4.1. Correlation of Green Fruit Drop in ‘Draper’ Blueberry with Fruit Calcium and Plant Vigor
The correlation between reduced GFD and higher fruit Ca resulting from foliar applications in the current study, relates to the physiology of plant Ca nutrition in numerous crop species. Since Ca has little mobility in the phloem, its movement from the root system to the above-ground parts of the plant is primarily through the xylem, driven by transpiration [
34]. Compared to other cationic nutrients that can move in the phloem (e.g., K and Mg), Ca transport to fruit is often much lower than to leaves, resulting in deficiencies in many crops e.g., [
5,
6,
7]. Environmental conditions that result in low transpiration, low uptake of Ca and high competition between floral and vegetative organs lead to these disorders, especially in crops with separate floral and vegetative buds as in blueberry [
20]. While flowers and fruits are typically stronger sinks for phloem transported elements and carbohydrates than shoot apices, the movement of xylem transported elements is dictated by transpirative demand. A high transpiring organ (e.g., leaves with many active stomata) will draw more water from the roots than a low transpiring organ (e.g., fruit with relatively few stomata that decrease in function as the fruit develop) [
34]. Competition for Ca as it moves by mass flow in the xylem is not a complete explanation of the uptake and differential allocation of this mineral because water transport to the fruit does not always correlate well with allocation of Ca, as seen in apple [
13]. However, competition with other ions such as K may be aggravated under low transpiration and high competition [
35].
The association between severity of GFD and plant vigor evaluated in the current study through comparison of Ca treatment under different rates of N management, is in accordance with findings for Ca disorders in other crops. Specifically, several crops demonstrate an interaction between Ca disorders and N management, with high levels of N resulting in increased vigor and often triggering the Ca deficiency or exacerbating the related disorder [
19]. In apple, for which these relations have been most extensively studied, the competition between vigorous shoot growth and fruit Ca was proposed more than sixty years ago [
36]. This interaction is most likely due to competition between growing vegetative shoot apices and developing fruit rather than inhibition of Ca uptake from the roots by excessive N in the root zone [
37]. Not just vigor of shoot growth but the timing of onset for vigorous growth relative to fruit development is important in some Ca disorders in other crops. Various rates and durations of Ca uptake to the fruit have been document in apple, ranging from primarily in the first part of fruit growth to occurring with a decreasing linear trend that extends until harvest [
16,
38]. In any case, Ca concentration in apple tends to reach its maximum soon after bloom before dropping due to dilution as the fruit grow through cell enlargement [
39]. Therefore, the key Ca loading phase is early in development for many types of fruit and is highly influenced by variation in transpiration [
34], but the reasons for the decline in xylem transport of Ca into fruit after this phase are still only partially understood, likely relating to a decline in xylem function [
40].
4.2. Calcium Deficiency as a Genetic Pre-Disposition to Green Fruit Drop in ‘Draper’ Blueberry
The current finding that foliar Ca corrects GFD in ‘Draper’ builds on the current knowledge of Ca nutrition in blueberry. In Oregon, a high leaf Ca and low fruit Ca were noted in a replicated survey of tissue nutrient content across a range of cultivars [
8], which validates that an interaction between Ca and N is observed and may be due to competition between these plant organs for Ca in the xylem sap. These authors explain the higher leaf Ca in ‘Draper’ as being related to the high vigor and many growing shoot tips of ‘Draper’ in Oregon. The severity of GFD in Southwestern BC compared with the near lack of GFD in Oregon indicates differential performance across environments. Specifically, this belies marginal adaptation of ‘Draper’ to Southwestern BC in comparison to a highly adaptive region like Oregon. Consequently, Ca uptake, adequacy of allocation to the fruit or both factors are in a range of marginal sufficiency in this region, entering deficiency under certain field- and season-specific conditions, subsequently resulting in GFD.
In apple, the occurrence and severity of bitter pit is related, among many factors, to the specific cultivar and where it is grown. Pre-disposition of ‘Draper’ to GFD is similar, a physiological condition with an important genetic component, manifesting under certain environmental conditions. While ‘Draper’ is the first highbush blueberry commercial cultivar to manifest this condition, GFD has also been observed in progenies of ‘Draper’ in breeding plots in the Willamette Valley of Oregon (C. Finn, personal communication). As with apple bitter pit, GFD symptoms include decayed internal tissues with no visible symptoms to the exterior. The internal browning is likely related to action of degradation enzymes. Lack of damage to the exterior of the fruit is due to the ability of the soluble fraction of fruit Ca to move within the fruit [
41], leading to deficiencies in some parts and not in others. Detailed comparison of skin, pulp and seed Ca levels have not been conducted to study a presumed gradient in fruit Ca relating to GFD in ‘Draper’.
Therefore, with a genetic pre-disposition toward relatively greater allocation of Ca toward vegetative structures at the expense of reproductive structures [
8], ‘Draper’ responds to the environmental conditions of Southwestern BC with a deficiency that can be characterized as an environmental maladaptation. With Ca deficiency as a proximate cause of GFD, interacting with plant vigor and presumably other factors, the ultimate cause of this maladaptation could be due to any number of physiological mechanisms or disfunctions, likely relating to the high leaf/fruit Ca ratio for the cultivar. For example, one could speculate that there is low expression of a cell membrane Ca pump gene under environmental conditions conducive to GFD. As contributing factors, edaphic soil properties and horticultural management practices may aggravate the effects of the climatic conditions on plant phenology that reduce Ca uptake or result in greater competition for Ca allocation to the fruit.
Temperature, light intensity, precipitation, soil moisture, and relative humidity are all important factors that affect transpiration. Climatic conditions in Southwestern BC are generally cooler, wetter and more often overcast during spring phenological development of blueberry in comparison with Oregon. In most years, Southwestern BC is likely to have higher relative humidity, wetter soils, lower light levels, and cooler temperatures, indicating generally lower transpirative demand, in comparison to Oregon with minimal GFD. High relative humidity decreases Ca uptake in either the fruit or vegetative tissues of sweet pepper (
Capsicum annuum L.), tomato, and apple [
6,
42,
43]. Further, well-watered plants have reduced transpiration, which could be made worse if leaf development is relatively delayed very early after floral bud break, as observed in the field in the case of ‘Draper’. Later in the season, when fruit development is in its first stage of cell division and the shoots are in a rapid phase of elongation, excessive guttation on the leaves is sometimes observed in the field under overcast conditions with high relative humidity, indicative of positive root pressure for xylem sap. Tomato and apple demonstrate much reduced allocation of Ca to fruit under experimental conditions of high relative humidity [
44]. From these field observations, one or both potential environmental factors could be involved in triggering GFD in ‘Draper’.
Parsing the effects of these putative environmental triggers would require detailed documentation of fruit Ca loading over time and a comparative study of phenological and climatic factors across regions with and without GFD. A companion survey of tissue and soil nutrient values across BC, WA, and OR in 2015 and 2016 was conducted as a preliminary investigation of these fundamental issues (unpublished data). Nevertheless, the current explanation of GFD is consistent with what is known of Ca disorders in other fruit such as apple and pear, the severity of which tend to vary from year-to-year and site-to-site by directly impacting plant parts, indirectly interfering with the interactions between plant parts or otherwise responding to cultural management practices [
20].
4.3. Mitigation of Green Fruit Drop in ‘Draper’ Blueberry Using Corrective Foliar Calcium Applications
In BC, GFD is observed in fields with a broad range of soil Ca (data not shown), with some severely affected fields having very high levels of soil Ca based on established nutrient management recommendations for the Pacific Northwest [
30]. For this reason, and since blueberry is a calcifuge that is highly efficient at taking up Ca under low pH conditions [
11], soil applications are unlikely to impact the incidence of GFD. Whereas application of lime or gypsum to the soil may benefit overall crop health in the long-term management of soil pH of blueberry plantings where pH is dropping below the recommended range, soil applications of these fertilizers demonstrated little effect on fruit Ca and no effect on fruit yield, size or firmness in studies in Michigan [
45].
As seen in other crops, loading of Ca into blueberry fruit occurs primarily when shoot growth is limited, and low transpiring fruit accumulate less Ca than high transpiring leaves overall [
18]. This results in a high DW leaf/fruit Ca ratio, which increases several-fold as the season progresses [
26]. While Ca is more likely than more mobile elements to be affected by foliar application due to its immobility within the plant, affecting a substantial change in fruit Ca via exogenous treatment is challenging for this same reason. That is, without translocation of Ca from leaves to fruit by the phloem, effective correction of acute fruit deficiencies requires movement of exogenous Ca into fruit tissues, primarily through stomata, as described in apple [
46]. This is problematic because fruit stomata are only functional for a short period of time during early fruit development, the waxy cuticle decreasing their functionality as it develops [
47]. In apple, uptake of exogenous Ca occurs in very small in quantities (<1 ug·cm
−2) during foliar management of bitter pit, necessitating as many as ten or eleven applications to be even partially successful in some varieties [
38,
48]. Notwithstanding, uptake of exogenous Ca is known to occur by first penetrating the cuticle and then moving apoplastically through the flesh [
49]. Evidence for this ability is based on Ca concentration changes within different parts of the apple fruit during maturation, and even post-harvest [
50]. With the availability of the mobile, physiologically-active forms of water-soluble Ca increasing as the fruit develop [
41], the small quantity of Ca that effectively penetrates the fruit surface can redistribute to portions of the fruit that are deficient.
In blueberry, fruit Ca levels impact fruit quality [
51], but attempts to use foliar Ca to increase fruit Ca concentration, usually for the purpose of improving fruit quality, have been met with varying degrees of success. In one study in Michigan, a 0.08% Ca solution was applied to ‘Bluecrop’, ‘Blueray’, and ‘Ivanhoe’, but fruit Ca and quality parameters such as firmness were not affected [
28]. In comparison, Experiment 3 used 0.136% and 0.272% Ca, which were 1.7× and 3.4× higher, respectively, and were effective at increasing ripe fruit Ca significantly for all timing treatments except for L-CaCl
2 at Early timing (
Table 5). In Chile, increases in Ca levels in fruit skin and seeds were seen at a low rate of 0.0475%, but increases in fruit pulp Ca required 0.09% or 0.18% to be detected [
52]. Experiment 3, which did not compare Ca concentrations in different parts of the fruit, used rates of CaCl
2 that were 1.5× and 3.0× higher, respectively, than the lowest amount needed by these researchers to affect an increase in Ca content in the pulp in addition to the skin and seeds. In another Chilean study, increases in fruit Ca were achieved following two applications at 0.06% Ca but not 0.036% Ca or 0.006% Ca [
26]. The rates in Experiment 3 were 2.3× 4.5× higher.
In contrast, work in Oregon used a range of Ca products (chloride, silicate, acetate, and chelate) with 3–5 applications from late bloom to early green fruit development (1–4 weeks before harvest) on ‘Spartan’, ‘Draper’, ‘Liberty’, and ‘Legacy’ [
53]. The highest rate of CaCl
2 in this study was applied at 0.09% Ca in 748 L water·Ha
−1, and the researchers saw no significant effect on fruit Ca or fruit quality. While the concentrations used for Experiment 3 were maintained as constants, the amount of spray volume applied to experimental plots varied from 371 to 914 L water·Ha
−1, depending on the field, year, and stage of plant development. Comparing the total amount of Ca applied would require a nuanced comparison of the plant sizes used in these studies, but with applications being made in the current study to the point of runoff, it is the actual concentration of the spray solution that contacts the fruit surfaces that is most relevant to uptake. Therefore, the concentrations applied in the current study were 1.5× and 3.0× higher than the highest rate applied in Oregon, which largely explains the difference in results. Further, while the timings of application in Oregon started at approximately the same time as the effective Mid timing used in BC, the Oregon study did not use a surfactant, based on supplier recommendations (B. Strik, personal communication), which may also have resulted in lower uptake of Ca across fruit tissues.
Foliar Ca treatment has long been used as a standard production practice by apple growers to decrease bitter pit [
54]. Foliar applications are effective in reducing bitter pit, and this is often associated with a measurable increase in fruit Ca but not in all studies [
55,
56]. Determining significant differences in fruit Ca is often problematic in experimental bitter pit research due to a high degree of between-plant and within-plant variation in mean Ca [
57,
58]. Fruit Ca was determined in the current study on both a FW and DW basis to remove the impact of differences in fruit size across samples based on the same approach taken in apple research [
59]. However, the results on a FW and DW basis showed very similar effects across treatments in the current study.
As seen in bitter pit control [
59], CaCl
2 applications demonstrated risk of phytotoxicity (i.e., leaf marginal necrosis) in blueberry when applied at the higher rate in the current study. More phytotoxicity is seen in apple in response to early applications [
60], which was also observed in the current study. In Michigan, a very low rate (0.08% Ca) resulted in similar phytotoxicity [
28], but no such damage was observed at more than twice this rate (0.18% Ca) in Chile [
52], a rate that was intermediate to the low and high rates used in Experiment 3.
In summary, foliar application at sufficiently early stages of fruit development is important but so is use of the correct material. Likewise, early applications are most effective for bitter pit control in apple [
59] and CaCl
2 is generally the most effective material [
60]. Moreover, the amount of exogenous Ca uptake by fruit is not just related to the permeability of the fruit tissues at the time of application but to the quantity of spray volume that contacts the fruit tissues and its concentration at the time of contact [
61]. For these reasons, the success of the current study in increasing fruit Ca and mitigating GFD in ‘Draper’ is attributed to five factors: (1) use of a Ca material that has been demonstrated to affect high uptake in other crops; (2) use of high concentrations of Ca, afforded by the use of a basic fertilizer product (CaCl
2) rather than a formulated product with a restricted label rate; (3) application during early stages of fruit development when stomata are most functional and the cuticle least developed; (4) repeated application, three being substantially as effective as seven; and (5) use of a surfactant in tank mixes to decrease the surface tension of water droplets as they contacted fruit tissue, extending the period of time over which Ca was absorbed. Further qualification of this final point is that applications were made to the point of run-off under slow drying conditions that were conducive to optimal foliar uptake.
4.4. Future Research Directions for Green Fruit Drop in Blueberry
For decades, the research in apple and tomato have indicated that low Ca predisposes these crops to bitter pit and blossom-end rot, respectively, but that there are other factors which require consideration [
62,
63]. While mitigation of GFD with foliar Ca applications is highly effective and straight-forward, investigation into additional factors contributing to GFD in ‘Draper’ highbush blueberry should be part of an integrated approach to combatting this economically devastating disorder for commercial growers. Specifically, the antagonistic relationships of Ca with K and Mg seen in apple bitter pit and strawberry tip burn, respectively, are due to the ability of these other cations to move freely via the phloem and compete with Ca, influencing allocation to different organs [
14,
64]. Moreover, in apple, the role of K, Mg, Zn, and Mn, and ratios between these cations and Ca, have been known for more than two decades though their role is still not fully understood [
65].
Climatic maladaption resulting in poor carbohydrate partitioning is also a potential area of future research relating to the underlying physiological cause of GFD in ‘Draper’. Unbalance partitioning plays a role in poor Ca allocation and contributes to bitter pit in apple [
66]. In ‘Draper’, shade studies have not, however, demonstrated a significant effect on severity of GFD [
67]. Alternatively, the use of plant growth regulators (PGRs) to modify the uptake of Ca, or enhance its allocation to fruit, is established as strategy for decreasing bitter pit in apple. For example, PGRs such as Prohexadion-Calcium (a gibberellic acid inhibitor), 1-methylcyclopropene (1-MCP), or diphenylamine (DPA) have been used in apple [
68,
69]. The potential negative effect of the types of PGRs on fruit set make this avenue of investigation problematic.
Seeds are important for the accumulation of fruit Ca [
70], and ‘Draper’ does demonstrate problematic pollination under some conditions, perhaps related to a squat floral morphology or some other cause of low attractiveness to honey bees (
Apis mellifera L.) [
67]. However, dissection of fruit during extensive field observations across the region has demonstrated that dropped fruit consistently contain numerous developing seeds, and the pattern of GFD severity across fields and years does not present as being related to poor pollination or low fruit set. Consequently, the role of enhanced pollination as an additional GFD mitigation strategy is unlikely to be worthwhile.
In apple, Ca is remobilized in the xylem sap from storage in tissues such as bark prior to significant root growth [
35]. The same is seen in blueberry, and it is possible that Ca levels in these storage structures is low for ‘Draper’ in BC. Fall foliar Ca applications to increase the amount that is stored by the plant during the dormant season, and that will be available for remobilization in the spring, is a potential strategy for reducing GFD pre-disposition. Thus far, our initial attempts to reduce GFD by increasing plant Ca through fall foliar applications in the previous year have not been effective (data not shown), and a subsequent study by researchers in northwestern WA has shown no effect of fall Ca on GFD in the following year [
71].
Finally, the genetics of predisposition to GFD is the most important avenue of future research because the disorder is not just a feature of ‘Draper’. It appears to be a heritable condition in highbush blueberry. ‘Draper’ is being used in many northern highbush breeding programs around the world due to its ability to transmit high fruit quality to its progeny. A few cultivars with ‘Draper’ as a parent (e.g., ‘Calypso’) have already been commercialized and are being widely planted in regions where GFD is observed. Therefore, evaluating the genetic tendency for GFD is an important objective for breeding new cultivars of blueberry.