Abstract
Background and goals Tissue analysis is used as a diagnostic tool to identify grapevine mineral nutrient status. Leaf blade and petiole samples are commonly collected for routine analysis at bloom or at veraison, which are stages of the growing season when fungicides may be applied. This project compared the potential for fungicide contamination of leaf blades and petioles and evaluated different decontamination methods to identify a practical procedure for removing unwanted residues without leaching endogenous elemental nutrients.
Methods and key findings Across six tests, fungicide contamination was observed on unwashed blades for seven nutrients (manganese, phosphorus, calcium, potassium, iron, sulfur, and zinc) and sodium after a fungicide application. Unwashed petioles had less fungicide contamination compared to blades, likely due to their surface area, texture, and/or orientation in the canopy, but contamination was observed in five out of six tests for the same nutrients as blades. Washing whole leaves in water only and in solutions of citric or hydrochloric acid, phosphate-free dish soap, and plant-based oils and organic acids did not completely remove fungicide contaminants, particularly from blades. A liquid, phosphate-free laundry detergent was most effective at removing fungicide contaminants at a rate of 3 mL/L, with an agitation time of 30 sec by hand per 30 whole leaves, followed by a double rinse in reverse osmosis water with hand agitation for 5 sec per five leaves.
Conclusions and significance The results of this project highlight the importance of prudent tissue decontamination (particularly of leaf blades) for mineral nutrient analysis, and if the described protocols are adopted, these results may extend to improving methods at tissue testing labs.
Introduction
Tissue analysis is widely recognized both as the most accurate method of monitoring plant nutrition (Bryson et al. 2014) and as a diagnostic tool used to identify nutrient deficiencies and toxicities (Bates and Wolf 2008). Leaf petioles and blades are the preferred tissues to monitor grapevine nutrition, although each is recognized as having limitations. Leaf petioles have been recommended for nutrient analysis because they are less likely to require washing than leaf blades and are easier to handle in large quantities, offering greater potential to sample larger vineyard acreage and better represent nutrient status (Christensen 1984). However, more recent research has suggested that leaf blades may provide greater accuracy for certain nutrients such as nitrogen (Davenport et al. 2012, Schreiner and Scagel 2017), potassium, phosphorous, calcium, magnesium, and zinc (Romero et al. 2013, 2014).
The specific method used to prepare tissue samples for nutrient analysis is critical to obtain useful data (Campbell and Plank 1998). To ensure that analysis results reflect endogenous nutrients, it has long been recommended to decontaminate tissue samples prior to analysis if dust, nutrient spray, and/or pesticide contamination is suspected (Ashby 1969, Jacques et al. 1974).
Decontamination procedures for grapevines are not consistently reported. The most common recommendation published on university websites in the United States involves washing samples in distilled water or a dilute phosphate-free detergent followed by a double-rinse in distilled water, but details regarding washing conditions are inconsistent (Christinsen 2005, Dami and Smith 2019, Hickey et al. 2021, Klodd and Rosen 2021, Skinkis and Schreiner 2024). An informal survey of 15 public and private plant analysis laboratories across the U.S., Australia, and New Zealand yielded inconsistent recommendations regarding tissue sample volume, timing, and sample preparation.
It has been reported that fresh plant tissue samples should be washed in a mild 2% phosphate-free dilute detergent solution while fully turgid, yet done rapidly so as not to leach soluble nutrients from the tissue itself (Bryson et al. 2014). The same study indicated that washing with water only or with dilute acid will not remove many contaminants. However, other research has reported more successful decontamination with detergent and dilute hydrochloric acid, either mixed in a single washing solution or as consecutive washes (Smith 1962, Orphanos 1975, Smith and Storey 1976, Crowley et al. 1996, Peryea 2005, Sonneveld and van Dijk 2008). Other authors have also reported that the efficacy of a particular decontamination method varies by plant type (Jacques et al. 1974, Wallace et al. 1980, Hargrove et al. 1985, Sabina Rossini and Vales 2004) as well as by the specific tissue of an individual plant (Ashby 1969).
Because excessive washing can leach endogenous soluble nutrients from plant tissue (Bhan et al. 1959, Bosell 1972, Wallace et al. 1982, Moraghan 1991), one study recommended dipping tissue samples in a wash solution for only 10 to 15 sec, while mixing vigorously by hand (Sonneveld and van Dijk 2008). Brushing samples with cleaning solution resulted in excessive tissue damage to several vegetable crops and was not recommended. Alternatively, plant tissue may be gently rubbed between the fingers while washing to remove dust and dirt particles, but this must be done quickly to minimize exposure to the washing solution and to minimize the risk of leaching soluble elements (Bryson et al. 2014). This same study also reported that the presence of wax, pubescence, and concentration of contaminant(s) influence the efficacy of a particular washing method.
In commercial viticulture, grapevine tissue testing is widely practiced, and the most common sampling times often coincide with foliar pesticide applications, likely increasing the potential for contamination and subsequent false readings in the analysis from the lab if tissue is not decontaminated properly. For example, common vineyard fungicides may contain significant concentrations of macronutrients (e.g., phosphorous from phosphorous acid, potassium from potassium bicarbonate) and micronutrients (e.g., manganese, zinc from mancozeb). Grape leaf blades and petioles may require different decontamination procedures to remove unwanted residues because of the differences in their surface area and texture. This study evaluated the potential for pesticide contamination of grape leaf petioles versus blades, and nine tissue washing procedures were compared to identify a simple, practical method to effectively remove contaminants from grapevine tissues.
Materials and Methods
Plant material and fungicide application
To evaluate the potential of contamination from fungicide residues on leaves and the efficacy of various washing treatments, fungicides were applied as a tank mix to mature (year 5 and older) Vitis spp. cultivars Blanc Du Bois, Camminare noir, and Champanel, using a Jacto Arbus 400 (Jacto Inc.) air blast sprayer at a delivery rate of 468 L/ha (Table 1). The study was conducted from 2020 to 2024 at the Texas A&M Horticulture Teaching, Research, and Extension Vineyard in Snook, TX (30°53′N; 96°W; 103 m asl) and at a commercial vineyard in Harleton, TX (32°38′N; 94°W; 97 m asl). Fungicide contamination testing was initially performed to compare the potential for contamination of leaf blades versus petioles. However, additional decontamination treatments were added through five additional iterations of testing in an effort to identify a simple and effective decontamination procedure.
Fungicide product name, active ingredient, and rate by cultivar applied in Tests 1 to 6 for fungicide decontamination trials.
Immediately prior to the fungicide application, 1 hr after application, and 7 days after application (2019 only), 400 to 600 whole leaves were collected from randomly selected shoots on both sides of the canopy of 25 consecutive vines to increase homogeneity of nutrient concentrations among leaf samples. Leaves subtending clusters and the most recently mature leaves (five to seven nodes from the shoot tip) were sampled at bloom and from veraison to postharvest, respectively. After collection, samples were immediately placed into air-tight plastic bags and stored at room temperature until decontamination treatments were performed. No other plant protectant or fertilizer sprays were applied to the vines within 2 wk prior to sampling except for Champanel in Test 6, which was sprayed with Dithane F-45 at a rate of 7.48 L/ha, 10 days prior.
Decontamination treatments
Explanation and abbreviation of all decontamination treatments is provided (Table 2). Approximately 30 min after tissue collection, the whole leaf samples were removed from storage bags and pooled together to increase sample homogeneity. A subsample of 100 whole leaves was collected from each decontamination treatment and divided into four replicates of 25 leaves. Washing was performed in reverse osmosis (RO) water-based solutions (Table 2) in 7.57-L buckets. Agitation by hand was performed by submerging samples and gently rubbing them individually between gloved fingers to remove contaminants for the specified amount of time. Rinsing was performed by submerging five whole leaves with agitation by hand for 5 sec. Washing and rinse solutions were replaced between each replication to avoid cross-contamination. After washing, samples were pat dry with paper towels, then the petiole was separated from the leaf blade using a razor blade. Samples were then transferred to paper bags and placed in a drying oven at 80°C for 72 hr. Dried samples were sent to the Texas A&M AgriLife Extension Soil, Water and Forage Testing Laboratory for mineral analysis, using inductively coupled plasma-mass spectrometry (Issac and Johnson 1975, Havlin and Soltanpour 1980).
Decontamination treatment abbreviation, rate, and agitation time for Tests 1 to 6 for fungicide decontamination trials.
Statistical analysis
The experimental design for the decontamination treatments was a randomized complete block with four replications. Data were subjected to the Proc GLM procedure using SAS statistical software (SAS Institute, Inc.) and means were separated using Tukey’s honestly significant difference test. Data from each test were analyzed separately except for three treatments (not sprayed, not washed [NSNW]; sprayed, not washed [NW], and washed with laundry detergent 3 days post-spray [LD3]) in Tests 3 to 5, which were also pooled over test (year, cultivar). Data for the NSNW and NW treatments were also pooled over the first five tests and analyzed using unpaired t-tests.
Results
Test 1
Bloom-time tissue sampling in vineyards frequently corresponds with pesticide applications to control fungal disease and insect pests. Many pesticide products contain elemental nutrients that could artificially inflate nutrient values on tissue test results, and in the first iteration of testing, a tank mix application of Dithane F-45 and Pristine 38 WG resulted in significant contamination on leaf blades compared to unsprayed leaves for four nutrients (Zn, Fe, S, and Mn) and Na when samples were collected ~1 hr after application (Tables 1 to 3). One week after application, Zn, Mn, and S contamination was still present on leaf blades, although 12.7 mm rain fell prior to the sample collection. In contrast, the petiole treatments did not differ for any nutrient except N at either sample timing. The only difference observed for N was between the samples collected 1 hr after application and those collected 7 days later. This may have been a result of natural temporal fluctuations in nutrient concentrations (Keller 2020) rather than fungicide contamination.
Mineral nutrients in leaf blades and petioles for Tests 1 to 6 from fungicide decontamination trials.
In the leaf blade samples, the greatest contamination observed was for Mn, with a range of 54.66 to 474.06 mg/kg for the NSNW and NW treatments, respectively. The washing treatment performed with dish soap (DS) within 2 hr of the spray application contained less Mn than the NW treatment but still had a greater than 4.5-fold concentration (258.94 mg/kg) of Mn than the NSNW treatment. Similar results were observed for the washing treatment performed 1 wk after spraying (DS7), but subjecting the leaf blade samples to a second washing and rinsing regime (DS7×2) removed additional Mn. Although the DS7×2 treatment still contained a concentration of Mn that was 3-fold higher (177.47 mg/kg) than the NSNW treatment, it was not statistically different.
Test 2
In the second test, difficulties in completely removing visible soap residue with a double rinse in RO water resulted in replacing the phosphate-free dish soap with a phosphate-free liquid laundry detergent. Additional washing treatments (citric acid [CA], RO, and running tap water [RW]) were also added and the agitation time for all treatments was extended from 15 to 20 sec to increase the efficacy of decontamination.
Similar to Test 1, four nutrients (P, Zn, S, and Mn) and Na were higher in the NW blade treatment compared to NSNW, indicating that contamination was present. Contamination was also observed in the NW petiole samples in this test, but only for Mn. The RW treatment was not effective at reducing fungicide contamination in either tissue type, but the other washing treatments did not differ from NSNW, with the exception of the RO and CA treatments in the petiole samples, which contained 24.3 and 25.3% more Mn, respectively.
Test 3
In a continuing effort to identify an effective and practical decontamination option, additional wash solutions (essential oils-organic acids [AO] and RW) and longer agitation times (30 sec) were incorporated in Test 3. An unsprayed treatment washed with liquid detergent (NSLD) was also added to determine if surface contamination was already present on the unsprayed samples and if the wash solution itself may serve as a potential contaminant, as reported previously (Peryea 2005).
In this test, NW leaf blades that were sprayed with Dithane F-45 and Rampart fungicides had higher P, Ca, Fe, Zn, S, Mn, and Na compared to the treatments that were not sprayed (NSNW and NSLD). The contamination of additional elemental nutrients observed in this test (P, Ca, and Fe) may at least be partially attributed to the new fungicide combination used. In previous tests, Dithane F-45 and Pristine 38WG were applied, but in Tests 3 to 5, Rampart was added in place of Pristine 38WG. By weight, Rampart contains 53% mono- and dipotassium salts of phosphorous acid. Dithane F-45, which was used in all six test iterations, contains high concentrations of Mn (2.74% by weight), Zn (0.33% by weight), and S (28.7% ethylene bisdithiocarbamate ion), which were present as a contaminant in the NW blade treatment in all tests. It should also be noted that inert or carrier ingredients may also serve as a source of contamination.
In Test 3, tissue contamination was also observed as a result of some of the washing solutions. The AO washing solution caused Na contamination in leaf blade samples, with eight times the concentration of Na (163.60 mg/kg) compared to the NSLD and NSNW treatments (20.17 mg/kg for both treatments). In Tests 4 and 5, the LD3 treatment also caused Na contamination, indicating that the rinsing procedure used was not adequate. After washing, samples were double rinsed in RO water for a duration of 5 sec per batch of five leaves for each rinse and the wash-and-rinse basins were replaced with new water between treatment replications to reduce the potential for cross-contamination. Although RO water was used in this study rather than distilled water, it was not suspected to have caused the contamination, as the mean Na concentration of two separate water samples was 3 mg/L.
In Test 3, the LD3 with drill agitation (LD3D), DS, and RW treatments were not completely effective at removing fungicide contamination from leaf blades, as samples contained higher Mn and Zn (only DS and RW) than the NW and NSNW treatments. The best decontamination treatments in this test were LD and LD3. However, the paint stirrer attachment with drill agitation used in LD3D was not as effective as agitation by hand combined with gentle rubbing of leaves between fingers.
Tests 4 and 5
In the fourth and fifth iterations of testing, the primary objective was to confirm the efficacy of the LD3 treatment. Other washing treatments (hydrochloric acid [HCl], LD3D) were still included in Test 4 for comparison, but the goal was to determine if the LD3 treatment was consistent across different non-pubescent grape cultivars (Blanc Du Bois and Camminare noir). Similar to the previous test, the LD3 treatment effectively removed contaminants from both tissue types (blades and petioles) when compared to the NSNW treatment, but the LD3D and HCl treatments did not decontaminate leaf blades or petioles.
Test 6
In the sixth test, Champanel, a grape cultivar with dense leaf pubescence, was added to determine if the LD3 treatment parameters were sufficient for cultivars with characteristics different than those previously tested. The tissues sampled from Champanel were either sprayed with Dithane F-45 the day of sampling or 10 days prior (considered unsprayed; defined in Table 2 as "NSNW"). When the LD3 treatment was applied to the either sprayed or unsprayed tissue, several nutrient elements (K, Ca, Zn, Fe, and Mn) were reduced, indicating the presence of contamination. Similar results were observed for petioles. In comparison, the NSLD treatment, when performed 3 days after spraying, had lower Mn and Fe than LD3, indicating that contamination still remained in the LD3 treatment. However, a second washing (LD3×2) did not further reduce Mn or Fe.
Pooled data
Data for the LD3, NSNW, and NW treatments from Tests 3 to 5 were pooled for analysis to determine if the efficacy of LD3 on leaf blades and petioles was consistent over the three iterations of testing (Figures 1 and 2). Unwashed leaf blades (NW) showed contamination for five nutrient elements (Zn, P, S, Mn, and Fe) and Na. The LD3 treatment did not differ from the NSNW treatment in terms of nutrient elements, indicating decontamination was successful. However, Na was higher in the LD3 treatment than in the NSNW and NW treatments, indicating that detergent remained on the leaves after rinsing. Although the same rinse procedure was used for all tests, the method of double rinsing was not sufficient for the LD3 treatment in two out of three tests, indicating that additional rinsing is necessary to remove the Na contributed by the detergent.
Mean leaf blade concentration of zinc, sodium, manganese, phosphorus, and sulfur for Tests 3 to 5 for blades that were not sprayed (NS), sprayed and not washed (NW), and sprayed and washed in a 3 mL/L laundry detergent solution followed by a double rinse in reverse osmosis water (LD). ** and *** indicate statistically significant differences at p < 0.001 and 0.0001, respectively. Means followed by different letters are significantly different according to Tukey’s honestly significant difference test. Error bars represent standard error.
Mean leaf petiole concentration of zinc, manganese, and sulfur for Tests 3 to 5 for petioles that were not sprayed (NS), sprayed and not washed (NW), and sprayed and washed in a 3 mL/L laundry detergent solution followed by a double rinse in reverse osmosis water (LD). ** and *** indicate statistically significant differences at p < 0.001 and 0.0001, respectively. Means followed by different letters are significantly different according to Tukey’s honestly significant difference test. Error bars represent standard error.
Fewer elemental nutrient contaminants were found in the petiole samples (Mn, Zn, and S) over the three tests compared to leaf blades, but contamination was present in unwashed samples. The LD3 treatment was not different than the NSNW treatment, indicating that the decontamination procedure was successful on the petiole samples.
A direct comparison of the contamination potential of leaf petioles versus leaf blades was made by comparing mean nutrient concentrations of NW samples over the five tests, expressed as a percentage of the NSNW treatment for the respective tissue (Figure 3). Concentrations of the elemental nutrients that were elevated in most tests in the unwashed tissue samples (Zn, Mn, and S) and Na were higher in the blade samples than in the petioles by 172, 493, 36, and 239%, respectively. The greater potential for contamination of leaf blades may warrant more deliberate and careful decontamination to reduce the potential for artificially inflated nutrient values in tissue tests.
Mean tissue sodium, zinc, manganese, and sulfur in fungicide-sprayed leaf petioles and blades that were not washed from Tests 1 to 5, expressed as a percentage of the unsprayed treatment. *** indicates statistically significant differences at p < 0.0001. Error bars represent standard error.
Discussion
The initial objective of this study was to compare the potential for fungicide contamination of leaf petioles versus blades. Over the past decade, multiple studies have suggested that leaf blades may be more reflective of overall vine nutrient status (Davenport et al. 2012, Romero et al. 2013, 2014, Schreiner and Scagel 2017) and thus, increased use of leaf blades for tissue analysis has been observed in some regions. Based on their size, texture, pubescence, and orientation in the canopy, leaf blades were expected to be more prone to contamination from pesticides and nutrient sprays than leaf petioles. This was evident when comparing unwashed samples that had been sprayed with fungicides to those that had not been sprayed. Likewise, leaf blades were expected to be much more difficult to decontaminate because of these same characteristics. This was also observed in this study, however, several of the tested washing methods proved to be ineffective for either tissue. The tested solutions were selected based on previous research and observation, as well as product availability to producers. Water-only treatments were also included in Test 2 to mimic practices reported by some grapegrowers. Ultimately, the goal was to identify a practical method of decontamination that grapegrowers could adopt that does not leach endogenous nutrients from tissue samples, as reported in other plant tissue studies (Bhan et al. 1959, Bosell 1972, Moraghan 1991). This goal may also extend to the laboratory setting, as some growers under time and labor constraints prefer decontamination to be performed by a plant tissue testing laboratory.
Across all tests, a phosphate-free liquid laundry detergent solution proved to be the best at removing fungicide residue. However, leaf blade samples were not contaminant-free when the phosphate-free liquid laundry detergent was used with 20 sec of agitation, as Mn in the LD treatment was 28% higher than in the unsprayed treatment. Therefore, a third round of testing was conducted with longer agitation times. In Tests 3 and 4, a plastic paint stirrer drill attachment was used for more vigorous agitation, but without success. The additional agitation time by hand proved to be more effective, but it should be noted that the grape cultivars used in this study, Blanc Du Bois and Camminare noir, had medium to small leaves, depending on sample location on the shoot (basal or distal), and are not densely pubescent. Larger and more pubescent leaves such as those on Vitis labrusca hybrids would likely require greater effort to decontaminate the blades and washing may need to be performed on fewer blades at a time. This was observed in the pubescent grape cultivar Champanel, although doubling the washing treatment did not result in greater efficacy. Further research is needed to identify more effective washing conditions for similar pubescent cultivars, possibly also when using other common pesticides.
In this study, 25 whole leaves for each sample replication were placed in a washing basin which was filled with 8 L of any of the described washing solutions. Agitation was performed by hand, rubbing each leaf quickly, but individually, except in the treatments in which a paint stirrer was used for agitation. A similar action was performed in each of the rinse steps, but with only five leaves at a time for 5 sec. In three tests, Na contamination was observed that resulted from incomplete removal of the washing solution. For larger leaves or in cases of significant contamination, washing and rinse solutions may require frequent replacement to ensure proper removal of contaminants and to avoid cross-contamination (Piper 1942). Alternatively, running water may be used for rinsing, but the last rinse step should be pure water to avoid contamination from the rinse water.
In Test 1, leaf samples that were collected 1 wk after a fungicide application (NW7, DS7, and DS7×2) still contained significant contamination of several nutrients even though the vineyard had received 12.7 mm of rainfall. It has been suggested that unless leaf tissue is visually coated with dust or other foreign substances, washing is usually not required (Jones and Case 1990). However, the widespread use of tenacious fungicide materials in viticulture likely warrants tissue washing, particularly for samples collected at bloom time, when fungal disease prevention is most critical. While the persistence of a fungicide is typically a desirable property, it may present a significant challenge for proper decontamination when collecting samples for nutrient analysis. It is also possible that systemic fungicides within leaf tissue can serve as a source of contamination if they contain elemental nutrients that are not in a plant’s useable form. Although some researchers have recommended dipping plant tissue in a wash solution for only 15 sec to prevent leaching of soluble nutrients (Sonneveld and van Dijk 2008), we were unable to completely decontaminate grape leaf blades with 15 sec of washing with agitation included.
In Tests 4 to 6, the LD3 treatment was the primary focus due to its relative effectiveness observed in Tests 2 and 3. While there may be alternative detergents or methods of decontamination that are equally or even more effective, this study demonstrates that the washing technique used for grapevine leaf samples is important. Careful and deliberate tissue washing is recommended when pesticide or nutrient sprays have been applied prior to sampling, to prevent possible overrepresentation of certain plant nutrients during analysis.
Conclusion
This study demonstrates the need to methodically decontaminate grapevine tissue samples collected for nutrient analysis by comparing leaf blade and petiole samples sprayed with fungicides, compared to tissue samples that were not treated with fungicide. Leaf samples were also subjected to a series of washing treatments that varied by solution type, agitation time, and agitation type, to identify a simple and effective decontamination method that could be easily adopted by growers with minimal costs. Overall, petioles were less prone to fungicide contamination than leaf blades, but contamination was observed on petioles in five out of six tests, indicating a need to decontaminate all tissue types.
The specific decontamination procedure used with respect to solution type, length of agitation time, and method of agitation proved to be important. A solution of liquid phosphate-free laundry detergent at 3 mL/L with an agitation time of 30 sec by hand removed fungicide contaminants from leaf blade and petiole samples, while shorter agitation times and solutions of phosphate-free dish soap, citric acid, hydrochloric acid, and plant-based essential oils and organic acids were less effective. In two tests, Na contamination from the detergent wash solution was observed, indicating that the double-rinse procedure (5 sec of agitation per five leaf blades) was not consistently effective and the wash solution itself could serve as a source of contamination. In many vineyards, especially those in higher rainfall areas where an increased number of fungicide applications are necessary throughout the growing season, grapevine tissue samples should be carefully washed prior to nutrient analysis to remove possible contaminants from pesticides, nutrient sprays, or dust. The potential for contamination is expected to vary based on the specific products applied and application timing relative to sampling. While some plant protectant products may not contain significant concentrations of elemental nutrients in their active ingredients, carrier materials may serve as a source of contamination. The specific washing and rinse procedure used may need to be adjusted based on tissue surface area, the presence of pubescence or a rough texture, and the concentration and type of contaminants present.
Data Availability
All data underlying this study are included in the article.
Footnotes
This research was partially funded by the Gulf Coast Grape Growers Association.
Scheiner JJ, Pontasch F, Cook M and O’Brien P. 2025. Fungicide contamination on grape leaf blade and petiole samples used for nutrient analysis. Am J Enol Vitic 76:0760016. DOI: 10.5344/ajev.2025.24065
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- Received November 2024.
- Accepted April 2025.
- Published online July 2025
This is an open access article distributed under the CC BY 4.0 license.
References
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