Utilizing mycorrhizal responses to guide selective breeding for agricultural sustainability

This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. © 2021 The Authors. Plants, People, Planet © New Phytologist Foundation. 1Department of Natural Resource Ecology and Management, Oklahoma State University, Stillwater, OK, USA 2School of Earth and Sustainability, Northern Arizona University, Flagstaff, AZ, USA 3Environmental Science Division, Argonne National Laboratory, Lemont, IL, USA 4Department of Plant and Soil Sciences, Oklahoma State University, Stillwater, OK, USA


Societal Impact Statement
Agriculture touches all aspects of society and global environmental health. Dwindling phosphorous reserves are a looming crisis for civilization, and soil erosion typically far outpaces pedogenesis. Improving plant-mycorrhizal symbioses can enhance sustainable agriculture because mycorrhizas typically improve host-plant nutrition and stabilize soils. Selective breeding of plants that gain greater benefits from mycorrhizas can provide considerable economic and environmental benefits. Our assessments demonstrate switchgrass genetic improvement increased or maintained production of two populations, and low-input breeding increased mycorrhizal responsiveness, compared to parent lines. Selecting for increased mycorrhizal reliance may be an effective strategy for more sustainable and economical agricultural production.

Summary
• Plant-mycorrhizal interactions are not typically assessed in crop breeding programs. Our experiment addresses this by determining host-plant outcomes of newly developed synthetic (agronomic) populations compared with parent lines, following low-input selective breeding. Assessing the potential of low-input breeding to enhance crop mycorrhizal benefits is a critical step toward more sustainable agricultural production.
• We compared four synthetic populations of Panicum virgatum, from a low-input biofuel breeding program at Oklahoma State University, to corresponding parent lines. Plants were grown in a greenhouse in native prairie soils that were either steam-pasteurized (nonmycorrhizal) or non-steamed (mycorrhizal).
• We assessed shoot and root biomass, shoot P concentration and P content, mycorrhizal growth response (MGR), and mycorrhizal phosphorous response (MPR). Importantly, we provide novel evidence that low-input selective breeding increased mycorrhizal reliance of switchgrass synthetics compared to parent lines, with implications for global agricultural systems.
• There are substantial opportunities for plant traits associated with increased MGR and MPR to be transferred to a wide array of crops. Our findings indicate

| INTRODUC TI ON
Humanity faces looming environmental challenges that are linked with current agricultural production strategies and addressing the negative impacts of food systems on planetary health is vital (Duhamel & Vanderkoornhuyse, 2013;Garcia et al., 2020). For example, one of the most pressing concerns for global food security is loss of productive soils, with mounting evidence that agricultural mismanagement leads to vicious cycles of environmental degradation (Wuepper et al., 2020). To meet these challenges, we need sustainable agricultural systems that optimize fertilizer use while regenerating soil quality and maintaining or increasing agroecosystem productivity (Edwards, 2020). Indeed, healthy carbon-rich soils are foundational to most food production systems and to achieving the United Nation's Sustainable Development Goals (Lal, 2020).
North American prairie grasses represent a strategy to help reduce agricultural inputs and improve soil health, particularly as a source of a low-input biofuel feedstock with potential under proper management to increase soil C stocks (de Graaff et al., 2014;McGowan et al., 2019;Revillini et al., 2019). Switchgrass (Panicum virgatum) is a North American native perennial species that shows great promise as a low-input biofuel feedstock source and as a model crop for improving agroecosystem efficiency due to its general adaptability, yield potential, and soil benefits (Emery et al., 2018;Parrish & Fike, 2005). Perennial crop cultivation and mutualistic microbial interactions are broadly transferable to multiple agricultural settings, with potential to decrease fertilizer inputs while enhancing soil quality. Previous research indicates enhancing host-plant reliance on mycorrhizal symbioses can facilitate efforts to improve soil stability and agricultural sustainability Duhamel & Vanderkoornhuyse, 2013;Galván et al., 2011;Hetrick et al., 1995).
Researchers are examining strategies for integrating mycorrhizal symbioses across varied agricultural landscapes, to enhance efficient food, fiber, and biofuel feedstock production (Mitter et al., 2019;. Mycorrhizas represent a critical mutualism for the vast majority of global plant species, including North American warm-season grasses (Miller et al., 2012;Wilson & Hartnett, 1998). Therefore, our current research focuses on assessing switchgrass populations, following low-input breeding, as a potential strategy to improve sustainability of biofuel production systems. The emerging holobiont conceptual framework, identifying host-plants as both primary drivers and scaffolding for complexes of diverse microbial communities that function as an evolutionary unit, can be used by breeders to enhance plant production (Gopal & Gupta, 2016;Ravanbakhsh et al., 2021;Wei & Jousset, 2017;Ye & Siemann, 2020).
A holobiont describes multipartite relationships where a host-plant genome is tightly linked with an associated microbiome, forming a nascent and complex hologenome (Theis et al., 2016). In crop breeding, it is critical to directly assess binary interactions between host-plants and key mutualists, such as AM fungi, that likely drive production outcomes. Such assessments can establish breeding benchmarks toward further elucidation and utilization of hologenome dynamics. We propose the holobiont framework can inform alternative selection criteria and strategies for the development of genetic resources that can enhance agricultural sustainability (see Selective breeding for improved host-plant partnership with mycorrhizal fungi is a necessary step, setting the stage for additional efforts to use soil amendments and agroecosystem management to support effective microbial symbioses Cobb, Wilson, Goad, & Grusak, 2018;Duhamel & Vanderkoornhuyse, 2013;Smith et al., 1992). Mycorrhizal symbioses are widespread and typically beneficial, suggesting that evaluation of mycorrhizal relationships is critical groundwork for disentangling additional plant-associated microbial dynamics of the total holobiont. For example, switchgrass breeders are also assessing the influence of nitrogen-fixing bacteria on switchgrass growth in low-N soils (Rodrigues et al., 2017;Roley et al., 2020).
As mycorrhizas enhance nutrient uptake, water-use efficiency, and soil quality in native ecosystems, plant breeding programs that select for increased reliance on mycorrhizas in agroecosystems can potentially deliver synthetic (agronomic) populations that reduce fertilizer requirements (Galván et al., 2011;Porter & Sachs, 2020).
AM fungi contribute to resource-use optimization, regeneration of degraded soils, and enhanced sustainability in food, fiber, and bioenergy production systems (Cobb et al., 2017;Zhang et al., 2019). Plant breeders are making clear progress by selecting traits that improve aboveground biomass yields, yet plantmycorrhizal interactions were not historically incorporated into breeding programs and breeding under high-input conditions can profoundly reduce reliance on resources supplied by mycorrhizal low-input selective breeding can improve MGR and MPR. We propose these traits serve as a useful proxy for host-plant mycorrhizal reliance, facilitating successful hologenome breeding to reduce fertilizer requirements.

K E Y W O R D S
biofuel, hologenome breeding, mycorrhizal responsiveness, Panicum virgatum, switchgrass, synthetic populations relationships (Cobb et al., 2016;Hetrick et al., 1995). Increasing multiple ecosystem services, such as soil carbon storage and downstream water quality, is an emerging paradigm in agriculture, and AM fungi represent a strategic component of improved agroecosystem management .
Hence, incorporating host-plant reliance on resources supplied by AM fungi should be an important component for crop breeding programs concerned with improving agricultural sustainability, including mycorrhizal contributions to soil regeneration and fertilizer use optimization.
It is critical to assess breeding strategies, such as hologenome breeding, that potentially enhance mycorrhizal growth responses (MGRs) and can be scaled up to improved agroecosystem efficiency. Switchgrass is known to associate with AM fungi, and advanced molecular tools have provided valuable insights into the genetic diversity and evolution of switchgrass across geographic regions (Evans et al., 2018;Zhang et al., 2011). However, linking switchgrass breeding practices with mycorrhizal responsiveness is underexplored. These knowledge gaps constrain our capacity to advise crop breeders on incorporating key rhizosphere traits, such as mycorrhizal symbioses into selective breeding programs. Our current experiment addresses these gaps by comparing host-plant outcomes of newly developed switchgrass synthetics with parent lines, following low-input selective breeding, to examine potential alternations in mycorrhizal reliance. The Oklahoma State University (OSU) switchgrass breeding program is focused on the development of high-yielding cultivars targeted for bioenergy feedstock production.
Genotypic and phenotypic recurrent selection has been used to advance populations, whereas selection and evaluation are conducted in low-input environments. Assessing the potential of low-input breeding, as part of a hologenome breeding framework, to enhance mycorrhizal benefits is a step toward more sustainable agricultural production, and ecological insights gained are transferable to a wide array of agroecosystems.

| Soil and inoculum preparation
Prairie soil, a Chase silty clay loam, fine mont-morillonitic, mesic Aquic Argiudoll (pH = 7.5; plant-available N = 9.6 mg/kg plantavailable p = 10 mg/kg [Bray test I]), was freshly collected from Konza Prairie Biological Station (KPBS), Manhattan, Kansas, USA, and transported to OSU. To eliminate soil microorganisms, including AM fungi, soil was steam-pasteurized at 80°C for 2 hr and allowed F I G U R E 1 Conceptual diagram comparing and contrasting selection criteria and strategies used in plant genome breeding versus hologenome breeding. While both approaches seek to develop new genetic resources for sustainable agricultural production, differences in assessments, focus, and systematic design across key categories, such as (1) microbial ecology, (2) soil amendments, and (3) agroecosystem management can lead to substantially different outcomes to cool for 72 hr with no measurable changes in soil chemistry (pH, NH 4 , NO 3 , plant-available P). Communities of indigenous AM fungi and other soil organisms were added back in a controlled manner by inoculating one-half of the pots (mycorrhizal) with 20 g of living soil from KPBS. The living soil inoculum was added directly below seedling roots during transplantation. The remaining half of the pots (nonmycorrhizal) filled with only sterile soil as a control for each switchgrass population. All pots were also amended with 60 ml nonsterile soil sievate. Sievates were prepared by blending soil:water in a 1:2 ratio and passing the slurry through a 38µm sieve. The relatively large AM fungal spores were trapped on the sieve, whereas smaller organisms pass through, allowing for the addition of the majority of soil microbes while excluding AM fungi (Koide & Li, 1989).

| Plant preparation
Panicum virgatum, including four synthetics from the OSU switchgrass breeding program, SL93 C2-2, NL94 C2-3, NL94 C2-4, and NLSL 2009-1, and two parental cultivars, Alamo and Kanlow, were selected. These populations originate in the southern great plains and are commonly planted for biofuel feedstock and selected for increased production. Lowland switchgrass germplasm was used to initiate two populations, South Lowland population in 1993 (SL93) and North Lowland population in 1994 (NL94; Bartley et al., 2013;Wu, 2014). All four OSU experimental synthetics were developed at Cimarron Valley Research Station, Perkins, OK, by crossing elite individuals selected from cycle two evaluation nurseries that were given low-input fertilization: 112 kg/ha (18-46-0, N-P-K) at establishment and 56 kg/ha plant-available N in subsequent years. Seeds were pregerminated in vermiculite and 14-day-old seedlings (second-leaf stage) were transplanted into pots (6 cm × 25 cm) containing 600 g of soil (one seedling per pot). were harvested, roots washed free of soil, and biomass was ovendried at 60°C for 48 hr. Subsamples of dried roots were stained in trypan blue (Phillips & Hayman, 1970) and examined microscopically to assess percentage root colonization (McGonigle et al., 1990).

| Experimental design and maintenance
Shoot tissue P concentrations were determined using a Spectro Blue ICP following acid digestion (NFTA, 1993).

| Statistical analyses
Prior to analyses, all data were tested for normality and homogeneity of variances using Shapiro-Wilk and Levene's tests, respectively.
To assess effects of population on biomass production, shoot P concentration and content, and AM fungal root colonization, one-way analyses of variance (ANOVAs) were conducted, with population as the sole factor. Within populations, mycorrhizal and nonmycorrhizal replications were also compared using one-way ANOVAs. Tukey's honest significant differences (HSDs) tests were performed post hoc to assess differences among populations, and mycorrhizal status, with α = 0.05. Due to normal distribution of proportional data, shoot phosphorus and root colonization were not transformed.
We determined MGR and mycorrhizal phosphorus response (MPR) using equations from Cavagnaro et al. (2003).

| Mycorrhizal growth response and mycorrhizal phosphorus response following low-input selective breeding
Synthetics developed by OSU's switchgrass biofuel breeding program originated from lowland populations in the Southern Great Plains; however, these modified populations express substantially greater MGR than parents (Figure 2). We also found evidence selective breeding under low-input nutrient conditions influenced MPR, with synthetics expressing substantially greater MPR than parents ( Figure 3).

| Plant biomass, arbuscular mycorrhizal fungal root colonization, and shoot P
There was at least one synthetic within each mycorrhizal comparison that produced equivalent or greater shoot, root, and total biomass, than the corresponding parent line; however, nonmycorrhizal synthetics generally produced less biomass compared with corresponding parent lines (Table 1). In particular, SL93 C2-2 and NLSL 2009-1 produced significantly greater total biomass when grown with AM fungi, compared with corresponding parent lines, and significantly reduced total biomass when grown in the absence of AM fungi, compared with corresponding parent lines (Table 1). For mycorrhizal plants, AM fungal root colonization and shoot P concentration or P content did not differ between synthetic and parent lines (Table 2).
However, nonmycorrhizal synthetic plants derived from Kanlow were characterized by lower shoot P content, compared with the parent line (Table 2).

F I G U R E 2 Mycorrhizal growth response (MGR) of Oklahoma State University (OSU) Panicumvirgatum populations and parents.
Comparison A = SL93 C2-2 with Alamo (parent); Comparison B = NL94 C2-3, NL94 C2-4, and NLSL 2009-1 with Kanlow (parent). We determined MGR using the equation from Cavagnaro et al., (2003). All calculations are expressed as %MGR = (dry wt. mycorrhizal plant) -(mean dry wt. nonmycorrhizal plant) / (mean dry wt. nonmycorrhizal plant) × 100, such that all values indicate a percentage change in biomass of mycorrhizal plants, compared with mean biomass of nonmycorrhizal plants. Results expressed as means ± standard error (n = 6), based on one-way ANOVA and Tukey's honest significant difference. Bars that do not share a letter are significantly different (p < .05)

F I G U R E 3 Mycorrhizal phosphorus response (MPR) of Oklahoma State University (OSU) Panicumvirgatum populations and parents.
Comparison A = SL93 C2-2 with Alamo (parent); Comparison B = NL94 C2-3, NL94 C2-4, and NLSL 2009-1 with Kanlow (parent). All MPR calculations are expressed as %MPR = (shoot P content mycorrhizal plant) -(mean shoot P content nonmycorrhizal plant) / (mean shoot P content nonmycorrhizal plant) × 100, such that all values indicate a percentage change in shoot P content of mycorrhizal plants, compared with mean shoot P content of nonmycorrhizal plants. Results expressed as means ± standard error (n = 6), based on one-way ANOVA and Tukey's honest significant difference. Bars that do not share a letter are significantly different (p < .05)

| D ISCUSS I ON
The most important and novel findings of our study are low-input selective breeding generally increased host-plant reliance on AM symbioses, and, when grown with AM fungi, two lines from OSU's breeding program produced ~39% greater total biomass, compared with corresponding parent lines. Our results suggest that breeders can develop agronomic populations that outperform less reliant parent lines. If low-input breeding improves MGR and MPR of synthetic populations compared with parent lines, the high nutrient and water-use efficiency associated with effective mycorrhizal symbioses can facilitate economical switchgrass biomass production as a cellulosic bioenergy crop. Additionally, our results indicate low-input selective breeding has substantial potential to improve mycorrhizal reliance, with applications across a diverse array of agricultural crops.
With increasing recognition that plants and associated microbes should be assessed together as a holobiont, and that crop breeding strategies must account for complex symbiotic amalgams (Gopal & Gupta, 2016;Ravanbakhsh et al., 2021;Wei & Jousset, 2017; Ye & Siemann, 2020), our findings add an important dimension to switchgrass research that can be extrapolated to multiple agricultural production systems. We assessed plant responses to AM fungi in conditions where additional holobiont components (i.e., local soil microbes) were consistent across the experiment. We used this approach because AM fungi represent a flagship mutualism within TA B L E 1 Shoot biomass, root biomass, and total biomass (dry wt. g) for OSU switchgrass and corresponding parent lines Note: Oklahoma State University (OSU) switchgrass synthetic populations compared with corresponding parents. Comparison A = SL93 C2-2 with Alamo (Parent); Comparison B = NL94 C2-3, NL94 C2-4, and NLSL 2009-1 with Kanlow (Parent). Grown with (M) or without (NM) arbuscular mycorrhizal fungi; results expressed as means ±standard error (n = 6), based on one-way ANOVA and Tukey's honest significant difference. All mycorrhizal replications produced significantly greater biomass than corresponding nonmycorrhizal replications. Within a column, for each parent line comparison, values that do not share a letter are significantly different (p < .05). † AM fungal structures were not present in nonmycorrhizal roots.
root-associated microbial communities, making them a convenient target for hologenome breeding. Future research and breeding efforts will likely discover multiple selection criteria within holobiont consortia, as diverse microbiota have both complementary and competitive influences on the hologenome (Theis et al., 2016). By considering the influence of AM fungi, among the most widespread symbiotic partners in both natural and agroecosystems, our work sets a baseline for further study involving additional mutualistic microbes such as nitrogen-fixing bacteria (Roley et al., 2020).
Agricultural breeding generally focuses on aboveground performance traits and qualities, whereas belowground traits and microbial partnerships receive less attention, impacting the potential utility of crops for use in agricultural systems designed around microbial-sensitive practices. Selective breeding in highly fertilized and irrigated systems reduced MGR of modern Sorghum bicolor genotypes compared with less modified varieties, presumably due to inadvertent decoupling of host-plant dialogue with AM fungi (Cobb et al., 2016). Similarly, ancestral land races of wheat often respond very positively to mycorrhizas, whereas modern cultivars respond negatively-even in low-P soils (Hetrick et al., 1993). The authors concluded wheat cultivars are selectively bred under high P input, profoundly reducing reliance on mycorrhizas. In fact, genetic inheritance studies identified alleles on several chromosomes in wheat linked with responsiveness to mycorrhizas (Hetrick et al., 1995). Switchgrass breeding must avoid negative unintended outcomes associated with high-input breeding. Understanding the influence of breeding protocols on mycorrhizal dynamics may enable optimization of agricultural production in low-input conditions. Indeed, we previously proposed targeted breeding for increased MGR, especially for C 4 grass species (Cobb et al., 2017;Wilson et al., 2015). It is important to note that reduced nonmycorrhizal biomass in synthetic populations had an influence on MGR and MPR calculations. This type of selective alteration is not always considered an effective breeding target (Galván et al., 2011); however, in our study, two synthetic populations also produced greater total biomass than parent lines, when grown with AM fungi. Therefore, improvement of MGR  (Cortinovis et al., 2020), and development of CRISPR gene-editing provides a reason for optimism regarding the future of appropriate crop development (Zhang et al., 2020). Gene-editing enables additional options to stack multiple beneficial traits (Khan et al., 2021), for example genes associated with greater mycorrhizal reliance, into highly productive lines. Considering the findings of our current study, we advocate for eventual application of gene-editing technology to use traits associated with locally adapted plant germplasm in agronomic varieties that meet the economic requirements of producers while simultaneously enhancing the nutrient-use efficiency benefits of mycorrhizal associations in agroecosystems.
A meta-analysis by Zhang et al. (2019) describes mycorrhizal contributions to commodity crop production and grain quality but highlights persistent variation in mycorrhizal-mediated outcomes across agricultural systems and management practices. We propose that inconsistencies in management are potentially driven by study-  (Khan et al., 2021). As an attractive model and production crop, switchgrass represents another exciting opportunity to enhance mycorrhizal contributions across a wide array of agroecosystems.

| CON CLUS IONS
Our strategy of identifying more mycorrhizal reliant populations of P. virgatum elucidates a path forward, as P. virgatum serves as a model plant for sustainable biofuel production (Emery et al., 2018), with implications for numerous additional crops. Developing in situ management of mycorrhizas using MGR and MPR crop genotypes can potentially reduce cost to farmers (Benami et al., 2020). Many natural ecosystems display stability and resilience, in large part due to interactions such as AM symbioses (Jia et al., 2020;Yang et al., 2018). Discovery and utilization of mycorrhizal mechanisms and modification of plant genetics underlying holobiont interactions have potential to help us meet key agricultural sustainability goals, such as enhancing soil health and improving fertilizer efficiency. We propose low-input breeding creates selective pressure for switchgrass and other crops to invest more resources in chemical dialog with AM fungi, potentially enhancing both production efficiency and soil quality. If loss of symbiotic potential follows high-input plant breeding, our current study indicates an opportunity to harness the benefits of AM fungi under low-input breeding and other conditions that are based on a hologenome breeding framework.

CO N FLI C T O F I NTE R E S T
The authors declare research was conducted without commercial or financial relationships that can be construed as a conflict of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
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