Arbuscular Mycorrhizal Fungal Communities Associated with Vitis ...

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Taylor C. Holland,1 Pat Bowen,2 Carl Bogdanoff,2 and Miranda Hart1*. Abstract: ..... Bever, J.D., S.C. Richardson, B.M. Lawrence, J. Holmes, and M. Wat- son.
Arbuscular Mycorrhizal Fungal Communities Associated with Vitis vinifera Vines under Different Frequencies of Irrigation Taylor C. Holland,1 Pat Bowen,2 Carl Bogdanoff,2 and Miranda Hart1* Abstract: The effects of irrigating daily or every three days on arbuscular mycorrhizal fungal communities associated with grapevine roots were determined in vineyard blocks of Merlot and Syrah on 3309 rootstock. After exposure to treatments for four growing seasons, root samples were analyzed for changes in AM fungal colonization, species richness, and community composition. AM fungal colonization was higher in response to irrigating every three days compared with daily irrigation, indicating a treatment effect on the physiology of the fungal communities. Using a pyrosequencing approach, no difference in AM fungal community composition was found in response to irrigation frequency. Species richness, identity and dispersion were consistent across the two treatments. A difference in AM fungal communities between the two varietal blocks was associated with differences in soil chemistry and plant physiological traits. In particular, soil carbon and extractable copper levels along with vine vigor and photosynthesis were correlated with community variation. This indicates environmental factors other than irrigation frequency influence the fungal community structure in vineyards. Key words: mycorrhizal, fungi, soil, community, deficit irrigation, Vitis vinifera

Altering moisture levels in the soil profile through irrigation practices can influence the abundance of soil organisms (Holland et al. 2013a). This may contribute to irrigation effects on crop performance, since altering the structure and function of soil communities can impact crop health. The importance of arbuscular mycorrhizal (AM) fungal communities in natural and agricultural ecosystems is well established. These fungal symbionts associate with the roots of most plant species and are linked to increases in plant nutrient acquisition, most notably phosphorus, sulfur, and copper (Schreiner 2007), soil aggregation (Rillig 2004), water uptake (Augé 2001), and plant productivity (Linderman and Davis 2001). It is therefore important to understand how management practices such as irrigation regimes may affect AM communities. Grapevines should be highly responsive to AM fungi, since plants growing under stressful conditions, such as those imposed in vineyards to reduce vigor, experience major benefits from AM fungi (Harris-Valle et al. 2009). In fact, grapevines (Vitis spp.) are known to form associations with mycorrhizas, which influence vine physiology in different ways. For

example, grapevines show increased biomass (Linderman and Davis 2001, Nogales et al. 2009), nutrient uptake (Schreiner 2003, Schreiner et al. 2007), and water-use efficiency in association with AM fungi (Valentine et al. 2006). Deficit irrigation is commonly used in viticulture to reduce water use and grapevine vigor by altering the amount and timing of water application (Dry et al. 2001, Chaves et al. 2010, Acevedo-Opazo et al. 2010). In winegrapes, water stress resulting from deficit irrigation has been associated with increased quality of fruit and wine (Qian et al. 2009, Acevedo-Opazo et al. 2010). Increasing the frequency of irrigation while maintaining the same deficit application rate has been shown to reduce water stress and improve fruit composition (Bowen et al. 2012). While increasing irrigation frequency can improve winegrape quality, its effects on other vineyard biota, including AM fungi, have not been explored. Changes to soil moisture have been shown to alter AM fungal colonization of Vitis spp. roots. Drought stress has been shown to decrease AM fungal colonization in young vines under greenhouse conditions (Valentine et al. 2006), but can also stimulate arbuscular colonization in older field-grown plants (Schreiner et al. 2007). Previously it was found that less frequent irrigation enhanced total fungal biomass yet reduced the biomass of other soil organisms (including microarthropods, nematodes, fungi, and bacteria) (Holland et al. 2013a). Frequency of irrigation may also affect AM fungal communities in vineyards. A fluctuating dry/wet soil environment may allow for a greater range of fungi to persist (i.e., both drought-tolerant and mesic-tolerant fungi could establish and persist), resulting in increased fungal diversity. Conversely, soils kept constantly moist may select for a specific subset of the community tolerant of those conditions (phylogenetic conservatism), resulting in reduced diversity. In this study, AM fungal communities were characterized in vineyard soils

University of British Columbia, 3333 University Way, Kelowna, BC V1V 1V7, Canada; and 2Pacific Agri-Food Research Centre, Agriculture and Agri-Food Canada, Summerland, BC VOH 1Z0, Canada. *Corresponding author ([email protected]) Acknowledgments: The authors acknowledge NSERC Discovery Grant Program (MH), the BC Wine Grape Council and Agriculture & Agri-Food Canada’s Matching Investment Initiative (PB and CB), NSERC CGS scholarship (TH), and Constellation Brands for hosting the field research. Supplemental data is freely available with the online version of this article. Manuscript submitted Sept 2013, revised Jan 2014, accepted Feb 2014 Publication costs of this article defrayed in part by page fees. Copyright © 2014 by the American Society for Enology and Viticulture. All rights reserved. doi: 10.5344/ajev.2014.13101 1

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that had been exposed to two frequencies of deficit irrigation over four growing seasons to determine the effects of irrigation frequency on AM fungal community richness.

Materials and Methods Experimental design and sampling. Soil samples were collected following harvest in mid-October 2010, from a fouryear-old irrigation study at Sunrock Vineyard, Osoyoos, BC, Canada (49°5´N, 119°31´W). The study consisted of identical experiments with irrigation frequency treatments in two adjacent blocks of Merlot and Syrah on 3309C rootstock. Both blocks shared the same Osoyoos soil series, a deep, loamy sand with a water-holding capacity of ~13% (v/v) (Bowen et al. 2011). For the purposes of this study, the two experiments were treated as two replicate blocks, with the main differences being scion and row direction. Though the row direction may introduce unforeseen differences in the blocks, this information was incorporated into our analyses through the use of multivariate statistics, which factor for differences in vine physiology and variations in soil parameters. The initial irrigation experiment incorporated two frequencies of drip irrigation, either 5.33 L daily or 16 L every third day, starting at the beginning of July and running until harvest: 7 Oct for Merlot and 21 Oct for Syrah. These irrigation regimes were applied to five-vine plots in a randomized block design with four replicates. The end vines in each plot were considered guards. Both irrigation treatments provided on average 5.33 L/vine per day, which was determined previously to be a deficit rate under the site conditions (Bowen et al. 2011). Moisture levels within the 0 to 15 cm depth interval fluctuated on average from 12% (v/v) to 9% water content in response to daily irrigation, compared with a 13% range in response to the three-day interval (decreasing from 19% after irrigating to 6%) (Bowen et al. 2012). For both regimes, soil was replenished before the wilting point was reached, as indicated by leaf gas exchange and stomatal conductance measured just before the irrigations (Bowen et al. 2012). Nine soil cores (2.5 cm in diameter) were sampled from each plot, which included three cores taken 15 cm from the base of each of the three vines (this point was also the location of the drip emitter) in a spatially explicit manner to compensate for heterogeneity in the soil moisture profile. Initially the cores taken from individual vines were homogenized and kept at 20°C. These were processed separately (soil preparation, DNA extraction, PCR) then pooled to form one composite sample from each experimental unit for sequencing and chemical analyses. Community analysis. DNA extraction, PCR amplification, and 454 pyrosequencing. For all experimental units, Vitis roots were separated from soil before DNA was extracted in duplicate from 0.05 g of root tissue following the protocol outline in the Mo Bio PowerSoil Kit (Mo Bio Laboratories, Carlsbad, CA). From each sample triplicate, PCR amplifications were performed using the Glomeromycotan specific primers FLR3/FLR4 (Gollotte et al. 2004), which amplify a 400 bp fragment of the large subunit of rDNA. Primers were modified for 454 pyrosequencing with the addition of Roche ligating adaptor and multiplex identification (MID) regions. A 23 mL

mixture of 13.25 mL ddH 20, 5 mL 5x PCR buffer (Promega, Madison, WI), 2 mL MgCl2 (New England BioLabs, Ipswich, MA), 0.5 mL dNPTs (Amresco, Solon, OH), 1 mL BSA (New England BioLabs), 0.25 mL GoTaq (Promega), and 0.5 mL of each primer was used per reaction. Two 1-mL aliquots of DNA template (1 mL from each DNA extraction) were added to this mixture, making a total volume of 25 mL. Cycling conditions were 95°C for 3 min followed by 35 cycles of (95°C for 30 sec, 52.5°C for 30 sec, 72°C for 60 sec), 72°C for 10 min, and a hold at 4°C. PCR products were standardized to 1.25 ng/mL using the Invitrogen SequalPrep kit before amplicon sequencing with Roche 454 pyrosequencing GS FLX+ Titanium chemistry (UBC Prostrate Center, Vancouver, Canada). Sequence analysis. The sequencing data were analyzed using quantitative insights into microbial ecology (QIIME) (Caporaso et al. 2010). Sequences were filtered under the default parameters with the following exceptions: 370 to 410 bp fragment length, a maximum of six homopolymers allowed, zero mismatches in MID tags, and an average quality score of 30. Sequences were then organized into their respective samples using the MID identifier tags, rarified to 1250 sequences per sample, and then grouped into operational taxonomic units (OTU) based on 95% similarity using the UCLUST algorithm (Edgar 2010). This 95% similarity was used to group taxa because this target gene (LSU) cannot resolve species level differences, leading to inflated diversity measures at 97% (Rosendahl 2008). Further, to ensure identified OTUs were not due to sequencing error, each OTU was represented by a minimum of six sequences or occurred in at least three separate samples for retention in the analysis. While this approach might bias against rare taxa, it ensures that OTUs in the analysis were real entities. AM fungal root colonization. Percent colonization of the Vitis roots was measured using a modified version of a previous technique (Klironomos et al. 1993). Briefly, roots were washed using dH2O, cleared by autoclaving for 15 min in 10% KOH, further bleached in room temperature 3% H 2O2 for 30 min, acidified in boiling 10% HCl for 10 min, and stained in 0.5% Trypan blue (5% lactic acid, 50% glycerol) in the autoclave for 15 min. Roots were then destained overnight and stored in lactoglycerol for further analysis. The magnified intersections method (McGonigle et al. 1990) was applied to quantify the proportion of roots that contained arbuscules, vesicles, and hyphal colonization. Environmental measurements. Soil chemistry. A subsample of each soil core was analyzed for extractable elements (Al, B, Ca, Cu, Fe, K, Mg, Mn, Na, P, S, and Zn) by inductively coupled plasma–optical emission spectrometry in combination with the Mehlich III extractant (0.2N CH3COOH + 0.25N NH4NO3 + 0.013N HNO3 + 0.015N NH4F + 0.001M EDTA). Soil pH was determined from a 1:1 (weight) mixture of soil:water measured using a pH electrode. Total carbon and nitrogen were quantified using combustion elemental analysis (Wolfgang 1983). Plant physiology. Grapevine growth and physiological characteristics were reported previously including leaf nitrogen and dry weight, stomatal conductance, transpiration,

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photosynthesis, pruning mass, shoot length, and berry yield (Bowen et al. 2012). Leaf gas exchange parameters were measured using a portable system (model 6400; LI-COR, Lincoln, NE), equipped with an artificial light source (6400-02B) to provide a photosynthetic photon flux of 1000 μmol m -2 s -1. Average vine shoot length was based on five randomly chosen shoots per vine measured in early July before the vines were lightly hedged. Percent leaf N was determined from a 0.25 g dried sample of fully mature leaves collected randomly (27 Sept 2010) from each vine and measured using a LECO FP528 analyzer (Leco Corp., St. Joseph, MI). Data analysis. AM fungal colonization and alpha diversity. Irrigation treatment effects on mycorrhizal root colonization or alpha diversity were determined using R (ver. 2.8.1, R Development Core Team, Foundation for Statistical Computing, Vienna) to perform two-way analyses of variance (ANOVA) with irrigation as factor and variety as block. This was done separately on both arbuscular and vesicular colonization, along with species richness (determined as the number of OTUs present in each sample). AM fungal community analysis. In order to account for differences in AM fungal taxa between communities, weighted UniFrac distances were calculated. Weighted UniFrac is a measure of community similarity based on the amount of phylogenetic overlap calculated between two samples, taking into account the taxa present and the abundance of these taxa (Lozupone and Knight 2005). Sørensen similarity index (Sørensen 1957) was also used to measure distance between communities, based on OTU presence without considering phylogenic relationships or relative abundance. In order to test for community-level differences attributable to irrigation frequency, PERMANOVA was performed with irrigation (three times day and daily) and block (Shiraz and Merlot) as fixed factors (9999 permutations, Type I SS) (PERMANOVA+ for PRIMER, PRIMER-E, Lutton, UK) (Anderson et al. 2008). This was done for both the weighted UniFrac and Sørensen distances. Soil chemistry and plant physiology were also likely affected by irrigation treatments, thus these were used as covariates in the PERMANOVA analysis. Data were first transformed to satisfy normality requirements (log transforming soil S and Zn) and then normalized. Redundant variables were removed using draftsman plots in PERMANOVA+ to detect colinearity. The resulting data set included soil Al, B, C, Cu, Fe, Na, PO4, S, Zn, and pH and the seasonal average for each of crop yield, vine shoot length, leaf dry weight, photosynthesis, and stomatal conductance. Given the high number of soil and physiological variables, a PCA axis was performed on each set of variables separately, and the first PCA axis was used to reduce the number of covariates in the analysis (Supplemental Tables 1 and 2). AM fungal community dispersion. To determine if irrigation frequency affected the turnover of AM fungal taxa (i.e., spatial heterogeneity), differences in dispersion of our samples from their treatment means (or centroids) were analyzed (PERMADISP) (Anderson et al. 2008). The magnitude of spread around the group centroid reflects greater compositional turnover among samples. Here, Sørensen similarity

distance matrix was used (9999 permutations) in PRIMER-E+ (PRIMER, ver. 6) (Clark and Gorely 2006). Environmental effects. In order to assess the relative contributions of environmental factors to the observed variation in the AM fungal communities, the DISTLM function of Primer-E+ was used (Anderson et al. 2008). This linear model partitions variance between environmental predictor variables using distance-based community data sets. Both soil and plant characteristics were included to determine their relative contributions to changes in the AM fungal community. The Best selection routine was used to select models made up of all possible combinations of soil chemistry and plant physiological predictor variables, which were most correlated with community variance. 9999 permutations were performed and models were selected based on their AICc values, a measure of quality for statistical models that includes a correction factor for the number of variables included in the model (Anderson et al. 2008). Using the DISTLM model with the lowest AICc value, a distance-based redundancy analysis (dbRDA) was conducted in PRIMER-E+ to depict the community variance after taking into account the environmental variables in the model (Clark and Gorely 2006).

Results

Root colonization and AM fungal community. Irrigation frequency affected AM fungal colonization. Root arbuscular colonization (F1,28 = 6.324, p = 0.018) was higher in response to lower irrigation frequency, but there was no effect on the vesicular colonization (F1,28 = 1.795, p = 0.208) (Figure 1).

Figure 1 Arbuscular (A) and vesicular (B) colonization in Vitis roots irrigated every three days (third day) or daily (daily). Measures represent gridline-intersect proportion for 100 fields of view (* signifies statistical significance with α = 0.05, n = 32).

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There was no evidence that irrigation frequency affected AM fungal species richness (Figure 2) (F1,12 = 1.331, p = 0.271) or community composition (weighted UniFrac; Fpseudo 1,15 = 0.396, p = 0.828) (Sørensen; Fpseudo 1,6 = 0.698, p = 0.718) (Figure 3). Similarly, the beta diversity of samples revealed no difference in dispersion (i.e., turnover between samples) associated with irrigation frequency (F1,14 = 0.903, p = 0.369). Community differences between blocks. Arbuscular mycorrhizal fungal community structure differed between the two varietal blocks. Vine roots had fewer fungal species in the Syrah block than in the Merlot block (Figure 2) (F1,12 = 4.834, p = 0.048). The varietal block effect was also detected based on both Sørensen similarity (Fpseudo 1,15 = 3.065, p = 0.007) and UniFrac values (Fpseudo 1,15 = 3.407, p = 0.023). Between PC1 (47.7% total variation) and PC2 (29.1% total variation) (Figure 3), the communities associated with the two varietal blocks had minimal overlap, indicating they differed in taxonomic composition. However, soil chemistry and plant

physiological characteristics were both covariables, separately contributing to large portions of the community variation, at 60.1% (Table 1, 38.1% PC1 and 22% PC2) and 72% (Table 2, 43.3% PC1 and 29.4% PC2), respectively. After including these factors as covariates in the PERMANOVA, there was no difference in AM fungal community structure between the two blocks based on either Sørensen similarity (Fpseudo 1,15 = 1.345, p = 0. 244) or UniFrac values (Fpseudo 1,15 = 0.530, p = 0. 711). Relative contribution of soil and plant factors to AM fungal variation. Soil carbon (Fpseudo 1,15 = 3.194, p = 0.028)

Figure 3 The dispersion of arbuscular mycorrhizal fungal species for Merlot and Syrah blocks that were subjected to irrigation every three days or daily, Osoyoos BC, Canada. Each point represents a Sørensen distance value composed from OTU presence/absence data (n = 22).

Table 1 Results from principal component analysis (PCA) for soil chemistry variables associated with Vitis roots. The first two eigenvalues were used as proxies for plant physiology covariates in PERMANOVA analyses and the percent variation contributed by each PC axis is given. PC

Eigenvalue

% Variation

Cumulative % variation

1 2 3 4 5

3.81 2.2 1.6 0.768 0.585

38.1 22.0 16.0 7.7 5.9

38.1 60.1 76.1 83.8 89.7

Table 2 Results from principal component analysis (PCA) for vine physiological traits with the percent variation contributed by each PC axis. The first two eigenvalues were used as proxies for plant physiology covariates in PERMANOVA analyses.

Figure 2 Arbuscular mycorrhizal fungal alpha-diversity in roots of Vitis rootstock 3309C in response to irrigation either every three days or daily (A) and varietal block (B), Osoyoos BC, Canada. Values are based on species richness, defined as the number of OTUs (confidence level of 95%) per sample (* signifies significance at α = 0.05, n = 16).

PC

Eigenvalue

% Variation

Cumulative % variation

1 2 3 4 5

2.16 1.47 0.917 0.307 0.14

43.3 29.4 18.3 6.1 2.8

43.3 72.7 91.0 97.2 100.0

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and vine shoot length (Fpseudo 1,15 = 2.902, p = 0.038) were predictors of differences in AM fungal community structure (Table 3). Photosynthesis was weakly associated with community composition (Fpseudo 1,15 = 2.340, p = 0.074), indicating a possible influence of plant primary production on AM fungal community composition. Overall, models incorporating soil carbon, vine shoot length, or photosynthesis in combination with boron, copper, or sodium best explained the observed variation among the AM fungal communities (Table 4). The variables from the best model were incorporated into a distance-based redundancy analysis (dbRDA) and the resulting ordination (Figure 4) illustrated that carbon was associated with dbRDA1 (58% fitted, 18.7% of total variation) and that copper was associated with dbRDA2 (39.3% fitted, 12.7% of total variation).

Discussion Irrigation frequency effects on AM fungi. Despite the substantial effects of irrigation frequency on moisture in the Table 3 Marginal tests from DISTLM analysis showing the proportion of variation among AM fungal communities attributed to soil chemistry and plant physiology. Variable Aluminum Boron Copper Iron Sodium Sulfur Zinc pH Phosphorous Carbon Berry yield Vine shoot length Leaf dry weight Photosynthesis Stomatal conductance

Pseudo F

p value

Proportion

1.172 1.213 1.988 0.904 1.952 0.842 1.729 1.532 0.340 3.194 0.339 2.902 1.203 2.340 1.110

0.315 0.302 0.120 0.450 0.118 0.492 0.157 0.204 0.868 0.028 0.870 0.038 0.315 0.074 0.344

7.72E-02 7.98E-02 0.12435 6.07E-02 0.12238 5.67E-02 0.10992 9.87E-02 2.37E-02 0.18575 2.36E-02 0.1717 7.91E-02 0.1432 7.35E-02

soil profile during the growing season, AM fungal community composition was insensitive to the irrigation treatments in this study. Instead, soil nutrients and plant physiology were more important in explaining observed differences in AM fungal composition. There was no evidence to support our hypothesis that fluctuating moisture conditions in response to less frequent irrigation enhances AM fungal species richness or alters species composition. Previous studies have found differential AM fungi growth and abundance (Valentine et al. 2006, Schreiner et al. 2007) in response to water stress, but these studies represent a variety of experimental conditions and did not consider community composition. Water availability, both limiting and excess, has been shown to influence the diversity of AM fungi. Under increasing xeric (Jacobson 1997) or mesic (Ipsilantis and Sylvia 2007) conditions, there is a tendency for communities to be less diverse, likely due to differential stress tolerance among fungi. For example, decreases in soil water availability have correlated with decreased hyphal growth and colonization of roots (Clark et al. 2009), yet this depends on the AM fungal species tested (Klironomos et al. 2001). As the semiarid soils of the Okanagan region (BC, Canada) represent an extreme growth habitat for AM fungi, it may be that soils are naturally so dry that any irrigation, regardless of the frequency, alleviates drought stress. Differences may be greater between naturally arid and irrigated soil, rather than among different frequencies of irrigation which provided the same amount of water. Even though differences in AM f ungal community structure were not detected between treatments, arbuscular

Table 4 Best models of soil chemistry and plant physiology traits for explaining community variance. Included are the approximate proportion of community variance that each model explains and the quality of each model, represented by the R2 and AICc values, respectively. AICc -91.997 -91.723 -91.591 -91.367 -91.247 -91.182 -91.172 -90.834 -90.82 -90.798

R2

Variable (n)

Selection

0.1857 0.1717 0.3109 0.3012 0.2960 0.1432 0.2927 0.1243 0.2769 0.1224

1 1 2 2 2 1 2 1 2 1

Carbon Vine length Copper, carbon Copper, vine length Boron, vine length Photosynthesis Copper, photosynthesis Copper Boron, carbon Sodium

Figure 4 Distance-based redundancy analysis (dbRDA) plot depicting arbuscular mycorrhizal fungal community variance in vine roots of Merlot and Syrah blocks that were subjected to irrigation every three days or daily, Osoyoos BC, Canada. Each point represents a UniFrac distance value after incorporating carbon, copper, and irrigation in the community variance (n = 22). Vectors depict the direction of each variable and its contribution to the axes, with variables closer to the circle parameter indicating stronger contribution.

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colonization of roots was higher in response to the lower than higher frequency irrigation, perhaps signifying a functional change in the roots. Since arbuscules are known to be the site of carbon and nutrient exchange (Smith and Read 2008), the fungi may have altered the allocation of resources under the different irrigation regimes. Similar results have been found in grapevines and other host plants in response to soil moisture stress. Schreiner et al. (2007) observed that vines had increased arbuscular levels in response to deficit irrigation as compared with nondeficit irrigation. A field study using Lythrum salicaria grown in a gradient of soil moisture conditions also found root colonization was highest in plants grown in the driest soils (Stevens and Peterson 1996). This response to moisture was also seen in Boswellia papyrifera subjected to irregular pulses of water instead of regular continual watering regimes (Birhane et al. 2012). It is still unclear whether this pattern results from direct effects on soil moisture on the fungi or whether plant stress response to irrigation mediates this response. In any case, AM fungal growth patterns can impact resource allocation, which may affect the quality of the symbiosis. Environmental effects on AM fungi. The differences observed between the blocks in the species richness and community composition of AM fungi were not significant after accounting for differences in soil chemistry and vine physiological covariates, indicating that soil chemistry and host physiological differences played a large role in determining AM fungal community structure. However, it is important to note that differences in vine vigor and photosynthesis together with possible unmeasured aspects such as soil temperature and physical properties may have resulted from differences in sunlight exposure due to the perpendicular row directions of the two varietal blocks. There is increasing evidence that physiochemical properties of soil (i.e., pH gradients (An et al. 2008), water status (Jacobson 1997), and temperature (Klironomos et al. 2001) are important determinants of AM fungal communities, but there is little consensus as to which factors are most influential. Such differences could exert selection for AM fungal taxa well adapted to specific soil conditions. Soil carbon was the only soil variable measured that explained a significant proportion of the community variation. While the types of soil carbon were not differentiated, it is possible that some carbon was comprised of host exudates, which are significant drivers of AM fungal communities (Hartmann et al. 2008). It is also possible that soil carbon and the soil communities were affected by other factors not measured here, such as soil temperature or the community composition of surrounding plants (Holland et al. 2013b). In addition to carbon, soil levels of boron, copper, and sodium were also important for explaining variation among AM fungal communities. Micronutrients are rarely linked to changes in fungal community structure, as most studies have been focused on phosphorous, nitrogen, and carbon (Johnson 2010). Boron, copper, and sodium are essential plant nutrients (Arnon and Stout 1939), and there is evidence that AM fungi are able to mediate the uptake of these nutrients

(Marschner and Dell 1994, Schreiner 2007, Tseng et al. 2009). This influence on nutrient uptake may cause hosts to preferentially associate with fungi that can improve access to limiting nutrients (Bever et al. 2009). Alternatively, fungi may be affected directly by micronutrient levels. In extreme cases of sewage and heavy metal contaminated soils, it was shown that the infection rate of AM fungi is often lowered with increasing levels of Cu, Zn, and other heavy metals (Karagiannidis and Nikolaou 2000). If certain fungal species can tolerate higher levels of micronutrients and heavy metals, then environmental selection may alter community composition. Vine shoot length and photosynthesis were correlated with a significant proportion of the fungal community variance. These plant physiological traits could be proxies for the amount of sugar available for root allocation, which can affect fungal abundance, community structure, and function (Bever et al. 2009, Fellbaum et al. 2012). This can occur through the regulated release of root exudates that soil communities rely on or through the allocation of carbon within the roots that has been shown to maintain spatial structure within AM fungal communities. The amount of carbon available from the host plants also affects the functional aspect of the symbiosis, determining nutrient transported within the AM fungal network (Fellbaum et al. 2012). Fundamentally, the amount of carbon available below ground has a major influence on the abundance and the diversity of fungi that are able to colonize and persist on host roots. Thus, in this study, irrigation practices that limit below-ground carbon transfer may result in AM fungal communities that are less abundant and/or have different composition.

Conclusions Results indicated that soil moisture fluctuations as influenced by two commercially acceptable irrigation frequencies had little influence on AM fungal communities, whereas site conditions associated with soil chemistry, vine growth, or physiological traits caused a shift in community structure. Further study is needed to identify these conditions and to determine possible influence of AM community structure on vineyard ecology and commercial performance. Relatively little is known about how viticultural management influences soil communities, which are known to provide ecosystem services important to grapegrowing and soil maintenance. Irrigation frequency did not influence the species composition of AM fungi, but there may be longterm functional responses to this variable since, after four years, higher frequency irrigation had reduced the level of colonization by fungal functional structures (i.e., arbuscule density). Differences in soil micronutrients and plant vigor and physiology were associated with variation among communities. Further studies should explore the response of AM fungal communities to micronutrients, how soil communities are influenced by vineyard factors such as natural and supplemented soil nutrient levels, and the effect of these communities on grapevine physiology factors that control fruit yield and compositional quality.

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