Effects of a Companion Plant on the Formation of Mycorrhizal Propagules in Artemisia tridentata Seedlings

Rangeland Ecology & Management 73 (2020) 138e146

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Effects of a Companion Plant on the Formation of Mycorrhizal Propagules in Artemisia tridentata Seedlings* Marcelo D. Serpe*, Adam Thompson, Erika Petzinger Department of Biological Sciences, Boise State University, Boise, ID 83725-1515, USA

a r t i c l e i n f o

a b s t r a c t

Article history: Received 19 June 2019 Received in revised form 31 August 2019 Accepted 9 September 2019

Inoculation of seedlings with arbuscular mycorrhizal fungi (AMF) can increase their establishment after outplanting. The success of this practice depends partly on the extent of root colonization and abundance of AMF propagules in the outplanted seedlings. We conducted a greenhouse experiment to investigate the effects of a companion plant, the native grass Poa secunda J Presl (Sandberg bluegrass), on the formation of spores and vesicles, AMF colonization, and AMF taxa present in the roots of the shrub Artemisia tridentata Nutt (big sagebrush). These effects were tested at two phosphorus (P) fertilization levels, 5 mM and 250 mM. Neither coplanting nor differences in P had an effect on spore density in the potting mix. In contrast, coplanting increased vesicular colonization of A. tridentata from 5% to 18%, but only at low P. Differences in P also affected vesicular colonization of P. secunda, which was 10% and 30% at high and low P, respectively. Arbuscular colonization of A. tridentata was not affected by the treatments and ranged between 12% and 20%. In P. secunda, arbuscular colonization was lower but increased from high to low P. Coplanted seedlings exposed to low P also had the highest levels of total AMF colonization, 70% for A. tridentata and 63% for P. secunda. On the basis of partial sequences of the 28S ribosomal RNA gene, coplanting did not affect the AMF taxa, which were within the Glomeraceae. In some taxa within this family, root fragments containing vesicles are the main propagules. Particularly in this situation, increases in vesicle density caused by coplanting and low P are likely to facilitate mycorrhization of A. tridentata after outplanting, resulting in higher levels of colonization than those naturally occurring in the soil. Such outcomes are critical for assessing the extent to which A. tridentata establishment is limited by insufficient AMF colonization. © 2019 The Society for Range Management. Published by Elsevier Inc. All rights reserved.

Key Words: big sagebrush coplanting mycorrhiza Poa secunda phosphorus vesicles

Introduction Arbuscular mycorrhizal fungi (AMF) are widespread in terrestrial ecosystem, where they form symbioses with the roots of about 80% of plant species (Brundrett and Tedersoo 2018). These fungi (phylum Glomeromycota) depend on the plant host for organic carbon and in return promote plant growth by facilitating the uptake of nutrients, particularly those with low diffusivity in the soil such as phosphorus (P) and zinc (Smith et al. 2011). In addition, AMF colonization tends to increase plant growth under water
2001; Jayne and deficits and improve plant water status (Auge Quigley 2014). Although AMF are common in semiarid and arid lands, their propagule density tends to be low when compared with that of

* This work was supported by a grant from the US Dept of Agriculture
NIFA (grant 2018-67020-27857). * Correspondence: Marcelo D. Serpe, Dept of Biological Sciences, Boise State University, 1910 University Dr, Boise, ID 83725-1515, USA. E-mail address: [email protected] (M.D. Serpe).

more mesic environments (Requena et al. 1996; Stutz and Morton 1996). Furthermore, disturbances that cause changes or removal of vegetation can disrupt mycorrhizal networks, reducing their infectivity and the inoculum potential of the soil (Mummey and Rillig 2006; Buscardo et al. 2012; Dove and Hart 2017). Low abundance of AMF can limit the success of revegetation efforts, and in this situation addition of AMF inoculum often leads to increases in plant survival and early growth (Allen 1989; Caravaca et al. 2005; Ouahmane et al. 2007; Barea et al. 2011). A common semiarid habitat in western North America is the sagebrush steppe. This habitat is characterized by a vegetative community composed of perennial grasses, forbs, biological soil crusts, and several subspecies of the shrub Artemisia tridentata Nutt (big sagebrush) (Anderson and Inouye 2001). Over the past century, sagebrush habitats have been disturbed by a variety of factors including invasion by exotic grasses and subsequent increases in wildfire frequency. Fires tend to eliminate A. tridentata and other components of the native vegetation (Brooks et al. 2004). A. tridentata provides soil stability, as well as habitat and forage for local animals (Aldridge and Boyce 2007; Davies et al. 2007). Due to

https://doi.org/10.1016/j.rama.2019.09.002 1550-7424/© 2019 The Society for Range Management. Published by Elsevier Inc. All rights reserved.

M.D. Serpe et al. / Rangeland Ecology & Management 73 (2020) 138e146

the critical functions of A. tridentata, there is considerable interest in reestablishing this species after fires. This, however, has proven difficult due to high seedling mortality (Brabec et al. 2015). As in other plants, the mycorrhizal dependency of A. tridentata is likely to vary with environmental conditions, but this shrub is considered mycorrhizal (Allen et al. 1993; Johnson and Graham 2013). AMF colonizes A. tridentata in natural habitats (Trent et al. 1994; Busby et al. 2013; Carter et al. 2014), and the symbiosis has led to enhancements in P uptake, plant growth, or drought tolerance (Stahl et al. 1988, 1998; Allen et al. 1993). A major problem in reestablishing of A. tridentata is high seedling mortality due to summer drought (Hovland et al. 2019). Through various mechanisms, AMF have been shown to alleviate plant drought stress in many species including A. tridentata (Stahl et al. 1998; Ruiz-Lozano et al. 2012; Jayne and Quigley 2014). Stahl et al. (1998) showed that mycorrhizal seedlings of this shrub survived lower soil water potentials than nonmycorrhizal ones. More recently, Reinhart et al. (2017) also reported that AMF increased the root-to-shoot ratio of A. tridentata seedlings; an increase in this ratio is a well-known response to cope with drought (Ruiz-Lozano et al. 2012). Notwithstanding the potential benefits of AMF colonization, attempts to improve establishment of outplanted seedlings via AMF inoculation have found mixed results with either neutral or positive effects (Dettweiler-Robinson et al. 2013; Davidson et al. 2016). These differences may be attributed to dissimilarities in environmental conditions or disturbance histories among sites that affect the mycorrhizal dependency of the plants or the soil inoculum potential (Doerr et al. 1984; Reinhart and Callaway 2006; Reinhart et al. 2016). Alternatively, lack of consistency in the response to inoculation may be associated with differences in the origin and density of viable propagules in the added AMF (Weinbaum et al. 1996; Davidson et al. 2016). The effectiveness of inoculation with AMF on improving colonization and seedling establishment depends on the ability of the inoculum to survive and spread after outplanting (Weinbaum et al. 1996). An important factor affecting the survival of the introduced AMF is their origin (Maltz and Treseder 2015). Native AMF are adapted to local climatic and edaphic conditions and tend to have higher rates of survival and dispersal than non-native AMF (Caravaca et al. 2005; Rowe et al. 2007). Although less studied, another factor that may be important for increasing colonization is the number of AMF propagules present in the inoculum (Lekberg and Koide 2005). For shrubs and trees, the most common method to increase colonization involves inoculation in the nursery (Asmelash et al. 2016). Upon outplanting, the hyphae and spores present in the potting medium, as well as vesicles and hyphae within roots, serve as propagules for the growing root system (Klironomos and Hart 2002; Müller et al. 2017). The contribution of each of these propagules to colonization varies with AMF taxa, but in semiarid environments where Glomerales are common, both extraradical and intraradical propagules can be important sources for AMF colonization (Biermann and Linderman 1983; Klironomos and Hart 2002). To increase AMF colonization after outplanting, it would be valuable to produce container-grown seedlings that have a high density of infective propagules from native AMF. In the nursery, however, certain common cultivation practices such as the use of potting mixtures with high peat moss content and the application of phosphorus-rich fertilizers tend to reduce AMF colonization and the formation of propagules (Ma et al. 2007; Fleege 2010; Smith et al. 2011). In addition, for A. tridentata, the length of the root system that can host AMF may be relatively short due to exclusion of these fungi by secondary growth and lack of extensive branching (Welch and Jacobson 1988). Limited branching, particularly in medial portions of the tap root, can also result in a root ball that breaks apart during transplanting, potentially reducing outplanting

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success and the infectivity of extraradical hyphae (Jasper et al. 1989). In the present study, we investigated two approaches to increase AMF colonization and the density of AMF propagules in greenhouse-grown seedlings of A. tridentata Nutt. ssp. wyomingensis Beetle & Young (Wyoming big sagebrush, henceforth referred as A. tridentata). Specifically, we analyzed the effects of coplanting with Poa secunda J. Presl (Sandberg bluegrass) and differences in P fertilization on AMF colonization of roots, density of spores in the potting mix, and firmness of the root ball. P. secunda was selected because it is a native perennial grass with a shallow and fibrous root system that is known to host AMF (Spence 1937). We hypothesized that the increase in root density associated with the presence of P. secunda would enhance the development of mycorrhizal networks and/or intensify competition for P, leading to higher AMF colonization and formation of propagules. This notion is supported by results using nurse plants or mix-cultures, which usually show higher rates of AMF propagules and colonization than monocultures (Ingleby et al. 2007; Walder et al. 2012; MontesinosNavarro et al. 2019). One possible effect of the coplanting treatment is that it changes the quality of the inoculum by altering the AMF community composition of the roots (Klironomos 2000). Even though AMF have low host specificity, several studies have shown that distinct plant species co-occurring in the same habitat and even the same pot can harbor different AMF communities (Hausmann and Hawkes 2009; Torrecillas et al. 2012). With the aim of determining the extent to which coplanting with P. secunda affected the AMF community, we compared the AMF taxa present in A. tridentata seedlings growing alone with those coplanted with P. secunda. Methods Fungal and Plant Material To produce mycorrhizal inoculum, we collected silty-loam soil from a sagebrush community near Big Foot Butte, Idaho (43180 48.4300 N, 116 21048.5700 W). Subsequently, the AMF in the soil were multiplied in trap cultures using Sudan grass as a host (Davidson et al. 2016). Before the initiation of the experiment, we planted seeds of A. tridentata in 3.8  21 cm (164 mL) cone-tainers filled with a potting mix consisting of 2 parts fine vermiculite, 2 parts perlite, and 1 part of roots and soil from the trap cultures. Seedlings were grown for 8 mo under a 15-h photoperiod and day/ night conditions of 23/18 C ± 3 C. They were fertilized biweekly with a modified Hoagland’s solution having one quarter the strength of all essential elements, except for P, which was present at one eighth the strength or 125 mM. Total, vesicular, and arbuscular colonization before the initiation of the experiment was 45.0 (± 5.0), 4.5 (± 1.7), and 12.1 (± 2.6)%, respectively. Noninoculated seedlings were not included in the experiment because the purpose of the experiment was to analyze the effect of coplanting and P fertilization on AMF colonization and the abundance of propagules. Neither coplanting nor P fertilization can affect the response variables when AMF are not present. The potting mix used lacks AMF and, based on routine observations, noninoculated seedlings in this mix show negligible colonization. Experimental Approach Eight months after sowing, the A. tridentata seedlings were used to conduct a completely randomized factorial combination experiment consisting of two planting treatments (without and with P. secunda) and two fertilization treatments (low and high P). Twelve replications (each consisting of a 164-mL cone-tainer) were used for each of the four treatments. We added P. secunda seeds to

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the cone-tainers in the coplanting treatment and after the initial germination thinned them to 3 seedlings per cone-tainer. We fertilized the seedlings under the low P treatment with a Hoagland’s solution having one quarter the strength of all essential elements, except for P that was present at 1/50 strength or 5 mM. Likewise, we fertilized the seedlings under the high treatment with a one quarter Hoagland’s solution, which had a P concentration of 250 mM. We applied the Hoagland’s solutions biweekly at a rate of about 100 mL per cone-tainer. We harvested seedlings 3
5 mo after planting P. secunda, at z10-d intervals. At each of the six harvest times, we sampled two cone-tainers per treatment. For each treatment, we used one conetainer to estimate percent loss of potting mix during removal from the cone-tainer and spore density and the other to determine fresh weight of roots and shoots and AMF colonization of A. tridentata and P. secunda roots. Loss of potting mix during removal of seedlings from the cone-tainers was simply estimated by weighing the potting mixture that separated from the roots (detached potting mixture), as well as the remaining potting mix with embedded roots. The percent loss was then calculated as D  100/T, where D represents the weight of the detached potting mix and T represents the weight of the detached and attached potting mix plus roots. For quantification of spores, 50 cm3 of the potting mix and roots were mixed with 3 volumes of water, blended, and wet sieved through 400 and 45 mm mesh sieves. Subsequently, the material collected from the 45-mm mesh was decanted into a 60% sucrose solution in water and left to sediment overnight. The supernatant was poured through a 45-mm mesh and rinsed and resuspended in deionized water. Spores were quantified in aliquots of this suspension, and these values used to estimate the spore density in each cone-tainer. To estimate fresh weight and AMF colonization, the potting mix was washed from the roots and for the coplanted treatments the roots of the different species were separated from one another. Although this process allowed us to obtain sufficient roots that clearly were from A. tridentata or P. secunda, complete separation of the root system was not possible due to extensive intertwining of roots among the seedlings. This prevented us from making a correct estimate of the root fresh weight of each species and thus we opted for measuring the combined root weight, which includes the weight of roots that we could identify as A. tridentata or P. secunda, as well as broken and heavily entangled roots. Similarly, for comparison of plant biomass, measurements of dry weight would have been preferable over those of fresh weight. However, portions of fresh roots were used for colonization and deoxyribonucleic acid (DNA) extraction. Recovery of these roots after the staining and grinding steps was highly variable, and the fraction of the total root system used for colonization and DNA extraction differed among samples. Due to these complications, we reasoned that, for comparison of biomass, measurements of fresh weight would be less susceptible to errors than those of dry weight. Analysis of AMF colonization was based on sampling z100, 2cm-long root segments for each cone-tainer and species. We sampled these segments at random from fine roots (< 2 mm in diameter) and subsequently stained them with Chlorazol Black E as previously described (Davidson et al. 2016). We estimated total mycorrhizal, vesicular, and arbuscular colonization by the intersection method using 200 intersections per sample (McGonigle et al. 1990). Any mycorrhizal structure crossing the intersection was counted to estimate total AMF colonization, while only vesicles or arbuscules were counted for vesicular and arbuscular colonization. After the analysis of colonization, we used additional root segments to extract DNA. The DNA was purified separately from the root segments of five A. tridentata seedlings growing alone, five A. tridentanta seedlings coplanted with P. secunda, and from P. secunda seedlings coplanted with A. tridentata. These extractions

were only made from plants under the low P treatments (without and with P. secunda). Only these treatments were used to reduce sequencing cost because the goal was simply to determine the effect of coplanting on AMF composition. Roots were ground in a cell disrupter (Thermo Savant FastPrep FP120) and the DNA extracted using the FastDNA Green Spin Kit as described by the manufacturer (MP Biochemicals). DNA was amplified via nested polymerase chain reaction (PCR) using the general fungal primers LR1 and FLR2 in the first amplification and the Glomeromycota specific primers, FLR3 and FLR4 in the second amplification (Gollotte et al. 2004). This last pair of primers had been modified by the addition of Illumina adapters at their 50 ends. The PCR procedure amplifies the D2 region of the 28S ribosomal RNA gene (LSU-D2). PCR reactions were conducted as previously described (Carter et al. 2014). After the second amplification, PCR products were cleaned using ExoSAP-IT (USB Corporation), adjusted to a DNA concentration of 20 ng mL
1, and sent for sequencing to a commercial facility (Genewiz, Inc.). Data Analysis The effect of planting treatments and P fertilization on percent soil loss, spore density, fresh weight, and AMF colonization of A. tridentata roots was analyzed using a linear mixed model with the package lmer4 in R 3.5 (R-Development-Core-Team 2018). Planting, P fertilization, and the interaction of planting treatments  P fertilization were treated as fixed factors while sampling time was treated as a random factor. The effect of P fertilization on AMF colonization of P. secunda was analyzed using the same approach but with only P fertilization as a fixed factor. The significance of the fixed factors on soil loss, AMF colonization, and spore density was analyzed using a likelihood ratio test comparing a model without the factor of interest to that of a model including this factor. Significant differences between treatments were determined using the lsmeans function (lsmeans package in R) with P values adjusted for multiple comparisons by the Tukey method. All estimates of treatment variability are reported as standard errors. The DNA sequences obtained were resolved into amplicon sequence variants (ASV) after the DADA2 pipeline (Callahan et al. 2016). ASVs represent a newer and presumably more accurate alternative to the traditional grouping of sequences into operational taxonomic units (Callahan et al. 2017). The taxonomic identity of the ASVs was inferred to the genus level by comparing their sequences to those from the phylogenetic reference data for molecular systematic of arbuscular mycorrhizal fungi presented by Krüger et al. (2012) plus additional sequences from AMF isolates and vouchers downloaded from the NCBI GenBank (http://www. ncbi.nih.gov). The comparison was made using the assignTaxonomy function in DADA2. Before the analysis, the reference sequences were trimmed to the LSU-D2 region and formatted to be compatible with the assignTaxonomy function (S1, available online at [https://doi.org/10.1016/j.rama.2019.09.002]). Possible differences in ASV composition among A. tridentata seedlings growing alone, A. tridentata coplanted with P. secunda, and P. secunda coplanted with A. tridentata were assessed using nonmetric multidimensional scaling (NMDS). This analysis was conducted using the ordinate function in the Phyloseq package (McMurdie and Holmes 2013) in R (The R Foundation, Vienna, Austria) based on either the presence/absence of ASVs or their relative abundance. Results Loss of Potting Mix Loss of potting mix during removal of seedlings from the conetainers was not affected by difference in P fertilization (c2 ¼ 0.45,

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P ¼ 0.50). In contrast, co-planting of A. tridentata with P. secunda reduced soil loss (c2 ¼ 19.55, P < 0.0001) and no significant interaction was observed between P fertilization and planting treatments. Without P. secunda approximately a quarter of the potting mix (26.2% ± 3.3%) separated from the rest of the root ball during removal of the seedlings from the cone-tainers. With P. secunda, this loss was 4.3% (± 3.3%). Fresh Weight The shoot fresh weight of A. tridentata seedlings was not significantly affected by the planting treatments (c2 ¼ 0.96, P ¼ 0.33) or differences in P fertilization (c2 ¼ 0.65, P ¼ 0.42), even though average values were slightly higher at high than low P (Table 1). Similarly, differences in P fertilization did not affect the shoot fresh weigh of P. secunda (c2¼ 2.0, p ¼ 0.15). Significant differences in fresh weight were only observed when comparing the total plant fresh weight between cone-tainers with A. tridentata only and those with the two species. Total fresh weight was higher in cone-tainers with the two species (c2 ¼ 15.41, P < 0.001), and this difference was more marked for roots than shoots. The total root biomass was almost threefolds higher in cone-tainers with the two species than in those with A. tridentata only, 1.7 g and 0.6 g, respectively (P < 0.001). AMF Colonization The low P treatment increased total AMF colonization of A. tridentata roots when compared with the high P treatment (c2 ¼ 5.57, P ¼ 0.018). However, there was a significant interaction between P fertilization and planting treatments (c2 ¼ 7.11, P ¼ 0.0077). The effect of low P on increasing total colonization was only apparent in A. tridentata seedlings coplanted with P. secunda (Fig. 1A). For these seedlings, total colonization under high and low P was 46% and 70%, respectively (see Fig. 1A, P ¼ 0.006). An interaction between P fertilization and planting treatments was also observed for vesicular colonization (c2 ¼ 19.98, P < 0.0001). For A. tridentata seedlings growing alone, low P had a negligible effect on vesicular colonization (see Fig. 1C). In contrast, for A. tridentata seedlings coplanted with P. secunda, vesicular colonization was reduced under high P (P < 0.0001), with values of 7.4% and 18% (±1.8%) at high and low P, respectively. Furthermore, this last value was significantly higher than the average values measured in the other three treatments (see Fig. 1C). In contrast to total and vesicular colonization, none of the fixed factors had an effect on arbuscular colonization, which ranged from 12% to 20% (±4.3%) between the different treatments (see Fig. 1B). During the analysis of colonization, we also searched for intraradical spores, but the occurrence of these structures was negligible for all treatments. Table 1 Effect of coplanting with Poa secunda and phosphorus fertilization on shoot and root fresh weight of seedlings (g). Values represent least square means (± standard of error) of six replications. Treatment

Artemisia shoot

Artemisia Artemisia Artemisia Artemisia

0.68 0.78 0.52 0.67

without Poa low P without Poa high P with Poa low P with Poa high P

(±.17)a2 (±.17)a (±.17)a (±.17)a

Poa shoot

Roots1

NA NA 0.46 (±.15)a 0.75 (±.17)a

0.56 (±.12)b 0.68 (±.12)b 1.46 (±.17)a 1.9 (±.17)a

NA, not applicable. 1 For the coplanted treatments, root weight represents the combined root weight of roots that we could identify as A. tridentata or P. secunda, as well as broken and heavily entangled roots. Intertwining of roots prevented us from making a correct estimate of the root fresh weight of each species. 2 Within a column, values followed by the same letter are not statistically significant (P > 0.05).

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In response to differences in P fertilization, the P. secunda seedlings coplanted with A. tridentata responded somewhat similarly to the latter; P fertilization had a significant effect on total (c2 ¼ 14.61, P < 0.001) and vesicular colonization (c2 ¼ 6.5846, P ¼ 0.01). Total AMF and vesicular colonization of P. secunda seedlings was lower under high than low P (Fig. 2A and C). In addition, differences in P fertilization affected arbuscular colonization (c2 ¼ 12.71, P < 0.001). Under high P, P. secunda seedlings had lower rates of arbuscular colonization than those under low P (see Fig. 2B). Spores in Potting Mixture Neither coplanting with P. secunda (c2 ¼ 0.06, P ¼ 0.81) nor differences in fertilization (c2 ¼ 0.002, P ¼ 0.96) affected the spore density in the potting mix. Furthermore, the interaction between these factors was not significant. Without and with P. secunda the spore density was 18.6 (±3.0) and 18.0 (±2.9) spores per cm3 of mix, respectively. Similarly, for the low and high P treatment, the spore density was 18.2 (±3.0) and 18.4 (±2.9) spores per cm3 of potting mix, respectively. AMF Community Composition Sequences were obtained from 13 out of the 15 samples sent for analysis. From these sequences, the DADA2 pipeline resolved 105 ASVs (NCBI GenBank accession numbers MN388353-MN388457), which, based on the taxonomic comparison using the assignTaxonomy function, were in four Glomeracea genera: Funnileformis, Glomus, Rhizophagus, and Sclerocystis. Of the 105 ASVs, 7 were found in > 90% of the samples analyzed and were within the Funneliformis or Glomus genus. In contrast, 87 ASVs appear to be rare and were found in only one sample. We did not detect differences in the number of ASV per sample (richness) between A. tridentata growing alone, A. tridentata coplanted with P. secunda, and P. secunda coplanted with A. tridentata (Fig. 3A, P ¼ 0.08). In addition, the NMDS analysis based on either relative abundance or presence/absence of ASVs indicated no significance difference between the AMF communities of these three groups of plants (see Fig. 3B, P ¼ 0.44). Discussion Effects of Coplanting and Phosphorus Fertilization on AMF Propagules and Colonization In this study, coplanting with the grass P. secunda in combination with low P fertilization increased the abundance of AMF vesicles in A. tridentata roots. Low P fertilization also increased total, vesicular, and arbuscular colonization of P. secunda roots. Root fragments containing vesicles and, to a lesser extent, intraradical hyphae can act as infective propagules (Biermann and Linderman 1983; Klironomos and Hart 2002). Consequently, the results of this study support the notion that coplanting increases the density of mycorrhizal inoculum. Contrary to expectations, however, neither coplanting nor differences in P fertilization affected the spore density in the potting mix. The combined root weight of A. tridentata and P. secunda in the coplanted treatments was about three times higher than the root weight in the treatments without P. secunda. Assuming that the specific root length was similar between the treatments, the intraradical hyphal length and number of vesicles were then at least three times higher with than without P. secunda. Notwithstanding these differences, all treatments showed similar spore density. This result indicates that as roots were colonized by AMF, the allocation of carbon to vesicles took precedence over that to spores. Furthermore, spore density was overall low and similar or

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Figure 1. Effect of phosphorus availability and coplanting with Poa secunda on arbuscular mycorrhizal fungi (AMF) colonization of Artemisia tridentata roots. A, Total AMF colonization. B, Arbuscular colonization. C, Vesicular colonization. Bars represent least square means (± standard of error) of six replications. Bars labeled by different letters are significantly different (P < 0.05).

lower to that reported for several sagebrush habitats (Stahl and Christensen 1982; Allen and West 1993). The low spore density strongly suggests that spore formation was minimal during the experiment. Low sporulation may be a characteristic of the AMF taxa identified in this study, which were all within the Glomeraceae. In some taxa within this family, the main propagules are colonized root fragments presumably containing vesicles (Biermann and Linderman 1983; Schalamuk and Cabello 2010). Taxa with this characteristic often require repeated periods of cultivation and slow soil drying to build up increases in spore density (Brundrett et al. 1999; Busby et al. 2013). Although these practices are useful to increase spore density in trap cultures, they are not suitable for seedlings intended for field transplanting. The factors that trigger the formation of AMF storage organs (vesicles and spores) are not fully understood (Müller et al. 2017). However, it has been observed that conditions less favorable for plant growth such as low temperature, water stress, and waterlogging tend to increase the proportion of carbon allocated to storage organs (Mendoza et al. 2005; Hawkes et al. 2008). Changes in the allocation of carbon have also been reported for differences in P. Carrot roots growing in vitro with AMF allocated more carbon to storage lipids under low than high P availability (Olsson et al. 2006). Furthermore, this difference was more noticeable for storage lipids in the intraradical mycelium than those outside the roots (Olsson et al. 2006). For a given P treatment, the P applied to conetainers without and with P. secunda was the same. However, the

higher density of roots in the latter may have increased competition for P. In the low P-coplanted treatment, this competition could have decreased tissue P concentrations below a threshold that promoted vesicle development. In addition to differences in vesicular colonization, A. tridentata seedlings coplanted with P. secunda had higher total colonization under low P conditions than at high P. In contrast, in cone-tainers without P. secunda, total colonization under low P was almost identical to that at high P. Furthermore, the percent total colonization without P. secunda was intermediate and not statically different from those of A. tridentata coplanted with P. secunda. Thus, coplanting seemed to have augmented the sensitivity of A. tridentata seedlings to P. For coplanted seedlings, the higher colonization at low P could have been attributed to high synthesis
pez-Ra
ez et al. and exudation of strigolactones (Liu et al. 2013; Lo 2017). The secretion of these hormones is enhanced by P starvation and varies among plant species (Liu et al. 2013). Low P levels in the coplanted
low P treatment may have increased strigolactone secretion due in part to P. secunda. Such increase would have ten
pez-Ra
ez et al. ded to promote hyphal branching and infectivity (Lo 2017). On the other hand, lower colonization at high P could have been attributed to increased competition among roots for the hyphae, especially if hyphal branching was limited by inhibitory effects of high P on strigolactone synthesis. The P levels that inhibit AMF colonization depend on various factors including the plant species considered (Amijee et al. 1993).

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Figure 2. Arbuscular mycorrhizal fungi (AMF) colonization of P. secunda growing with A. tridentata under different phosphorus fertilization. A, Total AMF colonization. B, Arbuscular colonization. C, Vesicular colonization. Bars represent least square means (± standard of error) of six replications. Bars labeled by an asterisk (*) are significantly different (P < 0.05).

In our experiment, A. tridentata and P. secunda plants growing in the same pots responded similarly to the P treatments, except for arbuscular colonization. Arbuscules are ephemeral structures, and

consequently their presence is likely to reflect colonization events that occurred within a few days of sampling (Kobae et al. 2016). In A. tridentata seedlings, the P concentration present in the one

Figure 3. Analysis of arbuscular mycorrhizal communities in Artemisia tridentata growing without Poa secunda, A. tridentata growing with P. secunda, and P. secunda growing with A. tridentata, all under low phosphorus. A, Richness of amplicon sequence variants (ASVs). B, Comparison of the arbuscular mycorrhizal fungi communities by nonmetric multidimensional scaling based on presence/absence of ASVs; dots indicate the position of each sample in the ordination space, and ellipses represent 95% confidence limits around the centroids of each species or treatment. No significant differences were detected in richness or arbuscular mycorrhizal fungi composition.

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fourth Hoagland solution (250 mM) did not reduce arbuscular colonization when compared with the 5-mM treatment. In contrast, arbuscular colonization in P. secunda was lower at high than low P and overall lower than arbuscular colonization in A. tridentata. On the basis of these results, the P level that inhibits arbuscule development appears to be lower in P. secunda than A. tridentata. Apart from its effects on vesicles and total colonization, coplanting reduced losses of potting mixture during removal of seedlings from the cone-tainers. The increase in root ball firmness caused by coplanting helps to preserve the integrity and infectivity of the extraradical hyphae (Jasper et al. 1989). Colonization from hyphae extending from P. secunda and A. tridentata roots could be particularly important immediately after outplanting. Vesicles and spores may require turnover of fine roots or a dormancy breaking period to become infective (Tommerup 1983; Klironomos and Hart 2002). Under these situations, the hyphal network present in the root ball would be the readiest source of new infections. It is also likely that the presence of P. secunda increased the density of extraradical hyphae. This would tend to enhance the rate of mycorrhizal spread and probability of producing new entry points in uncolonized root fragments (Schnepf et al. 2016). Effect of Coplanting on AMF Community Composition All the AMF taxa identified in this study were within the Glomerales. This is consistent with results in other semiarid and arid environments, where AMF within this order are dominant (Torrecillas et al. 2012; Busby et al. 2013). However, we cannot discard the possibility that our results were affected by the primers used for amplification, FLR3 and FLR4. These primers discriminate against some AMF taxa and tend to bias the AMF composition towards the Glomerales (Krüeger et al. 2009). Within this limitation, the results of our study indicate that the presence of P. secunda did not affect the AMF community composition of A. tridentata roots and that the AMF composition of these two plants was similar. These results may be partly attributed to the order of planting. A. tridentata seedlings were established first and thus had the opportunity to establish a mycorrhizal network. This network would have been better positioned to colonize P. secunda, thereby limiting the possibilities of this host to select specific taxa (Hausmann and Hawkes 2009). The similarity in the AMF community composition of the two species may also be attributed to the taxa present in the pots. Even though the sequencing analysis revealed 105 ASVs, this is not necessarily indicative of high taxonomic diversity because AMF display substantial within-species genetic variability (Koch et al. 2006; Egan et al. 2018). This variability can be the result of variants of the same gene within a nucleus, the presence of genetically different nuclei in an individual mycelium, and genetic variation within a population (Koch et al. 2006; Young 2015; Egan et al. 2018). Consequently, different ASVs could have originated from the same individual or different individuals within the same species. A low number of AMF species in the cone-tainers and/or the presence of generalist taxa may have further reduced the possibilities for host selectivity (Davison et al. 2011). Can Poa secunda Act as Nurse Plant? In sagebrush habitats, as well as other semiarid and arid environments, interactions between plants can facilitate seedling establishment and growth (Callaway 1995; Padilla and Pugnaire 2006; Hovland et al. 2019). This is well documented for nurse plants that through changes in the microenvironment improve survival of neighboring seedlings (Padilla and Pugnaire 2006). Adult A. tridentata plants are known to serve as nurse plants for other vegetation, but perhaps as seedlings they benefit by the

presence of heterospecific neighbors and specifically bunchgrasses such as P. secunda (Callaway et al. 1996; Maestre et al. 2001). One mechanism by which nurse plants facilitate seedling establishment is via enhancement of associations with beneficial microorganisms (Rodríguez-Echeverría et al. 2016; Montesinos-Navarro et al. 2019). Within this context, a desirable outcome would be for P. secunda to act as a nurse plant of A. tridentata by increasing AMF colonization of the latter after outplanting. In the short term, AMF colonization would be favored by the high density of propagules in the outplanted root ball, while in the medium and long term the spread and proliferation of the initial inoculum could be facilitated by the fibrous root system of P. secunda. It is also plausible that P. secunda could act as a nurse plant even without AMF inoculation. In our potting mix, attempts to increase AMF colonization of A. tridentata roots by simply coplanting with P. secunda would not have been useful because the mix lacked AMF. However, in the field, the soil most often has some AMF (Requena et al. 1996; Weinbaum et al. 1996). Coplanting of noninoculated seedlings may facilitate propagation of these AMF and subsequent colonization of A. tridentata. In addition, the close proximity of the two species may increase establishment of A. tridentata by AMFindependent mechanisms, such as by decreasing competition with weeds. Experiments comparing the effects of coplanting with and without inoculation may help to identify potential nursing roles of P. secunda. Whether through coplanting or other approaches, practices that increase AMF colonization to higher levels than those naturally occurring in the soil would be valuable to assess the effect of the symbiosis on seedling establishment and the need for inoculation. Such need is likely to vary depending on edaphic and climatic conditions, as well as disturbance histories (Doerr et al. 1984; Reinhart and Callaway 2006). A disturbance in sagebrush habitats that often precedes seeding or outplanting of A. tridentata is fires (Dettweiler-Robinson et al. 2013; Brabec et al. 2015). Fires tend to increase the levels of P, which may lessen the mycorrhizal dependency of A. tridentata and plants in general (Johnson and Graham 2013; Reinhart et al. 2016). However, this may be offset by diminishing soil moisture. Decreases in soil moisture lengthen the pathway for nutrient movement to the root surface (Sardans and Penuelas 2007; Suriyagoda et al. 2014). For nutrients with low mobility in the soil, such as P, decreases in soil moisture can severely limit nutrient uptake even with adequate levels of P in the soil (Suriyagoda et al. 2014). Symbioses with AMF facilitate and prolong the uptake of P as soil moisture declines and through various mechanisms also increase drought tolerance (Ruiz-Lozano et al. 2012; Bowles et al. 2018). Thus, during postfire planting, increases in AMF colonization of A. tridentata seedlings due to coplanting or other inoculation methods may prove to be beneficial despite increases in P. This is most likely to be the case at the more xeric sites of the sagebrush range, as well as sites with low AMF density due to, for example, high density of nonmycorrhizal plants before the fire or lengthy postfire periods with little vegetation (Pringle et al. 2009; Dove and Hart 2017). Implications The effectiveness of inoculation with AMF on improving colonization and seedling establishment depends on the ability of the inoculum to survive and spread after outplanting (Weinbaum et al. 1996; Rowe et al. 2007). The coplanting treatment provides a new approach to attempt to achieve and sustain high AMF colonization in seedlings of A. tridentata. Coplanting combined with low P fertilization increased the firmness of the root ball, as well as vesicular colonization of A. tridentata and P. secunda roots. Thus, in comparison with the other treatments, the root ball of seedlings under the low P
coplanting treatment had better structure to

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maintain the infectivity of extraradical hyphae and higher density of AMF propagules. These characteristics are likely to facilitate continued AMF colonization of A. tridentata roots after outplanting. Furthermore, an intact root ball may reduce transplant shock and planting mortality. A concern when outplanting the coplanted seedlings is that P. secunda may compete with A. tridentata for nutrients and water, thereby reducing establishment of the latter. Competition for nutrients could occur in spring when both species have maximum rates of growth. Competition for water seems less likely. A. tridentata can extract water from deep soil layers that P. secunda does not reach, and this grass becomes dormant during summer drought (Germino and Reinhardt 2014). Even if competition occurs, this may be outweighed by other benefits of the proximity. The presence of P. secunda may reduce weed invasion in the immediate vicinity of A. tridentata seedlings. Common weeds in sagebrush habitats such as tumble mustard (Sisymbrium altissimum L.), Russian thistle (Salsola tragus L.), and cheatgrass (Bromus tectorum L.) can reduce the AMF density in the soil or alter the AMF community with negative effects for A. tridentata (Dierks et al. 2019; Hovland et al. 2019). Via effects on AMF or due to other growth characteristics, weeds are likely to be more aggressive competitors than P. secunda. Thus, by increasing the density of AMF inoculum and reducing competition, coplanting with P. secunda may be a valuable approach to increase outplanting success of A. tridentata seedlings. Experiments are needed to test this notion. Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi.org/10.1016/j.rama.2019.09.002. References Aldridge, C.L., Boyce, M.S., 2007. Linking occurrence and fitness to persistence: habitat-based approach for endangered Greater Sage-Grouse. Ecological Applications 17, 508e526. Allen, E.B., 1989. The restoration of disturbed arid landscapes with special reference to mycorrhizal fungi. Journal of Arid Environments 17, 279e286. Allen, E.B., Cannon, J.P., Allen, M.F., 1993. Controls for rhizosphere microorganisms to study effects of vesicular-arbuscular mycorrhizae on Artemisia tridentata. Mycorrhiza 2, 147e152. Allen, E.B., West, N.E., 1993. Nontarget effects of the herbicide tebuthiuron on mycorrhizal fungi in sagebrush semidesert. Mycorrhiza 3, 75e78. Amijee, B.F., Stribley, D.P., Lane, P.W., 1993. The susceptibility of roots to infection by an arbuscular mycorrhizal fungus in relation to age and phosphorus supply. New Phytology 125, 581e586. Anderson, J.E., Inouye, R.S., 2001. Landscape-scale changes in plant species abundance and biodiversity of a sagebrush steppe over 45 years. Ecological Monographs 71, 531e556. Asmelash, F., Bekele, T., Birhane, E., 2016. The potential role of arbuscular mycorrhizal fungi in the restoration of degraded lands. Frontiers in Microbiology 7, 1e15.
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