Impact of Vine Vigor, Nitrogen, and Carbohydrate Status on Fruitfulness of Pinot Noir

The first year of a two-year research project was completed in 2014. The project involves three components, all analyzed within main plot vines managed with different vineyard floor management practices and different levels of vine vigor and nitrogen status. Preliminary results show that bud fruitfulness is not reduced at basal nodes for canes assessed within any of the vineyard floor treatments. Vines grown with cultivated alleyways (Tilled) were the most vigorous with the greatest leaf area and lowest light infiltration compared to vines with grass alleyways (Grass). Dormant buds collected in winter 2014 from Tilled treatment vines had the highest fruitfulness at several nodes along the cane. Primary bud necrosis was rarely found and did not differ between Tilled and Grass treatments. Grass vines had less leaf area, lower yields and higher fruit total soluble solids at harvest as was found in prior years of research in this block. Vine tissue N and carbohydrate analysis are still pending as of this reporting. The differences in nitrogen ({aed9a53339cdfc54d53cc0c4af03c96668ab007d9c364a7466e3349a91bf0a23}N) and total non-structural carbohydrates of bud, cane, shoot, trunk and root samples will be compared to bud fruitfulness data to understand the dynamic role of N and carbohydrates on bud development and fruitfulness. Within the main plot vineyard floor management study, two experiments were conducted within sub-plots to evaluate the effect of canopy management practices on bud fruitfulness, including lateral removal and cluster zone leaf removal. Both included a time course study of lateral and leaf removal.

Lateral shoot removal treatments were imposed during one of three different time points (fruit set, pea-size and bunch closure) in 2014 and compared to a no lateral removal treatment. Timing of lateral removal did not increase light exposure to the upper canopy where laterals were removed, indicating that any differences in bud fruitfulness within apical sections of dormant canes may be due to internal differences such as nitrogen or carbohydrates rather than light or temperature effects. The main differences in canopy light were due to the main plot effect (Grass, Alternate and Tilled). There were no differences in photoassimilation rates of primary leaves with or without a lateral. Data collected from these experiments will be compared with bud fruitfulness that is currently being assessed for winter 2015. Cluster zone leaf removal was applied to vines during one of three different time points during 2014 (bloom, fruit set, and pea-size), and these were compared to a no leaf removal treatment. Leaf removal increased light exposure to the buds in the fruit zone where leaves were pulled. Within the Grass vines, yields were higher with no leaf removal compared to those with leaf removal. Grass vines had lower total vine leaf area than Alternate and Tilled vines, and removal of leaves early season could have affected fruit set. Fruit set data are still being analyzed. Data collected from these experiments will be compared with bud fruitfulness that is currently being assessed for winter 2015.

Cultural Practices to Modify Berry Physical Properties and Susceptibility to Cracking

Ethrel sprays, irrigation regimes, and girdling all had a significant influence on Flame Seedless cracking in the 2010 field trials at Arvin, CA. Among the 64 treatment combinations of these factors, ethrel sprays had the largest effects, particularly the first ethrel spray, which increased cracking rate by six-fold compared to plots with no spray. This suggests that the dose and timing of the ethrel spray may be a key issue in balancing the advantages for color development with disadvantages of cracking. The four irrigation treatments, 0.6X, 1X (grower standard), 1.7X and 2.3X, caused clear differences in plant water status as measured by stem water potential (SWP), with berries from the higher irrigation treatments having significantly increased berry size, but also increased cracking, and berries from the lowest irrigation having decreased size, firmness, and cracking. Cell turgor was also well correlated with berry firmness. The correlation between irrigation and cracking justifies the prospect of future usage of SWP and other linked berry properties to monitor and minimize cracking, and we will continue research to resolve these factors in order to provide growers with information about threshold values for cracking. Girdling also significantly increased cracking with the late girdle having higher effects than the early girdle. Late girdle may not be as necessary for this field considering its negative impact on cracking but minor effects on color development. Tracking of individual berry growth showed that berries at around veraison expanded with an average rate of about 2.5{aed9a53339cdfc54d53cc0c4af03c96668ab007d9c364a7466e3349a91bf0a23} per day, but most of the expansion occurred at night. A peak short-term rate equivalent to 6.4-8.5{aed9a53339cdfc54d53cc0c4af03c96668ab007d9c364a7466e3349a91bf0a23} per day was exhibited at 7-8am, but the monitored berries that showed these rates did not crack. Skin mechanical properties in the standard and frequent irrigation treatments were tested with custom equipment (berry balloon system, BBS), and in both treatments, pressure, stress and skin strain at failure (cracking) progressively decreased over berry development, indicating, as expected, that cracking susceptibility increases over time. In the scanning electron microscope (SEM) the surfaces of field-cracked, soaking-cracked, and BBS-cracked berries all showed clear evidence of failure by cell wall breaking and not cell separation, contrary to current scientific thinking. The BBS also detected varietal differences (Flame seedless vs Syrah) in skin mechanical properties, and given the strong impacts of ethrel spray on cracking, it will be important to quantify skin properties with BBS for the different ethrel treatments in 2011. Flame seedless berries were soaked in water with only the stylar end or the longitudinal side immersed, and berries cracked at the stylar end in the former case but remained intact in the latter. Soaking of entire flame seedless berries in water confirmed that berries crack at a fairly low strain (<5{aed9a53339cdfc54d53cc0c4af03c96668ab007d9c364a7466e3349a91bf0a23}). These results and our SEM images showing broken cell walls, indicate that cracking is most likely a local event that is initiated by failure of a few cells rather than a consequence of an overall expansion of the flesh, as previously believed.

Growth and Sugar Transport in Grapes: Early Events in Ripening

In this project we show advances in both our understanding of ‘what happens when’ during berry development and in new technologies and methodologies to study berry growth and development. Ultimately, we intend to discover what veraison is and how the onset of sugar accumulation is regulated in the vine. In the process, we are discovering what veraison is not. Two prevalent ideas about veraison are that: the xylem gets torn apart and stops being conductive and that cell membranes breakdown and cell lose viability (start dieing). Krasnow et al. (J. Exp. Bot. 2008) shows that grape cells are not dieing nor losing membrane function at the beginning of veraison. And in fact cells remain intact and viable until harvest. The two Chatelet papers (Chatelet et al. 2008a,b, J. Exp. Bot.) show that the xylem in the berry remains intact and functional throughout development. We also discovered in the course of that work that the xylem in the berry is comprised of vessels, not tracheids as has been consistently reported (i.e. assumed).

What is veraison? We don’t know yet, but we found that sugar and color accumulation are closely linked, even when water deficits separated the resumption of growth from the onset of those ripening phenomena and caused increased color development. (Castellerin et al. 2008, Planta). We also found that flesh cell turgor pressure decreases in advance of the increased growth, sugar, and color that typify veraison. This together with earlier reports that the stress hormone ABA accumulates at veraison have caused us to speculate that turgor loss and ABA increase may be important in getting ripening started. We sought and were awarded a USDA grant to study the xylem connections of the berry and the how it is that turgor is lost prior to veraison. We report Wada et al. (2008, Planta) that the turgor loss is associated with solutes accumulating outside of the flesh cells. We hypothesize that veraison and the onset of high rates of sugar import is dependent upon this loss of turgor.

To understand the cellular physiological events regarding softening and the onset of berry ripening, we completed development of algorithms that measure and control cell volume and pressure on the automatically-controlled cell pressure probe that this project has funded the development of for three years. We show here application of the instrument to a ‘model system’ that has large cells that are easy to work with. In the no-cost extension period that we are requesting, we will use this new technology to make similar measurements in developing berries. This technological development will put us at the forefront of cell water relations studies and has allowed us to begin work on another USDA grant proposal to investigate the interactions of sugars, cell turgor, and ABA in causing veraison and anthocyanin synthesis. That proposal also exploits our recent progress in developing molecular methods to investigate the genetic basis of veraison. We hope to learn which genes are important in the onset of ripening. We intend to resume work with industry support on sugar accumulation in the near future.

RLF Immunocromatographic Devices for Wuantification of Aspergillis and Laccase in Grape Juice at Harvest Time

Over 850 grape juice samples (approx 10ml) were collected, by CWIAB inspectors from the test stand at Gallo Livingston, throughout the 2006 harvest. They were collected from every 10th load graded for rot. In addition, 123 juice samples were collected, by Gallo personnel, from juice prepared on the test stand from loads that were not being graded for rot but were suspected of containing a significant amount of rot. Samples were collected by Dr Molly Dewey and her research assistant twice a week and tested in five different ways at UC Davis. Samples were tested separately for levels Botrytis and Aspergillus antigens by Lateral Flow immunoassay Devices from two different companies, Central Science Lab, York, UK and Envirologix, Portland, Maine and for laccase by the deScenzo method and by the pilot CSL Lacccase Lateral Flow Devices..

As in 2005, only low levels of rot were found by visual handsort tests but, as expected, levels of rot determined by both the CSL and EnvirologixLateral Flow devices indicated that there was a significant level of Botrytis rot in a number of the 2006 samples particularly toward the end of the season. Results from tests for Aspergillus antigens were suspiciously high but this may, in part, be due to the overly high sensitivity of the new devices. Nevertheless, tests indicated that there was far more Aspergillus rot in 2006 than in previous years. Almost all juice samples with high levels of Aspergillus antigens had high levels of Botrytis. There was a reasonable level of agreement between the fieldbased Lateral Flow devices produced by Central Science labs and the labbased devices produced by Envirologix for quantification of Botrytis and Aspergillus rot. Batch to batch variation in the devices from Central Science Labs was a problem but this should be rectified once their new spin off company ?Foresite? is launched in January 2007. The new company will have the necessary equipment for the manufacture and automatic assembly of devices in large batches. Tests for laccase, by the DeScenzo high through put method, confirmed results from 2005, in that, correlation between levels of Botrytis and laccase was poor at low levels of Botrytis but greater at high levels of Botrytis. The prototype laccaseLateral Flow devices from CSL, gave promising results.

Inception, Diagnosis and Consequences of the Berry Shrivel Disorder

Berry shrivel (BS) is a ripening disorder of unknown cause and sporadic appearance that has been increasingly observed in vineyards around California. BS has been mistaken for another disorder, bunchstem necrosis (BSN), but we have found that the disorders can be distinguished by the rachis of affected clusters. The rachises on BS clusters are green and healthy looking, while BSN rachises are brown necrotic. Much of the data presented here is from the 2005 growing season. This data had not been generated by report time last year. BS fruit has less sugar, lower pH, and reduced coloration several weeks before visible shriveling of the fruit. These differences are maintained throughout the ripening period. BS fruit stops sugar accumulation about two weeks prior to symptom appearance. The apparent rise in Brix of BS fruit after this point is due to fruit dehydration and solute concentration. BS affects a whole vine, as nonshriveled clusters on a vine with shriveled clusters are also compositionally different than fruit from a healthy vine. These ?likely to shrivel? (LTS) clusters have compositions intermediate between BS and healthy fruit. Nonshriveled clusters on a vine with BS clusters also stop, or slow, sugar accumulation at the same time as BS fruit does.

Since BS appears to be due to phloem dysfunction, girdling treatments were set up at different times in Davis and at the Oakville Experimental Vineyard (OEV) in Napa County. Artificially stopping sugar accumulation by girdling caused the berries to develop with reduced coloration and they eventually (3-4 weeks post girdling) began to shrivel.

There has been a trend at OEV for vines with a history of BS to be less water stressed (determined by leaf water potential) than control vines. To test the hypothesis that vine water status exacerbates or mitigates BS, an irrigation trial was set up in the Alexander Valley in Sonoma County. One treatment applied water at a rate that vastly exceeded the grower?s standard irrigation (approximately 5 gph as compared to 0.5 gph), and vines in the other treatment were not irrigated. These treatments affected vine water status, but they did not affect BS incidence in the vines.

The hormone CPPU (Prestige®) was sprayed at two concentrations (2 and 4 ppm) after set (8mm berry size) at two sites (OEV and Alexander Valley) to assess its effect on the incidence of BS and BSN. None of the hormone treatments had an effect on the incidence of BS or BSN.

BS can be propagated by chip buds. Budding was done in 2003, thus there was fruit for analysis in 2006. Vines propagated with buds from vines with a history of BS had reduced sugar accumulation, shorter shoots after budbreak, and reduced berry weight compared to vines propagated from healthy material.

Wines were made with the addition of different amounts of BS and BSN fruit in 2006 (0{aed9a53339cdfc54d53cc0c4af03c96668ab007d9c364a7466e3349a91bf0a23}, 5{aed9a53339cdfc54d53cc0c4af03c96668ab007d9c364a7466e3349a91bf0a23} and 10{aed9a53339cdfc54d53cc0c4af03c96668ab007d9c364a7466e3349a91bf0a23} by weight). The wines fermented to dryness and will be used for difference testing and descriptive analysis.

The Measurement of Vascular Flow into Syrah Clusters

This project attempted to measure the vascular flow (xylem and phloem collectively) through the peduncle of ripening Syrah fruit clusters. Measurements were made on eight separate clusters beginning on July 31, 2006, and continuing until October 16, 2006; measurements were made at 30-minute intervals 24 hours a day. Companion measurements of fruit zone evaporation rates, reference evapotranspiration, irrigation system operation, and berry weights over time were also taken.

Due to unforeseen problems with the datalogging equipment, data was only collected from Sept. 7 onwards, and from only four of the eight clusters. This limited data did not appear to be of the same consistency that was attained the season prior with the same equipment at the same site. Analyses with this limited and possibly non-representative data indicated that the daily sap flow into the clusters was not correlated to daily fruit zone evaporation rates. The parameter ?grams of sap flow per berry, per mm of evaporation? was hypothesized to change over the berry ripening period, reflecting changes in berry physiology. However, the limited data did not show a trend in this parameter, but a proper evaluation was not possible due to the lack of data over the entire ripening period as had been proposed.

Effect of Cluster Temperature on the Composition of Berries Grown Under Field Conditions

The objectives of this project are consistent with the highest priority research objective as outlined by the National Grape and Wine Initiative (Research Priority 1.1.1, “Using modern analytical and sensory techniques identify and quantify the components of grapes and grape products that impact key sensory quality attributes.”) The specific goals of this project are the following:

  • To precisely manipulate the pre-véraison and post-véraison temperature of clusters grown under field conditions to achieve the following:
    • Variance in daytime and nighttime temperature separately
    • Variance in diurnal temperature amplitude (constant mean temperature)
  • To determine the composition of the following compounds in clusters grown under the various temperature treatments:
    • Tannins, anthocyanins, flavonols, flavan-3-ol monomers, polysaccharides, pH, sugars, organic acids

In order to more completely understand the influence of temperature on the growth of the berry and the accumulation of solutes, this field experiment is being conducted with clusters that are growing under different temperature environments under otherwise normal growing conditions. Of specific interest in this study is understanding the effect of diurnal temperature variation on berry growth and development (the integrated temperature was the same but the temperature variation was dampened).

To date and for pre-veraison growth, when one compares the data collected during the first year to images of the clusters collected there are some clear relationships. Overall, a reduction in diurnal temperature variation increased berry size and color suggesting that these berries were temporally advanced in development. In contrast to the influence of diurnal temperature variation, there was a relationship between overall temperature and tannin accumulation, with higher temperatures associated with more tannin production.

Additional analyses for pre-veraison experiments are currently in progress in addition to a separate experiment that was conducted during fruit ripening.

Growth and Sugar Transport in Grape: Early Events in Ripening

Our most important observation is that there is a significant loss of cell turgor early in the onset of ripening in the grape berry. Berry cell turgor was evaluated using two developmental time scales. The full report shows the pattern of turgor during berry development as indicated by Brix. The turgor data are for cells in the outer mesocarp below the hypodermis and dorsal vasculature. The Brix data are for the juice of the same berry after turgor measurements were completed. The data show that turgor declines several bars to a stable turgor of about 1 bar or less during ripening. For intact clusters, the highest cell turgor occurred early in development and was less than 4 bars. Values of turgor shown that are greater that 4 bars are from cells of clusters that were excised and hydrated with water. Thus, turgor could be increased 2 to 4 bars by supplying water prior to veraison. For any stage of development beyond 6 Brix, turgor was about 1 bar. The hydration experiments failed to increase turgor in these berries to as much as 2 bars except in one case at 7.5 Brix.

We observed a “rapid” loss of turgor prior to or coincident with “veraison” on both scales. On a Brix scale, veraison is usually about 7 Brix; on the deformability scale, it is usually somewhere above 10{aed9a53339cdfc54d53cc0c4af03c96668ab007d9c364a7466e3349a91bf0a23}. However, both scales are problematic in testing for earlier events in the onset of ripening. Brix is of limited use as a developmental scale before “veraison” – Brix for all of our green berries was between 4 and 5 regardless of “age”. In addition, Brix determination generally requires destructive sampling of the berry, and it is not possible to know the “age” of the berry before sampling it. It may be possible to measure deformability nondestructively, i.e. on the vine, but it is not clear whether the measurement itself disrupts cell turgor and water relations.

We have had limited success with a vital staining technique for cells of grape berries using fluoresceni diacetate and ethidium bromide. Images of berry sections exposed to the dyes were visualized with a fluorescence microscope equipped with a digital camera and image processing system. The fluorescein stain has been used by Prof. Shackel to visualize impact injury in grape (both red and green varieties). Uptake of this stain has been used by Prof. Vito Polito (Dept. Pomology, UCD) as a measure of membrane integrity and cell vitality in pollen. The ethidium bromide has been used in bacterial and animal studies as a dead cell indicator. When we supplied fresh berry segments with the stains, getting sufficient uptake has been difficult.

We determined that there is a loss of several bars of turgor in the outer mesocarp near of at the beginning of veraison. We determined that there is an accumulation of apoplastic solutes in ripening berries and that the concentration of malate in the apoplast becomes similar to that in the berry as a whole as ripening progresses. We need to learn more about the geography of turgor and water transport in the berry in order to discover the site and timing of the origin of veraison. We need to increase our resolution of berry age and of the changes in apoplastic solutes that occur near veraison in order to draw conclusions about the source of apoplastic solutes and the cause of turgor loss at veraison.

PDF: Growth and Sugar Transport in Grape: Early Events in Ripening

Growth and Sugar Transport in Grape: Early Events in Ripening

Our most important observation is that there is a significant loss of cell turgor early in the onset of ripening in the grape berry. Berry cell turgor was evaluated using two developmental time scales. The full report shows the pattern of turgor during berry development as indicated by Brix. The turgor data are for cells in the outer mesocarp below the hypodermis and dorsal vasculature. The Brix data are for the juice of the same berry after turgor measurements were completed. The data show that turgor declines several bars to a stable turgor of about 1 bar or less during ripening. For intact clusters, the highest cell turgor occurred early in development and was less than 4 bars. Values of turgor shown that are greater that 4 bars are from cells of clusters that were excised and hydrated with water. Thus, turgor could be increased 2 to 4 bars by supplying water prior to veraison. For any stage of development beyond 6 Brix, turgor was about 1 bar. The hydration experiments failed to increase turgor in these berries to as much as 2 bars except in one case at 7.5 Brix. We observed a “rapid” loss of turgor prior to or coincident with “veraison” on both scales. On a Brix scale, veraison is usually about 7 Brix; on the deformability scale, it is usually somewhere above 10{aed9a53339cdfc54d53cc0c4af03c96668ab007d9c364a7466e3349a91bf0a23}. However, both scales are problematic in testing for earlier events in the onset of ripening. Brix is of limited use as a developmental scale before “veraison” – Brix for all of our green berries was between 4 and 5 regardless of “age”. In addition, Brix determination generally requires destructive sampling of the berry, and it is not possible to know the “age” of the berry before sampling it. It may be possible to measure deformability nondestructively, i.e. on the vine, but it is not clear whether the measurement itself disrupts cell turgor and water relations. We have had limited success with a vital staining technique for cells of grape berries using fluoresceni diacetate and ethidium bromide. Images of berry sections exposed to the dyes were visualized with a fluorescence microscope equipped with a digital camera and image processing system. The fluorescein stain has been used by Prof. Shackel to visualize impact injury in grape (both red and green varieties). Uptake of this stain has been used by Prof. Vito Polito (Dept. Pomology, UCD) as a measure of membrane integrity and cell vitality in pollen. The ethidium bromide has been used in bacterial and animal studies as a dead cell indicator. When we supplied fresh berry segments with the stains, getting sufficient uptake has been difficult. We determined that there is a loss of several bars of turgor in the outer mesocarp near of at the beginning of veraison. We determined that there is an accumulation of apoplastic solutes in ripening berries and that the concentration of malate in the apoplast becomes similar to that in the berry as a whole as ripening progresses. We need to learn more about the geography of turgor and water transport in the berry in order to discover the site and timing of the origin of veraison. We need to increase our resolution of berry age and of the changes in apoplastic solutes that occur near veraison in order to draw conclusions about the source of apoplastic solutes and the cause of turgor loss at veraison.

New Parameters to Measure Ripeness

The HPLC method has not been used in a routine analytical fashion. This method uses cacao bean tannin extract for the determination of grape seed tannin molecular weight. Cacao bean extract contains procyanidin polymers consisting solely of epicatechin subunits which results in well separated, easily identifiable molecular weight markers. Since grape based tannins contain the same class of subunit, their retention characteristics are similar to cacao bean tannins. Quantification was achieved by comparing the peak areas of the grape extracts with that of an epicatechin standard. In order to apply this method in a routine fashion, additional method validation was performed and the reproducibility was determined. Based on the cacao bean extract, this method is capable of achieving a reproducibility of between 3.4{aed9a53339cdfc54d53cc0c4af03c96668ab007d9c364a7466e3349a91bf0a23} and 10{aed9a53339cdfc54d53cc0c4af03c96668ab007d9c364a7466e3349a91bf0a23} in the molecular weight range of 300-2100. Using this method, grape seed extracts have been analyzed. This method has the ability to quantify phenolics over a large molecular weight range, from 290 molecular weight units to about 3800 molecular weight units. This translates to a vast amount of information that can be obtained from extracts. From the vineyards studied this year, several trends can be seen in the extracts. First, the overall extractibility of the seeds decreases over time in both vineyards. Second, the amount of monomeric material extracted into solution decreased over time. Finally, the proportion of the extract which is made up of polymeric material (material having >6 subunits) increases as the grapes mature. Information on sample reproducibility was obtained using data gathered on all samples. In general, it was determined that the reproducibility was very good using the sampling technique used. In the worst case, berry weight and sugar reproducibility was 8.8{aed9a53339cdfc54d53cc0c4af03c96668ab007d9c364a7466e3349a91bf0a23} and 1.4{aed9a53339cdfc54d53cc0c4af03c96668ab007d9c364a7466e3349a91bf0a23} respectively. The reproducibility of grape seed extracts was determined also, and ranged form 3.9{aed9a53339cdfc54d53cc0c4af03c96668ab007d9c364a7466e3349a91bf0a23} for monomeric material to 27{aed9a53339cdfc54d53cc0c4af03c96668ab007d9c364a7466e3349a91bf0a23} for the polymeric material. Sensory analysis of wines made this year indicated that there was a significant increase (p=0.05) in astringency in both vineyards as the maturity of the grapes increased. When these wines were analyzed by HPLC, polymer concentration increased as astringency perception increased. Bitterness also increased in the wines as the grapes matured although the differences were much less significant. These results are inconsistent with the grape seed data, where the extractability of the seeds decreased as grapes matured. One possible explanation is that the high sugar levels in the grapes result in a higher alcohol concentration which can extract more tannin material from the seeds. Additional work is needed to study this possible relationship.