Commercial standards of epicatechin, malvidin-3-O-glucoside, and quercetin-3-O-glucoside were used for the quantification of flavan-3-ols, anthocyanins, and flavonols, respectively.Cost estimates on labor operations and gross income per hectare were calculated based on yield and net returns per hectare . Water footprint was calculated as described by Zotou and Tsihrintzis . Briefly, for the green component of the WF , precipitation data during the growing season was obtained from the CIMIS Station and estimated as m3 /ha to obtain the total green consumed water volume . Then the value was divided by the yield expressed as ton/ha. The blue component of the WF was calculated with the total irrigation water amount that grapevines received per hectare, and this blue consumed water volume value was divided by the yield . The gray component of the WF was not calculated given that our experimental conditions avoided the use of fertilizers. Then, the total WF was estimated as the sum of green WF and blue WF. statistical analyses were performed in R-Studio version 3.6.1 for Windows. All the monitored parameters were fit in linear mixed-effect models by using the lmer function from lme4 package with AMF inoculation , irrigation treatment , and their combination as fixed factors, and replicate as random factor . Then, pairwise contrasts were conducted with function lsmeans from lsmeans package using the Kenward–Roger method and Tukey adjustment for p-values. Previously, for gas exchange parameters, stem water potential, mycorrhizal colonization, and flavonoid contents a mixed-effect model including sampling date as fixed factor was run . However, as the treatment effect seemed to be independent in the sampling date , sampling date was removed from the analysis to assess the effect of treatments for each sampling date. Finally, blueberry packaging box correlations between the percentage of mycorrhizal inoculation and flavonoid contents were calculated with the Pearson’s test using the same software.
The comparison between the growing season of the experiment and the reference data for the same period within the last 20 years showed that 2019–2020 was warmer and drier . Thus, average daily temperature was 0.5◦C higher, especially in August, which reached 1.8◦C more, and precipitation of 530 mm less compared to the average, hence, the 2020 growing season was an extreme year regarding temperature and rainfall. Native mycorrhizal colonization was determined before treatment application and no differences between them were observed . The mycorrhizal colonization intensity was analyzed after 3 months of treatment application to ensure the establishment of the mycorrhizal symbiosis, which frequently take place after 2–4 months of inoculation. Similar patterns in AMF colonization intensity were observed in both, 3 months after inoculation and at harvest, where roots from inoculated grapevines showed percentages of colonization values threefold higher than non-inoculated ones . In addition, we observed increased AMF colonization rates along the growing season as shows the significant effect of the sampling date and its interaction with the AMF inoculation . Relative mycorrhizal dependency index allows assessing the dependency of a crop on the mycorrhizal symbiosis to achieve its maximum growth at a given environmental condition. Under FI conditions, RMD values were lower than 100% indicating that the mycorrhizal association impairs the vegetative growth of grapevines; however, RMD values for HI conditions highlighted the role of the mycorrhizal symbiosis for improving grapevine growth under water deficit conditions . Grapevine vegetative growth was also monitored during the 2020 growing season by measuring the green pruning weight, trunk diameter, and leaf area . Measurements before treatment showed no differences between the different plants concerning trunk diameter , corroborating the effect of treatments modulating vegetative growth of vines. Irrigation amount was the main factor affecting both vegetative growth and yield, with grapevines subjected to HI decreasing them . However, as RMD reported AMF inoculation impair the grapevine growth estimated as trunk diameter and as green pruning weight when vines were FI, whereas under conditions, inoculated vines improved their growth . Finally, the leaf area to fruit ratio was not affected by treatments applied. The contents of minerals measured in leaf blades were not affected by AMF inoculation or applied water amount in our experiment . Plant water status was determined by monitoring the SWP each 2 weeks at noon during the growing season.
The SWP values ranged between −0.8 and −1.3 MPa at harvest suggesting that the amount of applied water was successful in reaching the SWP target during the growing season. Irrigation amount was the main factor affecting the water status of vines especially at the end of the growing season. However, before veraison AMF inoculation could increase the grapevine water status under HI conditions . The calculation of the seasonal integral of SWP showed the same pattern; hence, siSWP was mainly affected by irrigation system with HI plants being the most stressed vines . Gas exchange parameters monitored during the season are shown in Figure 3. Carbon assimilation rates increased through the growing season, and were affected by the interaction between AMF inoculation and irrigation amounts . Thus, FII plants showed the highest values of AN at fruit set and harvest, while FINI grapevines increased AN after veraison. Leaf evapotranspiration was slightly modified by treatments at the beginning on the season but no effect was observed later in the season. On the other hand, although no differences in instantaneous water use efficiency were recorded at harvest, AMF inoculated plants showed a better WUE during berry development and ripening . Finally, stomatal conductance was highly affected by the interaction between AMF inoculation and irrigation system during the whole season . Thereby, AMF inoculation of HI plants mitigated the reduction of gs . Primary metabolites and berry fresh weight are presented in Table 4. Must pH, TA, and TSS were not affected by treatments. However, BFW was modified by treatments; hence, AMF inoculation increased BFW of FI plants and decreased in HI . Flavonols and anthocyanins were monitored through berry ripening. The effect of AMF inoculation and irrigation systems on berry skin flavonol content and composition was modulated during the growing season as indicated by the significant interaction of treatments with the sampling dates . At mid ripening, the berry skin flavonol content increased in HINI grapevines . Similarly, quercetin-3-O-glucoside and laricitrin- 3-O-glucoside decreased with AMF inoculation under HIconditions . At harvest, myricetin and quercetin derivatives were the most abundant flavonols found in Merlot berry skins, accounting for more than 40% of the total flavonols. Irrigation treatment was the main factor affecting flavonol content and composition as indicated by the decrease in quercetin, laricitrin, kaempferol, isorhanmetin, and syringetin derivative contents in HI grapevines . It is noteworthy to highlight the increased content of quercetin-3-O-galactoside in HII grapevines . At mid ripening the main anthocyanin was cyanidin-3- O-glucoside, which accounted for ca. 20% .
The total anthocyanin content of Merlot berry skins was not affected by treatments but HI treatment decreased the contents of some anthocyanin derivatives . At harvest, the total anthocyanin content in Merlot berry skins was not affected by different treatments . Malvidin was the most abundant anthocyanin detected in Merlot berry skins , with contents ranged between 23.1% for HINI plants and 28.7% from FII but none of the malvidin derivatives were affected by treatments . The main changes in anthocyanin composition were due to irrigation treatments, thus, HI ledto decreased contents of cyanidin and peonidin derivatives . Finally, an analysis of the relationship between the percentage of AMF colonization and the main flavonoid contents was conducted . The intensity of the AMF colonization had a significant positive relationship with total cyanidins , total peonidins , and total quercetins . In the last decades, warming trends in viticulture areas have been described worldwide . Likewise, weather data recorded during 2020 growing season in Oakville, CA, United States , suggested more stressful conditions for grapevines comparing to the average of last 20 years, blueberry packaging containers challenging their production and quality. Indeed, a recent study based on climate indices suggested a reduction of 8,700 km2 for the Califtornia land suitable for grapevine cultivation by mid-21st century . Within this scenario, smart-farming techniques are mandatory for adaptation and mitigation to guarantee the future of the wine making industry and for reducing potential water conservation issues. Colonization analysis of Merlot grapevine roots indicated that AMF inoculated integrated with the native communities colonizing grapevine roots . Thus, we found that the percentage of mycorrhizal colonization was two to three fold higher in mycorrhizal inoculated treatments compared to non-inoculated ones. However, no differences in mycorrhizal colonization due to water amount received by plants were evident in accordance with a previous study conducted on fruit bearing cuttings . In contrast, a study conducted on own-rooted Cabernet Sauvignon field grapevines reported increased frequency of arbuscules and reduced fine root production when an additional water deficit was applied to the regulated deficit irrigation plot, suggesting that plants could compensate the lower density of fine roots in vines facing water deficit by increasing AMF colonization . These discrepancies between studies may be explained by the fact that grapevines responded to the degree of water deficit from the previous growing season. Thus, Schreiner et al. observed increased arbuscular colonization at bloom, before the onset of differences between the treatments they applied whereas under our experimental conditions, water amounts received by Merlot grapevines the previous season did not differ. AMF colonization data also confirmed the seasonality effect on mycorrhizal colonization and the reinforcement that AMF inoculation exerts on native mycorrhizal colonization . Without imposed water stress, AMF inoculation impaired vegetative growth as indicated in the RMD index. However, when grapevines were subjected to HI treatment, AMF-inoculated vines grew better as indicated by the RMD, green pruning, and trunk diameter. Nevertheless, leaf area was not enhanced after AMF inoculation according to previous studies , which would explain that AMF inoculation was not sufficient to avoid the yield loss due to HI treatment. It is well established that AMF inoculation enhances mineral nutrition of grapevines presumably by a greater exploration of soil by the external hyphal network of the AMF resulting in more efficient roots for obtaining nutrients from soils . Moreover, it was recently reported that the inoculation of grapevines with AMF under controlled conditions led to the upregulation of nutrient transport genes . In spite of the consensus about AMF enhancing grapevine nutrient uptake, contradictory results are reported about increased mineral nutrient content due to the symbiosis . Leaf or petiole mineral nutrient content might be useful for the diagnostic of soil mineral deficiencies allowing growers to manage them. However, concentration of mineral nutrients does not provide accurate information on nutrient uptake or allocation of nutrient in various organs . Therefore, although no differences on the mineral nutrient content in leaf blades were observed, mineral uptake was presumably enhanced by AMF inoculation given the growth promotion recorded in mycorrhizal plants under HI conditions. Furthermore, Balestrini et al. recently reported that although mineral nutrient uptake genes were upregulated after inoculation with different inoculants , the degree of upregulation differed between them, suggesting a specific response to a specific inoculum. Similarly, Nogales et al. did not find accumulation of minerals in grapevine leaves after AMF inoculation with the exception of P, which was enhanced and decreased after F. mosseae and R. irregulare inoculations, respectively. Grapevine water status monitored during the growing season showed that irrigation amounts were the main factor affecting the plant water status. Thus, according to previous work FI plants were maintained under well-watered conditions with values of midday SWP higher than −0.9 MPa and/or gs higher than 200 mmol m−2 s −1 . On the other hand, grapevines subjected to HI were not exposed to a severe water stress as they never reached values of SWP and gs lower than −1.5 MPa and 50 mmol m−2 s −1 , respectively, considered detrimental for grapevine development . We did not measure any SWP differences due to the AMF inoculation when plants were subjected to FI. However, within HI plants, AMF inoculation tended to result in higher SWP values in accordance with previous studies . Therefore, a higher AMF occurrence in the root zone has been related to improve water status of vines by increasing water uptake presumably by increasing the mycorrhizal structures, mainly arbuscules . Accordingly, we observed that photosynthetic performance of AMF inoculated Merlot grapevines was improved .