit nutrient uptake for plants, increased P, N, and K provided by AMF under drought conditions improve plants resilience to even severe drought conditions (Chandrasekaran 2022, Wu 2013). AMF also stimulate production of phytohormones such as ABA (an abiotic stress hormone) which can be protective of plants under drought conditions (Bahadur 2019). AM fungal infection improves plant stomatal conductance under drought conditions (Augé et al 2014, Jayne & Quigley 2014). AM fungal isolates have been shown to vary in their conference of drought tolerance (Ruiz-Lozano et al. 1995, Ruiz- Lozano et al. 2012, Duc et al.2018), even though relatively few AM fungal species have been tested. A general problem in utilization of AMF, is identifying beneficial species for conditions and stresses that a given plant host may experience. Several approaches have been proposed. 1. Characteristics of the site of origin of the microbe.Symbionts derived from a stressful environment can confer greater resilience to thatstress than those from less stressful environments. For example, in a reciprocal transplant study, Johnson et al. (2010) found that a native prairie grass performed best with soil microbial inoculations from their resident soils. A meta-analysis of AMF inoculations generally supported this pattern (Rua et al. 2016). In a recent study, soil microbial inocula from arid sites conferred greater drought tolerance, than inocula from mesic sites (Allsup et al. 2023). While this study did not confirm that AMF conferred this effect, drought tolerance was correlated with AM fungal diversity. Other studies showed that AMF isolated from drier areas could confer greater drought tolerance, as measured by leaf resistance to water vapor loss, than isolates from moremesic sites (Stahl and Smith 1984). Lubin et al. (2021) found evidence for adaptation toclimate in the plant-mycorrhizal symbioses for xeric-origin plants matched with andxeric-origin AMF. These results suggest that site of origin could be important to consider when determining which isolates to include in inocula aimed at improving drought tolerance. 2. Experimental manipulation of stress (Host Selection under Stress).Other environmental differences might confound expected adaptation to stress. Experimental manipulation of stress starts with the same initial inocula and controls for other environmental factors. If plant hosts select the best symbionts for their local environment, as expected from their ability to preferentially allocate to the locally best symbiont (Bever 2015, Ji and Bever 2016), then plant microbiome dynamics under stress may yield communities enriched in symbionts capable of alleviating that stress. Indeed, experimental manipulations of water availability over three growing seasons in the greenhouse produced microbiomes capable of improving plant fitness in low water conditions, respectively (Lau and Lennon 2012). However, manipulations of water in the field have not always produced a beneficial soil microbiome (Monohon et al. 2021). Whether this proves a viable strategy requires additional research. 3. Trait-based identification of mediators of stress resilience. An alternative approach to identification of symbionts best able to improve drought tolerance to their host is to test more easily measured traits of the microbial community. This trait focused approach to identify functional differences has proven useful for plants (Volaire et al. 2014) and has been suggested as an important avenue to develop microbes, including AMF (Chaudhary et al. 2022). Recent work (Hopkins and Bennett 2023) found shifts in AM fungal spore traits (size and pigment) with fire frequency, suggesting functional consequences of spore characteristics. Spore traits such as wall thickness could be important for fungal resistance to desiccation during drought. Moreover, given the potential importance of increasing surface area of absorption for conference of drought tolerance, it is likely that characteristics of the internal and external hyphae will be important to this function.We will compare and contrast these approaches to identify AMF which bestconfer drought tolerance to plant hosts. Our experiments will test the meritsof each approach and identify symbionts with the potential to mitigate climate impacts onproduction in agriculture and rangelands. We do this by integrating across existing data setscharacterizing AM fungal composition along precipitation gradients in grasslands of central US(2 data sets) or along experimental manipulation of precipitation (2 data sets from 1 experiment) to identify AM fungal taxa that are differentially abundant in arid versus mesic environments. We then leverage the International Collection of (Vesicular-) Arbuscular Mycorrhizal Fungi (INVAM), to test the drought tolerance of these taxa. We will select INVAM isolates originally derived from arid locations and more mesic locations. We will be able to group isolates into four categories corresponding to all combinations of 1) AMF taxa being differentially abundant in arid sites (yes or no), and 2) AM fungal isolates originating from arid sites (yes or no). This allows us to test whether isolate origin or taxonomy is a better predictor of function. We also test if functional traits are phylogenetically conserved within lineages. AMF are a monophyletic clade with species being discriminated by both spore morphology (wall structure, size, and color) and rDNA sequence variation. Some traits (e.g., spore characteristics) tightly covary with taxonomy, others (e.g., host growth promotion) are less constrained (Hoeksoma et al. 2018). We will test the correspondence of taxonomy with the function of conferring drought tolerance to host. If taxonomy corresponds to a particular function like drought tolerance, then environmental patterns should link this correspondence to function, if not, then characteristics of sites of origin will be the best predictor. It is also possible that both factors are at play as predictors. Finally, we analyze whether measured morphological traits of the fungal isolates predict their ability to confer drought tolerance.Our specific objectives are:1. Identify candidate AMF from sites of origin and taxonomic distribution.2. Test candidate AMF isolates and taxa that could confer drought tolerance.3. Test traits of AMF species that best promote drought tolerance.
Project Methods
Methods1: Identify candidate AMF from sites of origin and taxonomic distribution.Rationale: We will identify candidate AMF isolates to confer drought tolerance using the criteriaof isolate origin or taxonomic distribution along precipitation gradients.Approach: We use three types of information to identify candidate isolates: 1) distribution oftaxa along precipitation gradients in grasslands as determined by amplicon sequencing; 2)distribution of taxa across experimental manipulation of precipitation in grasslands; 3) aridity ofsite of origin of isolates in INVAM and lab cultures of AMF.Details: We will use bioinformatics pipeline outlined in Delavaux et al. (2020, 2022) which usesa phylogenetic approach to identify OTUs to genera and species in the datasets. These methodswere developed in our lab, and we are practiced in their implementation.Evaluation: We will analyze the amplicon data using permanova in R to find compositionaldifferences between communities. We will use mixed models to test for consistency in responseto precipitation of OTUs within family and genera. We will test individual species response toprecipitation using the DESeq2 package in R. We will test for consistency across data sets bytesting individual log response ratios using regression approaches. Consistency acrossexperiments would be evidence that drought response was a property of the taxonomic unit.2: Test candidate AM fungal isolates and taxa that could confer drought tolerance.Rationale: We will test identified candidate AMF isolate's ability to confer drought tolerance oftwo important grasses: Sorghum bicolor, a globally cultivated, economically important crop, andAndropogon gerardii (Big bluestem), an important rangeland species, widely used in theplantings. We expect that both Sorghum bicolor and Andropogon will be most drought resilientwhen inoculated with AM fungal isolates which have originated in drylands and whose taxaidentified as potentially drought adapted from our previous analyses.Approach: We will test 40 isolates chosen from Obj. 1 to represent a range of combinations ofspecies and genera that are, or are not, differentially abundant in arid conditions, and for isolatesthat are, or are not, derived from arid conditions. We will include taxa that are differentiallyabundant in arid sites within all three amplicon datasets, to those being differentially abundantonly in natural or experimental gradients. We will include multiple isolates of the same speciesfrom contrasting origins when possible.Details: Because of limitations imposed by photosynthesis measurement time, we divide the testsinto two tests of 20 isolates each. Each experiment will have five replicates of both Sorghum andAndropogon inoculated with each of 20 inoculation isolates, and 10 uninoculated controls, grown in two watering levels for, planted in a full factorial, blocked, experimental design (440plants/experiment). High water will consist of 5 minutes of watering (full pot capacity) everytwo days. Low water will consist of a 60% reduction in watering (4 minutes every 4 days). Allplants will be watered as needed during the 2 weeks following planting to facilitateestablishment. We will monitor gas exchange and photosynthetic rate measurements between 9am and 12 pm on the 3rd expanded leaf of each plant weekly throughout the experiments using aLI-6800 Li-Cor as well as soil moisture using a TDR 150 moisture meter. We will then terminatewatering and monitor photosynthetic rates and soil moisture three times per week as the pots drydown completely.Evaluation: We will test for differential sensitivity to drought using multiple approaches. We willtest for isolate influence on differential accumulation of biomass in the high vs low precipitationtreatments by analyzing final dry biomass of shoot and root using general linear models (GLMs).We will directly analyze gas exchange metrics to test for differential impacts on transpirationusing GLMs. We will analyze the AMF isolate impacts on the rate of assimilation decline withsoil moisture during the pot dry down (as in Figs 1,2) using regression. For each isolate, we willthen have three measures of drought tolerance: sensitivity of biomass accumulation, sensitivityof stomatal conductance, and sensitivity of assimilation to soil moisture. We will then testwhether pattern of taxa along gradients, site of origin, or phylogeny predict these droughttolerance metrics using multiple regression. Analyses will be performed in R and SAS.3: Test traits of AM fungal species that best promote drought tolerance.Rationale: We will test whether measurable morphological traits of AMF predict its abilityto confer drought tolerance to their host.Approach: We will measure spore and hyphal traits of each of the 40 AMF isolates evaluated inObj 2. We expect that we will find commonalities in morphological traits or trait variations inspores within species, of drought-adapted AMF.Details: We will measure traits of intra-and extra-radical hyphae and of the asexually producedspores. We will collect root and soil samples at harvest from the greenhouse experiment in Obj 2and analyze the infection density of roots and external hyphal density in soil using standardmethods. We will extract spores from our cultures to measure spore size and color, we willmount spores on slides to measure wall layers and thickness. We will create images at 60x on aNikon compound microscope with DIC optics, paired with Nikon NIS elements software tomake measurements.Evaluation: We will test whether these morphological traits predict the three metrics of drought tolerance measured in Obj. 2. We will compare the predictive power of the best morphological traits to that of information on the isolate origin and the AM fungal taxa's response to precipitation gradients as described in Obj. 1.