Phosphite-Based Nutrients Impact Mineral Elements, Bitter Pit, and Fruit Quality Attributes of ‘Braeburn’ Apple

Preharvest nutrient applications have major impacts on apple (Malus domestica Bork) tree mineral status, productivity, and fruit quality attributes. Despite the wide use of calcium chloride (CaCl2) for improving apple fruit quality attributes particularly bitter pit, application of amino acid, polyamine and phosphite-based nutrients (Sysstem series) have become popular in the past decade. Susceptibility to bitter pit disorder varies greatly in different apples, among which ‘Braeburn’ is an extremely susceptible cultivars to this disorder. Thus, the impacts of Sysstem nutrients on leaf and fruit mineral status in one year and yield and fruit bitter pit and quality attributes in two years were compared with those of commercial “Grower’s nutrients” in ‘Braeburn’ apple (Malus domestica Bork), each year according to a randomized complete block design. Trees receiving Sysstem nutrients had lower leaf nitrogen (N), phosphorus (P), potassium (K) and boron (B) but slightly higher leaf magnesium (Mg), iron (Fe) and zinc (Zn) than those with Grower’s nutrients. Sysstem nutrients applications reduced and improved fruit N/Ca, K/Ca, (K + Mg)/Ca ratios over Grower nutrients. The improvement of mineral concentrations and certain mineral ratios resulted in better color, higher soluble solids concentration, more advanced starch hydrolysis, and lower bitter pit than those receiving Grower’s nutrients. Sysstem nutrients drastic reduction in the bitter pit disorder was the main advantage of this nutrient regime over traditional Grower nutrient applications in this study.


Introduction 'Braeburn' apple and its challenges
Braeburn' apple has gained popularity among consumers in the past decade, while it is sensitive to bitter pit disorder. However, limited information is available on the effects of foliar nutrients on 'Braeburn' apple yield and fruit quality attributes. The physiological and biochemical roles of calcium (Ca), nitrogen (N), potassium (K), and magnesium (Mg) on postharvest physiology of pome fruit have been extensively studied, because these elements are known to be more involved in various fruit quality attributes, particularly bitter pit incidence than other mineral elements.
activities of polyphenol oxidase and peroxidase enzymes [19][20][21]. Wang, et al. [20] reported that activities of catalase, peroxidase, ascorbate peroxidase and superoxide dismutase in apple fruit with bitter pit was significantly lower than those without this disorder. Lipoxygenase activity had a negative correlation with fruit Ca concentration, and thus a positive correlation with occurrence of bitter pit disorder [21][22][23]. De Freitas, et al. [24] suggested a connection between bitter pit and Ca 2+ binding to cell walls as well as accumulation of Ca 2+ in storage organelles. They later found an association between higher levels of water-soluble pectin Ca 2+ and bitter pit [25].
Imbalances between ratios of certain minerals to Ca have a greater impact than merely low levels of Ca in the occurrence of bitter pit in some apple cultivars. For instance, we have frequently observed imbalances in fruit K/Ca, N/Ca, and Mg/Ca ratios in the bitter-pitted fruit of 'Honeycrisp', 'Desert Rose Fuji', 'Autumn Rose Fuji', and 'Starkspur Golden Delicious' apples (Fallahi, et al. unpublished data). Also, Cheng, et al. [11] reported that 'Honeycrisp' apple fruit had lower Ca and much higher K/Ca ratio than 'Gala' apple fruit. The imbalances between ratios of certain minerals to Ca and their impacts on bitter pit in apples are also reported by other researchers [13,14,26,28,30,35,36]. Ben, et al. [37] reported ratios of Na/Ca, K/Ca, P/Ca and Mg/Ca had stronger association with bitter pit disorder than the concentrations of each of these elements by itself. Wińska-Krysiak and Lata, et al. [22] reported that high ratio of K/Ca increases the activity of lipoxygenase enzyme in fruits, leading to higher bitter pit disorder.
High fruit K and Mg are also believed to increase bitter pit incidence regardless of Ca content [38]. Fallahi, et al. [10] were able to induce severe bitter pit symptoms in 'Golden Delicious' apple fruit by vacuum infiltration of Mg, and thus increasing Mg/Ca ratio in the fruit tissue. Baugher, et al. [39] reported a strong correlation between fruit N/Ca ratio and bitter pit. They also revealed that peel nutrients had stronger correlations with bitter pit than did the flesh nutrients in 'Honeycrisp' apple. We found that fruit Ca and K/Ca ratio in the calyx end tissue had stronger association with bitter pit than did those in other portions of apple fruit tissues in 'Honeycrisp' apples (Fallahi, et al. data not published).

Calcium spray to reduce bitter pit and improve fruit quality
Calcium is often considered to be the most important mineral element determining apple fruit quality and reducing metabolic disorders. Foliar sprays can increase the Ca content of apple fruit [40] and firmness [41] and reduce bitter pit and postharvest decay caused by Gloeosporium perennans and Alternaria spp [41,42].
Calcium chloride Ca(Cl) 2 sprays are widely used on 'York', 'Fuji', 'Golden Delicious' apples to reduce bitter pit in Pennsylvania orchards [43]. Despite the use of CaCl 2 and Calcium nitrate [Ca(NO 3 ) 2 ] for improving apple fruit quality attributes, application of amino acid, polyamine, and phosphite-based nutrients have become popular in the past decade. "Sysstem" Series products are manufactured by Agri-K Science Driven Nutrition (Minneapolis, MN) and have phosphites leverage which may rapidly penetrate plant vascular tissue and be a vehicle to deliver critical nutrients like Ca, Zn, Mg, Mn, and K for maximum effect. In this research, the influence of Sysstem nutrients on leaf and fruit mineral status in one season, and fruit bitter pit and quality attributes in two seasons in 'Braeburn' apple was studied.

Materials and Methods
The experiment was conducted over two seasons in an 18-year-old 'Braeburn' apple orchard, where trees were budded onto M.7 rootstock and planted at 3.05 m between trees and 5.18 m between rows in Sunny Slope region of Caldwell, Idaho, U.S.A. The orchard soil was sandy loam with pH of near 7.5. Two nutritional regimes were applied in this experiment as 1) Grower's nutrients (GS) ( Table 1), 2) Sysstem nutrients ( Table 2).
Seventeen rows of uniform trees were used for this study in each year. Two guard rows were kept on either side of each experimental row and these guard trees did not receive any nutrient treatments. Thus, we had five experimental rows in each year. In the first year, five adjacent trees with uniform canopy size and crop load on each experimental row were tagged and all five trees received Grower's nutrients, while five uniform trees on 10 trees away on the same row received Sysstem nutrients. In the second year (Year 2), the experimental design was the same as Year 1, except that an additional group of five adjacent trees on each of the experimental row was selected as Un-Treated control (total of three treatments). Therefore, the experimental design was randomized block design with five-tree blocks or replications, Starch degradation pattern (SDP) of equatorial slices of each fruit was recorded by comparison with the SDP standard chart developed for apples. Percentages of fruit with disorders were determined from the collected sub samples at harvest and after storage. The percentage of bitter-pitted fruit was calculated based on number of affected fruits in the sub-samples as well as a percentage of affected fruit in the entire tree. To estimate the percentage of bitter pit in the entire tree, the actual numbers of infected and healthy fruits in two major limbs per tree were counted. In this article, percentage of bitter pit based on the entire tree is reported. Generally, methodologies for fruit quality measurements were accordingly to the procedures described by Fallahi, et al. [45]. Data was analyzed using SAS statistical packages and means were separated according to Fisher Protected Least Significant Differences (LSD) at 5% level.

General trends in mineral nutrients
In general, leaf concentrations of N, K, Zn, Cu, and B declined but Ca and Mn increased, and P, Mg, S, and Fe remained approximately the same as the growing season progressed in both Grower's nutrients and Sysstem nutrients in Year 1 (Table 3 and Table 4). Based on this study, late-July to early August would be an optimum period for leaf sampling as most mineral elements seemed to be at the "minimal flux" stage (Table 3 and Table 4).

Leaf and fruit minerals concentrations
Trees receiving Sysstem treatment had lower leaf N, P, with two treatments in Year 1 and three treatments in Year 2. In this study, all five trees in each plot received the same treatment or designated as Un-treated Control, but only the middle three trees of each plot were used for data collection and the two outer trees were considered as in-row guard trees. Therefore, we had a total of 15 trees (five blocks x three middle trees) for collecting data in each treatment each year. Other than nutritional treatments, all cultural practices in the Grower's nutrients and Sysstem nutrients were similar to those of commercial orchards in the Pacific Northwest [44].
The three "data trees" in each plot were flagged and used for repeated leaf and fruit samplings in Year 1. Fruits were sampled in September and November. Theses sampling dates in leaf and fruit were chosen because they were considered as critical developmental stages and would unveil a fluctuating pattern that could eventually assist us in finding an optimum time of sampling. Leaf area was measured, and leaf and fruit tissues were immediately analyzed for mineral analyses, similar to the procedures described by Fallahi, et al. [45]. Since the experimental trees were mature with large volume and had high yield in both years, three leaf and two fruit samplings did not have any effects on the final mineral nutrient status or fruit quality attributes in this study.
In early November of each year, 35 fruits per tree were sampled for quality analyses at harvest and after three months of 0 °C regular atmosphere storage. Bitter pit and quality attributes were measured at the University of Idaho Pomology and Viticulture Laboratory. For quality evaluations, fruits were weighed, and fruit color was visually ranked on a scale of 1 to 5, with 1 = 20% red, progressively to 5 = 100% red. Soluble solids concentration (SSC) was measured by temperature-compensated refractometer (Atago N1, To-   (Table 5). Based on author's personal experience, N/Ca ratio between 4 to 9 is considered normal and ratios above 10 may result in reduction of fruit firmness. In this study, N/Ca did not exceed this optimum range (Table 5). Nevertheless, fruit (K + Mg)/Ca ratio must remain between 10 to 30. For sensitive cultivars, fruit (K + Mg)/ Ca ratios exceeding 25 to 30 is alarming as values above these ranges may increase the chance of bitter pit incidence. In this study, application of Sysstem nutrients kept these values at significantly lower levels than those of Grower's nutrients ( Table 5), resulting in significantly lower bitter pit incidence at harvest and after storage ( Table 7, Table 8 and Table 9).

Impacts on fruit quality attributes
Major differences existed in some quality attributes between the fruits from Growers nutrients and Sysstem treatments in both years ( Table 7, Table 8 and Table 9). Fruits from the Sysstem-treated trees had better color, more soluble solids concentration (SSC), more starch degradation pattern K, and B but higher leaf Mg, Fe and Zn than those receiving Grower's nutrients during sampling dates in Year 1 (Table 3 and Table 4). Trees with Grower's nutrients were more vigorous (Data not shown) perhaps because of their higher N uptake (Table 3), leading to higher transpiration and thus uptake. Trees with Sysstem nutrients had larger leaves (data not shown), perhaps due to their higher Zn and Mg concentrations. Larger leaves can reduce blank wood, and higher leaf Mg may increase chlorophyll content, resulting in production of more carbohydrate [45]. Improvement in the leaf Fe uptake in the trees with Sysstem nutrients may also increase photosynthetic metabolites.
Trees receiving Sysstem treatment had higher fruit Ca (up to 12%) during all sampling dates (Table 5) and fruit iron (100%) in November (Table 6) but lower fruit K (up to 9.8%), and fruit S (up to 44%) in September and November (Table 5). Fruit K/Ca and (K + Mg)/Ca ratios in November were greater than those in September in both treatments (Table 5). Sysstem nutrients applications improved fruit N/Ca ratio by up Table 3: 'Braeburn' apple leaf macro-elements during the growing season (July-November) in Year 1 z .     Table 8), indicating that fruit ripened and reached or passed climacteric peak in the storage. Post-storage weight loss in the fruit from trees receiving Sysstem treatments was significantly (about 53%) less than those receiving Grower's Nutrients treatments (Table 8).

N (% dwt) P (%dwt) K (% dwt) Ca (% dwt) Mg (% dwt) S (% dwt)
Significant reduction in fruit weight loss, bitter pit and "pale spot" (Figure 3) has a major impact to commercial apple growers as improvement in these quality attributes would lead to a higher pack-out and more net profit.
In Year 2, trees receiving Sysstem treatments had higher (SDP), and lower bitter pit, sunburn, and "pale spot" than those from Grower's nutrients at harvest in Year 1 (Table 7) (Figure 1, Figure 2 and Figure 3). No difference was observed in SDP after the storage in Year 1 (Table 8). Otherwise, the pattern in treatment differences after storage was similar to those at harvest in first year.
The bitter pit difference between nutrient treatments after storage was greater than that at harvest in the first year ( Table 7 and Table 8). Water core and SSC of both treatments were greater after storage compared to harvest time ( Table   Table 7: 'Braeburn' apple fruit quality attributes at harvest in Year 1 z . Abbreviations: Color scales: 1= green, progressively to 5 = red, Starch degradation pattern (SDP) was ranked from 1= very starchy to 6 = most mature fruit. Abbreviations: Color scales: 1= green, progressively to 5 = red; Starch degradation pattern (SDP) was ranked from 1= very starchy to 6 = most mature fruit. Perhaps the most important impact of Sysstem regime is its reduction of fruit bitter pit incidence. 'Braeburn', 'Honeycrisp', and 'Golden Delicious' apples are among the most sensitive apple cultivars to bitter pit. When compared to Grower's nutrients, Sysstem treatments reduced bitter pit incidence by about 91% at harvest and by 87% after storage in Year 1 (Table 7 and Table 8) and by 26% at harvest and 60% after har-fruit size, color, and yield per tree but lower fruit water core, sunburn, russet, bitter pit, cracking, and internal browning than those receiving Grower's nutrients, although differences were not always significant (Table 9).
We believe that better quality attributes in the Sysstem-treated trees could be due to the better absorption of some essential nutrients and the lower N in their leaves as reported earlier [8]. Leaf area of trees with Sysstem nutrients was slightly increased during some sampling dates (data not shown). This factor, in addition to an improvement in Mg, Fe, and Zn accumulation in the leaves of Sysstem-treated trees could have contributed to an improved photosynthesis and thus better fruit quality in these trees. Sysstem nutrients   (Table 9). In Year 2, fruit from un-treated control trees had lower bitter pit than trees receiving Growers nutrient because they had smaller size, leading to higher fruit Ca concentration, which is consistent with a previous report in 'Spark Spur Golden Delicious' apple [38].
Based on author's observations and experience with different apple cultivars, the formulation of the calcium and windows of applications are crucial to uptake and performance, while the cost of chemical also plays an important role in a sustainable production system. Thus, apple growers need to emphasize on supplying Ca during and after fruit cell division stages and use various formulations of Ca as tools in the toolbox and pick the best one for each timing, variety and purpose. Application of Ca in a phophite formulation at certain stage of apple development could be more advantages over the nitrate and chloride forms. In addition to be an effective source of Ca, the phosphite portion of calcium phosphite formulation (Sysstem Cal, Sysstem SeaCal) may have an impact on the reduction of phythophora (Phytophthora spp.) and fire blight (Erwinia amylovora), although this aspect of phosphite potential impacts requires further studies. In some modern orchards, growers use a multiple-tool approach to control Ca-related issues such as bitter pit. In these orchards, foliar anti-bitter pit programs could start with Sysstem Cal as the Ca source and then moves to Vigor Cal (a dextrose-lactose formulation) that has efficient uptake and safety like the amino acid (Metalosate) formulations but at a lower cost than amino acid or than phosphite. During the mid-season, foliar application Vigor Cal can supply Ca to the trees, with or without growth regulator Apogee, and without having the potential negative impacts of calcium nitrate [Ca(NO 3 ) 2 ]. Calcium nitrate increases Ca uptake, but negatively impacts the fruit N/Ca ratio because its nitrate can promote bitter pit, reduce fruit color, firmness and storability. The nitrate form of Ca(NO 3 ) 2 may also promote fire blight and in some cases powdery mildew (Podosphaera leucotricha) diseases. Foliar calcium chloride (CaCl 2 ) is well documented to be an effective and inexpensive source of Ca [42]. However, frequent applications of CaCl 2 throughout the season in the western United States will induce stress. Thus, limiting the number of applications of CaCl 2 and delaying the starting dates for application may be a better strategy.

Conclusions and Recommendations
In our study, phosphite-based nutrients appeared to show promising results by reducing bitter pit and improving fruit quality attributes at harvest and after storage. Ratio of fruit minerals, especially K/Ca and (K + Mg)/Ca, are extremely crucial in predicting bitter pit occurrence. Despite our promising results, the following studies are recommended to understand the efficiency and cost of applications of nutrient products under high density systems with modern tree canopy architectures and rootstocks: 1) Application of Phosphite formulation during early growing season, followed by Vigor Cal in the middle season; 2) Early season applications of Ca(-NO 3 ) 2 and CaCl 2 followed by late applications of Sysstem nutrients; 3) Comparing amino acid-based and phosphite-based nutrients in each region; 4) Studying the impacts of nutrients Ha.

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