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Article

Inadequate Pollination Is a Key Factor Determining Low Fruit-to-Flower Ratios in Avocado

by
María L. Alcaraz
and
Jose I. Hormaza
*
IHSM La Mayora-CSIC-UMA, Algarrobo-Costa, 29750 Málaga, Spain
*
Author to whom correspondence should be addressed.
Horticulturae 2024, 10(2), 140; https://doi.org/10.3390/horticulturae10020140
Submission received: 19 December 2023 / Revised: 23 January 2024 / Accepted: 29 January 2024 / Published: 31 January 2024
(This article belongs to the Section Fruit Production Systems)
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Abstract

:
Avocado is an evergreen tree that exhibits protogynous dichogamy and displays a massive flower production, characterized by a high abscission of unfertilized flowers and developing fruitlets, ultimately leading to a low final fruit set. A common practice to improve avocado pollination involves introducing honey bee (Apis mellifera L.) hives during the flowering season. To evaluate the extent of inadequate pollination in avocado, the effect of different beehive densities on the percentage of flowers receiving pollen during the female flower stage was examined for seven years in an experimental orchard located in Southern Spain. A total of 17,288 flowers were observed under the microscope for this purpose. Additionally, pollen deposition was evaluated in five “Hass” avocado commercial orchards under different management strategies situated in the Malaga province (3960 flowers). The results obtained reveal that the percentage of flowers with pollen at the end of the female stage ranged from 2.85 (0.13% fruits retained at the end of June) in orchards without honey bee hives to 4.34 (0.11% fruits retained) in orchards in which 10 beehives per ha were placed. This percentage increased significantly to 13.79 after introducing 24 honey beehives per ha (0.21% fruits retained). Furthermore, the percentage of pollinated flowers in the commercial orchards remained below 15% even in those orchards in which pollen donors and honey bee hives were present. Thus, insufficient pollination could be considered as a primary limiting factor in avocado production under Mediterranean climates. Enhancing pollinator diversity and increasing their numbers could be a viable strategy to improve the percentage of avocado flowers receiving pollen during the female stage.

1. Introduction

The avocado (Persea americana Mill.) is a subtropical evergreen fruit tree crop that originated in Mesoamerica. Avocado belongs to the family Lauraceae in the order Laurales, which, along with the Canellales, Magnoliales, and Piperales, forms the Magnoliid complex, sister to the eudicot and monocot clades of angiosperms [1]. In recent years, avocado production and market demand have experienced exponential growth worldwide, reaching global production close to nine million tons in 2022 [2]. While avocado is cultivated in more than 60 countries worldwide, most of the production is concentrated in a few countries (Mexico, Dominican Republic, Peru, Indonesia, Colombia, and Brazil). Mexico stands out as the largest producer, with over 2.5 million tons in 2022, accounting for nearly 30% of the total world production [2]. Spain represents a particular case in avocado production since it accounts for more than 90% of the avocado production in Europe, of approximately 105,000 tons in 2022 [2]. Most of the avocado global market is dominated by the variety “Hass” [3], which originated from a chance seedling in California (USA) nearly 100 years ago.
Avocado is characterized by a synchronized dichogamy with two types of genotypes exhibiting reciprocal flowering behavior [4]. Consequently, each avocado flower opens and closes twice during two consecutive days for several hours each time. During the first day of the cycle, the avocado flower opens functionally as female, whereas during the second day it opens functionally as male (dehiscence of anthers). Avocado cultivars are classified into two different groups, A and B, based on their floral behavior. In type A cultivars, flowers open in the morning functionally as females, close at midday, and reopen the next day in the afternoon functionally as males. On the other hand, flowers of type B cultivars open in the afternoon as females, close overnight, and reopen the following morning functionally as males [5,6]. This floral behavior is highly dependent on temperature and, consequently, the cycle can vary under warmer or colder conditions [7,8]. In addition, studies performed in Mediterranean climates show that pollination during the male stage of the flower cycle does not result in fertilization and, consequently, pollination is only effective during the female stage [9]. Therefore, the flowering behavior of avocado promotes outcrossing, since close-pollination between flowers of the same tree or different trees of the same cultivar can only occur during the limited hours when closing female flowers and opening male flowers coincide on the same inflorescence, or in other inflorescences of the same tree or of nearby trees of the same variety. The duration of this overlap between the two floral stages is highly dependent on environmental conditions, mainly temperature [8].
In avocado, only a very small fraction of the flowers produced (less than 1%) is able to develop into fruits, primarily due to a massive drop of flowers and developing fruitlets mainly occurring during the two months following flowering [10,11,12]. Several factors, including inadequate pollination [9], extreme high or low temperatures [13,14,15], the absence of or an insufficient number of pollinizer trees [16,17,18], and alternate bearing [19,20] have been suggested as non-mutually exclusive contributors to the low avocado fruit set. In addition, results obtained in different plant species, including avocado, suggest that the events that occur between pollination and early fruit set depend on the flower quality at anthesis [21,22,23,24,25,26,27], underscoring the importance of factors inherent to the pistil in the premature abscission of avocado flowers [27,28,29].
Nevertheless, the initial and crucial step to ensure fruit production is adequate pollination. Previous findings in this species indicate that the deposition of a high number of pollen grains is necessary to achieve fertilization [9]. Inadequate pollination may result from a deficit of pollinating insects and/or limited pollen availability in the orchard. In its region of origin in Central America, avocado flowers are visited by different native insect species including bees, wasps, and flies [30,31,32,33]. However, as in most crops, avocado commercial production worldwide relies on the availability of honey bees (Apis mellifera), which were not present in the Americas until the arrival of Europeans [34], and the introduction of beehives during the flowering season is recommended in most avocado-producing regions [35,36,37,38,39]. However, the effectiveness of honey bees for avocado pollination is not entirely clear due to irregular visits, as honey bees often show a preference for flowers of other species over avocado flowers [36,38,40,41,42,43]. In addition to honey bees, a diverse range of pollinating insects is commonly observed visiting avocado flowers worldwide, suggesting their potential significant role in avocado pollination [34].
In order to understand the main limiting factors affecting avocado pollination in a Mediterranean climate, three different objectives were analyzed in this work. (1) Estimate the percentage of female-stage flowers of the cv. Hass receiving pollen during the flowering season. (2) Evaluate the impact of increasing honey bee hives on pollen deposition and fruit set in avocado cv. Hass. (3) Evaluate pollen deposition in a “Hass” orchard where cultivars of different floral groups were planted.

2. Materials and Methods

2.1. Pollen Deposition in a “Hass” Avocado Orchard with a Different Number of Beehives

The experiments were carried out in a 1 ha experimental avocado orchard located at the IHSM La Mayora (Málaga, Spain) at latitude 36°45′ N, longitude 4°4′ W, and 35 m altitude above sea level. The trees were 40 years old from the cultivar Hass (floral type A) grafted on “Topa-Topa” seedling rootstocks. Tree spacing was 8 m between rows and 8 m within rows. Under our environmental conditions, “Hass” flowers in March/April and most of the fruits are harvested from November to April.
To assess the impact of beehive density on the percentage of flowers receiving pollen during the female stage, three different situations were studied using the same trees in the same orchard across different years: (1) absence of beehives in the orchard (2021 flowering season); (2) introduction of 10 beehives in the orchard (2013 and 2020 flowering seasons); (3) introduction of 24 beehives in the orchard (2014, 2015, 2016, and 2019 flowering seasons). In the latter two treatments, beehives were introduced along the south side of the orchard at the beginning of the flowering season when approximately 20% of the flowers were open. The beehives were positioned perpendicularly to the first tree row arranged in one or two lines (when 24 beehives were introduced) side by side. Each beehive contained approximately 50,000 bees and the colony strength was monitored weekly throughout the flowering season.
To evaluate pollen deposition on the stigmas during the female stage, flowers at the beginning of their male-stage opening cycle, when anthers were still undehisced, were randomly collected from several inflorescences of different trees in the orchard. Thus, each year, at the beginning of the flowering season, 40 different trees exhibiting similar physiological conditions and comparable flowering intensity, placed in 13 rows (from 3 to 4 trees in each row) at increasing distance from the beehives (the distance between rows was 8 m), were selected and labeled. Each tree in the orchard was identified by a numbered metal label on the trunk. For ease of identification during sample collection, each tree was also labeled with a colored label on one branch. From each of those labeled trees, a minimum of fifteen flowers opening at the male stage were randomly collected. Sampling was conducted at least two or three times per week over the approximately four weeks of the flowering season from at least five different panicles placed on the north and south sides of the tree. The flowers were fixed in formalin-aceto-alcohol (FAA) [44]. The flowers fixed from each tree were identified with a label including the number of the tree and collection date.
Fixed pistils were washed in water three times, 1 h per wash, autoclaved for 10 min at 1 kg/cm2 in 5% Na2SO3, squashed between the slide and the coverslip, and stained with 0.1% aniline blue in 0.1 N K3PO4. Preparations were examined under a Leica DM LB2 microscope with UV epifluorescence using a BP 515–560 exciter filter and a LP 590 barrier filter. During the fixation process, most ungerminated pollen grains would be washed away, leaving only those that adhered and germinated present at the stigma. Consequently, although total pollen deposition could be underestimated as non-adhered pollen grains deposited in the female phase were not counted, only those pollen grains deposited during the female stage with capacity to germinate are relevant for reproductive success. Pollen adhesion and germination were evaluated and the number of pollen tubes was counted at different levels of the pistil.
With the aim of determining the total percentage of flowers receiving pollen throughout the entire “Hass” avocado flowering period and investigating a potential impact of temperature variation on pollen deposition, flowers were systematically collected over the entire flowering season. Temperature and relative humidity were monitored in the field at hourly intervals using a HOBO Pro v2 (ONSET Computer Corporation, Bourne, MA, USA) data logger.
In the 2021 flowering season, the study of pollen deposition was carried out without introducing honey bee hives into the orchard. To estimate the percentage of flowers that develop into fruits and remain on the trees at harvest, 64 inflorescences were labeled, and the initial number of flowers was counted and flower/fruit drop was monitored until harvest time.
Initial fruit set was calculated as the number of fruitlets remaining on the trees at the end of June (approximately two months after the end of the flowering period), divided by the total number of flowers produced in each inflorescence × 100. The final fruit set is the number of fruits remaining on the trees at harvest time (approximately 40 weeks after the end of the flowering season) divided by the total number of flowers produced in each inflorescence × 100. The main period of flower/fruit drop occurs during the two months following the end of the flowering season due to a high abscission of, initially, unfertilized flowers and, several weeks later, developing fruitlets.
In those years in which 10 beehives were placed in the orchard, a total of 6084 flowers were processed and visualized under the microscope (2896 collected in 2013 and 3188 in 2020). Moreover, 31 and 61 inflorescences, respectively, were labeled each year. The number of flowers in these inflorescences was recorded and the numbers of flowers and fruits retained by the trees were monitored from the end of the flowering season until fruit maturity to estimate the final fruit set.
Finally, in those years in which 24 beehives were introduced in the orchard, a total of 3115 (2014), 1368 (2015), 2066 (2016), and 2293 (2019) pistils were examined under the microscope. Additionally, during these years, 110, 50, 60, and 72 inflorescences, respectively, were labeled and the initial number of flowers was recorded. To estimate fruit set, fruit drop was monitored monthly until harvest time.

2.2. Effect of Different Pollen Donors on Pollen Deposition and Paternity on Avocado cv. Hass

To evaluate the effect of pollen donors, during the 2016 flowering season, pollen deposition was analyzed in a 0.5 ha 40-year-old “Hass” avocado orchard grafted on “Topa Topa” seedling rootstock where a high density of pollen donors (40%) of small size were planted two years earlier. The genotypes introduced in the orchard included four type B commercial varieties (“Bacon”, “Ettinger”, “Fuerte”, and “Zutano”), three type B non-commercial accessions (“LMAL”, “LMMA”, and “LMNO”), and three type A commercial varieties (“Gem”, “Lamb Hass”, and “Maluma”). Twice a week, during the flowering season, 15 “Hass” flowers at the male stage were collected from 15 different trees and fixed in FAA for the evaluation of pollen deposition. Fruit drop was monitored during the month following the flowering season to estimate initial fruit set. Upon fruit maturity, 20 fruits were collected from each of 18 different “Hass” trees for paternity analyses as previously described [45]. In this plot, the number of fruits collected by tree ranged from 0 to 988 with an average of 307.07 ± 38 fruits/tree. Paternity analyses were carried out using six different microsatellite loci previously developed in avocado (Avag21, Avd001, Avd003, Avd006, Avd102, and AvMix 03) by Sharon et al. [46] and Ashworth et al. [47]. The combination of these microsatellites allowed us to unequivocally distinguish between the different pollen donors. Male parent assignment was conducted with the Cervus 3 program [48].

2.3. Pollen Limitation in Flowers of cv. Hass in Commercial Avocado Orchards

To evaluate pollen deposition in commercial avocado orchards, during the 2018 flowering season, five “Hass” orchards were studied. These orchards were chosen among those with the historically highest productivity in the main avocado-producing region in Spain. In each orchard, 12 to 15 trees were selected, and flowers at the beginning of the male phase were collected at least two times during the flowering season (at full bloom and when more than 70% of flowers had opened). The commercial orchards showed differences in management practices such as the presence of pollen donors and the introduction of beehives (in orchards using beehives, the density was 6 per ha). The characteristics of each orchard are described in Table 1.
Pollen deposition was evaluated and, when the fruits reached maturity, a minimum of ten fruits from the labeled trees were collected for paternity analysis. A total of 728 “Hass” fruits were collected and DNA was extracted from the embryo of the fruits following the method described in Section 2.3.

2.4. Statistical Analyses

The impact of introducing different numbers of beehives in the orchard was assessed by estimating the percentage of flowers with pollen and initial fruit retention. Comparisons between years of study were conducted using a chi-square test for 2 × 2 contingency tables, with Yate’s correction for continuity.
The effect of the distance between the sampled trees and the honey bee hives on the percentage of flowers receiving pollen was analyzed using the results obtained in the 2014, 2016, and 2019 flowering seasons. Each collection date for each of the trees analyzed in each row in the three years of study was considered a replicate (with the number of replicates per treatment ranging from 41 to 71). Data were analyzed using a univariate generalized linear model (Univariate-GLM), with the percentage of flowers receiving pollen as the dependent variable and the distance to honey bee hives as the explanatory variable. Flower percentages were arcsine transformed to meet the assumption of normality and homogeneity of variances. Furthermore, the percentage of flowers with pollen was compared among the four weeks of the flowering season (2018, 2019, 2020, and 2021) using a univariate generalized linear model with percentage of pollen as the dependent variable and time as the independent variable. Every collecting day of every tree sampled was considered a replicate for each week of the flowering period in the different years of study.
To compare the percentage of fruits derived from crosses with the different pollen donor genotypes in the orchard that combined “Hass” with different genotypes, a chi-square test for 2 × 2 contingency tables, with Yate’s correction for continuity, was used. Similarly, the percentage of flowers receiving pollen in this orchard was compared with the results obtained in the monovarietal “Hass” orchard using the Student’s t-test at the 0.05 significance level. Finally, in the commercial orchards, the percentage of flowers with pollen deposition during the female stage was compared among orchards using a GLM with the collection day for each tree considered as a replicate. Flower percentages were arcsine transformed to meet the assumption of normality and homogeneity of variances.
Statistical analyses were performed using SPSS 27.0 statistical software (SPSS Inc., Chicago, IL, USA).

3. Results

3.1. Pollen Deposition and Fruit Set with Different Numbers of Honey Bee Hives

The total number of samples analyzed and the main results related to the percentage of flowers receiving pollen and the percentage of fruits retained by the trees at the end of June are summarized in Table 2.
In 2021, a year in which no honey bee hives were introduced in the orchard, out of a total of 2279 flowers analyzed, only 2.9% showed at least one pollen grain on the stigma, with an average of 3.68 pollen grains per stigma in the pollinated flowers (considering only flowers with at least one pollen grain; see Figure 1). Additionally, from a total of 76,024 flowers, only 96 developed into fruits and were retained on the trees two months after the end of the flowering season (0.13%).
During the 2013 and 2020 flowering seasons, 10 beehives were introduced into the monovarietal “Hass” avocado orchard. In 2013, a total of 2979 pistils were examined under the microscope, and only 4.77% of the flowers showed pollen grains on the stigmas. In 2020, out of a total of 3188 pistils analyzed, only 3.92% of the flowers showed pollen grains. Considering only those flowers that received at least one pollen grain, the average number of pollen grains adhered to the stigma was 6.79 during the 2013 flowering season and 3.33 during the 2020 flowering season.
Regarding fruit set, in 2013, out of the 11,642 flowers counted, only 11 fruits were retained by the trees at the end of June (0.09%), and 8 fruits remained at harvest time (0.07%). In 2020, the initial number of flowers was 29,404, with 35 fruits retained at the end of June (0.12%) and 27 fruits at harvest time (0.09%).
No significant differences were observed in either the percentage of flowers receiving pollen at the female stage (χ2 = 2.66, p = 0.103) or in the initial (χ2 = 0.45, p = 0.503) and final (χ2 = 0.52, p = 0.470) fruit set between the two years of study.
During the 2014, 2015, 2016, and 2019 flowering seasons, 24 honey bee hives were introduced in the orchard. In 2014, a total of 3115 pistils collected were visualized under the microscope. The percentage of flowers that received pollen during the female stage was 13.68 (426 pistils), with an average of 13.8 pollen grains on the stigma (considering only flowers that received at least one pollen grain). Out of a total of 63,336 flowers, 200 fruits (0.32%) were retained on the trees at the end of June. In 2015, the percentage of flowers that received pollen during the female stage decreased to 6.65% (91 pistils from 1368) and the average number of pollen grains on the stigma was 6.99. The percentage of fruits retained at the end of June also decreased to 0.14% (from 23,506 flowers, 32 developed into fruits). In 2016, although the percentage of female flowers with pollen on their stigmas increased to 15.83% (327 of 2066), the initial fruit set was 0.11% (69 fruits from 61,337 flowers). In those flowers collected in the 2016 flowering season, the average number of pollen grains on the stigma was 7.37 (considering only those flowers that received at least one pollen grain). Finally, in 2019, 441 pistils out of 2293 (19%) collected during the flowering season received pollen during the female stage. In this year, the average number of pollen grains on the stigma was 5.63 and the initial fruit set was 0.28% (69 flowers from a total of 25,028 developed into fruits).
Significant differences in the percentage of female flowers receiving pollen during the female phase were found between 2014 and 2015 (χ2 = 45.96, d.f. = 1, p < 0.001), 2014 and 2016 (χ2 = 4.63, d.f. = 1, p = 0.031), 2015 and 2016 (χ2 = 64.81, d.f. = 1, p < 0.001), and also between 2019 and all the other years tested (p < 0.005). Regarding initial fruit set (at the end of June) significant differences were observed between 2014 and 2015 (χ2 = 20.76, d.f. = 1, p < 0.001), 2014 and 2016 (χ2 = 59.81, d.f. = 1, p < 0.001), 2015 and 2019 (χ2 = 11.368, d.f. = 1, p < 0.001), and 2016 and 2019 (χ2 = 29.675, d.f. = 1, p < 0.001).
No significant differences were found either in the percentage of female flowers receiving pollen (χ2 = 0.055, d.f. = 1, p = 0.814) or initial fruit set (χ2 = 0.444, d.f. = 1, p = 0.505) between the years when 10 beehives were placed in the orchard and the year without beehives. However, significant differences were observed in final fruit set (χ2 = 21.596, d.f. = 1, p < 0.001) and percentage of flowers with pollen (χ2 = 231.79, d.f. = 1, p < 0.001) between the year without beehives and the years when 24 beehives were introduced in the orchard.
To determine whether increasing the number of beehives from 10 to 24 affects the percentage of flowers receiving pollen and the initial fruit set, the results obtained in both treatments were compared. Thus, when the number of honey bees increased in the orchard, the percentage of female flowers with pollen grains on their stigmas increased significantly (χ2 = 398.08, d.f. = 1, p < 0.001). Likewise, the percentage of fruits retained two months after the flowering season was significantly higher when the number of honey bee hives in the orchard was increased (χ2 = 17.675, d.f. = 1, p < 0.001).
To evaluate the effect of the distance to the beehives on pollen deposition, the percentage of flowers with pollen on their stigmas collected from trees placed at 13 rows with increasing distance from the beehives during the flowering seasons of 2014, 2016, and 2019 was compared. No significant differences were observed based on the distance from the honey bee hives (F12,655 = 1.180, p = 0.294) (Figure 2).
On the other hand, differences were observed among weeks during the flowering season in the percentage of flowers receiving pollen during the four years evaluated: 2018 (F3,218 = 84.11, p < 0.001), 2019 (F3,234 = 19.27, p < 0.001), 2020 (F3,229 = 17.03, p < 0.001), and 2021 (F3,187 = 13.31, p < 0.001). In all years, the percentage of flowers receiving pollen during the female phase significantly increased at the end of the flowering season except in 2021, where the highest percentage was obtained not only in the last week but also in the first one. A general increase in the average of mean, maximum, and minimum temperatures over the flowering season was observed in the 4 years studied (Figure 3).

3.2. Pollen Deposition in a “Hass” Orchard Planted with Different Varieties of A and B Floral Groups

A common recommendation in different avocado-producing regions is to interplant varieties of complementary floral groups to increase yield. For this reason, the effect of introducing different pollen donors on the percentage of “Hass” flowers with pollen on their stigmas was evaluated. Out of a total of 553 pistils of “Hass:” collected and analyzed during the 2016 flowering season, 148 (27%) received pollen during the female stage, with an average of 5.35 pollen grains on the stigma, considering only those flowers that received at least one pollen grain.
These results were compared to those obtained in the monovarietal “Hass” orchard with high honey bee densities (Section 3.1) in the 2016 flowering season. Significant differences in the percentage of flowers receiving pollen during the female stage were found between both orchards (t = −2.57, d.f. = 31.71, p = 0.01). The percentage of flowers that received pollen in each orchard is shown in Figure 4.
Despite the small size of the trees used as pollen donors, a high percentage of the “Hass” fruits analyzed were derived from outcrossing. Out of a total of 180 “Hass” fruits tested for paternity, 56.11% derived from close-pollination (between flowers of the same tree or different trees of the same cultivar), while the rest of the fruits resulted from crossing with the other varieties interplanted in the orchard. These were mainly of floral type B but, in some cases, even with varieties of the same floral group (Figure 5).

3.3. Pollen Deposition in Commercial “Hass” Avocado Orchards

No previous works have analyzed the need and optimal percentage of pollen donors or honey bee hives to improve fruit set in avocado under the environmental conditions of Mediterranean Europe. For this reason, in this study, we evaluated the effect of different management strategies used in a commercial orchard under our environmental conditions (presence/absence of pollen donors, genotype, and introduction of honey bee hives) on the percentage of flowers receiving pollen during the female phase. The results obtained showed significant differences among orchards (F4,224 = 3.43, p = 0.01) in the mean percentage of flowers receiving pollen during the female phase, ranging from 6.6% in “Orchard B” to 14.3% in “Orchard A” (Figure 6a).
Considering the number of pollen grains on the stigmas of pistils collected during the male stage, only 3.61% of them received more than six pollen grains and only 0.7% of them received more than twenty pollen grains (only 24 flowers from 3251 analyzed). Figure 6b illustrates the percentage of flowers that received different pollen loads in the commercial orchards.
The percentage of flowers with pollen was significantly higher in the orchards with pollen donors and honey bee hives (Orchards A, B, and C) than in the orchards with neither pollen donors nor honey bee hives (Orchards D and E) (χ2 = 5.061 d.f. = 1, p = 0.024). Regarding only those flowers that received at least one pollen grain, the average number of pollen grains on the stigma ranged from 5.25 in orchard C to 3.1 in orchard B. The average number of pollen grains on the stigma of collected flowers was 5.04 in orchard E, 4.9 in orchard A, and 4.4 in orchard D.
Paternity analyses showed that the percentage of fruits derived from cross-pollination ranged from 29% in the monovarietal “Orchard D” to 63% in the mixed “Orchard C”. The paternity of fruits collected from the different orchards is shown in Figure 7. Even in those orchards without pollinizers (Orchards D and E), some fruits derived from cross-pollination were observed probably due to the presence of pollinizer varieties in neighboring orchards. In these two orchards, the percentage of “selfed” fruits (derived from close-pollination between flowers of the same tree or different trees of the same cultivar) was 71 and 59, whereas in those orchards with pollinizers, the percentage of fruit derived from close-pollination with pollen from the same variety ranged from 37 to 65. Among the mixed orchards, Orchard A, in which the cultivar Fuerte was used as the pollen donor, showed the highest percentage of “Hass” fruits derived from cross-pollination between flowers of the same tree or other “Hass” trees. Moreover, in this orchard, most of the “Hass” fruits derived from outcrossing came from crosses with “Bacon”, another B type variety not present in this orchard.
In all the orchards, the percentage of flowers that received pollen varied significantly among the different collection dates (F2,223 = 42.29, p < 0.001), showing an increase at the end of the flowering season except in one of them (Orchard E). At the end of the flowering season, the percentage of female flowers with pollen ranged from 6 (Orchard E) to 30.1 (Orchard A). The percentage of flowers with pollen in the commercial orchards is shown in Figure 8, and the statistical comparison between the different collection dates within each orchard is presented in Table 3.

4. Discussion

A common observation worldwide is that avocado trees produce a large number of flowers, yet only a small fraction of them develop into fruits. While several factors contribute to this phenomenon, the observation that the percentage of fruit set increases significantly when manual pollen application is carried out [49] suggests that pollen limitation at the stigma could be one of the main limiting factors for avocado fruit production.
While it is possible that the number of pollen grains deposited on the stigmas might be underestimated due to some of the pollen grains being washed away from the stigma during fixation and staining, the results obtained in this work show that a high percentage of flowers close at the end of the female stage without pollen grains on their stigmas. This occurs even when honey bee hives are introduced in the avocado orchards. As a result, the number of flowers that could develop into fruits is low, resulting in a very low avocado fruit production [9]. Several non-exclusive factors could explain this high percentage of flowers ending their female phase without pollen on their stigmas. One of the reasons could be insect deficit, due to a low number of insects or to unfavorable environmental conditions at flowering time for pollinating insects to efficiently transport the pollen from the anthers of male-stage flowers to the stigmas of female-stage flowers. Another potential reason might be pollen limitation in the field, due to the protogynous dichogamy that characterizes avocado, which, under some environmental conditions, requires the presence of complementary floral type pollinizers in the orchard [16,18].

4.1. The Effect of the Number of Honey Bee Hives in Avocado Pollination

To improve avocado pollination, a common practice in most avocado-producing countries is to introduce honey bee hives in the orchards at the beginning of the flowering season [30,34]. However, our results suggest that honey bees may not be the most efficient pollinators for avocado, as even with a substantial number of honey bee hives, the percentage of flowers receiving pollen during the female stage remains very low. In fact, avocado is native to Central America, where it coevolved with different species of local pollinating insects, particularly stingless bees of the genus Melipona, which play a crucial role in avocado pollination in its center or origin but are absent outside the Americas. The introduction of honey bees in the Americas occurred after the arrival of Europeans in 1492.
Honey bees show a low preference for avocado flowers and they show higher attraction to more fragrant flowers [39,50]. This has been linked to a low appeal of avocado nectar to honey bees due to its high mineral content and the presence of the sugar alcohol perseitol, which has repellent properties [51,52]. Honey bees typically visit avocado flowers to collect pollen or nectar, and bees collecting nectar have been suggested as being more efficient for avocado pollination [32,38,39,50,53] because they visit flowers during both the female and the male stages, facilitating pollen transfer from the anthers of male-stage flowers to the stigmas of female-stage flowers. In monovarietal avocado orchards under Mediterranean climate conditions, this pollen transfer can only occur during the brief overlap period between closing functionally female flowers and opening functionally male flowers. Thus, close-pollination (geitonogamy) (between flowers of the same tree or of different trees of the same cultivar) is the main process explaining fruit production in monovarietal avocado orchards. However, in orchards where varieties of complementary floral groups are interplanted, the period for pollen deposition on the stigma of female flowers is usually longer than that in monovarietal orchards. In addition, in some particular cases, mainly under subtropical humid conditions, efficient self-pollination or close-pollination during the male opening phase has been suggested [54], although studies in Mediterranean climates show that no fertilization occurs when flowers are pollinated during the male stage [9].
One possible strategy to enhance the number of flowers receiving pollen during the female phase and, consequently, improve fruit set, might be to increase the number of insects visiting flowers in the field. The results obtained in this work show that an increase in the number of honey bees present in avocado orchards during the flowering season leads to an increase in the percentage of flowers receiving pollen during the female stage, as well as in the number of pollen grains per stigma and, consequently, in the percentage of fruit set. Our results agree with those obtained in Israel, where pollination rates in different avocado cultivars were found to be correlated with the number of honey bees visiting the trees [55].
On the other hand, the capacity of mobility of the pollinators plays an important role in pollen transfer. Previous results in avocado in Israel suggested that honey bees predominantly carry pollen grains to the trees located in close proximity to the pollen source, with considerably reduced pollen transfer observed at increasing distances from the pollen source [56]. However, in this work, no differences were found in either pollen deposition or on fruit set in trees placed at different distances from the hives (up to 104 m). Our results coincide with those obtained in Colombia [53] where no differences on pollen deposition in trees placed up to 150 m from the honey bee hives were found. Based on our results, the beehives could be dispersed in the orchard at distances up to 100 m without affecting pollen deposition or fruit set. Moreover, the mobility of the honey bees is demonstrated in the year in which no bee hives were placed in the orchard, since honey bees were still the most common avocado flower visitors. These results are consistent with those obtained in the commercial orchards where neither pollen donors nor honey bee hives were introduced, and an important percentage of fruits derived from cross-pollination were observed.
In addition, honey bee activity considerably decreases with lower temperatures (below 12–14 °C) and solar radiation (under 500 lux) [30,57,58,59]. Temperature fluctuations under our environmental conditions are frequent during the avocado flowering season, mainly at its beginning. In this context, Monzón et al. [60] reported that the frequency of bees visiting avocado flowers is more closely associated with temperature than with the number of open flowers, with the highest frequency of visits recorded at higher temperatures. These findings support our observation of a higher proportion of flowers receiving pollen towards the end of the flowering season, when temperatures are usually higher, and also explain the preference of honey bees for flowers located in areas of the trees more exposed to sunlight.
Honey bees are important pollinators in many crop systems. However, due to the potential risks associated with depending only on a single pollinating species, it is essential to explore alternative taxa that can provide additional crop pollination and potentially be more effective than honey bees for avocado pollination. Moreover, while increasing the number of honey bee hives in the orchard contributes to increasing the percentage of flowers receiving pollen, this percentage remains very low, suggesting that honey bees might not be the most effective pollinators for avocado and, consequently, that the pollination effectiveness could be increased considerably by introducing additional pollinators in the orchard [34,61]. Studies in Israel have shown that introducing bumblebee (Bombus terrestris L.) hives together with honey bee hives results in a significant increase in avocado yield [61,62]. Implementing diverse management practices that increase pollinator diversity could include reducing pesticide use, maintaining an appropriate soil vegetable cover to provide additional floral resources for different pollinators, or creating nesting opportunities through the introduction of artificial nesting sites.
Additional studies are needed to thoroughly evaluate the impact of beehive density and environmental conditions, not only on honey bee activity but also on the effectiveness of flower visits in avocado pollination. Similarly, additional studies in different locations are essential to understand the diversity and efficiency of different insects in avocado pollination and, therefore, in avocado production.

4.2. The Effect of Pollinizers on Avocado Pollination

Another factor that could explain the low percentage of flowers receiving pollen at the end of the female phase in avocado is limited pollen availability. The results obtained in this work show that the percentage of “Hass” flowers with pollen was higher in the orchard with pollen donors, although previous works under the same climatic conditions have not found a clear correlation between the presence of “Fuerte” as pollinizer and increased final fruit set [45]. This suggests that, in monovarietal orchards under our environmental conditions, most of the fertilization events occur when the pollen is transferred during the time of overlap between the sexual stages. Temperature might affect not only the duration of each sexual stage but also the overlap between both sexual stages, influencing avocado production. In all the orchards, even in those in which no beehives were introduced, honey bees were observed visiting the flowers at the time of flower collection. These bees probably flew from neighboring orchards where honey bee hives were present.
Furthermore, the results obtained in commercial “Hass” avocado orchards showed that the percentage of flowers that received pollen was also higher in those orchards in which pollen donors were present. In addition, the pollen donor genotype plays an important role, since the cultivar Bacon was more represented in the outcrossing progeny, even in a commercial orchard where the only available pollinizer was “Fuerte”. Previous results have shown that “Fuerte” is not an effective pollinizer for “Hass” under our climatic conditions, mainly due to a limited overlap period in their flowering seasons [8].

5. Conclusions

The results of this work highlighted that pollination is one of the main factors limiting avocado production in the Mediterranean climate of Southern Spain, the most important avocado-producing region in Europe. The number of pollen grains adhering to the stigma and the percentage of flowers with pollen during the female stage varies not only across years but also throughout the flowering season. This variability is probably influenced by the fact that both floral behavior and insect activity are affected by environmental conditions. Different strategies could be adopted to increase the number of flowers receiving pollen and the number of pollen grains per stigma. Future research should focus on improving the pollination process by looking at and analyzing honey bee activity and how this is affected by different environmental conditions, together with the contribution and efficiency of pollen deposition of different insect taxa that are frequently observed visiting avocado flowers during the flowering season.

Author Contributions

Conceptualization, J.I.H. and M.L.A.; Formal analysis, M.L.A.; Methodology, J.I.H. and M.L.A.; Resources, J.I.H.; Supervision, J.I.H.; Validation, J.I.H. and M.L.A.; Writing—original draft, J.I.H. and M.L.A.; Writing—review and editing, J.I.H. and M.L.A. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the project PID2022-141851OB-I00 funded by MCIN/AEI and ERDF A way to make Europe (MCIN/AEI/10.13039/501100011033).

Data Availability Statement

Data are contained within the article.

Acknowledgments

The authors would like to thank Sonia Cívico, Yolanda Verdún, and Antonio Mendez for technical assistance, and also SAT TROPS for their cooperation in the studies in the commercial avocado orchards.

Conflicts of Interest

The authors declare no conflicts of interest.

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Figure 1. Stigmas and upper part of the style from avocado pistils collected in the field when the flowers started to open at the male phase. (a) Stigma with some pollen grains adhered and germinated. (b) Stigma that did not receive any pollen grains. Stain: aniline blue. Scale bars = 500 µm.
Figure 1. Stigmas and upper part of the style from avocado pistils collected in the field when the flowers started to open at the male phase. (a) Stigma with some pollen grains adhered and germinated. (b) Stigma that did not receive any pollen grains. Stain: aniline blue. Scale bars = 500 µm.
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Figure 2. Percentage of flowers of ”Hass” avocado receiving pollen during the female phase of the flowering cycle at different distances from honey bee hives (Rows). Each row represents 8 m. Data represent the means + SE. No significant differences were found among rows (GLM, p > 0.05).
Figure 2. Percentage of flowers of ”Hass” avocado receiving pollen during the female phase of the flowering cycle at different distances from honey bee hives (Rows). Each row represents 8 m. Data represent the means + SE. No significant differences were found among rows (GLM, p > 0.05).
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Figure 3. Mean + SE of mean, minimum, and maximum temperatures over the flowering period in those years in which variation in pollen deposition during the four weeks of the flowering period was evaluated (2018–2021).
Figure 3. Mean + SE of mean, minimum, and maximum temperatures over the flowering period in those years in which variation in pollen deposition during the four weeks of the flowering period was evaluated (2018–2021).
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Figure 4. Percentage of flowers receiving pollen during the female phase in monovarietal and mixed avocado orchards. Data represent the mean + SE. Means with different letters indicate the presence of significant differences (Student’s t-test p < 0.05).
Figure 4. Percentage of flowers receiving pollen during the female phase in monovarietal and mixed avocado orchards. Data represent the mean + SE. Means with different letters indicate the presence of significant differences (Student’s t-test p < 0.05).
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Figure 5. Percentage of “Hass” fruits derived from crossing with the different pollen donors present in the orchard. The letter in parentheses indicates the floral group. Means with different letters indicate the presence of significant differences (chi-square test, p < 0.05).
Figure 5. Percentage of “Hass” fruits derived from crossing with the different pollen donors present in the orchard. The letter in parentheses indicates the floral group. Means with different letters indicate the presence of significant differences (chi-square test, p < 0.05).
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Figure 6. Percentage of flowers receiving pollen grains in their stigmas and pollen load sizes. (a) Percentage of flowers with pollen in their stigmas in five “Hass” avocado commercial orchards. Means (+SE) followed by different letters indicate that they are significantly different (GLM analyses followed by Tukey’s HSD test, p < 0.05). (b) Histogram showing the percentage of avocado flowers in each range of pollen load sizes in each of the five “Hass” avocado commercial orchards evaluated in this study.
Figure 6. Percentage of flowers receiving pollen grains in their stigmas and pollen load sizes. (a) Percentage of flowers with pollen in their stigmas in five “Hass” avocado commercial orchards. Means (+SE) followed by different letters indicate that they are significantly different (GLM analyses followed by Tukey’s HSD test, p < 0.05). (b) Histogram showing the percentage of avocado flowers in each range of pollen load sizes in each of the five “Hass” avocado commercial orchards evaluated in this study.
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Figure 7. Percentage of cross-fertilization with different pollen donors and close-pollinated “Hass” fruits in five avocado commercial orchards. Fruits derived from fertilization between flowers of the same tree or different trees of the same cultivar are included in close pollinated group. Means of percentage of cross-fertilization (+SE) followed by different letters indicate that they are significantly different among orchards (GLM analyses followed by Tukey’s HSD test, p < 0.05). H = “Hass”, B = “Bacon”, F = “Fuerte”.
Figure 7. Percentage of cross-fertilization with different pollen donors and close-pollinated “Hass” fruits in five avocado commercial orchards. Fruits derived from fertilization between flowers of the same tree or different trees of the same cultivar are included in close pollinated group. Means of percentage of cross-fertilization (+SE) followed by different letters indicate that they are significantly different among orchards (GLM analyses followed by Tukey’s HSD test, p < 0.05). H = “Hass”, B = “Bacon”, F = “Fuerte”.
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Figure 8. Percentage of flowers with pollen at the different periods of collection in five “Hass” avocado commercial orchards. Bars (mean + SE) with different letters show significant differences at p < 0.05 (GLM).
Figure 8. Percentage of flowers with pollen at the different periods of collection in five “Hass” avocado commercial orchards. Bars (mean + SE) with different letters show significant differences at p < 0.05 (GLM).
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Table 1. Description of the different “Hass” avocado commercial orchards where pollen deposition was evaluated during the 2018 flowering season. In those orchards using beehives, 6 beehives/ha were introduced at the beginning of the flowering season.
Table 1. Description of the different “Hass” avocado commercial orchards where pollen deposition was evaluated during the 2018 flowering season. In those orchards using beehives, 6 beehives/ha were introduced at the beginning of the flowering season.
OrchardPollen DonorsBeehivesYield (Kg/ha)
Orchard_A“Fuerte”Presence11.64
Orchard_B“Bacon” and “Fuerte”Presence7.67
Orchard_C“Bacon”Presence14.74
Orchard_DAbsenceAbsence9.90
Orchard_EAbsenceAbsence15.96
Table 2. Number of pistils analyzed under the microscope, percentage of flowers receiving pollen, and percentage of fruits retained by the trees at the end of June for each treatment with different beehive numbers in different years.
Table 2. Number of pistils analyzed under the microscope, percentage of flowers receiving pollen, and percentage of fruits retained by the trees at the end of June for each treatment with different beehive numbers in different years.
TreatmentYearNo. Flowers Analyzed% Flowers with PollenInitial Number of Flowers% Fruits at the End of June
0 Beehives202122792.8576,0240.13
10 Beehives201329794.7711,6420.09
10 Beehives202031883.9229,4040.12
24 Beehives2014311513.6863,3360.32
24 Beehives201513686.6523,5060.14
24 Beehives2016206615.8361,3370.11
24 Beehives201922931925,0280.28
Table 3. Percentage of flowers receiving pollen during the female stages in five different avocado commercial orchards. Comparison between collection days was conducted in Orchards A, B, and C using a GLM followed by Tukey’s HSD test, and comparison between dates in Orchard D and E was determined using a Student’s t-test. X indicates that samples were not collected on this date.
Table 3. Percentage of flowers receiving pollen during the female stages in five different avocado commercial orchards. Comparison between collection days was conducted in Orchards A, B, and C using a GLM followed by Tukey’s HSD test, and comparison between dates in Orchard D and E was determined using a Student’s t-test. X indicates that samples were not collected on this date.
Orchard2nd Week3rd Week4th WeekComparison between Dates
A4.89 ± 2.297.91 ± 2.7130.08 ± 4.66F2,35 = 17.89, p < 0.001
B2.11 ± 0.644.08 ± 0.8213.45 ± 2.38F2,223 = 42.29, p < 0.001
C1.91 ± 0.943.91 ± 1.5725.06 ± 4.26F2,43 = 24.75, p < 0.001
DX1.84 ± 1.0713.65 ± 3.35T student = −3.355, d.f. = 13.221, p = 0.005
EX4.96 ± 1.796.04 ± 2.22T student = −0.379, d.f. = 22, p = 0.709
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Alcaraz, M.L.; Hormaza, J.I. Inadequate Pollination Is a Key Factor Determining Low Fruit-to-Flower Ratios in Avocado. Horticulturae 2024, 10, 140. https://doi.org/10.3390/horticulturae10020140

AMA Style

Alcaraz ML, Hormaza JI. Inadequate Pollination Is a Key Factor Determining Low Fruit-to-Flower Ratios in Avocado. Horticulturae. 2024; 10(2):140. https://doi.org/10.3390/horticulturae10020140

Chicago/Turabian Style

Alcaraz, María L., and Jose I. Hormaza. 2024. "Inadequate Pollination Is a Key Factor Determining Low Fruit-to-Flower Ratios in Avocado" Horticulturae 10, no. 2: 140. https://doi.org/10.3390/horticulturae10020140

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