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Article

Investigations of Multiple Approaches to Reduce Green Spot Incidence in ‘WA 38’ Apple

by
Ryan Sheick
1,
Sara Serra
1,2,
David Rudell
3 and
Stefano Musacchi
1,2,*
1
Tree Fruit Research and Extension Center, Washington State University, 1100 N. Western Ave., Wenatchee, WA 98801, USA
2
Department of Horticulture, Washington State University, 149 Johnson Hall, Pullman, WA 99164, USA
3
Tree Fruit Research Laboratory, Agricultural Research Service, United States Department of Agriculture, 1104 N. Western Ave., Wenatchee, WA 98801, USA
*
Author to whom correspondence should be addressed.
Agronomy 2022, 12(11), 2822; https://doi.org/10.3390/agronomy12112822
Submission received: 28 September 2022 / Revised: 27 October 2022 / Accepted: 8 November 2022 / Published: 11 November 2022
(This article belongs to the Section Horticultural and Floricultural Crops)
Browse Figures
Versions Notes

Abstract

:
A pre-harvest apple (Malus × domestica Borkh.) disorder named “green spot” (GS) was recently identified on ‘WA 38’ apples. Previous work indicated a tentative association between GS and fruit mineral imbalance, and an influence of rootstock selection on GS frequency; however, the specific causes, risk factors, and mitigation steps have not been explored. In this study, the role of microclimate, modified using netting or fruit bagging, on GS incidence in ‘WA 38’ apples over two years on two different dwarfing rootstocks, ‘Geneva 41’ (‘G.41’) and ‘Malling 9-Nic29’ (‘M.9’), was investigated. Early season fruit bagging reduced GS appearance, but netting showed mixed results between year and rootstock. Rootstock selection influenced GS incidence, especially in the 2021 season. Fruit mineral analyses highlighted some differences of mineral composition between symptomatic and asymptomatic apples, which helped to corroborate earlier reports; however, within the full context of these results, it is not clear that nutrient imbalance directly influences disorder incidence. Further work to understand the mechanisms behind GS emergence should consider environmental factors, including light, humidity, and wind, as well as anatomical features of ‘WA 38’ fruit, such as lenticel morphology and physiology.

1. Introduction

Physiological disorders continue to pose challenges to the fruit production and marketability of apple (Malus × domestica Borkh.). Over the last century, many reports have characterized symptoms, etiology, and mitigation steps for several apple disorders [1,2,3,4,5,6]. Some physiological disorders, such as lenticel breakdown, bitter pit, sunscald, and stain, typically emerge following harvest but are believed to be induced by preharvest factors [4,6,7,8,9]. Both pre-and postharvest disorder incidence and severity are influenced by interactions of environment, cultural practices, and genetics.
Unfavorable orchard environmental conditions, such as elevated temperatures and high solar irradiance, can induce some physiological disorders, such as sunburn, sunscald, stain, and watercore [10,11]. Other disorders may be related to one or more causal factors, such as environmental, chemical, or biotic stress [11,12,13]. Russeting, for example, is a common symptom of pome fruit peel which is the result of epidermal cracking and repair [14] caused by chemical applications [15,16], excess moisture [12,17], microorganisms [18,19] or other orchard pests [20]. Lenticel breakdown of apple has also been associated with various factors, including fruit maturity, preharvest environmental conditions, and postharvest handling and storage regimens [9,21,22].
Cultural practices can also influence the incidence of physiological disorders of apple. Boron and calcium deficiencies influence the incidence and severity of disorders such as internal cork, drought spot, and bitter pit [3,4]. Nutrient applications have been shown to ameliorate such disorders [23,24,25,26]. Furthermore, manipulating tree vigor and crop load impacts nutrient content of leaves and fruit [4,27,28,29,30], which has implications for managing nutrient-related disorders.
Cultivar-specific susceptibility to certain disorders has been well documented [9,31,32,33,34] and implies a genetic predisposition. The causes of physiological disorders in apple are broad and may vary depending on the growing region, cultivar, orchard management practices, and postharvest handling protocols. The multitude of factors influencing fruit physiology complicates the determination of specific causes of physiological disorders, mainly resulting from similarity in appearance and characteristics of unrelated disorders [4]. Indeed, many metabolic changes associated with cork-like disorders, such as elevated ethylene biosynthesis and respiration rates, are, according to Faust and Shear [4], “common to all disorganized tissues regardless of cause,” including pathogenesis by biological agents. Thus, the causes and etiology better define a single disorder than symptom appearance.
The proper identification of disorders is paramount to developing and implementing effective control strategies specific to a disorder. Preharvest cultural practices, including the application of sprays, chemical fertilizers, and other vigor management strategies can ameliorate nutritional disorders [4]. There is evidence that preharvest application of lipophilic protective coatings can reduce postharvest lenticel breakdown in apple [9]. Enclosing developing fruits in paper bags can protect against pest damage [35,36,37], reduce sunburn [35,38], and enhance fruit coloration [36,39,40]. Matsumoto et al. [41] demonstrated the benefit of fruit bagging and tree shading on reducing the incidence of a preharvest cork spot-like physiological disorder (CSPD) in ‘Kurenainoyume’ apples. Because they found a reduction in CSPD in both bagging and shading treatments, and the disorder was correlated with sunshine duration, they concluded that CSPD is likely induced by extreme light conditions in the orchard. The major apple-growing regions of the U.S. Pacific Northwest are characterized by high light intensity and high temperatures, and protective netting has been used to reduce fruit damage and improve marketable yield by protecting against environmental stressors like excessive solar radiation, wind and hail, and biological pests such as insects and birds [10,42,43].
‘WA 38’, a cross between ‘Enterprise’ and ‘Honeycrisp’, is an apple cultivar that was released by Washington State University [44] and was made available to be planted by Washington producers in 2017. Outstanding fruit quality traits of this cultivar were reported [44]. A preharvest physiological disorder affecting ‘WA 38’ apples called “green spot” (GS, for symptom appearance at its onset) was identified in some experimental and commercial orchard plantings [29,45,46]. Symptoms typically occur near the pedicel end of the fruit as superficial regions of altered peel color without an apparent impact on the underlying cortex tissue, or as sunken discolored peel underlain by darkened, cork-like areas of necrosis in the affected flesh. Superficial symptoms can appear as green flecking or spots with irregular margins, often merging into large patches that contrast with the peel color as asymptomatic regions begin to redden mid-season. These affected areas may still be evident late in the season, but mild cases are often masked on apples that achieve intense red coloration by harvest. Symptoms affecting both peel and cortex typically appear as spots with regular margins, previously described as “dark green halos in the epidermis, with necrotic, corky, and [oxidized] cortical tissue underneath the damaged epidermis” [46]. In some cases, spots may darken along the season and appear brown by harvest. Symptoms of GS emerge approximately three months after full bloom and worsen until harvest. Unlike other cork-like physiological disorders such as bitter pit and Jonathan spot [4,6], GS symptoms on ‘WA 38’ apples seem to develop exclusively preharvest; worsening of severity during storage has not been observed [46].
Although Sallato et al. [46] found that GS in ‘WA 38’ was associated with fruit nutrient profiles similar to bitter pit-afflicted fruit in other cultivars, as well as differential susceptibility risk by rootstock selection, the specific cause(s) of the disorder and processes to mitigate it remains unclear. We hypothesized that GS in ‘WA 38’ may be triggered by conditions in the orchard, such as extreme light, heat, or humidity. To test this hypothesis, we examined the influence of fruit bagging and whole-row netting [47], techniques designed to alter the fruit microenvironment and reduce environmental stress, on GS incidence over two years. In the second year, we also evaluated whether the application of Parka® [48], a phospholipid- and polysaccharide-based coating designed to promote plant cuticle health, would alter GS incidence or severity in ‘WA 38’. Additionally, to further supplement the previous work of Sallato et al. [46], we investigated the effect of rootstock selection and the role of mineral composition of peel on GS in ‘WA 38’ apples.

2. Materials and Methods

2.1. Orchard Location and Design

All experiments were conducted at the Washington State University Sunrise Research Orchard (Rock Island, WA, USA; 47.31°, −120.07°; elevation 267 m) in a block established in 2013. This represents one of the major apple-growing areas in Washington State (USA), and is defined by an arid climate with high temperatures and intense radiation in the summer [43].
‘WA 38’ budded on ‘Geneva 41’ (‘G.41’) and ‘Malling 9-Nic29’ (‘M.9’) rootstocks were planted in 2013 in a randomized block and trained to a spindle, spaced at 0.9 × 3 m (3700 trees/ha). These trees were used for all fruit bagging, drape netting, and cuticle supplement experiments (see Section 2.4, Section 2.5 and Section 2.6, respectively), as well as fruit mineral content analyses in the comparison of apples bagged at T1 and control apples, and the comparison between asymptomatic apples grown on both ‘G.41’ and ‘M.9’ (see Section 2.7).
‘WA 38’ budded on ‘G.41’ and ‘M.9’ rootstocks were similarly planted in neighboring rows in the same year, but trained to a V-trellis system and spaced at 0.45 × 3 m (7400 trees/ha). These trees were used for fruit mineral analyses in the comparison of “high” (V+) and “low” (V-) vigor trees, as well as the comparison of fruit with (GS+) and without (GS-) green spot symptoms in the 2020 season (see Section 2.7).
Drip irrigation was used daily, Monday through Friday, in all field experiments for a cumulative watering time of approximately 2 h per day. Additionally, sprinkler irrigation was used for a cumulative 90 min per day, Monday through Friday, to maintain the crop cover and for additional irrigation requirements as needed. Water status was monitored regularly by the farm manager, and water use was adjusted as needed, on the basis of evapotranspiration. Pest management in the block was handled as in a commercial orchard, with targeted applications of fungicides and insecticides at crucial phenological stages as needed. Foliar nutrient fertilizers were applied in spring and fall as formulations of zinc, gypsum, monoammonium phosphate, sulfur, and magnesium sulfate.

2.2. Timing of Green Spot (GS) Onset (2020–2021)

Following fruit set throughout the 2020 and 2021 seasons, the ‘WA 38’ experimental block (both in the spindle and the V-system rows) was scouted by a trained evaluator for signs of GS on a weekly basis. Once an apple with early signs of GS was found, it was tagged and monitored on weekly basis. Early symptoms of GS were first observed on July 29 and July 22 in 2020 and 2021, respectively, and examples of symptomatic fruit were tagged and photographed. Photographs were taken weekly to track GS progression over the growing season in both years. A visual reference of GS progression is presented for 2021 in Figure 1.

2.3. Assessment of Fruit Defects (2020–2021)

Harvested apples from all control and experimental treatments were transferred immediately from the orchard to a regular air (RA) cold storage at 1 °C until external fruit attributes were assessed. Green spot was visually evaluated by one trained operator, and apples were classified into one or more groups corresponding to the type and severity of the disorder. Green spot symptoms displaying no more than one large spot (6 mm in diameter or larger) and/or several smaller spots and superficial green flecking were classified as “mild” GS if the symptoms did not significantly detract from fruit appearance. Apples with more than one large green spot (6 mm in diameter or larger), several small spots that detracted from fruit appearance, green spots with epidermal cracks, or green spots turning brown were classified as “severe” (Figure 2A–C). The main difference between these major categories is that “mild” symptoms may not affect the marketability of the apples, whereas “severe” symptoms are likely to result in pack-out losses. Apples with both “mild” and “severe” symptoms were considered “severe” for the purposes of this classification system. Other external defects, including russet, limb rub, cracks, splits, insect damage, and bird damage were also noted, but did not impact GS classification.
In order to depict the disorder, ‘WA 38’ specimens with green spot symptoms were photographed in the laboratory with a Canon EOS 70D DSLR camera and EF-S 18–55 mm lens (Canon Inc., Tokyo, Japan). Stereomicroscopy was performed on select examples by slicing fruit sections with a razor blade and placing them on a glass petri dish bottom to be viewed with a Nikon SM18 (Nikon Instruments, Inc., Melville, NY, USA). Stereomicroscope images were captured with the NIS-Elements software version 5.3 (Nikon Instruments, Inc., Melville, NY, USA) and presented in Figure 2.

2.4. Fruit Bagging (2020–2021)

2.4.1. Tree Selection and Time Points Treatment Imposition

Trees included in the fruit bagging experiment were selected based on similar crop load within each rootstock and year and were randomized into either control (never bagged) or time-point bagging treatments in 2020 and 2021. In 2020, fruit bagging was carried out over six time points (T1 = June 1; T2 = June 17; T3 = July 6; T4 = July 24; T5 = August 11; T6 = August 28), and in 2021, over three time points (T1 = June 18; T2 = July 2; T3 = July 16), which were selected following results from the previous year that identified crucial time points covering the time range during which bagging influenced disorder appearance. At each bagging time point, three trees for each rootstock combination were bagged (3 ‘WA 38’/‘G.41’ and 3 ‘WA 38’/‘M.9’), while three trees for each rootstock were selected and left unbagged to serve as controls. Approximately three to five apples per each bagged tree were left unbagged (internal checks) to observe the development of the GS disorder and the red overcolor on the fruit in each tree. Fruit bagging was conducted by thinning clusters to a single fruitlet, then enclosing fruitlets in 2-layer apple bags (Kobayashi Bag Mfg. Co., Nagano, Japan) and securing the bags to the fruiting wood with wire twist ties. Bags applied during the growing season remained on the apples until they were harvested. At harvest, all bagged apples were picked together with the 3–5 internal check apples and boxed together as the entire crop of each experimental tree. The harvest dates for this trial were 17 September 2020, and 24 September 2021.
‘WA 38’ GS incidence in both years was calculated for each tree [3 trees/rootstock × 2 rootstocks × 7 time points (6 bagged + 1 non bagged) = 42 trees for the 2020 season] as a proportion of apples affected by the GS disorder on the total apples graded/tree and expressed as a percentage (as reported later in Section 3.3.1). Furthermore, the proportions of “mild” GS-affected apples and “severe” GS-affected apples were calculated starting from the number of apples classified in each category and expressing them as a percentage of the total apples graded.

2.4.2. Microclimate Monitoring of Bagged and Control Treatments (2020–2021)

Three trees corresponding to the ‘WA 38’/‘G.41’ (unbagged) control and three corresponding to the bagged ‘WA 38’/‘G.41’ T1 treatment (Section 2.4.1) were selected for microclimate monitoring in both the 2020 and 2021 seasons. At the T1 bagging time point (1 June 2020; 18 June 2021), two dataloggers (DS1923 Hygrochron iButton®, Maxim Integrated Products, Inc., Sunnyvale, CA, USA) were installed in each tree to monitor temperature (°C) and relative humidity (%RH) inside and outside of fruit bags. In each tree, one datalogger was designated to monitor the “mid-upper canopy,” and the other was designated to monitor the “mid-lower canopy.” Dataloggers were placed inside fine mesh tea bags before installation and then secured to a branch near a fruiting spur with a twist tie in the control treatment, or inside a bag, along with the fruit, in the T1 bagging treatment. Temperature and relative humidity were logged every two hours for the duration of the experiment in each season. A complete day of measurements included 12 readings that were averaged to obtain the daily average temperature, while, across the 12 readings per day the minimum and maximum daily temperatures were selected. For each month, means of daily average, minimum, and maximum temperature, and means of daily average, minimum, and maximum %RH, were reported. Each of the six trees (three ‘WA 38’/‘G.41’ unbagged control trees and three bagged ‘WA 38’/‘G.41’ T1 trees) had 2 iButton® sensors (mid-upper and mid-lower canopy), so the values reported in the results represent the average of two sensors/tree for each day.

2.5. Mono-Row Drape Netting (2020–2021)

Mono-row netting (Diamond V5® Monorang, 10% shading, 2.8 mm × 4.0 mm weave, Helios® anti-hail systems, Bergamo, Italy) was installed and deployed in two experimental rows following full bloom in both seasons. In 2020, nets were deployed 44 days after full bloom (DAFB) on June 1, and 45 DAFB in 2021 (June 4). Upon deployment, tree canopies were fully enclosed by tightly securing nets with twist ties under the canopy and around each trunk, as previously described [46]. Trees remained netted throughout the growing season until fruit were harvested. Green spot incidence on combinations with netted and control trees (3 trees/rootstock × 2 rootstocks × 2 treatments = 12 trees in 2020 and 9 trees/rootstock × 2 rootstocks × 2 treatments = 36 trees in 2021) was analyzed as described in the bagging trial in 2020 and 2021 (Section 2.4.1).

2.6. Application of Parka® Cuticle Supplement (2021)

In the same block described in Section 2.1 utilizing a different row trained as spindle of ‘WA 38’ grafted on ‘G.41’ and ‘M.9’ rootstocks, trees were sprayed with a phospholipid- and polysaccharide-based coating (Parka®; Cultiva LLC, Las Vegas, NV, USA) six times during the 2021 growing season: May 4 (at 100% petal fall), May 18, June 2, June 24, July 15, August 5. The product was applied according to the manufacturer’s recommended timing and rate (1 gallon per acre; 1% v/v) using a backpack sprayer [SR 430; STIHL Inc., Virginia Beach, VA, USA; maximum spray rate at 0° spray tube angle was 121 mL/min with ultra-low volume (ULV) nozzle]. Three treated trees from each ‘G.41’ and ‘M.9’ rootstock groups were harvested for the evaluation of external defects (September 28, 2021). Green spot incidence from these trees were compared to three untreated control trees on both ‘G.41’ and ‘M.9’ rootstocks in the same block. Green spot incidence for combinations with Parka® (3 trees/rootstock × 2 rootstocks = 6 trees) and control (9 trees/combination × 2 rootstocks = 18 trees) in the 2021 season was analyzed as described in the bagging trial in 2020 and 2021 (Section 2.4.1).

2.7. Peel Nutrient Analysis (2020)

Nutrient analysis was conducted on peel samples in the 2020 season in two separate experiments.

2.7.1. The Impact of Rootstock Selection and Bagging on ‘WA 38’ Fruit Mineral Content

In the first experiment, apples without visible defects (GS-) were harvested from control trees (never bagged) on ‘G.41’ and ‘M.9’ rootstock to understand the effect of rootstock selection on ‘WA 38’ peel nutrient composition. Additionally, within each rootstock (‘G.41’ and ‘M.9’), nutrient profiles of apples bagged at T1 (=June 1) were compared to control fruit (unbagged) to assess the effect of early season bagging on peel nutrient composition at harvest. Apples collected for this purpose were harvested on 17 September 2020 and refrigerated in cold storage at 1 °C until sampling on 5 November 2020. Nine apples were harvested from one tree representing each experimental and control group (nine bagged at T1 and nine unbagged), and peel samples from three apples were pooled to obtain three replicates per group (Figure S1). Sampling was conducted by peeling each apple’s entire pedicel-end hemisphere and freezing the peel tissue in liquid nitrogen. Frozen tissues were stored at −80 °C until further processing. Then, samples were lyophilized until dry weights stabilized. Dry peel tissue was milled using an A11 basic mill (IKA Works, Inc., Wilmington, NC, USA), and powdered tissue was submitted to a commercial laboratory for macro and micro elemental analysis.

2.7.2. ‘WA 38’ Fruit Peel Mineral Composition of GS and Relation to Vigor

In the second experiment, three V-trained trees displaying relative “low vigor” (V-) and three trees displaying relative “high vigor” (V+) were selected in ‘WA 38’/‘G.41’ in the same orchard block. From those trees, apples showing signs of GS (GS+) or not (GS-) were selected and pooled into three replicates, three apples per replication. The exception was the GS+ in “low vigor” (V-) combination, where we did not find example to justify sampling for that combination. An additional comparison in this experiment was made between GS+ and GS- fruit harvested from typical ‘WA 38’/‘G.41’ trees (without deficiency or excess of vigor) to better understand the differences in peel nutrient content between GS+ and GS- apples, regardless of tree vigor. Apples collected for nutrient analysis were harvested on 17 September 2020 and stored in RA cold storage at 1 °C until peel sampling was conducted on 30 September 2020, as described above.
Fruit mineral concentrations and the calculated element ratios for each sample (three replications per each treatment) were input and analyzed in SAS 9.4.

2.8. Statistical Analysis

All data sets presented in this manuscript were statistically analyzed in SAS 9.4 (SAS Institute Inc., Cary, NC, USA) by proc GLM using type III SS, with the significance of the model represented as: * = p ≤ 0.05; ** = p ≤ 0.01; *** = p ≤ 0.001; ns = not significant. The Student-Newman-Keuls (SNK) post hoc test was used to separate the means when the model resulted in significance. All percentage values falling outside the 30–70% range were transformed, either with arcsin or root square transformation, depending on the range for each parameter according to Gomez and Gomez [49]. Transformed percentages were then input in SAS 9.4.
Correlation analysis was performed with proc CORR in SAS 9.4, analyzing each season separately. Correlations were determined for the following eight variables: number of apples per tree at harvest, kg yield per tree, average fruit weight, trunk cross sectional area (TCSA) of each tree after harvest, total crop load at harvest (calculated as number of apples per tree divided by TCSA) and “mild”, “severe” and total GS incidence (%). This analysis was performed to assess the positive or negative relationships between productivity parameters and GS incidence in the 2020 (N = 6) and 2021 (N = 17) season, utilizing only control trees (no treatments included). These data are presented as a Pearson correlation coefficient (r) table.

3. Results

3.1. Onset and Progression of GS Symptoms

The first instances of GS symptoms in the 2020 and 2021 seasons were observed and photographed on July 29 and July 22, respectively. Photographs were taken weekly until harvest in September to track GS development. In both years, symptoms appeared at the end of July and generally worsened as the season progressed. Superficial symptoms did not appear to spread over time in terms of proportion of surface area of the fruit; however, the contrast between the red overcolor and the patches of superficial GS became more pronounced in the weeks following the onset of GS. In some cases, particularly in those fruit achieving an intense red color by harvest, superficial symptoms became inconspicuous (Figure 1, Fruit #5). In severe cases, spots became more numerous, darkened in color, and apparently increased in size between symptom onset and harvest in September. Surface cracking within symptoms in the weeks leading up to harvest was not uncommon. A summary of GS progression over the 2021 growing season is shown in Figure 1.

3.2. Visualization of Magnified GS Symptoms

In the current study of GS of ‘WA 38’, it was observed that superficial symptoms usually appeared as flecking or irregular patches of green coloration that became more evident as anthocyanins accumulated in the surrounding peel surface in late July (Figure 1). In Figure 2A an example of an apple affected by “severe” GS symptom is depicted; in Figure 2B and C two images at increasing magnification of the parenchyma cells affected by the disorder are shown. In some cases, GS symptoms appeared to be centered around lenticels. Magnified images of GS symptoms support a possible connection of the disorder with lenticels (Figure 2D–F).

3.3. Bagging

3.3.1. The Influence of Rootstock and Bagging on Total GS Incidence and Severity (2020–2021)

In both seasons, bagging reduced GS incidence at harvest, regardless of rootstock (Figure 3). Mitigation of GS was most effective by bagging in June. In 2020, no GS was observed following bagging at T1 (June 1) or T2 (June 18) in either rootstock combination, and when apples on ‘WA 38’/‘M.9’ trees were bagged at T3 (July 6), only “mild” symptoms were observed at harvest (Figure 3a). Bagging ‘WA 38’/‘M.9’ trees eliminated severe symptoms as late as July 24 (T4) in 2020 (Figure 3a) and July 16 (T2) in 2021 (Figure 3b), whereas fruit bagged at later time points developed symptoms by harvest. In 2021, bagging at T1 (June 18) and T2 (July 2) reduced GS incidence on apples grown on both rootstock combinations (Figure 3b). Regardless of year-to-year variations in GS incidence for early July bagging, early season bagging still reduced the disorder in both years and rootstocks.

3.3.2. Microclimate Monitoring of Bagged and Control Treatments (2020–2021)

Average RH differed between bagged and control treatments in June, July, and August, but not in September of both 2020 and 2021 seasons (Table 1). Daily minimum RH was different in all months between June and September in both years, with bagged treatments maintaining a higher RH. Maximum RH was only different in September 2020, where air around control fruit had an average maximum of 78.5% RH compared with 75.5% in the bagged treatment. However, in 2021, maximum RH was higher around bagged fruit only in June and July.
In 2020, the average daily maximum temperature in July was 0.9 °C lower around bagged treatments (32.8 °C) compared with control (33.7 °C). In August, this difference was 1.3 °C (32.2 °C around bagged treatments compared with 33.5 °C around the control). In July 2021, the average daily maxima were lower around the control (36.2 °C) compared with the bagged apples (37.1 °C), but were not different in August. Daily maximum temperature between treatments was not different in June or September of either year. Average daily minimum and overall average temperature were not different between bagged and control apples in 2020. In 2021, differences in overall daily average and average daily minimum temperatures were different in July (Table 1).

3.4. Mono-Row Drape Netting: Influence of Rootstock and Netting on Total GS Incidence and Severity (2020–2021)

Total GS incidence in either rootstock (‘G.41’ or ‘M.9’) was not impacted by netting in 2020 (Table 2). Green spot incidence on apples grown on ‘WA 38’/‘G.41’ was higher than ‘WA 38’/‘M.9’, regardless of netting treatment, but did not differ from the control (unnetted) ‘WA 38’/‘G.41’. None of the rootstock-netting combinations differed in terms of severity when expressed as incidence of “mild” or “severe” symptoms (Table 2). In the 2021 season, netting ‘WA 38’/‘G.41’ trees reduced “mild” and total GS incidence but did not impact “severe” GS incidence. However, GS incidence in netted ‘WA 38’/‘G.41’ was not different from ‘WA 38’/‘M.9’ in either netted or control groups in the 2021 season. Furthermore, GS was not reduced by netting in ‘WA 38’/‘M.9’ in the 2021 season (Table 2).
Although there was generally higher GS frequency in ‘WA 38’/‘G.41’ combinations, the effect of rootstock alone on “mild,” “severe,” or total GS incidence was not different between ‘G.41’ and ‘M.9’ controls in 2020 (Table 2). However, in 2021, GS incidences were lower for ‘WA 38’ control trees grafted on ‘M.9’ compared with ‘G.41’.

3.5. Application of Parka® Cuticle Supplement: Effect on GS Appearance in ‘WA 38’ (2021)

Application of a cuticle supplement (Parka®, Cultiva LLC, Las Vegas, NV, USA) did not reduce “mild,” “severe,” or total GS incidence in either rootstock combination with respect to untreated controls in 2021 (Table 3). Although incidence was not different between experimental and control groups of ‘WA 38’ on either rootstock, incidence was different when comparing treated groups between rootstocks, suggesting rootstock was more influential on GS incidence than Parka® treatment.

3.6. Peel Mineral Analysis

3.6.1. The Impact of Rootstock Selection and Bagging on ‘WA 38’ Fruit Mineral Content (2020)

Analysis of asymptomatic peel of ‘WA 38’ apples (GS-) harvested from trees on two different rootstocks revealed differences in mineral content (Table 4). ‘WA 38’ apple peels from trees grafted on ‘G.41’ were higher in nitrogen (N), potassium (K), sulfur (S), and boron (B) than those sampled from trees grafted on ‘M.9’, but did not differ in phosphorus (p), calcium (Ca), magnesium (Mg), zinc (Zn), manganese (Mn), copper (Cu), iron (Fe), or any of the Ca-related ratios outlined in Table 4. Nutrient content of asymptomatic peel was altered by bagging at T1, but these differences were not consistent between rootstocks. In the ‘WA 38’/‘G.41’ combination, fruit bagging resulted in higher peel B, but lower Ca. Of the ratios of Ca to other elements previously described, N:Ca was not significantly impacted by fruit bagging on the ‘WA 38’/‘G.41’ combination, although the value in the bagged fruit reached 10.2 compared with 7.9 of the control. The K:Ca, Mg:Ca, (K + Mg):Ca, and (N + Mg + K):Ca ratios were all higher in peel from apples bagged at T1, and no GS symptoms were observed in the bagged fruit. In the ‘WA 38’/‘M.9’ combination, bagging at T1 resulted in higher peel P and K, as well as K:Ca, and (K + Mg):Ca ratios, but lower Zn.

3.6.2. ‘WA 38’ Fruit Peel Mineral Composition of GS and Relation to Vigor (2020)

‘WA 38’/‘G.41’ peel with (GS+) and without (GS-) green spot symptoms differed in total N, P, K, S, Ca, and Mg, but did not differ in Zn, Mn, Cu, Fe or B levels (Table 5). Peel with green spot symptoms (GS+) was higher in N, P, K, S and Mg levels, but lower in Ca than asymptomatic peel.
Nutrient ratios were also different between symptomatic and asymptomatic peel, for instance, N:Ca, K:Ca, Mg:Ca, [K + Mg]:Ca, and [N + Mg + K]:Ca ratios were higher in the symptomatic peel compared to asymptomatic ones.
The impact of vigor and peel nutrition on GS incidence was less clear. Mineral content differences were inconsistent when comparing asymptomatic peel from apples harvested from low vigor and high vigor trees. None of the nutrients differed among vigor and disorder combinations except S, in which symptomatic peel from vigorous trees (V+ GS+) was higher than asymptomatic peel from less vigorous trees (V-GS-; Table 5).

3.7. Correlative Analysis of Productivity Parameters and GS Incidence

In the 2020 season the total GS incidence in untreated control ‘WA 38’ trees grafted on ‘G.41’ and ‘M.9’ rootstocks (combined) was inversely correlated with the number of fruits per tree, yield (kg per tree), and crop load (fruit per cm2 TCSA), indicating trees with fewer fruits and lower yields were more likely to produce symptomatic fruit (Table 6a).
In 2021, total GS incidence in untreated controls across both rootstocks was instead positively correlated with number of fruits per tree and was not strongly correlated with yield or crop load. The trunk cross-sectional area (TCSA) alone, but not the crop load, was positively correlated with “mild” and total GS incidence in 2021, suggesting that tree vigor contributed to the elevated rates of symptomatic fruit (Table 6b).

4. Discussion

4.1. The Influence of Bagging, Netting and Rootstock on Total GS Incidence and Severity

Fruit bagging is a technique that has been used in various fruit production systems around the world [8,36,50,51,52] to impart various benefits on the fruit and production system [53]. Bagging imposes a physical barrier between the developing fruit and the environment, which can serve as an effective form of pest control [15,35,37] and can reduce pesticide residues on fruit surfaces [15,36,54,55]. Bagging fruit early in the season and then removing bags in the days or weeks leading up to harvest can enhance fruit coloration, making fruit more attractive to consumers [36,39,40,51,53,56]. Bagging alters peel biochemistry and reduces russet in Asian pear (Pyrus pyrifolia Nak.) [57,58] and can impact the incidence of storage disorders in ‘Fuji’ apples [8]. Matsumoto et al. [41] demonstrated that fruit bagging reduces the incidence of a preharvest cork spot-like physiological disorder (CSPD) in ‘Kurenainoyume’ apples. They found that bagging apples early in the season and for a longer duration was the most effective approach for reducing CSPD incidence. The authors tested three different bags with different spectral permeability and found that the 2-layer bags, non-permeable to light, were the most effective for reducing CSPD compared to semi-permeable 1-layer bags. Shading also reduced CSPD in ‘Kurenainoyume’ apples, and because the authors did not find that bagging or shading significantly altered temperature, they concluded that the disorder is likely related to light conditions in the orchard [41]. Similarly, GS incidence in ‘WA 38’ was also significantly reduced by fruit bagging. Furthermore, earlier bagging treatments were the most effective at reducing the frequency of GS in ‘WA 38’, regardless of year or rootstock combination. Interestingly, there were also higher N:Ca, K:Ca, Mg:Ca, [K + Mg]:Ca, and [N + Mg + K]:Ca ratios and lower total Ca in the bagged asymptomatic fruit peels (T1) compared to the control (Table 4).
The current study indicated mixed effects of netting on GS incidence: in 2020, netting did not significantly reduce GS concerning the control group on either ‘G.41’ or ‘M.9’ rootstocks, whereas in 2021, netting had an effect of reducing GS incidence, but only in the ‘WA 38’/‘G.41’ combination. The effect of shading on disorder incidence was more dramatic in the study published by Matsumoto et al. [41] compared to the current study, which could be related to differences in the photoselective qualities of the materials used. Matsumoto et al. [41] reported that using a black cheese cloth for shading effectively reduced CSPD incidence, but also reduced soluble solids (Brix°). In the current study, we used a 10% shading crystal net that was likely less photorestrictive than the material used by Matsumoto et al. [41] but more similar to netting systems used in commercial fruit production in the apple growing regions of the U.S. Pacific Northwest. Different netting materials and installations can differentially influence physiological processes [10,43,59], which could partially explain differences in efficacy in reducing apple disorder.
In addition to manipulating light conditions, fruit bagging [53] and netting [10,60] may alter other environmental aspects, including wind, temperature, and humidity. Netting can reduce evapotranspiration in the orchard [42], which suggests its use can impact irrigation requirements. The range of environmental factors affected by fruit bagging and netting, and the varying degree to which they contribute to the physiological processes in developing fruit, complicate troubleshooting disorders like CSPD in ‘Kurenainoyume’ and GS in ‘WA 38’ apples. Fruit bagging altered daily average temperatures around the fruit in the current study only in July 2021; however, we did find differences in the maximum temperature in July (both years) and in August (2021). Relative humidity between bagging and control treatments during the 2020 and 2021 seasons were different. In general, bagging attenuated diurnal humidity fluctuations and maintained higher daily average RH compared to ambient conditions in the canopy (Table 1). Humidity and temperature influence stomatal conductance [10], which has implications for gas exchange, nutrient mobility, and drought stress responses [61,62]—all may be factors contributing to GS onset. Cline and Hanson [62] found that fruit Ca concentration of ‘Delicious’ apples grown in high RH microenvironments was often reduced, suggesting that it was a consequence of different transpiration rates throughout fruit growth and development. Witney et al. [63] found that fruit bagging promoted bitter pit development in ‘Sundale Spur Golden Delicious’ apples. Fallahi et al. [50] did not find differences in Ca but reported higher N and K levels in bagged ‘BC-2 Fuji’ apples compared with non-bagged fruit. In the current study, nutritional trends in bagged ‘WA 38’ apple peels were more similar to trends found in bitter pit apples than control (non-bagged) apples, where bagging ‘WA 38’/‘G.41’ resulted in lower peel Ca concentration and higher Ca-related ratios of K:Ca, Mg:Ca, (K + Mg):Ca, and (N + Mg + K):Ca. However, considering GS in ‘WA 38’ was effectively mitigated by fruit bagging at the beginning of June, the role of peel mineral composition on GS appearance in ‘WA 38’ does not seem directly analogous to the role of Ca nutrition on the appearance of bitter pit in susceptible cultivars.

4.2. The Effect of Rootstock and the Role of Mineral Nutrition on GS Appearance in ‘WA 38’

Rootstock influences the mineral content of apple leaves [13,64,65,66,67,68] and fruit [13,66,67]. Other cultural practices, such as crop load adjustment [28,29,64,69], pruning, or other vigor control strategies [4,30] can also impact leaf and overall fruit mineral concentration and distribution in vegetative and reproductive tissues. Our data showed a significant effect of rootstock on the incidence and severity of GS on ‘WA 38’ apples, with ‘G.41’ rootstocks developing more of this disorder than ‘M.9’ in 2021. These data are consistent with the findings reported by Sallato et al. [46], who found a similar trend in rootstock susceptibility in a ‘WA 38’ planting in Prosser, WA. Mineral nutrient uptake is influenced by rootstock [67,70], scion [71], and soil quality [72], which lends evidence to the argument proposed by Sallato et al. [46] who suggested that GS in ‘WA 38’ is another calcium-related disorder; differences in rootstock capacity to absorb and distribute mineral nutrients could explain why some plantings appear more susceptible than others. Previous authors have tried to clarify the relationship between nutritional status and the onset of physiological disorders in apple. Bitter pit is one of the most widespread [4,63] and widely studied [63,66,73,74,75,76] physiological disorders of apple. It is characterized by slight indentations on the fruit surface, which become discolored, brown, and desiccated as the disorder progresses [1,4,77]. Unlike GS in ‘WA 38’, which emerges mid-season and may worsen until harvest, early symptoms of bitter pit may be visible at harvest, but the disorder typically appears or worsens after harvest [4,33]. No progression of GS symptoms was observed after harvest in our experiments. Bitter pit in apple has been broadly associated with Ca deficiency and elevated K and/or Mg [31,63,66,69,70,73]. Some researchers have suggested that nutritional ratios, such as N:Ca, Mg:Ca, K:Ca, (Mg + K):Ca [31,33,66,69,70,78] may be better correlated to bitter pit than Ca concentrations alone. Fazio et al. [70] demonstrated the role of K concentration and the ratio of K:Ca in leaves and fruit as predictors of bitter pit in ‘Honeycrisp’. Amarante et al. [31] found relationships between Mg:Ca and bitter pit only in the peel of ‘Fuji’ apples and only in the flesh of ‘Catarina’ apples, suggesting that the reliability of using nutrition to predict bitter pit among cultivars may depend on the tissue type used for the analysis. A common nutrient sampling approach for bitter pit studies is to sample the distal, or calyx-end of the fruit [33,50] as it is usually the portion of the fruit that is most impacted by the disorder [78]. Sallato et al. [46] found that peel, rather than flesh nutrition, was most associated with GS in ‘WA 38’; in the current study, we sampled peel at the pedicel-end hemisphere of apples as it is the portion of the fruit where GS symptoms most commonly appear.
In the current study, peel mineral results revealed statistical differences between symptomatic and asymptomatic apples in total N, P, K, S, Ca, and Mg, as well as N:Ca, K:Ca, Mg:Ca, (K + Mg):Ca, and (N + Mg + K):Ca ratios (Table 5). Apples with green spot had peel nutrient profiles similar to bitter pit reported in previous studies, where low relative Ca is a critical factor. These results also generally agree with Sallato et al. [46], who found that N and Mg concentrations were typically higher in symptomatic apples. However, Table 4 shows that N:Ca, K:Ca, Mg:Ca, (K + Mg):Ca, and (N + Mg + K):Ca ratios of asymptomatic fruit (control GS-) were similar to those showing symptoms (GS+) in Table 5. Moreover, total Ca was lower and Ca-related ratios were higher in asymptomatic bagged fruit peels than in the asymptomatic control (Table 4), which undermines the hypothesis that low relative Ca leads to green spot. It has previously been reported that higher GS frequency in ‘WA 38’ is associated with lower crop load [29,46], which parallels the general trends reported in several bitter pit studies [69,74].
In the current study, trees varied in productivity within each rootstock and treatment combination; however, the number of fruits per tree, yield (mass per tree), and crop load at harvest (fruit per cm2 TCSA) were inversely correlated with total GS incidence across both rootstocks in untreated control trees in 2020 (Table 6a). Conversely, in 2021, there was a weak positive correlation between total GS incidence and number of fruits per tree, as well as TCSA alone, when analyzing untreated control trees on both rootstocks (Table 6b). Crop load at harvest (fruit per cm2 TCSA) did not correlate with GS incidence in 2021 (Table 6b). Interestingly, although the average number of fruits per tree and average mass harvested from each tree were statistically different between rootstocks in 2021 (data not shown), GS incidence was not correlated with any of the productivity parameters in either rootstock when considered individually (data not shown). However, ‘WA 38’/‘G.41’ trees produced more fruits and yielded greater total fruit mass than ‘WA 38’/‘M.9’ trees in 2021 (data not shown), whereas ‘WA 38’/‘M.9’ trees produced more fruits and yielded greater total fruit mass than ‘WA 38’/G.41’ in 2020 (data not shown). Thus, it is unclear whether the tendency for ‘WA 38’/‘G.41’ trees to produce GS+ apples at a higher frequency than ‘WA 38’/‘M.9’ trees (Figure 3) was offset by the effect of crop load and bienniality in 2021.
Following observations that GS frequency is more prevalent in vigorous trees, we sought to further determine how tree vigor impacts fruit nutritional level by comparing symptomatic and asymptomatic apples from highly vigorous trees with fruit from trees of relatively low vigor. It has been proposed that vigor contributes to nutrient deficiency-related disorders by favoring the flux of nutrients into actively growing shoots and away from fruit [33]. However, the peel nutrient content of apples was not different between vigorous and less vigorous ‘WA 38’ trees (Table 5).
Calcium plays important structural and functional roles in cells, and insufficient Ca delivery into developing fruitlets is indirectly associated with apple disorders like bitter pit [34,79]. Magnesium is also a divalent cation, and it has been postulated that relative Mg overabundance could lead to competitive binding to structurally important substrates, resulting in localized cellular collapse in those affected areas [33]. However, it is notable that Ca concentration, and the relationship between physiological disorders like bitter pit and mineral nutrient status, are not consistent in the reported literature [73,74]. In some cases, Ca concentrations are reported to be higher in tissues with bitter pit symptoms. Despite the general agreement of an association between Ca deficiency and bitter pit, a mechanistic explanation remains unclear. De Freitas et al. [33] suggested that total fruit Ca determines the first level of bitter pit susceptibility, while cellular compartmentalization of soluble or insoluble Ca further determines susceptibility based on demand and availability of Ca2+ ions used for a variety of cellular functions.
Although we identified nutritional differences between GS+ and GS- ‘WA 38’ apples, it is unclear whether these differences reflect causal factors of GS, or if they are merely consequences of other physiological processes that more directly provoke symptom appearance. Gomez and Kalcsits [34] found higher fruit Ca and a lower (Mg + K):Ca ratio in ‘WA 38’ fruit than in ‘Honeycrisp’ when grown on the same rootstock and reported greater susceptibility to bitter pit in ‘Honeycrisp.’ Furthermore, Ca uptake was higher in ‘Honeycrisp’ grown on ‘G.41’ rootstocks in comparison to ‘M.9’ rootstocks [67], yet GS appears to be more prevalent in ‘WA 38’/‘G.41’ than ‘WA 38’/‘M.9’ plantings [46]. Apple peels sampled from ‘WA 38’/‘G.41’ and ‘WA 38’/‘M.9’ did not differ with respect to Ca concentration (Table 4).
Drazeta et al. [79] reported diminished xylem function in ‘Braeburn’ and ‘Granny Smith’ apples over the course of the growing season, and suggested that the physical expansion of the flesh was responsible for constricting vascular Ca flux, and subsequently, the onset of Ca deficiency disorders like bitter pit. ‘WA 38’ fruit xylem maintained functionality longer than ‘Honeycrisp’ apples [34], which suggests, at least in relative terms, that restricted Ca delivery to the fruit may not be the underlying cause of GS onset in ‘WA 38’. Furthermore, GS in ‘WA 38’ presents morphologically distinct symptoms and patterns of emergence from bitter pit in other cultivars. Green spot symptoms most commonly appear in the pedicel-end hemisphere of ‘WA 38’ fruit; however, the appearance of bitter pit symptoms at the calyx-end of the fruit in most apple cultivars has been used as evidence to support the hypothesis that bitter pit is a disorder triggered by Ca deficiency [74]. Green spot symptoms appear first on the peel—only later in the season do symptoms impact the cortex. This is a completely different etiology than bitter pit. Moreover, the observation that GS symptoms do not worsen after harvest implies that nutritional depletion may not be a causal factor in its appearance. However, the role of nutrient imbalance in the appearance and development of GS symptoms cannot be ruled out.

4.3. The Potential Role of Lenticels and Cuticle Health

During the growing season, developing fruitlets undergo structural and functional changes to accommodate the physical expansion of the hypanthium. During this process, stomata lose their ability to open and close, and the trichomes associated with young fruitlets are shed [77,80,81]. These vestigial stomata and trichome scars, along with other surface tears stemming from epidermal stretching and expansion, are the origins of mature fruit lenticels [77,80]. Lenticels function as sites for gas exchange [14,80] but are weak points for mechanical stress as the fruits enlarge [82]. In apple, microscopy studies have shown lenticels and their surrounding cuticle are susceptible to microcracking [9,81,83,84] and can serve as points of entry for disease [81,83,85]. Lenticel desiccation during the growing season has been implicated in postharvest lenticel breakdown [9]. Curry et al. [9] showed preharvest application of some lipophilic protective coatings reduced lenticel breakdown in storage, an effect the authors attributed to the patching of cuticular microcracks that might otherwise lead to lenticel desiccation and necrosis. In the current study, we applied Parka® to ‘WA 38’ on ‘G.41’ and ‘M.9’ rootstocks at six time points over the 2021 growing season. However, the results did not reveal differences in “mild,” “severe,” or total GS incidence between treated and control groups within each rootstock, although statistical differences in GS incidence between rootstocks were observed (Table 3), indicating rootstock selection, but not Parka® treatment, influenced GS incidence.
Previous research has characterized lenticel and cuticle diversity among apple cultivars. Clements [80] made a distinction between “open” and “closed” lenticels but found average total lenticel counts ranged from 536 lenticels per fruit in ‘Winesap’ apples to 2196 lenticels per fruit in ‘Spitzenburger’ apples. Đurić et al. [86] also reported differences in lenticel density of apple and pear cultivars, and suggested that cultivars with higher lenticel density are associated with shorter storability. Singh et al. [21] correlated higher proportions of “open” lenticels with greater lenticel breakdown severity in storage; however, Tessmer et al. [81] did not find a relationship between lenticel permeability to dye and the incidence of lenticel breakdown. The relative lenticel density over the entire surface area is higher near the calyx end than the pedicel end of apples [14,77,80]. Although a deep investigation into the lenticel characteristics of ‘WA 38’ fruit is currently lacking, our general observations suggest a correlation between the positional emergence of GS symptoms (mainly top apple hemisphere) on ‘WA 38’ fruit and low lenticel density in that portion. This trend suggests lenticel characteristics during the growing season may be related to the emergence of GS in ‘WA 38’ apples, perhaps induced by environmental or nutritional factors, or an interaction between them. The role of K and sucrose in osmotic regulation of stomatal guard cells has previously been studied [87,88,89] and may be possible area of investigation to help to explain the interaction between nutritional and environmental factors affecting GS development in ‘WA 38’ fruit.

5. Conclusions

The incidence and severity of green spot, a disorder afflicting ‘WA 38’ apples, can be effectively reduced by early season fruit bagging. Rootstock selection can also have a measurable impact on GS, with ‘WA 38’/‘G.41’ trees exhibiting higher rates of GS than ‘WA 38’/‘M.9’. The effect of drape netting on GS incidence was inconclusive, and spray application of a cuticle supplement was not effective. The mechanistic basis by which rootstock selection and early season bagging influence GS disorder in ‘WA 38’ is not well understood. The cumulative data of this research do not rule out a possible role of nutritional imbalances on GS appearance in ‘WA 38’, but suggest other physiological triggers may be involved. The mitigative effect of bagging on GS incidence and severity may be influenced by alterations in the microclimate surrounding the developing fruit, such as changes in light exposure, airflow, and humidity. Environmental stress could thus be involved in stimulating disorder onset, perhaps interacting with other physiological predispositions, such as mineral nutrient uptake and mobility, crop load, or tree vigor. In conclusion, GS in ‘WA 38’ may be a synergistic manifestation of genetic, nutritional, and environmental factors, although its specific mechanistic origin remains unclear. Future studies investigating the physiological consequences of isolating the specific variables influenced by bagging, along with an in-depth microscopy study on lenticel structure and function over the course of fruit development, may help to better elucidate the mechanistic basis of GS in ‘WA 38’ apples.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/agronomy12112822/s1, Figure S1. ‘WA 38’ fruit mineral samples in 2020: comparison between asymptomatic bagged (bagged GS-) and asymptomatic control (control GS-) apples within each rootstock (‘G.41’ and ‘M.9’) after harvest. Bagged apples at T1 were enclosed in the bags on 1 June 2020, and they developed the entire season inside the bag until harvest on 17 September 2020. Sampling was performed by peeling the top hemisphere (pedicel-end) of each apple and freezing the peel tissue in liquid nitrogen. Each of three replications from each treatment consisted of pooled peels from three apples. Data regarding peel mineral analysis were reported in Table 4.

Author Contributions

Conceptualization, S.M.; methodology, S.M.; software, S.S.; validation, S.S.; formal analysis, S.S.; investigation, R.S., S.S. and S.M.; resources, S.M.; data curation, S.S. and R.S.; writing—original draft preparation, R.S.; writing—review and editing, S.S., R.S., D.R. and S.M.; visualization, S.S. and R.S.; supervision, S.M.; project administration, S.M.; funding acquisition, S.M. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by Washington Tree Fruit Research Commission (WTFRC), grant number AP-20-103A titled “WA 38: understanding green spot origin, timeline, and development” and by the USDA National Institute of Food and Agriculture Hatch project 1014919, titled “Crop Improvement and Sustainable Production Systems” (WSU reference 00011).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The data presented in this study are available upon request from the corresponding author.

Acknowledgments

The authors would like to thank the Sunrise Farm crew for their orchard support. We would also like to thank our technical staff for their technical contributions in particular Zachary Chapman for Figure 1 images.

Conflicts of Interest

The authors declare no conflict of interest. The funders had no role in the design of the study, in the collection, analyses, or interpretation of data, in the writing of the manuscript, or in the decision to publish the results.

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Agronomy 12 02822 g001 550
Figure 1. The progression of GS symptoms over time in ‘WA 38’ apples in 2021. On top of the fruit progression picture the dates of the photo are reported in days after full bloom (DAFB) and in calendar dates. The first GS symptoms were observed and photographed on 22 July 2021 (Fruit # 1–4) and were photographed weekly until they were harvested and photographed on 23 September 2021. Fruits # 1–4 developed severe green spot symptoms by harvest, while Fruit # 5. developed only mild symptoms that became less conspicuous in the weeks leading up to harvest. Early symptoms on Fruit # 5 were first observed on 29 July 2021.
Figure 1. The progression of GS symptoms over time in ‘WA 38’ apples in 2021. On top of the fruit progression picture the dates of the photo are reported in days after full bloom (DAFB) and in calendar dates. The first GS symptoms were observed and photographed on 22 July 2021 (Fruit # 1–4) and were photographed weekly until they were harvested and photographed on 23 September 2021. Fruits # 1–4 developed severe green spot symptoms by harvest, while Fruit # 5. developed only mild symptoms that became less conspicuous in the weeks leading up to harvest. Early symptoms on Fruit # 5 were first observed on 29 July 2021.
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Figure 2. Green spot symptoms on ‘WA 38’ fruit. Example of: (A) ‘WA 38’ apple with “severe” GS symptoms visible on the surface and in the underlying parenchyma; (B) at 1× and (C) 2× magnification (black arrow indicates the position of cut for magnified images); (B,C) note the darkened, cork-like tissue and areas of collapsed cells; (D) green spots centered around lenticels in two different locations on a ‘WA 38’ apple were visualized under (E) 1× and (F) 2× magnification. Whole fruit images (A,D) were taken with a Canon EOS 70D DSLR camera, and magnified images were taken with a Nikon SM18 stereomicroscope. Scale bars represent 1 mm at each respective magnification.
Figure 2. Green spot symptoms on ‘WA 38’ fruit. Example of: (A) ‘WA 38’ apple with “severe” GS symptoms visible on the surface and in the underlying parenchyma; (B) at 1× and (C) 2× magnification (black arrow indicates the position of cut for magnified images); (B,C) note the darkened, cork-like tissue and areas of collapsed cells; (D) green spots centered around lenticels in two different locations on a ‘WA 38’ apple were visualized under (E) 1× and (F) 2× magnification. Whole fruit images (A,D) were taken with a Canon EOS 70D DSLR camera, and magnified images were taken with a Nikon SM18 stereomicroscope. Scale bars represent 1 mm at each respective magnification.
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Figure 3. Green spot incidence at harvest by bagging time point and rootstock in (a) 2020; and (b) 2021. To facilitate the comparison between time points in the two seasons they are expressed in DAFB (for 2020: T1 = 44 DABF to T6 = 132 DAFB and for 2021: T1 = 59 DAFB and T3 = 87 DAFB). Light green shaded bars represent the proportion of “mild” GS, and dark green shaded bars represent the proportion of “severe” GS over the total incidence. Total incidence (%) in each treatment combination is cited above each bar and indicated by a round black marker. Separation of means are indicated by capital (‘G.41’) or lowercase italics (‘M.9’) letters within each rootstock and year, and the significance within each rootstock across time points (*** = p ≤ 0.001) is indicated in parentheses on the right side of the figure.
Figure 3. Green spot incidence at harvest by bagging time point and rootstock in (a) 2020; and (b) 2021. To facilitate the comparison between time points in the two seasons they are expressed in DAFB (for 2020: T1 = 44 DABF to T6 = 132 DAFB and for 2021: T1 = 59 DAFB and T3 = 87 DAFB). Light green shaded bars represent the proportion of “mild” GS, and dark green shaded bars represent the proportion of “severe” GS over the total incidence. Total incidence (%) in each treatment combination is cited above each bar and indicated by a round black marker. Separation of means are indicated by capital (‘G.41’) or lowercase italics (‘M.9’) letters within each rootstock and year, and the significance within each rootstock across time points (*** = p ≤ 0.001) is indicated in parentheses on the right side of the figure.
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Table
Table 1. Temperature (°C) and percent relative humidity (%RH) measurements inside fruit bags (bag) compared to the non-bagged (control) in the 2020 and 2021 seasons. Monthly averages of daily average, minimum and maximum temperatures (°C), and average, minimum and maximum RH (%), were compared. N = indicated the number of observations generating the means for each month and season. Different letters within comparisons indicate statistical differences between means (p = 0.05) by SNK. Significance: * = p ≤ 0.05; ** = p ≤ 0.01; *** = p ≤ 0.001; ns = not significant.
Table 1. Temperature (°C) and percent relative humidity (%RH) measurements inside fruit bags (bag) compared to the non-bagged (control) in the 2020 and 2021 seasons. Monthly averages of daily average, minimum and maximum temperatures (°C), and average, minimum and maximum RH (%), were compared. N = indicated the number of observations generating the means for each month and season. Different letters within comparisons indicate statistical differences between means (p = 0.05) by SNK. Significance: * = p ≤ 0.05; ** = p ≤ 0.01; *** = p ≤ 0.001; ns = not significant.
2020 Temperature
(°C)		 Relative
Humidity (%)
MonthTreatmentN =AverageMinimumMaximumAverageMinimumMaximum
Junebag18020.714.528.454.0 a38.3 a71.3
Junecontrol18020.914.429.047.6 b28.6 b69.1
Significance nsnsns******ns
Julybag18624.617.432.8 b45.1 a29.7 a62.9
Julycontrol18624.817.133.7 a41.7 b24.0 b62.4
Significance nsns*******ns
Augustbag18624.317.332.2 b45.2 a30.3 a62.5
Augustcontrol18624.516.833.5 a41.7 b23.6 b62.8
Significance nsns*********ns
Septemberbag13220.113.926.958.842.3 a75.5 b
Septembercontrol13220.213.528.157.837.4 b78.5 a
Significance nsnsns
(p = 0.059)		ns***
2021 Temperature
(°C)		 Relative
Humidity (%)
MonthTreatmentN =AverageMinimumMaximumAverageMinimumMaximum
Junebag7830.422.239.543.4 a31.5 a57.9 a
Junecontrol7829.921.839.235.6 b19.9 b53.5 b
Significance nsnsns********
Julybag18628.3 a20.8 a37.1 a41.7 a28.7 a56.4 a
Julycontrol18627.7 b20.3 b36.2 b37.4 b21.9 b54.4 b
Significance **************
Augustbag18624.518.532.248.9 a34.0 a63.7
Augustcontrol18624.017.731.846.6 b28.5 b65.4
Significance nsnsns*****ns
Septemberbag14418.712.6 a26.359.441.0 a77.6
Septembercontrol14418.211.9 b25.657.635.8 b79.9
Significance ns*nsns***ns
Table
Table 2. Effect of rootstock and netting treatments on ‘WA 38” trees grafted on ‘Geneva 41’ (‘G.41’) and ‘Malling 9-Nic29’ (‘M.9’) rootstocks on the green spot (GS) incidence (%) in the 2020 and 2021 seasons. Significance by treatment combination: * = p ≤ 0.05; *** = p ≤ 0.001; ns = not significant.
Table 2. Effect of rootstock and netting treatments on ‘WA 38” trees grafted on ‘Geneva 41’ (‘G.41’) and ‘Malling 9-Nic29’ (‘M.9’) rootstocks on the green spot (GS) incidence (%) in the 2020 and 2021 seasons. Significance by treatment combination: * = p ≤ 0.05; *** = p ≤ 0.001; ns = not significant.
2020
GS Incidence (%)
Treatment N Trees“Mild” 1“Severe” 2 Total 3
‘WA 38’/‘G.41’Control330.124.8ab54.9ab
‘WA 38’/‘G.41’Net323.441.5a64.9a
‘WA 38’/‘M.9’Control320.76.5b27.3b
‘WA 38’/‘M.9’Net315.810.9ab26.7b
Significance nsns (p = 0.0503)*
2021
GS Incidence (%)
TreatmentN Trees“Mild” 1“Severe” 2 Total 3
‘WA 38’/‘G.41’Control917.19.8a26.9a
‘WA 38’/‘G.41’Net99.96.9ab16.8b
‘WA 38’/‘M.9’Control97.73.9b11.6b
‘WA 38’/‘M.9’Net97.91.8b9.7b
Significance **** ***
1 Includes apples presenting only “mild” symptoms that did not significantly detract from the overall appearance. 2 Includes all apples presenting “severe” symptoms, including those presenting superficial GS symptoms considered unmarketable due to appearance. 3 Includes apples presenting one or more types of GS; apples were counted just once (presence/absence of GS regardless of the type).
Table
Table 3. ‘WA 38’ GS incidence (%) in 2021: comparison between apples receiving a cuticle supplement (Parka®) and apples harvested from control trees (control = no PARKA®) on ‘G.41’ and ‘M.9’ rootstocks. Different letters within comparisons indicate statistical differences between means for p = 0.05 by SNK. Significance by treatment combination: *** = p ≤ 0.001; ** = p ≤ 0.01.
Table 3. ‘WA 38’ GS incidence (%) in 2021: comparison between apples receiving a cuticle supplement (Parka®) and apples harvested from control trees (control = no PARKA®) on ‘G.41’ and ‘M.9’ rootstocks. Different letters within comparisons indicate statistical differences between means for p = 0.05 by SNK. Significance by treatment combination: *** = p ≤ 0.001; ** = p ≤ 0.01.
2021
GS Incidence (%)
Treatment N Trees“Mild” 1 “Severe” 2 Total 3
‘WA 38’/‘G.41’control917.1a9.8ab26.9a
‘WA 38’/‘G.41’PARKA®316.9a17.3a34.2a
‘WA 38’/‘M.9’control97.7b3.9b11.6b
‘WA 38’/‘M.9’PARKA®36.2b1.5b7.7b
Significance *** ** ***
1 Includes apples presenting only “mild” symptoms that did not significantly detract from the overall appearance. 2 Includes all apples presenting “severe” symptoms, including those presenting superficial GS symptoms that were considered unmarketable due to appearance. 3 Includes apples presenting one or more types of GS; apples were counted just once (presence/absence of GS regardless of the type).
Table
Table 4. ‘WA 38’ fruit mineral composition in 2020: comparison between T1-bagged (bag) and asymptomatic control (control GS-) apples within each rootstock (‘G.41’ and ‘M.9’), and direct comparison of element composition between asymptomatic fruit (GS-) from both rootstocks. Different capital letters (comparison between bagged and control GS- apples within each rootstock) and lowercase letters (comparison between control GS- apples across rootstock combinations; p = 0.05) indicate statistical differences and means separations by SNK. Significance by treatment combination within each rootstock: * = p ≤ 0.05, ** = p ≤ 0.01, ns = not significant.
Table 4. ‘WA 38’ fruit mineral composition in 2020: comparison between T1-bagged (bag) and asymptomatic control (control GS-) apples within each rootstock (‘G.41’ and ‘M.9’), and direct comparison of element composition between asymptomatic fruit (GS-) from both rootstocks. Different capital letters (comparison between bagged and control GS- apples within each rootstock) and lowercase letters (comparison between control GS- apples across rootstock combinations; p = 0.05) indicate statistical differences and means separations by SNK. Significance by treatment combination within each rootstock: * = p ≤ 0.05, ** = p ≤ 0.01, ns = not significant.
Macro-Nutrients
Treatment Total N (%) P (%) K (%) S (%) Ca (%) Mg (%)
‘WA 38’/‘G.41’bag0.392 0.077 0.784 0.040 0.039B0.094
control (GS-)0.383a0.061 0.693a0.036a0.049A0.089
Significancens ns ns ns * ns
‘WA 38’/‘M.9’bag0.346 0.079A0.801A0.032 0.045 0.088
control (GS-)0.320b0.048B0.598Bb0.028b0.045 0.086
Significancens ** ** ns ns ns
Micro-Nutrients
TreatmentZn (ppm) Mn (ppm) Cu (ppm) Fe (ppm) B (ppm)
‘WA 38’/‘G.41’bag2.92 2.76 2.19 9.38 63.92A
control (GS-)1.91 3.82 2.01 11.97 47.79Ba
Significancens ns ns ns *
‘WA 38’/‘M.9’bag0.93B2.46 2.51 8.43 34.09
control (GS-)3.03A3.49 1.94 9.87 28.36b
Significance* ns ns ns ns
Ratios
TreatmentN:Ca K:Ca Mg:Ca (K+Mg):Ca (N+Mg+K):Ca
‘WA 38’/‘G.41’bag10.2 20.3A2.4A22.7A32.9A
control (GS-)7.9 14.3B1.8B16.2B24.1B
Significancens ** ** ** *
‘WA 38’/‘M.9’bag7.7 17.8A2.0 19.8A27.5
control (GS-)7.2 13.4B1.9 15.4B22.6
Significancens * ns * ns
Table
Table 5. ‘WA 38’/‘G.41’ peel mineral composition in 2020: comparison between symptomatic (GS+) and asymptomatic (GS-) apples, and comparison between combinations of high vigor with (V+ GS+) and without (V+ GS-) GS symptoms and low vigor without GS symptoms (V- GS-). Significance by treatment combination: * = p ≤ 0.05, ** = p ≤ 0.01, *** = p ≤ 0.001, ns = not significant. Different letters within comparisons indicate statistical differences and means separations by SNK.
Table 5. ‘WA 38’/‘G.41’ peel mineral composition in 2020: comparison between symptomatic (GS+) and asymptomatic (GS-) apples, and comparison between combinations of high vigor with (V+ GS+) and without (V+ GS-) GS symptoms and low vigor without GS symptoms (V- GS-). Significance by treatment combination: * = p ≤ 0.05, ** = p ≤ 0.01, *** = p ≤ 0.001, ns = not significant. Different letters within comparisons indicate statistical differences and means separations by SNK.
Macro-Nutrients
Treatment Total N (%) P (%) K (%) S (%) Ca (%) Mg (%)
‘WA 38’/‘G.41’GS-0.342b0.059b0.727b0.034b0.112a0.120b
GS+0.484a0.076a0.872a0.046a0.065b0.141a
Significance *** ** ** ** ** **
‘WA 38’/‘G.41’V+ GS-0.379 0.057 0.721 0.037ab0.072 0.128
V+ GS+0.416 0.064 0.811 0.041a0.075 0.132
V- GS-0.342 0.050 0.693 0.031b0.060 0.108
Significance ns ns ns * (0.0430) ns ns
Micro-Nutrients
TreatmentZn (ppm) Mn (ppm) Cu (ppm) Fe (ppm) B (ppm)
‘WA 38’/‘G.41’GS-2.01 4.56 2.88 14.56 55.27
GS+2.00 6.16 2.16 13.68 58.70
Significance ns ns ns ns ns
‘WA 38’/‘G.41’V+ GS-2.04 5.13 1.50 14.79 44.42
V+ GS+2.04 6.32 2.95 15.58 49.23
V- GS-1.91 3.92 1.69 10.36 46.39
Significance ns ns ns ns ns
Ratios
TreatmentN:CaK:CaMg:Ca(K+Mg):Ca(N+Mg+K):Ca
‘WA 38’/‘G.41’GS-3.1b6.5b1.1b7.6b10.6b
GS+7.7a14.0a2.3a16.2a24.0a
Significance * * * * *
‘WA 38’/‘G.41’V+ GS-5.4 10.3 1.8 12.1 17.4
V+ GS+5.7 11.1 1.8 12.9 18.6
V- GS-5.7 11.5 1.8 13.3 19.0
Significance ns ns ns ns ns
Table
Table 6. Correlation of “mild”, “severe”, and total green spot (GS) incidence (%) and productivity parameters in (a) 2020 and (b) 2021. Pearson correlation coefficients are in bold, with respective p-values below for significance.
Table 6. Correlation of “mild”, “severe”, and total green spot (GS) incidence (%) and productivity parameters in (a) 2020 and (b) 2021. Pearson correlation coefficients are in bold, with respective p-values below for significance.
GS Incidence (%)
(a) 2020 Number of Fruits Per TreeYield (kg Per Tree)Avg. Fruit WeightTCSACrop Load at Harvest
(Fruit per cm2 TCSA)		“Mild” “Severe”Total
Number of fruits per tree 10.90509−0.48653−0.313430.75001−0.53144−0.75764−0.83332
0.01310.32780.54520.08590.27790.0810.0394
Yield (kg per tree) 1−0.07064−0.547240.86191−0.82239−0.66836−0.8756
0.89420.26110.02730.04450.14670.0223
Avg. fruit weight 1−0.37263−0.00356−0.445730.450130.19255
0.46690.99470.37570.37040.7147
TCSA 1−0.846740.549660.548160.66853
0.03340.25850.26010.1466
Crop load at harvest (fruit per cm2 TCSA) 1−0.63558−0.80897−0.91685
0.1750.05130.0101
GS incidence (%)“Mild” 10.257130.60846
0.62280.1999
“Severe” 10.92335
0.0086
Total 1
GS incidence (%)
(b) 2021 Number of Fruits Per TreeYield (kg Per Tree)Avg. Fruit WeightTCSACrop Load at Harvest (Fruit Per cm2 TCSA)“Mild”“Severe”Total
Number of fruits per tree 10.92947−0.463760.636420.667780.559410.327260.51971
<0.00010.06080.0060.00340.01960.19980.0325
Yield (kg per tree) 1−0.115010.613140.567850.490070.258090.44217
0.66030.00890.01740.04580.31720.0755
Avg. fruit weight 1−0.28121−0.39342−0.36469−0.28623−0.37226
0.27420.11820.15010.26540.1412
TCSA 1−0.127250.776870.342710.67045
0.62650.00020.17810.0032
Crop load at harvest (fruit per cm2 TCSA) 10.001920.165880.07739
0.99420.52460.7678
GS incidence (%)“Mild” 10.582340.92781
0.0142<0.0001
“Severe” 10.84357
<0.0001
Total 1
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Sheick, R.; Serra, S.; Rudell, D.; Musacchi, S. Investigations of Multiple Approaches to Reduce Green Spot Incidence in ‘WA 38’ Apple. Agronomy 2022, 12, 2822. https://doi.org/10.3390/agronomy12112822

AMA Style

Sheick R, Serra S, Rudell D, Musacchi S. Investigations of Multiple Approaches to Reduce Green Spot Incidence in ‘WA 38’ Apple. Agronomy. 2022; 12(11):2822. https://doi.org/10.3390/agronomy12112822

Chicago/Turabian Style

Sheick, Ryan, Sara Serra, David Rudell, and Stefano Musacchi. 2022. "Investigations of Multiple Approaches to Reduce Green Spot Incidence in ‘WA 38’ Apple" Agronomy 12, no. 11: 2822. https://doi.org/10.3390/agronomy12112822

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