Month: September 2024

Determining how long grape berries are competent to induce the expression of anthocyanin biosynthetic genes may help determine the optimal time, number, and frequency of S-ABA applications. Currently, little is known about the potential benefits of multiple applications, which may be desirable if a single application results in an insufficient response. The aim of the present study was to determine the effects of S-ABA applications at different concentrations and times on the quality and biochemical properties of berries from the seedless grape Selection 21 hybrid during three growing seasons in the region of Paraná, Brazil. We evaluated a variety of parameters including: grape color development, berry phenolic profiles, and gene expression of transcriptional regulators and biosynthetic enzymes of the anthocyanin pathway after treatments with S-ABA. The results of this report indicate that two S-ABA applications during and after véraison extend the competency of grape berries to respond to ABA and induce the accumulation of anthocyanins, resulting in higher grape berry coloration. The study was conducted during three consecutive seasons in a commercial vineyard located in Marialva, state of Paraná , Brazil , using 4-year-old vines of hybrid seedless grape Selection 21 grafted onto IAC 766 Campinas rootstock. According to the Köppen classification, growing bags the climate of the region is Cfa , with an average temperature below 18◦C in the coldest month and above 22◦C in the hottest month and an average annual rainfall of 1,596 mm. The region’s soil is classified as dystroferric red latosol .

The vines were trained using a bilateral overhead trellis system, where vines were spaced at 2.5 m × 2.5 m , and each vine had 6.25 m2 total canopy area. Cane pruning was performed during the 2013, 2014, and 2015 seasons and was followed by application of 3% hydrogenated cyanamide to the two apical buds to induce and standardize sprouting. The number of canes per vine was evenly adjusted to 40 and the number of shoots per vine was also established to 40 . Considering that a grape bunch of the Selection 21 weighs on average 460 g, the load per vine is 18.40 kg, which represents an estimated yield of 29.44 tons/ha. Furthermore, to avoid drifting, a non-treated vine was left as side border between two treated vines, which almost duplicated the experimental area. In each plot, all grape bunches were treated , and the bunch samples were collected from random positions at each side of the canopy to account for intracanopy variability. Control plants were not subjected to any treatment, instead, they were sprayedwith water at the same time and following the same procedures as the S-ABA treatments.The effects of applying S-ABA isomer at different concentrations and times were evaluated in terms of berry quality traits. ProTone R, the commercial growth regulator used in this study, has an active ingredient concentration of 100 g/L S-ABA. As shown in Figure 1, the initial treatments tested in the 2013 and 2014 seasons corresponded to: control or water spray, 200 mg/L S-ABA application at 7 days after véraison , 400 mg/L S-ABA application at 7 DAV, 200 mg/L S-ABA application at 7 DAV plus an additional application at 21 DAV, and 400 mg/L S-ABA at 7 DAV plus an additional application at 21 DAV. In the 2015 season, only the control and treatments of 400 mg/L S-ABA with one or two applications were performed and analyzed.

Berry samples from the 2015 season were collected from each treatment at four different times: 7 DAV , 14 DAV, 28 DAV, and 35 DAV for further targeted analyses . For all seasons, a randomized complete block experimental design was used, with five treatments and three to four replicates, and with each plot consisting of one vine . Véraison was determined by measuring soluble solid content and firmness of grape berries randomly sampled in the experimental vineyard. At véraison, the mean grape SSC concentration was 9◦Bx, and 20% of the berries in more than 50% of the grape clusters presented softening . The berries presented a mean of 11◦Bx at 7 DAV, the time of the first S-ABA application, and a mean of 13◦Bx at 21 DAV, the time of the second S-ABA application. For treatment applications, grape clusters were sprayed in the morning using a knapsack sprayer at a pressure of 568.93 psi with JA1 hollow cone nozzle tips at a volume of 800 L/ha to provide complete and uniform coverage. In addition, 0.3 mL/L of Break-Thru R a non-ionic surfactant was added to all treatments. During the trials, the standard regional cultivation practices with regard to nutrition, weed control, and pest and disease management were used.Clusters of each plot were manually harvested when SSC stabilized . The clusters were cleaned, and damaged berries were discarded. Color coverage of the bunches was determined using 10 grape clusters per plot by visually rating the clusters on a scale of 1–5 using the following scale: 0–20%, 21–40%, 41–60%, 61–80%, and 81–100% coverage .

The same grape clusters used for evaluating color coverage measurements were used for berry sampling. For physicochemical analyses, two berries were collected from the upper, middle, and lower portion of each grape bunch, yielding a total of 70 berries per plot. Total anthocyanins and color index of red grapes were determined in berry samples from all seasons. The following variables were analyzed only for the 2013 and 2014 seasons: color coverage, total polyphenols, and berry firmness. All physiological analyzes were performed in the Laboratory of Fruit Analysis of the Agricultural Research Center, Londrina State University, Brazil. The total anthocyanin concentration of the berries was determined using 30 berries per plot, which were frozen and stored at −20◦C. The berry skins were removed using tweezers, taking care to remove only the skin, without pulp. The skins were washed once with water, followed by washing in deionized water and drying with absorbent paper. A 5-g skin sample was then placed in a polystyrene tube containing 50 mL of acidified methanol and stored in the dark for 48 h at room temperature. The tubes were then removed from the dark and stirred manually for 5 s. Absorbance was determined using a Genesys 10S spectrophotometer at 520 nm with the solvent as blank. The results were expressed in milligram malvidin-3-glucoside per gram of skin . The CIRG was determined using 10 berries per plot with a CR-10 colorimeter , using the CIELAB color system. The following variables were determined for the berry equatorial section: lightness , saturation , and hue . CIRG was then determined using the following equation: CIRG = / . Total polyphenol determination was performed using 30 berries per plot based on a modified Folin–Ciocalteu method. In summary, the absorbance of each sample was measured after 15 min at 765 nm using a Genesys 10S spectrophotometer against a blank sample prepared with water instead of the extract. Determination of total polyphenol was calculated from the calibration curve obtained with gallic acid. The results were expressed in milligram total polyphenols per 100 g of sample . The berry firmness was performed with a TA.XT2i Texture Analyzer , at 25 ± 1 ◦C, nursery grow bag analyzing the equatorial position of 10 berries with pedicels per plot. Each berry was placed on the base of the equipment and compressed using a cylindrical probe with a diameter of 35 mm parallel to the base. A constant force of 0.05 N at a speed of 1.0 mm/s was applied to promote the cracking of the sample. The berry firmness was then determined .Application of abscisic acid increased the total anthocyanin concentration in berry skins of the seedless grape Selection 21 during the 2013 and 2014 seasons, regardless of the S-ABA concentration and time of application . However, berries that received 400 mg/L of S-ABA at 7 and 21 DAV had significantly higher, almost two to three times more, anthocyanin concentrations than other treatments. According to the CIRG, berries from control treatments had a green to a yellow color in both seasons . In 2013, berries treated with one or two applications of 200 mg/L S-ABA or one application of 400 mg/L S-ABA at 7 DAV, and those in the 2014 season that were treated with one application of 200 mg/L S-ABA developed a pink color . Remarkably, berries of the 2013 season treated with two applications of 400 mg/L S-ABA and berries of the 2014 season treated with one or two 400 mg/L S-ABA applications, developed a stronger red color .

For both the 2013 and 2014 seasons, color coverage was lowest in control grapes and highest in grapes treated with two applications of 400 mg/L S-ABA. Increase in total polyphenols was evident in grapes subjected to two 400 mg/L S-ABA applications during the 2013 and 2014 seasons. These berries also presented the lowest mean berry firmness . Importantly, the increased softening due to S-ABA application did not result in higher frequency of cracked berries in any of the studied seasons. Qualitative assessment of berry cracking was performed visually. Further analyses of the effect of 400 mg/L S-ABA treatments on CIRG, total and individual anthocyanins concentrations, and gene expression of transcription factors and biosynthetic enzymes were performed with grape berries collected from the 2015 trial. Measurements of CIRG confirmed previous results obtained during the 2013 and 2014 seasons, at the time of harvest , grapes treated with two S-ABA applications had the highest CIRG values . Grape bunches from the control treatment presented pink berries , whereas those treated with one or two applications of S-ABA had red berries . As determined in the previous seasons, berries treated with 400 mg/L S-ABA also presented higher total anthocyanin content than the control at 14 and 28 DAV . At 28 DAV, grapes treated with one or two applications of 400 mg/L S-ABA presented total anthocyanin concentrations almost three times higher than the control. Even 3 weeks after the first application , berries treated with only one S-ABA application showed a total anthocyanin content similar to those treated with two S-ABA applications. Nonetheless, the second application of 400 mg/L S-ABA significantly affected the total anthocyanin accumulation at the time of harvest . S-ABA altered the concentrations and proportions of individual anthocyanins in berries from the seedless grape Selection 21 . With the exception of petunidin- 3-glucoside, S-ABA application significantly improved the concentrations of all the measured anthocyanins. Cyanidin-3- glucoside and peonidin-3-glucoside levels increased at 14 DAV, 1 week after the first S-ABA application. The second S-ABA application stimulated the accumulation of the anthocyanin delphinidin-3-glucoside at 28 DAV, yielding differences relative to both the control and to the samples treated with only one S-ABA application. At 28 DAV, the concentrations of peonidin-3-glucoside and malvidin-3-glucoside increased after exogenous S-ABA application but were not further increased by the second application. At the time of harvest , peonidin-3-glucoside and cyanidin-3-glucoside were the dominant pigments present after all treatments. Delphinidin- 3-glucoside, cyanidin-3-glucoside, and peonidin-3-glucoside presented higher accumulation following the second application of 400 mg/L S-ABA, but the number of applications did not affect the accumulation of malvidin-3-glucoside. As presented in Figure 4, treatment with 400 mg/L S-ABA significantly increased the expression of the transcription factors VvMYBA1 and VvMYBA2 and the expression of the bio-synthetic genes CHI, F3H, DFR, and UFGT 1 week after the first application . Three weeks after the first S-ABA application , expression of CHI, F3H, and DFR genes remained high, but this was not observed for the transcription factors VvMYBA1 and VvMYBA2 or the UFGT gene. Four weeks after the first S-ABA application , no significant differences were observed in the expression of genes or transcription factors between berries that received one S-ABA application and those that received the control treatment. The two applications of 400 mg/L S-ABA induced expression of the genes CHI, F3H, DFR, and UFGT and the transcription factors VvMYBA1 and VvMYBA2 at 14 and 28 DAV . F3H expression was the most affected by S-ABA application, displaying higher levels than the control until the final stages of berry maturation at 35 DAV,whereas the remaining genes presented no differences from the control at harvest. Overall, the gene expression results indicate that a second S-ABA application contributed to the maintenance of the expression of the transcription factors VvMYBA1 and VvMYBA2 and the genes F3H and UFGT at higher levels than in the control for an extended period of time.

Although string connections did increase ant resource recruitment efficiency and pest removal rates compared to the control treatments, A. sericeasur exhibited a clear preference for natural vegetation over string connections. Interestingly, while distance from the A. sericeasur nest tree did negatively impact ant activity, recruitment to baits, and ant-mediated CBB removal on the control and string treatments, distance did not affect these response variables on the vegetation treatment plants. Vegetation connectivity influences the distribution, diversity, and interspecific competition of arboreal ant species by affecting the availability of nesting habitats, foraging ranges, and resource availability. Higher degrees of vegetation connectivity provide a range of food resources to arboreal ants, including access to honeydew-producing insects, extrafloral nectaries, and other insects. Arboreal ants can take advantage of these resources more efficiently when connected vegetation provides a network of foraging opportunities, which increases access to patchy resources while enhancing predator avoidance capability. In contrast, disconnected vegetation may limit ant distribution to isolated tree patches. Observed increases in ant activity on plants with vegetation connectivity suggest that structural connectivity facilitates ant mobility and movement efficiency on foraging paths. Between the control, string, and vegetation treatments, ant activity was highest on vegetatively connected plants . After string placement, ant activity did increase on the string plants, indicating that A. sericeasur learned to use strings as foraging paths over time; however, nusery pots the overall ant activity levels on the string treatment plants were not significantly different from the control treatment.

The significant positive interaction between the string treatment and time explains the observed increase in ant activity on the string treatment plants. On the control and vegetation treatment plants, there was no change in ant activity throughout the 5-week duration of the experiment. Consistent with Jiménez-Soto et al., ant activity decreased on coffee plants with increasing distance from A. sericeasur nest trees on the control and string plants. Notably, our additional treatment of naturally occurring vegetation connectivity overrode the effect of distance from ant nest trees, with no impact of distance on the amount of ant activity on the vegetation treatment plants. This important finding suggests that vegetation pathways can facilitate A. sericeasur foraging activity on coffee plants that are farther away from their nest tree. Additionally, increasing their foraging range may help the ants to avoid parasitic phorid flies in the genus Pseudacteon, which parasitize A. sericeasur and decrease in density with increasing distance from the A. sericeasur nests. A. sericeasur activity may be highest on coffee plants with naturally occurring vegetation connections because vegetation connections are generally larger and more structurally complex. Additionally, on the existing vegetation pathways, the ants had more time to establish foraging trails and chemical cues as compared to the string connections. Furthermore, in addition to providing linear foraging trails, vegetation bridges may also contain useful resources including extrafloral nectaries and plant fluids that the strings do not provide. Vegetation pathways can also offer protection from phorid flies beneath the leaves, whereas strings are open and unprotected foraging paths. Studies have also suggested that ants have preferences for foraging on certain surfaces, and that surface characteristics impact foraging speed and chemical communication on the ants’ trails.

The A. sericeasur preference for vegetation surfaces may therefore result from texture-based foraging efficiency differences between vegetation and string. Yanoviak et al. studied ant recruitment to baits on bare vs. moss-covered tree trunk surfaces and observed the Azteca spp. actively avoiding baits on moss-covered trunks, indicating a clear surface preference for smoother pathways. In our study, we observed A. sericeasur walking around stray threads on the jute strings , decreasing their foraging efficiency compared to smoother thread-free vine surfaces. In some instances, we observed A. sericeasur “cleaning” the string pathways by biting off jute string threads from the connections to minimize obstacles and enhance their efficiency on these pathways. Another explanation for higher A. sericeasur activity on the vegetation treatment coffee plants is that A. sericeasur may already be tending established green scale colonies on vegetatively connected plants, drawing their activity to these plants over the string treatment plants. C. viridis, a sessile coffee scale insect, has been linked to increased A. sericeasur activity. In a mutualistic relationship, A. sericeasur protect C. viridis from predation in exchange for the honeydew that these scales produce. Increased connectivity, by increasing ant mobility, may also increase the scale tending activity by A. sericeasur. Notably, interactions between A. sericeasur and CBB on coffee plants occur more frequently with higher densities of C. viridis, indicating a relationship between scale tending activity and CBB control services.Consistent with the ant activity results, the number of ants recruiting to baits on control and string plants declined with distance from the ant nest tree but remained consistent over all distances for vegetation treatment plants. These results confirm the A. sericeasur preference for vegetation foraging paths over artificial ones, as explained in Section 4.1.

Between treatments, the control treatment plants had the lowest ant recruitment to baits. Other ants in tropical systems similarly prefer vegetation pathways over ground and leaf litter, optimizing networks of vines, leaves, and branches in their foraging trails. Clay et al. suggest that ants may even favor vines over bark or moss because the linear nature of vines reduces the necessity for intensive chemical trail maintenance. Strings might similarly provide this linear path advantage, which reduces chemical trail maintenance and opportunities for path confusion compared to ground trails. Because ants account for energy efficiency when deciding between foraging paths, the control plant baits were likely the least attractive because they required the highest energy expenditure due to traveling over ground and leaf litter. Because none of the tuna baits were depleted within the 20 min observation period, recruiting to control baits while more energy-efficient paths were present is an inefficient use of ant workforce.Over time, the overall number of ants that recruited to the baits decreased with time post-string placement on both the control and vegetation plants, but there was no significant change in the number of ants that recruited to the baits on the string treatment. Decreases in ant recruitment rates on the control and vegetation treatment plants may have resulted from the presence of phorid flies, plastic planters which greatly reduce A. sericeasur activity. Phorid fly attacks may have curtailed ant recruitment along the pre-existing foraging routes, as phorids are likely more abundant in leaf litter along popular foraging routes. Because the strings were a novel foraging route, it is likely that fewer phorids frequent those routes and interfere less with ant recruitment to the baits. Between treatments, A. sericeasur removed the most CBB from vegetation treatment plants and removed more CBB on the string plants as compared to the control plants . The overall number of CBB removed on the vegetation plants decreased with time post-string placement. Decreases in CBB removal may similarly have resulted from phorid fly attacks inhibiting pest removal activity, as occurred in Philpott et al. and Pardee and Philpott. Consistent with the results of our ant activity and resource recruitment experiments, the number of CBB removed on the control and string plants declined with distance from the nest tree, but remained consistent with distance from the nest on the vegetation treatment plants. Interestingly, Jiménez-Soto et al. did not find any effect of distance from the nest tree on CBB removal for the control or string plants. Our contrasting results may be the result of the A. sericeasur preference for the vegetatively connected plants in our study; in the absence of vegetation pathways, ants may forage more on artificial connections. Our results reinforce how habitat complexity in the form of vegetation connectivity impacts interspecific interactions, specifically ant-mediated CBB removal at the local scale.Our results confirm the importance of naturally occurring vegetation connectivity and habitat complexity in facilitating arboreal ant mobility and ant-mediated CBB removal. Our findings have important implications for the practical application of ant-provided pest removal in coffee systems, indicating that A. sericeasur may most effectively control CBB on coffee plants with natural vegetation connectivity connected to their nest trees. In the absence of vegetation connectivity, implementing artificial connections between ant nests and coffee plants can increase CBB removal by A. sericeasur; however, with increasing distance from the ant nest tree, the strength of this pest removal service decreases on artificially connected plants. The observed preference of A. sericeasur for vegetation pathways underscores the importance of maintaining or promoting vegetation connectivity via habitat complexity and structural diversity within coffee agroecosystems. In managing agroecosystems in support of ant-mediated ecosystem services, artificial connectivity does not provide an equal substitute for the naturally occurring vegetation connectivity provided through forest conservation and structural complexity. Consistent with studies affirming the influence of vegetation connectivity on predatory arthropod movement and predation range, our results illustrate how vegetation connectivity facilitates A. sericeasur foraging mobility and pest removal. In coffee systems, higher degrees of vegetation connectivity are associated with shade trees, as well as more heterogeneous habitat complexity and variability in plant structure.

In other studies, ants generally increase predation services in shaded systems as compared to monocultures and, in coffee plants, more effectively remove CBB in shaded coffee systems as compared to sun monoculture systems. Interestingly, most studies find the opposite effect of structural complexity on parasitoid behavior, with higher degrees of plant structural complexity leading to decreased parasitoid foraging efficiency. This negative relationship between parasitism and habitat complexity transfers to coffee systems, where the parasitic phorid flies exert a greater inhibiting effect on Azteca ants in simple, low-shade farms thanin complex, high-shade farms. Together with the aforementioned study, our combined results illustrate how habitat complexity at the landscape scale and vegetation connectivity at the plot scale dually facilitate A. sericeasur-mediated pest removal: by facilitating ant mobility and by reducing the efficiency of the parasitoid that interferes with their pest removal ability. In order for A. sericeasur to provide ant-mediated pest removal services, coffee agroforests must include enough shade trees to provide sufficient habitats for ant nests. Planting coffee plants close enough to shade trees to allow for direct connectivity and leaving some vegetation connections between coffee plants and shade trees rather than chopping them or relying on herbicides can facilitate ant-provided ecosystem services by providing foraging paths through naturally occurring structural connectivity. By enhancing the A. sericeasur effectiveness in controlling CBB populations, vegetation connectivity can potentially reduce chemical pesticide use. Our results offer management insight into one piece of a complex ecological puzzle. Because A. sericeasur tend C. viridis, they could indirectly reduce coffee plant growth by contributing to high-scale densities and an associated damaging sooty mold. However, high densities of C. viridis also beneficially attract Lecanicillium lecanii, which attacks coffee leaf rust, a devastating coffee fungal disease. Moreover, the CBB is regarded as a far more damaging coffee pest than C. viridis. Furthermore, facilitating the mobility of A. sericeasur as a single ant species is not necessarily the most effective pest management approach, as higher ant diversity can improve pest control through the cooperation of complementary predatory species. Enhanced A. sericeasur activity on coffee plants could alter the behavior of other ant species, which could have positive or negative effects on overall pest control services due to spatial complementarity or potential negative interactions between predators. However, studies find that increasing connectivity generally increases species richness, and so, vegetation connections that increase A. sericeasur mobility likely facilitate the mobility of other predatory ants in coffee systems, even by providing alternative paths to avoid aggressive altercations with A. sericeasur. Although A. sericeasur occupies only 3–5% of the shade trees at our research site, other ants known to contribute to CBB regulation would likely also use vegetation pathways, facilitating additional pest control. Future research should examine how vegetation connectivity impacts the abundance and diversity of other ant species on coffee plants and the associated spatial complementarity between specific predators of the CBB. Future studies could also investigate how phorid attacks on Azteca vary on different foraging pathways to better understand the mechanisms behind their preference for vegetation pathways. The earliest cultivars of allo-octoploid garden strawberry originated approximately 300 years ago from spontaneous hybrids between ecotypes of non-sympatric wild octoploid species: Fragaria chiloensis subsp. chiloensis from South America and Fragaria virginiana subsp. virginiana from North America.

Nematicides have been commonly used to control PPNs in agriculture, but some nematicides such as methyl bromide and aldicarb are currently banned from use in many countries due to their negative effects on the environment and human health . It has therefore become important to understand the molecular mechanisms of plant immunity against PPNs to provide a foundation for the development of new environmentally friendly and effective control methods. In general, the plant immune system is represented by two inter-related tiers . The first is governed by cell surface-localized pattern recognition receptors that perceive pathogen associated molecular patterns , leading to pattern triggered immunity . Successful pathogens secrete effector molecules into the apoplast ordirectly into plant cells, which interfere with PTI, resulting in successful infection. Resistant plants recognize cell-invading effectors through recognition by intracellular nucleotide-binding domain leucine-rich repeat -type immune receptors, which are encoded by resistance genes. Similar mechanisms are also conserved in plant-PPN interactions. For example, the well conserved nematode pheromone ascaroside has been identified as a PAMP , but the corresponding PRR has not yet been found. PPN genome sequence analyses identified a number of candidate virulence effectors , and a handful of NLR protein-encoding R genes involved in PPN recognition have been well-studied and characterized, including tomato Mi-1.2, Mi-9, and Hero-A; potato Gpa2 and Gro1-4; pepper CaMi; and prune Ma . Mi-1.2, Mi-9, CaMi, and Ma confer resistance against RKNs, black flower buckets whereas Hero-A, Gpa2, and Gro1-4 provide resistance against CNs.

Although the PPN perception mechanism is somewhat clearer at the molecular level, it is still largely unknown what kind of downstream responses are induced after the recognition of avirulent PPNs. It is also unclear what kind of host responses are induced after infection with virulent PPNs, leading to susceptibility and infestation. There are several difficulties in working on plant responses against PPNs. First and foremost, most model plants, such as Arabidopsis, are susceptible to PPNs and therefore cannot be used to study the cascade of responses leading to resistance. Second, PPNs migrate long distances inside roots, inducing complicated responses as they go, triggered by mechanical stress and wounding, among others, making it difficult to pinpoint the key genes involved in resistance or susceptibility by transcriptome analyses. Some studies have used comparative transcriptomics using susceptible and resistant plants infected with a single genotype of nematode . However, it is difficult to rule out the possibility that differences in gene expression were due to resistance or susceptibility rather than to differences in the genetic backgrounds of host plants. Lastly, susceptible responses such as the formation of feeding sites are induced in specific cells targeted by PPNs, and defense responses are likely to be induced in the cells directly impacted by PPN activity. Thus, cells responding to PPNs are rather limited, making the analysis technically challenging. Here we have introduced Solanum torvum Sw “Torvum Vigor” to overcome these problems. S. torvum has been widely used as a rootstock of eggplant to prevent disease caused by PPNs, as well as the soil-borne pathogens Ralstonia solanacearum, Verticillium dahliae, and Fusarium oxysporum f. melongenae n. f. . S. torvum Sw “Torvum Vigor” is resistant to Meloidogyne arenaria pathotype A2-O , but susceptible to M. arenaria pathotype A2-J .

By using S. torvum and avirulent or virulent isolates, we established an in vitro infection system and performed comparative transcriptome analyses to identify genes whose expressions were associated with either resistance or susceptibility by carefully collecting only root tips attacked by RKNs, which allowed us to detect gene expression only in cells directly affected by nematodes. In addition, observation of infected root tip morphology suggests that the success or failure of the immune system against PPNs is determined within a few days of invasion. Thus, we decided to focus on the transcriptional changes that occurred in the very early stages of infection, which has not been studied in previous transcriptomic analyses . Comparative clustering analyses of gene expression identified a large number of novel genes, especially those involved in susceptibility through cell wall modification and transmembrane transport; resistance through lignin and isoprenoid biosynthesis and fatty acid metabolism; and suberin biosynthesis in mechanical wounding. Consistent with the transcriptional up-regulation of lignin biosynthetic genes from A2-O invasion, lignin is accumulated at the root tip of S. torvum infected with avirulent A2-O but not with virulent A2-J, suggesting that S. torvum reinforces the cell wall as a defense response against the avirulent RKN. M. arenaria pathotypes A2-J and A2-O were propagated on Solanum lycopersicum cultivar “Micro-Tom” in a greenhouse. Nematode eggs were isolated from infected roots and then hatched at 25 ◦C. Freshly hatched J2s were collected and transferred to a Kimwipe filter placed on the top of a glass beaker filled with sterilized distilled water containing 100 µg/ml streptomycin and 10 µg/ml nystatin. Only active J2s pass through the filter. Filtered J2s were surface sterilized with 0.002 % mercuric chloride, 0.002 % sodium azide, and 0.001 % Triton X-100 for 10 min, and then rinsed three times with SDW . Eleven-day-old S. torvum seedlings grown on the MS-Gelrite in 6-well plates were inoculated with 200–300 J2s resuspended in SDW.

The plates were wrapped in aluminum foil for 2–3 days after inoculation to promote nematode infection. When mature giant cells were observed 18 days post-inoculation , we used the MS-Gelrite media without sucrose to prevent the formation of callus-like structures. The difference in the number of normal galls formed by A2-J or A2-O at 4 DPI was statistically tested using the Mann-Whitney U test with R software . Nematodes resident in root tissues were stained with acid fuchsin 2–4 DPI , photographed by light microscopy , and the photomicrographs were processed using cellSens . For the observation of giant cells and developing nematodes at 18 DPI, infection sites were fixed with glutaraldehyde and cleaned with benzyl-alcohol/benzyl-benzoate . We observed BABB-cleaned samples by confocal laser scanning microscopy . Photomicrographs were processed using LAS X software . S. torvum seedlings were grown on half-strength MS-Gelrite medium containing 1% sucrose. Eleven-day-old seedlings were treated with SDW as a mock infection or with 200–300 J2s of M. arenaria A2-J for susceptible infection or A2-O for resistant infection. Root tips attacked by the nematodes were checked under microscopy, and more than 50 root tips were cut and collected for each treatment . Root tip samples were collected at 1, 2, and 3 DPI with four biological replicates. Whole shoot and root samples were collected at 1, 3, 6, and 9 DPI with four biological replicates. Root tip samples were used for de novo assembly and differential gene expression analyses, and whole shoot and root samples were used only for de novo assembly. RNA-seq libraries were prepared from the collected samples using a high-throughput RNA-seq method . The 85-bp paired-end reads for the root tip samples, and the 85-bp single-end reads for the whole shoot and root samples were sequenced on an Illumina NextSeq 500 platform . The FASTX toolkit 0.0.13.2 was used for quality filtering. Low-quality nucleotides were removed from the 30 ends, french flower bucket and short reads were excluded. Reads with at least 95% of nucleotides with Phred scores > 20 were kept and used for the downstream analyses . Filtered reads were mapped to the genome assembly of M. arenaria A2-J or A2-O using HISAT2 to exclude reads of nematode origin. Unmapped reads were used for de novo transcriptome assembly . Three different transcriptome assemblers were used for de novo assembly: SOAPdenovo-Trans v1.03 , Velvet v1.2.10 /Oases v0.2.09 and Trinity package v2.4.0 . Unmapped paired-end and single end reads were normalized using Trinity and assembled independently . Oases assembled scaffolds were split at gaps into contigs before merging with contigs from the other assemblies with the EvidentialGene tr2aacds pipeline. The tr2aacds pipeline produces ‘primary’ and ‘alternate’ sequences of non-redundant transcripts with ‘primary’ transcripts being the longest coding sequence for a predicted locus. Next, we used the evgmrna2tsa program from EvidentialGene to generate mRNA, coding, and protein sequences. BUSCO v3.0.2 was applied for quantitative assessment of assembly completeness. This assembly and one previously reported for S. torvum by Yang et al.were compared to the Embryophyta odb9 dataset, which contains 1,440 BUSCO groups. The homology of the contigs from the final assembly was searched against the NCBI non-redundant database using BLASTX with an e-value threshold of 1E-05.

We also compared the contigs with Arabidopsis genome annotation  using BLASTX at the e-value cutoff of 10. Results of the annotation are summarized in Supplementary Table 2. To group genes by expression pattern, we applied the self organizing map clustering method on genes within the top 25 % of the coefficient of variation for expression across samples as previously described . Scaled expression values, representing the average principal component values among each gene in a cluster were used for multilevel three-by-three hexagonal SOM . The final assignment of genes to winning units formed the basis of the gene clusters. The results of SOM clustering were visualized in a principal component analysis space where PC values were calculated based on gene expression across samples . We compared the contigs of our assembly with the NCBI non-redundant database using BLASTX with an e-value threshold of 1E-05. In addition, predicted amino acid sequences that begin with methionine were also annotated using InterProScan . BLASTX and InterProScan outputs were used for Blast2GO analysis to annotate the contigs with Gene Ontology terms . GO enrichment analyses of the sets of genes induced by A2-O infection at 1 DPI or that were assigned to each cluster generated by SOM was performed by comparison with all genes using GO terms generated by Blast2GO at the FDR cutoff of 1E-04 . We further used the “Reduce to most specific terms” option in Blast2GO to remove general GO terms and obtain only the most specific GO terms.Quantification of aliphatic suberin was performed as described previously . Eleven-day-old plants were treated with SDW or infected with A2-J or A2-O. At 4 DPI, root tips inoculated with nematodes were microscopically checked for infection, and more than 50 infected root tips were cut and collected for each treatment. To remove unbound lipids, samples were extracted in methanol for 24 h then in chloroform for 24 h, dried, and weighed. Samples were depolymerized and analyzed by gas chromatography-mass spectrometry for monomer identification and for quantitative analysis based on an internal standard using an identical gas chromatography system coupled with a flame ionization detector as described previously . To understand the differential responses of S. torvum to M. arenaria A2-J and A2-O, we first established an in vitro infection system. Seedlings of S. torvum were grown in MSGelrite plates for 11 days and then inoculated with 200–300 J2s of A2-J or A2-O. At 4 DPI, more than 90 % of root tips infected with A2-J induced the formation of gall-like structures ranging in size. These galls are classified here as “normal” galls, while the rest produced brown pigments. Normal galls lacked obvious brown pigment accumulation and were further classified based on the width of the gall into small , medium , and large . In contrast, about 60 % of A2-O-infected root tips accumulated at least some brown pigment. Some of these brownish root tips also had an abnormal appearance due to the formation of balloon-like structures, and others had many localized and highly pigmented spots. There were a very few small gall-like structures formed after infection with A2-O, but far fewer and smaller than in root tips infected with A2-J . RKN staining by acid fuchsin revealed that both A2-J and A2-O successfully invaded the roots . Interestingly, host cells invaded by A2- O uniformly accumulated brownish pigments, suggesting that the surrounding tissue is strongly responding to, and highly correlated with A2-O infection, a response that was absent from A2-J infected roots. It is generally known that browning of plant tissue is related to enzymatic or non-enzymatic oxidation of phenolic substances , but the identity of the brown pigments synthesized upon infection with A2-O is unknown. By 18 DPI, A2-J had induced the formation of mature multinucleate giant cells and developed into fourth stage juveniles . In contrast, A2-O did not induce the formation of giant cells nor develop past second stage juveniles.

A test run was conducted on 10 October 2013 to determine the optimal runoff sampling time intervals and the number of samples needed to capture the peak and total loadings. For the test run, the water soluble fertilizer was applied to both of the sites at a rate of 2.24 g m−2 . Grab samples were collected at a 10-min frequency over a two hour irrigation period from the control site. Water samples were collected immediately before the runoff was directed to the underground tank. The water samples were coarse filtered during sample collection with coffee filters to remove large tree leaves, grass clippings and large soil particles. Based on the results of the test run, the water sampling frequency for subsequent trials was extended to six hours with a variable sampling interval to better characterize the runoff pollutant pattern. The composite water sample from the treatment site was used for calculating the total loading of the treatment site where little surface runoff occurred in this study. A 5.1 cm diameter and 0.9 m long PVC drainage pipe was vertically installed into the middle of the treatment bioswale to collect a representative water sample for monitoring pollutants concentration dynamics in the bioswale. The treatment site water samples were not affected by successive flow from the control site because the treatment site was located upslope of the control site. The water sample collected from the control site was surface runoff, which was not affected by subsurface flow of the treatment site because of the site’s relatively flat surface. The oomycete pathogen Phytophthora cinnamomi Rands, procona buckets causal agent of Phytophthora root rot , is the most destructive disease of avocado worldwide .

In California, avocado PRR affects 60-75% of avocado growers who lose approximately $40 million annually . This globally distributed oomycete is called “the biological bulldozer” for its capacity to infect over 3000 plant species causing devastating impacts in natural ecosystems, forestry, agriculture, and the nursery industry . The economic impact due to P. cinnamomi infestation is evident in the forest and food industry, affecting eucalyptus, pine, oak , and other fruit crops such as pineapple, peach, and high bush blueberry . Losses include not only decreases in crop yield and product value, but also large amounts of money spent annually on control measures. There are no effective means to eradicate P. cinnamomi from infested areas as it survives in moist soil or dead plant material as chlamydospores for long periods under adverse conditions . Several PRR control strategies have been found to reduce the impact of this invasive pathogen including the use of chemical treatment , tolerant plants, and management practices . P. cinnamomi is a hemibiotrophic pathogen feeding initially from living host cells and then switching to necrotrophy by killing the host cells and feeding from the nutrients released by them . The entry into the plant is achieved by the adhesion of the motile zoospores to the host tissue, encystment, and germ tube formation. The germ tubes usually grow and penetrate the root surface via appressorium like swelling structures and then plant tissue is rapidly colonized . During its biotrophic stage, P. cinnamomi projects haustoria into the plant cells for the acquisition of nutrients and release of pathogen proteins to aid the infection process in the host . This is followed by a necrotrophic stage characterized by host cell death, hyphal proliferation, and production of numerous sporangia .

Currently, the molecular and genetic basis of P. cinnamomi pathogenicity, virulence, and plant immunity against this pathogen are largely unknown due to limitations associated with tree crop biology and the lack of tools available for functional studies in tree crops such as avocado . The model plant, Nicotiana benthamiana , has been widely used to study the pathogenicity and virulence of similar broad range and root Phytophthora pathogens such as P. capsici , P. palmivora , and P. parasitica . Moreover, several studies using model plants, crops, and tree crops to study pathogenicity, virulence, and fungicide efficacy of PRR pathogens such as P. sojae, P. capsici, P. parasitica, P. palmivora, P. cinnamomi, and P. ramorum have been done using detached-leaf assays . Phosphite is the most widely used chemical control method for managing PRR caused by several Phytophthora spp. including P. cinnamomi . Phosphorous acid dissociates to form the phosphonate ion , also called phosphite. Phosphorous acid and its ionized compounds are often referred to as phosphonate or phosphonite. The specific mode of action of potassium phosphite is largely unknown, however appears to involve both a direct and an indirect effect on the pathogen . Several studies have assessed the in vitro sensitivity of P. cinnamomi to phosphite using mycelial radial growth inhibition in solid and liquid media to identify sensitive and tolerant isolates . In California, avocado growers heavily rely on the use of phosphite products to control P. cinnamomi, however the phosphite sensitivity of California avocado isolates is largely unknown. In addition to phosphite, phenylamide fungicides such as metalaxyl and mefenoxam are also used for managing diseases caused by oomycetes including P. cinnamomi . Resistance to metalaxyl has developed in P. capsici, P. infestans, and P. nicotianae . Phenylamides usually do not inhibit germination of sporangia or encysted zoospores as effectively as they do mycelial growth . Consequently, inhibition of mycelial growth in vitro has been used as the primary method of determining the sensitivity to these fungicides among isolates of Phytophthora spp. .

The need for new oomycete-targeted fungicides to control diseases caused by these pathogens especially those that have developed resistance to phenylamide fungicides has resulted in the development of several new chemicals with varying modes of action such as fluopicolide and oxathiapiprolin . Fluopicolide is a pyridinylmethyl-benzamide fungicide that disrupts cell division and mitosis by acting on spectrin-like proteins . This fungicide is effective to control diseases caused by P. capsici and P. infestans . Oxathiapiprolin is the first of the new piperidinyl thiazole isoxazoline class fungicides discovered and developed by DuPont Co. in 2007. The molecular target of oxathiapiprolin is the oxysterol binding protein . This new fungicide exhibits strong inhibitory activity against a range of agriculturally important plant-pathogenic oomycetes including P. capsici, P. infestans, P. sojae, Peronospora belbahrii, and Pythium ultimun . However, its inhibitory activity against P. cinnamomi has not been tested. P. cinnamomi is a heterothallic species that requires the presence of both A1 and A2 mating types to undergo sexual reproduction. Despite that both mating types arepathogenic , avocado PRR disease in California is mainly associated with A2 mating type isolates . Previous P. cinnamomi population studies have revealed low levels of genotypic and phenotypic variation among isolates from different mating types, origin, isolation source, and host plants, however, only a few were conducted or have included isolates from avocado . These studies described the existence of three clonal lineages for P. cinnamomi, one corresponding to the A1 mating type isolates and two different clonal lineages for the A2 mating type isolates . Pagliaccia et al. conducted the first study to assess the genetic diversity of P. cinnamomi isolates from avocado in California and also found two genetically distinct clades of A2 mating type isolates . The A2 clade II consisted of isolates with unique genotypes collected only in Southern California. Interestingly, the authors in this study included isolates from P. cinnamomi previously identified as belonging to the A2 type 1 and A2 type 2 described by Dobrowolski et al. , however these isolates clustered within the A2 clade I group, suggesting that the A2 clade II group identified in Paglaccia et al. in California could be another clonal lineage. No studies have been conducted to assess the phenotype of avocado isolates corresponding to these genetically distinct A2 groups identified in California by Paglaccia et al. , therefore, procona florida container the objectives of this study were to i) assess the phenotype of several avocado isolates corresponding to these A2 clades regarding in vitro mycelial growth rate, optimal growth temperature, sensitivity to potassium phosphite and mefenoxam, and virulence, ii) test the sensitivity of avocado isolates to fluopicolide and oxathiapiprolin as alternative chemistries for controlling avocado PRR in California, and iii) develop and validate a detached leaf assay inoculation method using N. benthamiana to circumvent the difficulties associated with the avocado whole plant root inoculation method to assess the virulence of P. cinnamomi isolates. This information will help to design appropriate measures for managing avocado PRR in California and implement efficient and reliable screening methods towards the selection and development of new P. cinnamomi resistant avocado rootstocks effective against a more diverse pathogen population.A total of twelve P. cinnamomi isolates associated with avocado PRR were used in this study, including six isolates from Northern California corresponding to the A2 clade I group, four isolates corresponding to the unique A2 clade II subpopulation from Southern California, and two isolates collected from the same Southern California region from where the A2 clade II isolates were collected .

A2 clade I isolates from this study represent five of the ten total genotypes identified by Paglaccia et al. . Isolates representing the A2 clade II group covered three from the six unique genotypes identified in Southern California. All the isolates in this study were obtained from a P. cinnamomi small collection at the UCR Avocado Rootstock Breeding Program. Isolates were maintained as water agar plugs . Agar plugs were removed one at the time as needed for each experiment and plated on 10% clarified cV8A agar to resume growth. To ensure that isolates in the collection are free of contamination and avoid any problems with mix samples or not appropriate maintenance of the collection, we sequenced all the isolates using ITS and COX1 spacer regions. Mycelial DNA was extracted using the Qiagen DNeasy plant mini kit . Universal primers ITS1 and ITS4 were used for the ITS region, and primers OOM and FM85 were used for the COX1 spacer region, both sets of primers are described in Kroon et al. . Each 25-µl PCR reaction contained 2 µl of DNA , 2.5 µl of 10x PCR buffer , 200 µM dNTPs, 0.4 µM of each primer, and 1.25 units of Taq DNA polymerase . PCR reactions were performed using a Programmed Thermal Controller with conditions as follows: 95°C for 5 min; followed by 35 cycles at 95°C for 1 min, 50°C for 1 min, and 68°C for 1 min; and a final extension at 68°C for 10 min. PCR products were confirmed on 1% certified molecular biology agarose gel , stained with ethidium bromide , and visualized under UV light using a Universal Hood UV Light Table . PCR products were treated with Zymo DNA Clean and Concentrator according to the manufacturer’s instructions to remove excess primers and nucleotides before submitting the samples for Sanger sequencing. DNA sequencing was conducted in both directions with the same primers used for amplification at the UCR Genomics Core facility. Contiguous sequences were generated which were then subjected to a BLASTn search to determine the highest maximum identity to the sequence of the type isolates in GenBank.Two avocado rootstocks were selected based on their P. cinnamomi resistance phenotype. Clonal rootstocks corresponding to the moderate resistant Dusaâ and the susceptible PS.54 were obtained from Brokaw Nursery. The 6-month-old clonally propagated plants were removed from their bags and transplanted into pots after the nurse seed was removed. Plants were grown in a greenhouse with an average maximum temperature range of 25-28°C at 40-50% relative humidity. Plants were fertilized twice a week and watered every day. Nicotiana benthamiana seeds were germinated in trays and transferred to individual pots 2 weeks post germination. Plants were grown at 22°C with 16 h of light and 8 h dark cycles at 40-50% relative humidity. The effective concentration of potassium phosphite at which 50% of the mycelial growth of each isolate was inhibited was determined using the traditional agar dilution method as described in Adaskaveg et al. . Potassium phosphite was added to 10% cV8A to obtain final concentrations of 5, 25, and 100 µg/ml. Mycelial agar plugs from 6-day old cultures of P cinnamomi were placed at the center of the plates containing the corresponding chemical concentration. Three replicates were done per each treatment. After incubation for 3 days in the dark at 22°C, radial colony growth was measured. Colony growth inhibition of each isolate in the presence of potassium phosphite was then calculated as compared with their corresponding control plates without potassium phosphite.

The leaf positioned at the front of the cluster was specifically selected, and the petiole was immediately separated from the blade. The petioles were transported to the laboratory, where they were triple-washed with distilled water to remove any impurities before being sent to a private laboratory for nutrient analysis. In the winter, soil samples were collected at a depth of 30 cm and at a distance of 30 cm from the vine. These samples were transported immediately to the laboratory for analysis. The nutrient content was determined using the methods described in US Salinity Laboratory Staff .The taste panel evaluation of Scarlet Royal table grapes was conducted with the participation of twelve nonprofessional panelists. Astringent taste perception was assessed using a scale ranging from one, representing an extremely low level of astringency, to seven, indicating an extremely high level of astringency. The taste evaluation was performed on 24 clusters from each vineyard. Phenolic compounds analysis. Total phenolic analysis was performed on 250 grams of whole berries by ETS laboratory using a reversed-phase HPLC method adapted from Price et al. .Total RNA was isolated from whole berry samples following the protocol described by Boss et al. . To remove any residual DNA, RNase-free RQI treatment was performed according to the manufacturer’s instructions , and the samples were further purified using the RNeasy mini kit . For RNA-seq analysis, a total of 8 RNA-seq libraries were generated, plasit plant pot comprising four biological replicates from each of the two vineyards . The libraries were constructed as previously described using the NEBNext Ultra II RNA Library Prep Kit for Illumina .

Subsequently, these libraries were pooled in equal amounts and subjected to paired-end 150-base sequencing on two lanes of the NovaSeq 6000 platform at the Novogene Co., Ltd .Illumina sequencing of the multiplexed RNA-seq libraries resulted in 8 FASTQ files containing sequences, and the dataprocessing followed the methods described in our previous work . In summary, the quality of reads was assessed using FASTQ before and after trimming with Trimmomatic v0.39 . Subsequently, the trimmed reads were quantified using Salmon in non-alignment based mode to estimate transcript abundance . The transcripts were mapped to the Vitis transcriptome file “Vvinifera_457_v2.1.transcript_primaryTranscriptOnly.fa” extracted from Phytozome database , resulting in a mapping rate higher than 61.9% . To identify differentially expressed genes between V7 and V9 at the sampling point, we utilized the DESeq2 and EdgeR packages with default parameters . For convenience, the DEGs generated by both DESeq2 and EdgeR pipelines, with a threshold of PFDR<0.05 and log2fold change > 1.5 or < –1.5, were considered as being expressed . For the analysis of Gene Ontology terms, we employed the g:Profiler website with the g:SCS multiple testing correction method, using a significance threshold of 0.05 . Finally, to visualize the consensus result, the Web-based tool Venny was used .Despite the premium fruit quality of the variety, in some cases, an undesirable taste was observed under certain unknown circumstances. To gain comprehensive insights into the development of the occasional berry astringency of Scarlet Royal and understand the underlying mechanism of this phenomenon, berries were investigated at two contrasting vineyards , both following the same commercial cultural practices. However,leaf petioles analysis of grapes from both vineyards showed considerable differences in nutrient levels, especially in the primary macronutrients .

During both seasons, the amount of nitrogen in the form of nitrate in LP-V9 was roughly 2 to 3 times higher than the normal levels, in contrast to its counterpart in LP-V7, which slightly accumulated more or less N. Similarly, LP-V9 contained higher percentages of phosphorus and potassium compared to LP-V7 . Conversely, the amounts of secondary macronutrients, calcium and magnesium , in LP-V7 were within the normal range but greater than LP-V9, which showed Mg deficiency in the first year only. Regarding the micronutrients, their levels were mainly within or around the normal range at both vineyards and during both seasons, with some differences . For example, zinc was slightly higher in LP-V9, especially in the first year. On the contrary, manganese and chlorine were roughly 2 times higher in V7 . Similarly, soil analysis shoed a higher level of nitrogen, potassium and magnesium . However, no significant difference was observed in all other soil macro and micronutrients. During the two seasons of the study, we determined the total marketable yield and the number of clusters in both vineyards. Our data revealed a higher yield in V7 compared to V9 in 2016 and 2017, respectively. The lower yield in V9 can likely be attributed to the smaller number of clusters in V9 compared to V7 during 2016 and 2017. To monitor the changes in the biochemical composition of Scarlet Royal berries, V7 and V9 berries were periodically sampled at six time points from veraison until the end of the season . The obtained data showed that berry polyphenols exhibited discernible patterns in both vineyards, most notably during the ripening stage . Of special interest were the tannin compounds, which widely affect organoleptic properties such as astringency and bitterness . Our data showed that berries from both V7 and V9 vineyards maintained lower levels of tannin from veraison up to the middle of August . Subsequently, a significant gradual increment of tannin took place. However, only V9-berries showed consistent accumulation of tannin over the two studied seasons compared to V7-berries, where the significant induction occurred only during the first season.

It is worth noting that the levels of tannin were lower in both vineyards during the second year compared to the first season. Nevertheless, they were more pronounced in V9-berries compared to V7-berries, with roughly 2- to 4.5-fold increases by the end of the harvesting time during the two seasons, respectively . The patterns of catechin and quercetin glycosides were inconsistent during both seasons, particularly within V7-berries . During the first year, for instance, the levels of catechin were similar in both vineyards, showing a dramatic increase only by the end of the season . In contrast, during the second year, such induction of catechin was exclusively restricted to V9-berries, starting from time S3 . For quercetin glycosides, V7-berries exhibited significantly higher amounts at early stages during both seasons relative to V9-berries . However, subsequent amounts were comparable in both vineyards during the first season only , but not in the second one, where V7-berries showed a significant drop at the last sample S6 . Interestingly, the levels of quercetin glycosides were roughly equal at the last V9-berries sample between both seasons despite such inconsistency. For total anthocyanins , the levels in early samples were comparable in both vineyards and seasons . Afterwards, their pattern started to vary between V7 and V9 within the same season, as well as from the first season to the second, as the nutrient amounts fluctuated as well . Nevertheless, TAC accumulation was positively correlated with the progress of ripening in V7-berries, but not V9-berries. To further confirm our data, we measured these phenolic compounds for the third time in mid-September of the next year . Overall, the results showed that the patterns of tannins and TAC were reciprocally inverted between V7-berries and V9-berries as ripening advanced. In addition, both catechin and quercetin glycosides most likely followed the pattern of tannins despite their seasonal fluctuations. To further distinguish V7-berries and V9-berries and assess their astringency development, a panel test was performed using samples at three commercial harvest times . A group of 12 nontechnical panelists scored berry astringency on a scale from 1 to 7, plastic planter pot where 1 is extremely low and 7 is extremely high. The panelists were trained using samples from contrasting standard varieties, including Flame Seedless and Crimson as non-astringent and Vintage Red known for its astringent taste . The results showed that V7-berries exhibited lower intensity of astringency compared to V9-berries . As ripening proceeded, astringency levels increased in V9-berries, but decreased in V7-berries. Moreover, we collected samples from clusters with various astringent taste and measured its tannins content. We were able to determine that the threshold level of tannins that causes the Scarlet Royal astringency taste is around 400 mg/L . Taking into account the levels of polyphenol compounds and the taste panel data together , it is evident that astringency development is positively associated with tannins’ accumulation throughout the ripening process of V9-berries. Nevertheless, organoleptic analysis revealed a significant difference in the berries of the two vineyards, particularly in terms of total soluble solids and titratable acidity . Notably, V9 berries exhibited higher titratable acidity and lower total soluble solids, especially in the later stages . It’s worth noting that the weight of V9 berries is also higher than that of V7 .To better understand the molecular events associated with the induction of tannins and astringency upon ripening, the berry transcriptome profile was analyzed in both V7-berries and V9- berries at the late commercial harvest date . Following the quality and quantity check, extracted RNA from quadruplicate samples was deeply sequenced .

Of the 19.7 to 24.4 million high-quality clean reads per replicate, 61.9% to 66.1% were mapped against the V. vinifera transcriptome . Hierarchical clustering of the RNAseq data showed explicit changes in the berry transcriptome profile between V7- berries and V9-berries . The Principal Component Analysis showed high consistency among biological replicates . Samples were mainly separated along the first component , which was responsible for 97% of the variance, and was definitely associated with the site of cultivation; V7 and V9. In contrast, the second component was trivial, accounting for only 1% of the variance and was probably attributed to experimental error. Such results were expected, as berry samples came from the same cultivar, Scarlet Royal , and the only difference between them was the vineyard locations. To identify the differentially expressed genes in V7- berries and V9-berries at this specific time within the ripening window, the RNAseq data were analyzed using two different Bioconductor packages, DESeq2, and EdgeR . Subsequently, the DEGs with FDR < 0.05 and log2fold change > 1.5 or < –1.5 generated by both pipelines were considered . The pairwise comparison between berry transcriptomes resulted in 2134 DEGs, with 1514 up-regulated and 620 down-regulated . The data manifested the impact of the cultivation site on the transcriptional reprogramming of a large number of genes that ultimately affect berry quality. Most apparently, at the V9 vineyard, where roughly 2.5-fold higher number of berry transcripts were upregulated compared to V7 . Subsequently, the enrichment of Gene Ontology terms and Kyoto Encyclopedia of Genes and Genomes pathways were analyzed among the up- and down-regulated DEGs using the Vitis vinifera Ensembl GeneID . Among the significantly enriched GO terms, the up-regulated transcripts in V9-berries exhibited high enrichment in the molecular function GO terms for quercetin 3-O-glucosyltransferase activity and quercetin 7-Oglucosyltransferase activity . Additionally, the V9-berries induced DEGs were highly enriched in the biological process GO terms for the jasmonic acid signaling pathway and cellular response , Lphenylalanine metabolic process , L-phenylalanine biosynthetic process , and nitrogen compound metabolic process . Similarly, these DEGs were highly enriched in the KEGG pathways for the biosynthesis of secondary metabolites and phenylpropanoid biosynthesis . On the other hand, the down-regulated transcripts in V9-berries showed substantial augmentation in the MF GO terms for hormone binding , abscisic acid binding , and potassium ion transmembrane transporter activity . Correspondingly, the BP GO terms for hormone-mediated signaling pathway and response , auxin-activated signaling, cellular response, and homeostasis , abscisic acid-activated signaling, response, and cellular response , response to strigolactone , potassium ion transmembrane transport , and potassium ion transport , as well as the KEGG pathways for plant hormone signal transduction , brassinosteroid biosynthesis , and carotenoid biosynthesis were highly enriched in the down-regulated genes of V9-berries . Overall, the transcriptome analysis pointed out the substantial changes in transcript abundance that coordinate and reflect the observed induction of tannins/astringency during the maturation and ripening of V9-berries compared to the V7-berries .To elucidate which fundamental processes were altered during tannins/astringency induction within berries, the Weighted Gene CoExpression Network Analysis was applied to construct coexpression networks. Forty-two modules were identified based on pairwise correlations among the 17553 non-lowly expressed genes .

Binational workers, to be sure, own property in their home communities and view Mexico as their principal residence even when most household members may be in Santa Maria during the greater part of the year. Their domestic economy integrates resources and earnings on both sides of the border to, on one hand, support all family members and, on the other, to improve homes, farms, and businesses in Mexico. Typically, they save and accumulate earnings in Santa Maria to invest in Mexico in the hopes of developing a resource base that will eventually allow the family to live there permanently with security and in comfort. A few, however, are inadvertently becoming deeply rooted to Santa Maria and will likely end up forming part of the valley’s burgeoning community of immigrant farm workers. Most binational workers interviewed in 1991 and 1993 are from the same central states of Mexico where most of the immigrant settlers originate; only a few, 16 percent, are from the southern state of Oaxaca and none from the northern border states. Binational workers were at one time braceros who during the program years used earnings in California to assist their rural homes and families in Mexico. Even after the Bracero Program was cancelled, they continued to participate in the annual sojourn despite increased costs and risks brought about by the illegality of the practice. In fact, it is because travel and illegal border crossings became burdensome, expensive and risky that some ex-braceros who did not own farmland in Mexico decided to settle in the Santa Maria Valley with their families ; in contrast, plastic pots large exbraceros who did own farmland at home or had been awarded an ejido plot by the government’s land reform programs continued to migrate seasonally to California in search of earnings to improve their holdings in Mexico.

In the mid-to-late 70s, when high-value specialty crops took-off, migrant ex-braceros began to remain in the Santa Maria Valley during considerably longer periods of timeóup to nine or ten months rather than the former three to five months. In fact, under favorable climatic conditions, it was not unusual for a closing farm season to nearly overlap with the opening of a new one, forcing migrant farm workers to shorten their visits home or forgo them altogether. The successful development of specialty crops also created more job opportunities which were quickly filled primarily by ex-bracero relatives, often by the grown children of ex-braceros themselves. Although all this was a boon to migrant workers’ earning capacity, it also bore a painful hardship owing to difficult and prolonged family separations. Regularly employed migrant farm workers, as a result, began to establish temporary second homes in the valley to accommodate several related workers and to cut costs during the annual sojourn. They, moreover, transplanted other family members, mostly women, to provide a home environment and infrastructure, as well as to increase family wage earnings by placing them in occasional part-time farm jobs. Once installed in Santa Maria, children were also transported, among other reasons, to access better schools than those available to them in rural Mexico. An outcome of this behavior is the establishment of binational families who manage and share two sets of resources, one in each country, with members who shuttle back and forth between them with remarkable ease and frequency. Surveys conducted in 1991 and 1993 reveal that binational workers, like settled immigrants, enjoy the valley’s best farm jobs, especially the older more experienced workers who know the job market well and have developed good relations with local employers. Individual monthly earnings, as a result, range from $1,000 to $1,200. Although many are involved with vegetables, a sizeable number also are employed by strawberry farms as part of a core group of “privileged” workers who are the first to be hired when the harvest season begins in March/April and the last to be dismissed when the season ends in September/October. Binational families are large and complex. All of them, without exception, form extended family groups which operate as a single economic unit.

Typically, they include three to six nuclear families, three to four distinct generations, and as many as 25 to 40 individuals, more than one half of them being children under 15 years of age. Binational families work in teams; while one part, usually the least productive, remains in Mexico managing the homestead and caring for both the very young and very old, the most productive members and some school-age children journey to Santa Maria for variable periods of time. During spring and summer a sizable number are employed in the valley but in the autumn, when farm jobs begin to taper, unemployed members immediately trek back home to assist in the corn harvest there and to help keep costs down in Santa Maria. In late November only a skeleton group remains in the valley, along with some school children, and by Christmas it is oftenpossible to find the whole extended family group gathered in Mexico for a brief, fleeting instant. Soon afterwards, however, workers begin to drift back. In February, the northward movement begins in earnest and by May all employable members are back in Santa Maria. Binational families need to carefully and effectively coordinate the deployment and employment of their workers to ensure a maximization of the resources at their command. Because binational families place a large number of workers in the job market and, in the process, keep expenses down by maintaining a rural homestead in Mexico and temporary living quarters in Santa Maria, they are able to assemble a considerable family income even when individual wages are low or negligible. It is not common for binational workers to seek employment outside the Santa Maria Valley, away from their post; rather, they return to Mexico as soon as jobs become scarce. Binational households in Santa Maria contain a surprisingly large number of legal, documented migrants. Many of the first-generation ex-braceros still carry and use the micas issued to them in the late 60s by INS to commute across the border; others have subsequently exchanged these micas for “green cards” and, in the process, become legal residents.

Many of the undocumented, especially those who had evidence of employment, were able to legalize their status and obtain work authorization through IRCA’s General Amnesty and Special Agricultural Workers programs before the end of the 80s. The fact that so many workers are documented has not diminished their binational involvement; rather, documentation has just made it easier for them to shuttle between the Mexican homestead and the Santa Maria extension. Many, nonetheless, remain illegal. This is especially the case of women and children who did not qualify for the SAW program and, as a result, continue to cross the border clandestinely. It is not unusual for authorized workers to share their documents with undocumented kin to facilitate border crossings and to seek employment. The exact enumeration of binational migrants faces two inextricable complications: First, the extraordinary and often unpredictable mobility of household members may easily cause a house-to-house survey to elicit as few as 3 to 5 members one day and as many as 18 to 20 on another. Second, binational households contain a substantial number of undocumented/unauthorized residents, especially women and children, who need to be concealed and protected. Binational migrants are, to say the least, always apprehensive about providing correct, complete, and reliable information regarding the size and composition of their households. Not to be mistaken with the above-described binational workers are the approximately six to seven thousand migrants who regularly sojourn to the Santa Maria Valley to harvest fruit and vegetable crops. These are migrant workers who are firmly rooted to their Mexican home communities, where they maintain a permanent domicile, but who regularly migrate to California looking for seasonal farm jobs and wages to send back home. They are, in a sense, black plastic nursery pots the braceros of today without a Bracero Program. Many originally became involved in this annual trek in the 1940s when the Bracero Program was first established and have maintained the practice ever since by passing it from one generation to the next even after the program was terminated in 1964, converting the practice into a deeply embedded tradition. Families who participate in this tradition have organized their lives and households in a manner which enables workers to migrate and, as a result, wage remittances have become an intrinsic and indispensable part of the household economy . A key distinction of the seasonal migrant, vis-a-vis the binational worker, is that only the most productive and employable workers migrate. Less productive workers and dependents are always left behind in the home community to tend the family farm or just simply to survive on the basis of a, hopefully, steady flow of wage remittances arriving from the United States.

Many will return home as soon as the employment season ends or sooner if a pre-targeted goal of earnings is accomplished. They, in short, only come to work and earn wages, and they are with few exceptions always in a hurry to return home. Seasonal migrant workers occupy a particular niche in the farm-labor market and production cycle of Santa Maria Valley agriculture. They serve as a labor reserve which intermittently complements year-round vegetable harvest crews during the peak spring and summer months when crops tend to mature faster, even bolt, with the arrival of longer days and warmer temperatures. And they especially supply the bulk of the peak harvest labor for strawberries and wine grapes during the spring-to-summer and early autumn months, respectively. Although the presence of seasonal migrants in Santa Maria diminished considerably during the 80s as immigrant workers settled permanently in the valley, they began to increase again in the 90s as strawberry acreage expanded and farm employment practices changed owing to IRCA impacts. In effect, the recent proliferation of farm labor contractors has often placed migrant workers in direct competition against the stable but more expensive local immigrant laborer. Nonetheless, migrant workers do not enjoy the better paid, more stable, and skilled farm jobs which continue to be largely monopolized by immigrant and binational workers. Migrants, to be sure, hold the most seasonal, insecure and intermittent farm jobs with monthly earnings which range between $500 to $800 during the peak employment season. Interviews conducted among seasonal migrants during the 1993 campaign, especially among strawberry harvest crews, revealed that there are two distinct sub-types of migrants: first, the descendants of braceros, those who have established a tradition of migration, from the sending communities located in the central states of Guanajuato Jalisco, Michoacan, and Zacatecas; and second, new immigrants mostly from the southern states of Oaxaca and Guerrero. Traditional migrants have established effective networks and accrued experience which facilitate travel, border crossings, and employment. Some have kin and friends established permanently in the Santa Maria Valley who provide sanctuary and assistance during the annual trek. Others rent apartments or rooms for the season and share them with other migrants to cut living expenses during their stay in the valley. They, moreover, have considerable personal access to farm employers who hire them year after year. Many come to Santa Maria only to perform a specific job with a specific employer and return home with their savings as soon as the season concludes. Although many travel from Mexico alone, especially those who have kin in the valley, it is quite common to find cohort groups sojourning together, either groups of friends and neighbors of a similar age, or multi-generational kin-based groups. The presence of women workers among migrants is not uncommon, especially among family groups, but men continue to predominate in the ranks of the sojourn workers. In 1993, approximately forty percent of the interviewed migrants fit the description of traditional migrants. New migrants account for the remaining sixty percent of the migrant labor force observed in the Santa Maria Valley in 1993. As stated above, most come from the southern states of Oaxaca and Guerrero; many are Mixtec and Zapotec Indians. Few have a California migration experience of more than ten years, though most have lived the lives of migrants as seasonal farm workers laboring in the northwestern states of Sinaloa, Sonora, and Baja California in Mexico. In recent times they have included the United States’ west coast as part of their itinerary.

Like strawberries, raspberries and blackberries have benefitted from enhancements in cultural practices. When well-managed, both types of caneberries can produce crops for up to 20 years. However, to maintain acceptable quality and yield Central Coast growers typically manage raspberries and blackberries so that they produce two and five crops, respectively, prior to removal and replanting. In Santa Cruz County, raspberry production was relatively flat in the 1960s and 1970s, but began to increase substantially in the 1980s . This can be explained by a shift from floricane, or spring-bearing varieties, to the then newly developed proprietary primocane, or fall-bearing varieties, that do not carry the productivity constraints associated with the inadequate chill requirements along the Central Coast. Primocane-bearing varieties allow growers to successfully produce a high quality raspberry crop in low- or no-chill coastal locations, and further manipulate time to harvest and yield with pruning and other management practices . Between 1990 and 2014, the number of acres planted to Santa Cruz and Monterey area raspberries almost tripled, tons produced increased by about 350% and the value of production was up by over 400% in real dollars . Santa Cruz County raspberry growers began to experiment with and adopt field-scale semi-permanent protective structures or tunnels in the 1990s and 2000s . Initially developed in Europe, field-scale tunnels allow growers to extend their production seasons, enhance yield and fruit quality, and capture high off-season prices for fresh market fruit . The controlled environment, and resulting security of production, also allows for greater market stability. Tunnel culture is now a common practice in raspberry production. This shift away from open-field production to protected cropping, growing raspberries in container along with breeding improvements, has had lasting impacts on the raspberry industry and its expansion.

Cultural improvements geared towards fresh market blackberry production are more recent and include advances in breeding for thornless varieties and quality attributes . In 2011, a public primocane-bearing blackberry variety became commercially available for the first time and is now being planted in the area. Since that time, additional public and proprietary primocane-bearing varieties have been in development; some have already become available. Open-field production was the norm until recently, but to ensure marketable fruit of high quality, and as growers have shifted additional acreage to primocane-bearing varieties, tunnel culture has been more widely adopted and, based on discussions with growers, is now estimated at roughly 80% of the acreage. Like organic strawberries, remarkable growth in the statewide production of organic raspberries and blackberries was documented between 2000 and 2012 . Value of production was up over 3,000% in real dollars for organic raspberries and up by almost the same percentage for organic blackberries. It is important to note that although the organic raspberry and blackberry categories have demonstrated extraordinary growth, they still represent a relatively small percentage of all berry production in the area. Research points to several factors that have spurred consumer demand for all berries. Berries contain bioactive compounds, including essential vitamins, minerals, fiber and antioxidants that contribute to healthy diets, and that help to reduce the risks associated with some chronic diseases and cancers . This information has been widely shared with consumers through, for example, government programs promoting healthy eating , and more generic berry promotion programs . Per capita consumption of fresh strawberries in the United States almost doubled from 1994 to 2014, increasing from 4.1 to 8.0 pounds . U.S. per capita consumption of fresh raspberries was small by comparison, at just 0.5 pounds in 2014. Similar consumption data are not available for blackberries, but Cook notes that consumers generally view berries as complementary, and that sales for all berries have increased.

Indeed, in 2014, berry sales increased 5.8% over 2013; berries were the number one produce category for U.S. grocery retailers, at $5.7 billion in annual sales . Some berry operations also benefit from their proximity to the area’s urban centers, which have sizeable cohorts of educated, high-income consumers who generally demonstrate an interest in health and wellness, local agriculture and fresh and organic products. In addition to the more traditional grower shipper and direct marketing channels, new technology-driven food marketing companies — virtual food hubs — have evolved to cater to this demographic. They promote the values of sustainable communities, local food economies and business integrity and transparency, all important attributes for new 21st century consumers . These companies form relationships with local growers, provide some technical and market support, and enhance sales and engagement with consumers. It is not yet clear what impacts these still-niche marketing businesses may have on the industry in total. However, growers have responded to the various health and market signals by ramping up production of both conventional and organic products, berries included. Specialists and farm advisors with UC Cooperative Extension have performed economic analyses for Santa Cruz and Monterey county fresh market berry crops for decades . The studies estimate production costs for a representative enterprise based on characteristics common to the area’s farms. Data are collected from established growers, input suppliers and other industry experts so that a diversity of operations and practices are taken into account. Since 1990, UCCE researchers have used a farm budget software program to analyze the data and present results in several formats detailing costs for cultural and harvest practices, monthly cash costs and business and investment overhead costs. The studies also include an analysis estimating net returns to growers for several yield and price scenarios. Representative costs for food safety and environmental quality programs have been incorporated into more recent studies as they have evolved to become standard business practices.

The resulting production and economic information is specifically designed to assist growers, bankers, researchers and government agencies with business and policy decisions. The first economic analysis of fresh market strawberry production for Santa Cruz and Monterey counties was performed in 1969; at least one subsequent analysis has been conducted every decade since then. Though the level of detail and data included in each study has changed over time, some interesting trends can be noted. Annual land rent climbed from $150 per acre in 1969 to $2,700 in 2014, representing 2.5% and 5.5% of total production costs, respectively. The cost of soil fumigation for conventional strawberry production increased from $350 per acre in 1969 to $3,302 in 2010, representing 5.5% and 6.9% of total production costs, respectively. Production year water use gradually decreased from 80 acre-inches per acre in 1969 to 36 acre-inches by 1996 as drip irrigation became the standard. The amount of water used to bring a crop to harvest has remained roughly the same since that time; however, growers and researchers continue to investigate methods to increase water use efficiency even further. In some areas, soil types and fields, growers have been able to reduce per acre water use by several acre-inches more . When the above costs and water usage are assessed on a per ton rather than a per acre basis, production practice cost increases are less notable, and water savings even greater. Labor-intensive practices such as hand weeding and harvest are consistently shown as costly line items relative to other operations. Representative yields for conventionally produced fresh market strawberries rose from 20 tons per acre in the 1969 study to 30 tons in 2010, an increase of 50%. Even higher yields are discussed for some varieties and production conditions; county production statistics confirm that higher yields are indeed possible . Representative yields for organic strawberries, studied over a much shorter time period, raspberry container size rose from 15 tons per acre in 2006 to 17 tons in 2014, an increase of 13%. As more research is directed towards organic agriculture in general and strawberries in particular, yields will likely increase even more with time. Recent efforts include improvements in cultivar breeding, cultural practices and disease management, especially soil pathogen management. The most recent economic analyses for conventional, second year conventional and organic strawberry production were performed in 2010, 2011 and 2014, respectively. Second year conventional strawberries, or those producing a crop for a second year after having produced the first without replanting, represent about 15% of the total strawberry acreage in the area. Similarities and differences in total, cultural and pest management costs for the three management approaches are shown in figures 1 to 3. Total costs for conventional strawberries were $47,882 per acre and include expenses for all practices from land preparation to harvest . For the second year conventional strawberry crop, total costs were lower at $32,798 per acre, reflecting a reduction in expenditures for land preparation and reduced harvest costs because of lower yield.

For organic strawberries, total costs were $49,044 per acre, slightly higher than for conventional production, mostly due to higher soil fertility input costs. Harvest, a labor-intensive practice, clearly represents the lion’s share of total costs, at 58% in organic production, 60% in conventional production and 67% in second year conventional berries. Cultural costs represent 26% of total costs in the conventional and organic systems, but only 15% for second year strawberries because there were no associated planting costs, and because pest management costs were lower . Looking more closely at pest management, soil fumigation is the highest cost category for conventional production at $3,302 per acre, with weed control, another labor-intensive practice, the highest cost in second year and organic strawberries at $1,212 and $2,506 per acre, respectively . However, for organic strawberries the cost to control insects ran a close second at $2,488 per acre, which was dominated by control for lygus bug with a bug vacuum, and two-spotted spider mite with the release of predatory mites. By comparison, estimated costs for insect control in conventional strawberries were lower at $702 per acre and still lower at $579 in second year conventional berries. Raspberry and blackberry production were not routinely studied in years prior to 2003. Since then, several primocane-bearing raspberry and floricane-bearing blackberry cost and return analyses have been performed, with the most recent studies conducted in 2012 and 2013, respectively. Both studies detail establishment and first year production and harvest costs for not-yet-fully-mature crops. For raspberries, first year of production includes a $12,460 per acre construction, management and investment cost for protective tunnels. Costs for a mature raspberry crop are analyzed in the second production year and total $48,210 per acre . For blackberries, costs for a mature crop are shown for the second through fifth production years, and total $43,406 per acre per year. Harvest costs again represent the vast majority of total costs, at 81% and 71% of total costs for raspberries and blackberries, respectively. For raspberries, cultural costs represented a much smaller share of total costs at $4,656 per acre, roughly half of which was for trellis and tunnel management. Blackberry cultural costs totaled $5,709 per acre, of which over half was for pruning and training canes. Each study also includes an analysis of potential net returns to growers above operating, cash and total costs for a range of yields and prices. When evaluating net returns above total costs, gains are shown for higher yield and price points; losses are also documented at many lower yields and prices . Farms with productive soils, experienced managers, optimal production conditions and robust market plans generally realize higher net returns. In contrast, farms with less-than-optimal production conditions, reduced yields, poor fruit quality or inexperienced managers may contribute to lower net returns. Results from the strawberry analyses show that on a per acre basis, organic strawberries tend to be more profitable than conventional berries, even with lower yields. Organic price premiums explain the result; in this example price per tray for organic strawberries ranged from $12 to $18, while price per tray for conventional berries ranged from $7.30 to $11.30. Prices for second year conventional strawberries were slightly lower still to account for a portion of the crop that was diverted to the freezer market. Net returns for both caneberries were mostly positive. Other noteworthy entries in all recent berry studies include per acre costs for pest control advisers , management of invasive pests and food safety and regulatory programs for water and air quality. Though each alone represents a relatively small portion of total costs, they provide readers with insights into the changing nature of berry production activities and costs over time.Cultural practices in the berry industry have evolved to address changes in soil, water and pest management needs.

The alternative hypotheses we present in figure 2 could be addressed with herbarium specimens for plant species with long-term herbarium records and which vary in their phenological sensitivities to climate change. While collections may not always allow us to differentiate between alternative scenarios, they could reveal how herbivory changes with warming for plants across a range of phenological sensitivities, and inform field experiments to tease apart mechanisms. In some cases, it may be possible to test for herbivory by novel herbivores by quantifying types of damage that can be traced back to particular insect genera or species, such as galls and leaf mines , or chewing damage that is characteristic of certain insect orders, e.g. margin feeding, circular hole feeding, and skeletonization. Butterfly collections might also help in resolving alternative scenarios, although we suspect that larvae, responsible for most herbivore damage, may be under-represented in collections compared to adult Lepidoptera, and flight phenology may not be correlated with larval phenology . The mismatch between adult and larval butterfly life histories is a challenge for using butterfly collections to explore phenological asynchronies. However, there are also scenarios in which phenological change at the adult stage may affect herbivory, which may offer opportunities to use the extensive collections of butterflies and moths that are available. For example, some Lepidoptera species may develop ‘lost generations’, raspberry container growing in which warmer temperatures signal caterpillars to develop into adults rather than entering diapause. The adults of the last generation may suffer high mortality rates at the onset of winter; for a more thorough discussion of this topic, see.

Museum specimens of moths and butterflies could inform how common it is for Lepidoptera species to add another generation in response to climate change, and contrasting herbarium specimens of their host plants could reveal how herbivory is differentially impacted by species that have and have not added generations with climate change.One of the most supported predictions in global change biology is that species’ ranges will shift poleward and upward in elevation as the climate warms. For many insect species, poleward range expansion may be explained by increased over-winter survival and/or feeding owing to warmer winters. For multivoltine insects, longer growing seasons can also increase the number of generations completed per year, leading to population growth that might facilitate range expansion if host plants are available. Most predictions on plant species’ range shifts are predicated on the assumption that abiotic factors determine range edges; however, biotic factors can also contribute to range limits. There is also growing evidence that biotic factors, such as herbivores and disease can interact with abiotic factors to determine the trailing range edges of some plant species. However, the factors that drive range limits at leading and trailing edges remain unknown for most species. Biological collections typically have associated metadata describing when and where collections were made, and therefore provide rich data on species distributions and distributional shifts over time. Species distribution models are commonly used to map past and present distributions, but they are intrinsically limited by the number and representation of input records, and, in the case of global change research, the number of records available from before and after global change.

The extensive digitization efforts currently underway for insect and plant specimens will improve our predictions and ability to track changing distributions. For well sampled plant species, we might also be able to investigate changes in herbivory at poleward range edges to determine if it has declined over time as plant ranges expand into novel habitats—an extension of the enemy release hypothesis associated with species invasions, discussed below. Larger digital collections of insect herbivores will provide the opportunity to compare range shifts across insect clades and to identify traits that govern range expansion and contraction. For example, we might expect that warmer winters will disrupt winter diapause for many insect species, leading to range contraction and decline, while those that do not have diapause will benefit from higher rates of winter survival. However, it is also possible that insects with diapause are more likely to maintain phenological synchrony with on how herbivore damage will change over time. Species traits might also determine whether species shift over time or space, and how these two responses trade-off . The ability of insect herbivores to switch host plants may be another factor that constrains or facilitates herbivore range expansion, and thus plant –herbivore interaction strengths. Specialized insects that do not feed on newly encountered plant species may be limited in their geographical spread, whereas more generalist herbivores would be less constrained. Herbaria may capture such switches to novel hosts, showing up as new types of herbivore damage on specimens as host plants and their insect herbivores shift their distributions and provide the opportunity for novel plant –herbivore interactions. For example, leaf mines and galls—which are preserved on herbarium specimens—are made by a wide variety of insect herbivore taxa, including some of the most diverse groups of insects—Lepidoptera, Coleoptera , and Diptera —and are often specific to insect genera or species .

The Lepidoptera that make leaf mines are not well represented in long-term citizen science data because leaf miners are typically micromoths, which are not the focus of long-term observations, and leaf mining and galling damage are only rarely included in herbivory studies, which tend to focus on chewing damage. Thus, herbarium specimens provide a record of a unique insect herbivore fauna not represented in long-term herbivore monitoring or herbivory studies. Herbarium specimens may also provide data on a key hypothesis in global change biology that is based on theory which dates back to Darwin: the role of natural enemy release in species invasions. The enemy release hypothesis describes the escape from native predators and parasites when species are introduced into novel habitats. While there is evidence that introduced plants escape their native herbivores, it is unclear how long this ‘release’ persists. Herbarium specimens can provide rare long-term data on herbivory and disease pressure that allows us to resolve this question. In a well-documented example, Schilthuizen et al. used herbarium specimens to show that the non-native cherry tree, Prunus serotina, acquired higher rates of herbivory over time after its introduction to Europe, while its native congener, Prunus padus, had stable herbivory levels over the same time period. This led to field investigations into the contemporary herbivore communities for these congeners, which revealed that, surprisingly, P. serotina had a richer herbivore community than the native P. padus, and that P. serotina had acquired specialized herbivores from other native host genera. This supports the hypothesis that non-native plants accumulate herbivore taxa over time in their novel habitats, which might have significant implications for plants that shift in their geographical distributions.Urbanization affects insect herbivores via a variety of mechanisms, including habitat fragmentation, habitat and host plant loss, and introduction of novel host plants that attract and support non-native herbivore communities. Given these concurrent pressures, the effects of urbanization on plant – herbivore relationships are complex and varied . However, in recent years, it has become increasingly clear that a key aspect of urbanization, the urban heat island effect, can drive relationships between plants and herbivores and may uniquely inform climate change predictions. The urban heat-island effect—the local warming of urban areas relative to surrounding countryside—increases urban temperatures 1– 128C higher than rural temperatures. Thus, local warming caused by urban development is similar in magnitude to warming expected globally over the next 100þ years, and it has therefore been suggested that cities may provide insights into the future effects of climate change. Like global warming, urban warming drives phenological advance in plants and insect herbivores. For example, plants leaf out and flower earlier in cities than in nearby rural areas, and urban heat is associated with earlier egg production for certain insect herbivore species. While the effects of warming global temperatures on the synchrony of plant –herbivore interactions is still generally unresolved owing to a lack of data, these relationships can be studied across urban temperature gradients, blueberry plant pot and there is some evidence for reduced synchrony between insect herbivores and their natural enemies as a result of urban warming. Because of the parallels between the abiotic and biotic effects of urban and global warming, natural history collections from urban areas may allow us to more broadly predict how global climate warming will affect interactions between plants and their insect herbivores. Phenology data from specimens—e.g. flowering, leaf-out, insect flight— paired with data on urbanization intensity in the areas where specimens were collected could inform predictionson phenological change and synchrony for a broad range of plant and herbivore species. Specimen data from urban areas are, perhaps surprisingly, plentiful. A recent study shows that across three areas with large digitized herbarium collections—the US, South Africa and Australia—plant specimens are often collected close to natural history museums or roads. Thus, specimens could be used to explore urban natural gradients.

Like temperature, urbanization can be easily assigned to historical specimens via contemporary measurements or existing data. As a proxy for urbanization, we can use human population density data from censuses, which many countries have been collecting since the early 1900s, and in some places urbanization can be translated from historical maps or as impervious surface derived from satellite imagery. One novel approach might be to derive markers of urbanization from the herbarium specimens themselves, for example, signature pollutants, although disentangling the contributions of different drivers would then present additional challenges. In recent years, growing evidence shows that urban warming may increase abundance of certain herbivores, notably sapfeeders, potentially leading to more insect damage on urban than on rural plants, a pattern that has been documented by entomologists for over a century. Sapfeeding herbivores, such as scale insects and aphids, are often preserved on leaves and branches and thus may provide insights into changing herbivore pressure in response to urbanization. In a recent study, Youngsteadt et al. counted armoured scale insects on branches of herbarium specimens of red maple Acer rubrum and on branches of live trees across an urban warming gradient. Using these data, they showed that interannual warming and urban warming may have surprisingly congruent effects on scale insect prevalence. In box 2, we discuss how herbarium specimens might be used to investigate more complicated interactions between multiple trophic levels, relationships that could inform biological control efforts and management of urban plants. While the urban heat island effect benefits certain herbivores that survive within the urban matrix by advancing their phenolog and increasing their abundance, urbanization also excludes some insect species—a pattern which has been documented with insect museum specimens—making the effects of urbanization on herbivore damage to plants difficult to predict. Long-term records of butterfly flight from Britain showed that habitat loss is associated with butterfly decline, especially for species that are less mobile and are habitat specialists. Relatedly, a recent study across 16 European cities showed that leaf chewing damage was lower in cities relative to nearby rural areas, perhaps driven in part by higher rates of bird and ant predation on insect herbivores in cities than in rural areas. Thus, a pattern that might be emerging from the literature is that certain sap-feeding insects benefit from urban heat , while leaf chewing and the insects that cause this type of damage, notably Lepidoptera, decline in response to loss of habitat and host plants caused by urbanization. This finding suggests that the effects of bottom-up versus top-down forces driving insect herbivore fitness might differ among feeding guilds . Measurements of broad-scale chewing herbivory , presence of sap-feeders, and incidence of sooty mould as a proxy from herbarium specimens, along with insect herbivore occurrence data, could be used to test this hypothesis . In addition to describing the effects of urbanization at the local scale, museum specimens may also reveal how urbanization affects species distributions at broader spatial scales. For example, while urbanization may disrupt poleward range expansion for some species, it is possible that cities serve as warm habitat stepping stones for species with long-distance dispersal mechanisms, facilitating their poleward expansion. The insects that create leaf mines have been described as ‘aerial plankton’ because they tend to disperse long distances. Herbarium specimens might capture this rapid northern expansion of leaf mining insects and provide a record of shifting interactions with native plants that may be more likely to respond in time than space —see box 1.

A study in natural ecosystems comparing forest and savanna found species richness to be affected by habitat and strata ; the two environments clearly differentiated in terms of their species composition . In our study, canopy vegetation was not a strong driver for the community of twig-nesting ants since our best models did not include a VCI. However, species compositional differences observed across both vegetation layers could be an effect of microhabitat diversity and canopy connectivity . Providing complex vegetation not only promotes ant diversity but also other organisms that facilitate ant colonization into new twigs. Presumably ants often nest in hollow branches of trees that have been previously dwelled or inhabited by beetles . Moreover diversity of trees might also provide nesting resources that are different in terms of how difficult or attractive they are to dig cavities, for example studies have found that tropical woods can be different in terms of their structure, chemistry and biology ; this could suggest important drivers in the differentiation of ants that inhabit them. We found a large number of arboreal twig-nesting ant species in this coffee agroecosystem study supporting the notion that managed ecosystems, such as agroforestry systems in the tropics, blueberries in containers have the potential to host a great diversity of species. A number of previous studies have provided evidence that ant diversity increases control of pests and fungal diseases . We document here that increases in nest entrance size diversity on an individual tree relates to increases in ant diversity on trees. This may thus have important implications for promoting ants as biological control agents in agroforestry systems.

We conclude that the availability of a variety of nesting options and vegetation strata are important drivers of species diversity and support the idea that niche partitioning drives species coexistence . Future studies should further investigate the competitive hierarchies of the species colonizing twigs if we want to understand how species using similar resources interact with each other; and evaluate colony fitness in face of multiple resource use, as has been done in the past for colonies of Cephalotes persimilis . Since ants often engage in interactions that deliver ecosystem services future studies should focus on evaluating roles of different ant combinations using a diverse array of twig entrance sizes in agricultural pest control. Furthermore, we have learned from this study that the structuring of ant communities is multi-factorial and that local as well as regional factors should be considered when explaining species assemblages in the tropics. Habitat complexity is critical for the functioning of ecological communities in both terrestrial and aquatic systems. Processes such as resource foraging, colonization, and species interactions often depend on the level of heterogeneity in the configuration of physical elements in a habitat . Vegetation connectivity and structure are important components of habitat complexity and can influence species interactions and community patterns at local scales. In aquatic systems, more complex habitats made up of macrophytes support communities that are more diverse and abundant, and allow for greater food capture than systems without vegetation . In terrestrial systems, vegetation structure– such as the biomass of foliage and the variety of plant architectures– generally influences species composition, and increases species richness and abundance of numerous taxa . Additionally, vegetation structure can influence mobility and foraging success of vertebrates and invertebrates . In tropical ecosystems, ants are among the most abundant and bio-diverse of taxonomic groups , and are considered important predators, herbivores, and seed dispersers .

Ants are cursorial central-place foragers – organisms that forage from a central place to which they return with food to feed with the colony . Therefore, foraging and discovery of food resources is strongly constrained by the need to construct and follow trails along vegetation . This is particularly relevant for ants using the arboreal stratum as their primary foraging space . For instance, the availability of vegetation connections can maximize ants’ foraging efficiency, locomotion, and velocity , as well as contribute to changes in community composition and species richness . The availability of such resources can ultimately lead to differences in resource utilization by ant communities . In tropical agricultural systems, especially agroforests, ants play important ecological roles , and management practices can strongly influence ant behavior and their potential for providing biological pest control services . Indeed, one of the oldest known records of the use of ants for pest control dates to 304 A.D in citrus plantations in China. In these systems artificial connections made of bamboo were used by farmers to facilitate foraging by the Weaver Ant to suppress damaging phytophagous insects . In that same study, Huang and Yan report anecdotal evidence that suggests equal yields in orchards that use chemicals vs. orchards that use ant bridges to control for pests. Similarly, Peng et al. , report lower levels of fruit damage in cashew with the presence of weaver ants. However, as vegetation complexity declines in agroecosystems, tree density and diversity may also decrease , as well as the possibility to generate connections between the arboreal vegetation, which might impact arthropod populations . The lack of connectivity between trees in managed systems can have a significant impact on the mobility of worker ants and their ability to control resources. This impact may be particularly marked at greater distances from the nest, where ant dominance may be lower . This in turn may influence the ecosystem services provided by ants, particularly the suppression of pest outbreaks .

Shaded coffee plantations, which maintain high levels of shade and structural complexity can sustain complex networks of organisms, which can result in biological pest control . In coffee systems, ants are a functionally diverse and abundant group of ground and arboreal-nesting arthropods and are considered important biological control agents . Ants are predators of the most devastating coffee pest, the coffee berry borer , a beetle that drills cavities in coffee berries and severely damages the seed . Several species of arboreal ants, with nests attached to or inside tree trunks, branches, or twigs, control adult and immature stages of this pest either through direct predation or deterrence . Ants of the genus Azteca are numerically dominant in shaded coffee plantations. These ants forage intensively on coffee plants , and deter CBB adults by removing them from the coffee plant, therefore lowering fruit damage . In shaded coffee plantations, Azteca sericeasur ants nest on shade trees and access adjacent coffee plants through the leaf litter or available pathways, such as fallen branches, vines, and other vegetation , matching the description by Longino for this species in forest habitats. In more intensively managed coffee systems, with fewer and more distant nesting trees, connectivity may be sparse or absent and artificial connections might buffer against this loss. Vegetation structure and arboreal characteristics in coffee plantations are likely to be important factors influencing ant foraging behavior and nesting in arboreal ants . However, the influence of vegetation connectivity on the foraging of this dominant arboreal ant, and its effect on pest removal in coffee plantations has not yet been studied. Previous work has documented the importance of arboreal connections for ants and biological control in agricultural systems. For example, various studies and farmers’ manuals suggest that connecting nests to adjacent trees using bamboo strips enables weaver ants to colonize new trees, which increases ants’ efficiency in removing pests, including the pentatomid insect Tesserarotoma papillosa . However, there is little evidence about the effect of increasing arboreal connectivity on biological control using experimental data. We report an experiment testing the influence of adding connections between shade trees and coffee plants and its effects on CBB removal on coffee plants. To our knowledge, planting blueberries in pots this is the first study providing experimental data on the effect of adding connectivity on ant activity and pest removal in coffee agroecosystems. Specifically, we tested one hypothesis: connectivity affects CBB removal in this system by increasing recruitment rates of A. sericeasur ants to prey items; we predicted that 1) A. sericeasur ants use artificial connections between nesting trees and coffee plants; 2) plants with connectivity have higher ant activity than isolated plants; 3) plants with connections have grater removal rates of CBB by A. sericeasur ants; and 4) A. sericeasur activity and CBB removal rates by A. sericeasur ants decrease with increased distance from A. sericeasur nests. Within the farm, we haphazardly selected 20 non-overlapping sites located at least 10 m away from each other with one Inga micheliana tree containing an A. sericeasur carton nest on the tree trunk .

A. sericeasur is a polydomous, arboreal ant species , which occurs in ~13% of trees at our study site , and forages on coffee plants . Trees were selected only if ant nests were noticeably active. In each site, we quantified ant activity on the nest tree as the number of ants crossing a single point on the main trunk during one minute. This methodology has been used in previous studies to measure overall ant activity of a nest . We then selected the six coffee plants nearest to the nesting tree, making sure they were not directly touching each other or the tree by removing branches and vines . We then randomly assigned three of the coffee plants at each site to aconnection treatment and three as controls without connections, then measured ant activity on the plants by counting the number of ants passing a point on the central trunk for one minute. We connected treatment coffee plants to the nesting tree using jute string . Strings remained in the field for three days to allow for ant acclimation to disturbance and for ants to establish new foraging pathways. After three days, we returned to the sites and remeasured ant activity on the nesting tree and coffee plants. Observations took place between 10 am and 1 pm, and were immediately stopped as soon as it started raining, as this drastically decreases ant activity.To test how connectivity impacts potential biological control provided by ants, we added dead adult CBB onto connected and control coffee plants to directly assess ant removal rates. We collected CBB infested coffee berries from the field, dissected them, extracted female adult CBB individuals , and placed them in the freezer for up to 24 h, after which beetles were dead. Three days after placing strings and after reassessing ant activity, we placed 10 dead CBB adults on a small piece of white card on each coffee plant near the center of the trunk, left cards for 30 min, and then counted the number of CBB remaining. Restricting movement of sentinel prey, either by gluing them to observation sites or freezing them is a common technique for assessing predator behavior . We used frozen sentinel prey to increase the availability and similarity of beetles on cards and to reduce the potential for live prey to escape from the arena. To assess whether CBB removal was due to ant activity, we monitored cards across the plot over a period of 30 minutes and recorded any arthropods present. Only ants were observed on the cards, indicating that these were responsible for removing the CBB. Although we acknowledge that the use of dead prey may alter ant behavior, it is already well established that A. sericeasur both antagonizes and predates live CBB in the field, and reduces CBB infestation on plants . We used dead prey in this experiment to more readily assess ant removal rates and infer that these changes translate to changes in the bio-control efficiency of this ant on live prey. Immediately following each experiment, we characterized the vegetation in each site because several different environmental factors are known to influence ant foraging in coffee systems . We measured the percentage of canopy cover , coffee plant height, and distance from each coffee plant to the central Inga nest-tree.To test for statistical differences in ant activity on coffee plants before and after establishing connections we used a GLMM. We included time , treatment , coffee plant distance to nest tree, the interaction between time and treatment, and the interaction between time and distance as fixed effects . We also included coffee plant height and ant activity on nest tree as covariates.

While C0 wines in this study demonstrated lower alcohol content than shaded wines, previous literature corroborates cluster temperature reduction by partial solar radiation exclusion as an effective method to lessen sugar content in the grape berry and thus reduce alcohol content of wines . The effect of partial solar radiation exclusion in semi-arid climates on berry pH and TA is mixed. Previous work demonstrates partial solar radiation exclusion to reduce pH and increase TA in grape berries by reducing the thermal degradation of organic acids . However, in the present study, berry pH and TA at harvest were unaffected in either year by shade films . Nonetheless, there were apparent effects on wine pH and TA that were vintage dependent. In the present study, D3 wines had the lowest pH and highest TA, while C0 wines did not differ from the shade films D1, D4 or D5 in pH or TA in 2020. Differences observed in pH between the wines ultimately affect the colorimetric properties of these wines. In 2021, D4 and D5 wines showed the highest pH values. It is understood that the pH of the wines can shift the anthocyanin equilibrium in wine solution between the flavylium and quinoidal base forms . In the present study, D4 wines had the highest pH and the highest CI. In many cases, when pH rises, CI will decline as anthocyanin equilibrium shifts away from the flavylium form towards the colorless quinoidal forms . However, large plastic pots this was not the case in the present study. Rather, improved color intensity at elevated wine pH could be attributed to co-pigmentation in the wine matrix.

Co-pigmentation refers to non-covalent interactions between anthocyanins and cofactors such as flavonols, flavan-3-ols and proanthocyaninidins, that results in greater absorbance of the wine than color what would be indicated by anthocyanin content and pH conditions . Copigmentation in young wines was shown to increase color intensity in young red wines . In the hotter 2020 vintage, the total flavonols in grape berries were increased in D4 fruit compared to other treatments . This increased berry flavonol content was transmissible during winemaking, as D4 wines also showed the highest total flavonols with similar concentrations as C0 wines in 2020. TPI was also enhanced in D4 wines. As such, this increased the abundance of cofactors in the wine matrix. Thus, improved color intensity documented in D4 wines in both vintages could be due to the enhancement of absorbance from increased flavonol content by reducing thermal degradation in the vineyard . In the cooler 2021 growing season, shade films produced wines with less flavonols than C0, but greater anthocyanin content, thus leading to improved color intensity in D4 wines. The increase of phenolic cofactors in D4 wines not only enhanced color and hue, but also led to a higher percentage of polymeric anthocyanins when compared to other shade treatments. Phenolic and polyphenolic compounds from grape skins and seeds can form polymeric pigments in wine with anthocyanins. These polymeric anthocyanins are more stable than monomeric anthocyanins and help to stabilize wine color. This occurs as the proportion of monomeric anthocyanins decreases, leaving color to be maintained by polymeric anthocyanins . Across both vintages, the percentage of polymeric anthocyanins was maximized in D4 wines, indicating that these wines may have greater aging potential than wines from C0 and other shading treatments.In the present study, partial solar radiation exclusion modified the composition of anthocyanins in wine. Partial solar radiation exclusion resulted in increased anthocyanin glycosides in wine from shade film treatments except for D4 wines in 2020.

In 2021, D4 consistently showed the lowest cluster temperatures post-veraison and as a result, demonstrated the highest concentration of glucosides in resultant wines. Excessive berry temperatures post-veraison in both vintages led to C0 fruit with reduced total anthocyanin content at harvest and this carried over into resultant wines . The reduction of near-infrared radiation by at least 15% produced a cluster temperature conducive to anthocyanin accumulation, as these compounds are susceptible to thermal degradation above 35°C . When comparing total anthocyanin and flavonol concentrations between 2020 and 2021, regardless of treatment, 2020 wines had anthocyanin and flavonol concentrations six to seven times less than those in 2021 wines. As flavonoids are susceptible to thermal degradation, this drastic difference in total flavonoid concentrations may be attributed to hotter vintage air temperatures in 2020 compared to 2021. Previous works show berry sunlight exposure to alter the composition of anthocyanins, such as the proportion of acetylated and coumarylated forms . Modulation of acylated, methylated, and hydroxylated forms of anthocyanins result from the synergistic effect of solar radiation exposure and the coupled increases in berry temperature . Generally, high berry temperatures resulting from increased solar exposure results in increased acylated anthocyanins in the grape berry, particularly coumarylated forms . Also, high temperatures result in accumulation of highly methylated anthocyanins such as malvidin derivatives, as these compounds are less likely to degrade than their counterparts . In 2020, D1 and D5 wines demonstrated highest concentrations of acetylates, coumarylates, and methylated anthocyanins compared to C0 wines. While D1 and D5 treatments demonstrated cluster temperatures less than those from C0 treatments , the concomitant thermal degradation of total anthocyanins in C0 treatments proved to negate any modulation towards acylated or methylated forms in resultant wines.

Similarly in 2021, C0, D1 and D5 wines exhibited reduced acylation compared to D4 wines. Again, while D4 consistently exhibited less intense cluster temperatures, the thermal degradation in more exposed treatments eclipsed any identifiable acylation modulation from hot growing conditions. Acylated anthocyanins are more stable compounds and provide color stability and increase blueness in wine . However, an increase in methylated anthocyanins will lead to redder hues in wine . Therefore, the improvement in acylated and methylated anthocyanin content due to partial solar radiation exclusion may enhance color perception in young red wines through color stabilization and alteration of wine hue. Likewise, anthocyanin hydroxylation is also directly influenced by temperature and solar radiation exposure. Previous studies on berry exposure utilizing UV selective shade nets as well as leaf removal, demonstrated anthocyanin tri-hydroxylation increases with increasing berry temperature . Increases in tri-hydroxylation are driven by accumulation of malvidin derivatives and the temperature sensitivity of F3’H, the catalyzing enzyme for 3’- hydroxylated anthocyanin biosynthesis . The highest ratio of tri- to di-hydroxylated anthocyanins in 2020 C0 wines were driven by higher concentrations of 3-p-coumaroyl-glucoside derivatives of delphinidin, petunidin and malvidin, despite the ratio of tri- to di-hydroxylated anthocyanins being unaffected at harvest in the grape berry in 2020 . Among shade film treatments in 2020, the reduction of UV light exposure, was the determining factor in anthocyanin hydroxylation patterns rather than berry temperature. Previous shade net studies at the experimental site showed a reduction in UV radiation with black-40% and blue-40% shade nets led to higher anthocyanin tri-hydroxylation in the grape berry compared to control vines at harvest . With the reduction of UVB and UVC radiation in D4 and D5 vines, anthocyanin tri-hydroxylation was reduced, regardless of temperature. Ultimately, the upregulation of F3’H from sun exposure could be negated by the reduced catalytic activity of this enzyme under high temperatures experienced in 2020. In the cooler 2021 vintage, the ratio of tri- to di-hydroxylated anthocyanins was unaffected, due to non-significant effect of shade films on acetylated anthocyanins. Ultimately, increased tri-hydroxylation in young red wines will also impact wine hue, resulting in more purple wines . Flavonols in the grape berry skin act as photoprotectants and are strongly induced by ultraviolet radiation . Flavonol composition in the grape berry can be used to determine overexposure, raspberry container specifically by quantifying the molar abundance of kaempferol. C0 berries in this study were shown to be overexposed by surpassing the previously described threshold of approximately 7% molar abundance of kaempferol . In both years of the study, flavonol composition in grape berries was maximized in C0 fruit, but D4 and D5 fruit contained the most flavonols across the shade films with minimal thermal degradation of the compounds on the vine. Likewise in both winevintages, flavonol concentration was modulated by UV radiation exposure, proportional to the amount of UV radiation transmitted to the grapevine. Of the wines produced from shade films treatments, D4 allowed for the most UV transmission while subsequently reducing near infrared transmission by approximately 15%. These light conditions ultimately optimized flavonol content in D4 wines compared to the other shade treatments from both wine vintages. As such, this demonstrated the transmissibility of berry composition under shade treatments to directly improve wine flavonoid profiles. For hot viticulture regions, photo selective solar radiation exclusion provides a strategy to improve not only flavonoid profile but also wine color intensity through copigmentation with anthocyanins.

C6-alcohols such as 1-hexanol and -2-hexen-1-ol are often found in wines as fermentation products. These compounds are derived from microbial mediated cleavage of the C-C double bonds in linoleic and linolenic acids, by lipoxygenase and alcohol dehydrogenate enzymes in yeast . Compounds such as 1-hexanol and -2-hexen-1-ol are associated with aromas such as cut grass, green, fat, and herbaceous aromas and their OAV thresholds are 8000 and 400 ug/L, respectively . The effect of shade films on C6-alcohols was evident in both years; however, there was a yearly effect on which alcohol was altered by the treatment. In 2020, -2- hexen-1-ol was the lowest in D4. In 2021, -2-hexen-1-ol was unaffected by shade films, while 1-hexanol was highest in C0, D4 and D5. Although there was a statistical difference in C6 alcohols, the differences were not large enough between C0 and treatments to cross the OAV thresholds for these compounds. Increases of C6-alcohols in C0, D4, and D5 wines may be explained by solar radiation overexposure in the treated clusters. L. He etal. reported higher linoleic and linolenic acid biosynthesis with leaf removal at veraison. Subsequently, fruit exposed to increased solar radiation had elevated precursors for C6-alcohol production during yeast metabolism. Additionally, L. He et. al. showed higher initial concentration of C6-alcohols in grape berries from leaf removal treatments due to modulation of the volatile compound metabolome and transcriptome in grape berries exposed to sunlight under dry-hot conditions. Therefore, in our experiment which has similar climatic conditions to L. He et. al. , fruit from shade films with higher percentages of UV radiation may have both an increase in linoleic and linolenic acids to act as C6-alcohols aromas precursors and increased C6- alcohols in the exposed grape berries. Ultimately, overexposure of the grape berry led to more green and grassy aromas in wine, which may lead to an unripe perception of these wines. These compounds are generally pleasant aromas including mushroom, roses, honey, candy, and fruity notes. Of these compounds, shade treatments increased isoamyl alcohol concentration in 2020 and benzyl alcohol concentration in wines from both vintages. Isoamyl alcohol is associated with solvent and cheese aromas and, while benzyl alcohol is characterized as being citrusy and sweet . The odor active thresholds for these compounds are 30000 μg/L and 10000 μg/L, respectively . In 2020, C0 had the lowest concentration of isoamyl alcohol in wines. The effect of shading on the concentration of isoamyl alcohol in wines varies in literature . In hot growing regions, 75% of total solar radiation exclusion with black polyethylene canopy side shade nets resulted in wines with reduced isoamyl alcohol compared to the uncovered control vines . However, this experimental site was in a region that received approximately 704.5°C less growing degree days than the present experimental site in the hotter 2020 season, and 514.1°C growing degree days less than the cooler 2021 season. In the study by Lu et al. 2021, reduced solar radiation exposure in a cooler growing region may have resulted in reduced isoamyl alcohol in shaded fruit. When cluster temperatures exceed 42°C in exposed vines, there is a reduction in isoamyl alcohol in resultant wines compared to wines produced from fruit under red and black shade nets . With cluster temperatures of C0 fruit exceeding 42°C, excessive cluster temperatures may be prompting the reduction in isoamyl alcohol and overall wine fruitiness from those produced from overexposed clusters. However, while there was a statistical difference in isoamyl alcohol concentrations between C0 and treatment wines, the effect was not large enough to exceed the OAV threshold for this compound . Shade films affected the ester composition predominantly in 2020 wines.