Impacts of 1-Aminocyclopropane-1-Carboxylic Acid as a Late Post-Bloom Thinner on Fruit Set, Yield, and Fruit Quality in “Gala” and “Fuji” Apples

A few post-bloom thinners are available for apple (Malus domestica Borkh.) and the prospects for additional thinners appear to be limited. Application of blossom thinners may present a risk of overthining in the areas where weather is less predictable. Thus, we studied the impacts of various rates of 1-amin-ocyclopropane-1-carboxylic acid (ACC) in three strains of “Fuji” apple and different rates of ACC and one rate of Ethrel in “Buckeye Gala” apple, when fruitlet diameter was about 20 mm, on fruit set, yield and fruit quality attributes at harvest, and return bloom in southwest Idaho in the Intermoun-tain West region, USA. In 2013, application of Ethrel at 300 mg·L −1 did not affect fruit set, fruit weight, diameter (D), length(L), or L/D ratio 34 days after application, or yield, fruit weight, color, russet, and starch degradation pattern (SDP) at harvest, while application of ACC at 150 mg·L −1 or higher reduced fruit set by 19% to 34% in “Buckeye Gala” apple. In this cultivar, application of ACC at 350 mg·L −1 significantly increased fruit weight, diameter and length 34 days after application, and increased fruit weight, color, and and can be effective when applied fruitlet diameter is about 20 mm.

Even though these bloom thinners effectively reduced fruit sets of certain apple cultivars, other apple cultivars have shown ineffective thinning results [7] [8].
Carbaryl showed a synergistic effect when used in conjunction with a commercial 6-BA formulation (MaxCel, Valent BioSciences LLC). However, the use of carbaryl faces regulatory concerns in the USA and is banned in certain areas in Europe. Thus, there has been an increasing interest in finding new post bloom thinners for apples.
After it was discovered that the pathway of ethylene biosynthesis is methionine-SAM2-ACC-ethylene [9], various studies were conducted on the regulation of ethylene biosynthesis in fruit and they were reviewed by Yang and Hoffman [10]. They reported that the intact climacteric fruits have little ability to convert S-adenosyl-L-methionine (SAM) to ACC or ACC to ethylene and contain only low amounts of ACC [10]. Internal ethylene concentration, ability to convert 1-aminocyclopropane-1-carboxylic acid (ACC) to ethylene (ethyleneforming enzyme EFE activity), and ACC content in the peel of "Golden Delicious" increased only slightly during fruit maturation on the tree [11]. Treatment of immature apples with 100 mg·L −1 ethylene for 24 hours increased EFE activity in the peel tissue but did not induce an increase in ethylene production. This ability of apple peel tissue to respond to ethylene with elevated EFE activity increased exponentially during maturation on the tree [11].
McArtney [14] reported that ACC reduced fruit set in "GoldRush" apple, and the response was dose dependent. He also reported that the effects of combining ACC and naphthaleneacetic acid on fruit set in "GoldRush" apple were additive. McArtney [14] suggested that application of 200 mg·L −1 ACC when fruit is about 10 mm in diameter could be a useful chemical thinning treatment for apple fruit. Schupp et al. [15] reported that application of ACC at 300 mg·L −1 to 500 mg·L −1 to "Golden Delicious" apple, when fruit was 20 mm, reduced fruit set.
Theron et al. [16] evaluated ACC on "Laetitia", "Fortune", and "African Ro-seTM" plums. ACC consistently reduced the hand-thinning requirement in both seasons in "African RoseTM". In the second season, there was a linear decrease in yield and an increase in fruit size as the ACC rate increased from the low to medium rate before flattening off. They recommended that ACC at a rate of 600 mg·L −1 for "African RoseTM" plum and 400 mg·L −1 for "Laetitia" plum would be optimal rates. Valent BioSciences LLC announced registration of a new plant growth regulator, to be marketed under the brand name AccedeTM, utilizing the active ingredient 1-aminocyclopropane-1-carboxylic acid (ACC) [17].
The objective of this study was to study the impact of ACC as a late postbloom thinner on fruit set, yield, and fruit quality attributes of "Buckeye Gala", and "Fuji" apples. This study was conducted during 2013 and 2014 seasons.
However, we are publishing those critically useful data today, as the needs for a post-bloom thinner is becoming extremely crucial and leaving the world's apple growers with very limited choices. We chose "Gala" and "Fuji" in our study as these are the most commercially important cultivars in the western USA.

General Descriptions of Orchard, Treatment and Cultural Practices
The experimental orchards for this study were located in Fruitland, Sunny Slope, and Parma, Idaho, USA. Trees were irrigated with a micro-jet system and trained into a central leader vertical axis. In general, the cultural practices, other than applications of post-bloom thinners were according to the local recommenda-

Measurements and Hand Thinning
Four branches per tree, each 50-to 65-cm in length with about 1.0 to 2.0 cm diameter were selected and number of flower cluster in each of these tagged branches was counted before blooms were open. At the time of hand thinning in each year (about 20 to 30 days after applications of post bloom thinners, as described for each cultivar), fruit set was calculated as number of fruits in the selected branches/number of flower clusters × 100. After fruit set measurements, fruits in all trees including Untreated Control were hand-thinned to create a 15-cm spacing among fruits, if they were not already at that spacing after chemical thinning. The reason for hand thinning according to this protocol was that even Untreated Control trees must be hand-thinned to a 15-cm spacing among fruit to follow the existing commercial practice. If fruit spacing was more than 15 cm, they were not hand-thinned any further.

Measurement of Return Bloom
To measure return bloom, four 50 to 65-cm branches per tree on each tree were tagged, and the diameter of each branch at the 50 to 65-cm mark was measured with a digital caliper in mid-March each year. Number of flower cluster within that area in each tagged branch was counted before blooms were open on March 22, 2014 (the year after application of treatments). Return bloom was calculated as the number of flower buds/ branch cross sectional area at the 50 to 65-cm mark.

Measurement of Yield and Fruit Quality Attributes
In 2013 and 2014, fruit were harvested at commercial maturity dates as follows: "Buckeye Gala" on Aug. 20, 2013; "Sun Fuji" in Sunnyslope on Oct. 12, 2013; "Top Export Fuji" in Parma on Oct. 14, 2013, and "Aztec Fuji" in Parma on Oct. 16,2014. Total fruit yield per tree was harvested and recorded in each orchard. Twenty-five fruits per tree were sampled randomly from all sides of the tree and used for quality attributes measurements as described by Fallahi et al. [19].

Orchard and Treatments Descriptions in 2013
The maximum and minimum temperature fluctuations in the region during fruit thinner application and fruit set measurements (April and May) were recorded during 2013 and 2014 ( Figure 1). The following apple orchards and treatments were used in the 2013 experiments: 1) "Buckeye Gala": For this experiment, a seven-year-old orchard, where "Buckeye Gala" trees on RN-29 rootstock were planted at 1.8 × 4.2 m spacing at a north-south row orientation in Fruitland, Idaho was used. Number of mixed buds on each of the four tagged branches was counted and recorded on an attached plastic tag, and branch cross sectional area was calculated on April 23, 2013.

Experimental Design
The experimental design for all experiments in 2013 and 2014 was a randomized complete block with six blocks, each with six trees. The middle two trees of each segment were used for measurements. The assumption of normal data distribution was checked by computing univariate analyses for all responses in this study. Analyses of variance were conducted by General Linear Model, using SAS.
Means separation was analyzed by least significant difference (LSD) at 5%.  (Table 1), or yield, fruit weight, color, russet, and SDP at harvest in "Buckeye Gala" apple (Table 2). Nevertheless, application of ACC between 150 to 350 mg·L −1 had a linear dose response in fruit set and reduced fruit set by 19% to 34% (Table 1). Return bloom of "Buckeye Gala" in 2014 was inversely proportional to the concentrations of ACC applied in the 2013 season, so that trees receiving 350 mg·L −1 had the highest return bloom and those with 150 mg·L −1 had the least (Table 1).

Results and
"Buckeye Gala" trees of Untreated Control and those receiving Ethrel at 300 mg·L −1 in 2013 had similar levels of return bloom and these levels were significantly lower than trees receiving 250 mg·L −1 and 360 mg·L −1 ACC in 2014 (Table   1). This observation suggests that contrary to other post bloom thinners that need to be applied shortly after petal fall to assure return bloom, late application of ACC effectively thins the current seasons' fruit without adversely affecting next year's fruit induction in "Buckeye Gala" apple. Our fruit set results in "Buckeye Gala" apple are in general agreement with a study with ACC in "Golden Delicious" apple by Shupp et al. [15] who found a linear relation between rates of ACC applied at 300 -500 mg·L -1 and level of   [14] generally agrees with our results in "Buckeye Gala" apples. Application of ACC at 250 mg·L −1 or higher, increased fruit weight, diameter and length, and application of ACC at 150 mg·L −1 significantly increased fruit L/D ratio as compared to Untreated Control when fruitlets were sampled 34 days after application ( Table 1). Application of ACC at all rates reduced total yield per tree in "Buckeye Gala" ( Table 2). Our result is in agreement with McArtney [14] who found that ACC at a rate of 200 mg·L −1 to be an optimum rage for late fruitlet thinning when fruitlet size was at about 10 mm in "Gol-dRush" apple. Our report in "Buckeye Gala" is also consistent with McArtney and Obermiller's finding in "Gale Gala" apple [13].
Starch degradation pattern increased with every incremental increase in the ACC rate, although differences were not always significant. This effect is indirectly related to crop load, as reduced crop load on ACC-treated trees resulted in advanced maturity. Since we harvested trees of all treatments on the same day, fruit from trees thinned with ACC had advanced maturity. Even if all of the trees were hand thinned to a similar crop load, the fact that ACC reduced crop load much earlier compared to hand thinning could result in advanced fruit maturity. Application of ACC only at the rate of 350 mg·L −1 significantly increased fruit size, color, and SDP as compared to Untreated Control in "Buckeye Gala" apple in 2013 (Table 2).
Based on our research, although ACC at the range of 150 to 350 mg·L −1 effectively thinned "Buckeye Gala" apple, application of ACC at 350 mg·L −1 provided sufficient thinning so that fruitlet size between application time and 34 days later was higher (Table 1) and fruit size at harvest ( Table 2) was larger (heavier) than those from trees of Un-treated Control (Table 1 and Table 2). With an everincreasing cost of hand thinning and labor availability challenges, application of ACC at 350 mg·L −1 may be advisable for "Buckeye Gala" apple.

"Sun Fuji" Apple Experiment in 2013 and Return Bloom in 2014 in Sunny Slope
Application of ACC at 300 mg·L −1 significantly reduced fruit set in "Sun Fuji" apple in Sunny Slope in 2013 as compared to Untreated Control ( Table 3). Spray of ACC at 150 mg·L −1 tended to reduce fruit set as compared to Untreated Control but the difference was not significant (Table 3). Return bloom of "Sun Fuji" apples trees receiving ACC tended to be higher than that of Untreated Control, but differences were not significant (Table 3). Abbreviations: Bcm 2 = branch cross sectional area in cm 2 ; return bloom was calculated as the number of half-opened mixed buds/branch cross sectional area in cm 2 ; fruit color rating ranged from 1= up to 20% of fruit skin was red, progressively to 5 = the entire skin was red; SSC = soluble solids concentration in degree Brix; SDP = starch degradation pattern, ranging from 1 = very starchy to 6 = starch mostly hydrolyzed; Un. Control = Untreated Control. y Mean separation within columns by LSD at 5% levels.
Applications of ACC at 150 mg·L −1 or 300 mg·L −1 did not affect yield or quality attributes in 2013. The reason could be that trees in all treatments, including in Untreated Control, were hand-thinned to establish a 15-cm spacing, if fruits were not already at that spacing after chemical thinning.

"Top Export Fuji" Apple Experiment in 2013 and Return Bloom in 2014
Application of ACC reduced fruit set and increased fruit size in "Top Export Fuji", although differences were not always significant ( Table 4). The fruit set reduction in "Top Export Fuji" had a linear relation with the rate of applied ACC (Table 4). This observation is in agreement with Schupp et al. [15] who applied ACC at concentrations of 0, 100, 300, or 500 mg·L −1 to "Golden Delicious"/ Bud.9 apple trees at 20 mm fruit diameter and found a linear dose relationship between concentration of ACC and fruit thinning.
Reduction of fruit set between trees of Untreated Control and those receiving 150 mg·L −1 ACC, and between those with 300 mg·L −1 and 450 mg·L −1 were not significant in "Top Export Fuji" apple, but application at or greater than 300 mg·L −1 ACC significantly reduced fruit set (Table 4). ACC applications in 2013 did not affect the return bloom of trees in 2014 in "Top Export Fuji" apple (Table 4), which is another excellent feature of late ACC application in "Fuji" apples.
Other than the case of fruit weight, applications of ACC did not affect yield or fruit quality attributes. The reason could be that trees in all treatments were hand-thinned at the time of hand thinning.

"Aztec Fuji" Apple Experiment in 2014
Application of ACC at 300 mg·L −1 or greater significantly reduced fruit set in "Aztec Fuji" apple and the reduction was proportional to the dose of applied (Table 5).

General Discussion and Comments about the Study
General limitations in this study is the presence of a number of factors that could E. Fallahi, S. J. McArtney   Figure 1). We also found that ACC at the rate of 300 mg·L −1 was an effective rate for post-harvest thinning in all three strains of "Fuji" ("Sun Fuji", "Top Export Fuji" in 2013 and "Aztec Fuji" in 2014). Schupp at al. [15] reported that the impact of ACC on apple fruitlet thinning was consistent from year to year, while Jones and Koen [20] noted that the impact of Ethrel on fruit thinning varied between seasons, depending on the temperature. Relative independency of seasonal temperature effect is an outstanding feather for ACC.
Modern apple growers over the past three decades have been spraying post bloom thinners when fruit let diameter is between 5 mm (shortly after petal fall) Registration has been set for ACC and will be marketed as a naturally accruing compound, marketed under the brand name Accede, utilizing the active ingredient 1-aminocy clopropane-1-carboxylic acid [17].
Late application of ACC will have the following advantages to an apple grower: 1) The risk a freeze event is minimized at a later time in the growing season when fruit-lets are at 20 mm.
2) Natural fruit abortion is less, and growers will have a better idea about their final crop, and thus can make a better hand thinning strategy.
3) As we noticed in our study, the size of fruit in trees receiving ACC at higher rates were already larger than those of Untreated Control at about the handthinning time, often resulting in larger fruit size at harvest.

4)
In general, ACC gives growers a tool to have better predictability about their final crop load and fruit quality.

Conclusion
Based on the results of this study, the effective range of ACC for late post bloom thinning was between 150 and 350 mg·L −1 "in Buckeye Gala" and between 300 and 450 mg·L −1 in different strains of "Fuji" apples. The exact optimum rate of ACC within these ranges can be decided after crop load, purpose or objective of fruit production, weather, tree vigor, and stress conditions are taken into account.