Abstract
Due to a lack of knowledge on the pollination requirements of kiwifruit cultivars grown within the United States, farmers simultaneously implement multiple pollination methods, like the rental of managed bee species or artificial pollination to achieve high fruit yields. However, implementing multiple pollination methods is costly and possibly an inefficient use of resources. We assessed the contribution of two managed bees (Apis mellifera and Bombus impatiens) to the pollination of kiwifruit by i) determining the relative abundance of kiwifruit pollen collected by foragers of each bee species, and ii) comparing fruit set and fruit quality among insect and artificially pollinated flowers through an insect exclusion experiment.
▲Photo of red kiwi fruit pollination
A significant difference was observed between the mean relative abundance of kiwifruit pollen carried in the corbicula of A. mellifera and B. impatiens, with B. impatiens carrying on average 46% more kiwifruit pollen than A. mellifera. Artificially pollinated kiwifruit flowers set significantly greater numbers of fruit per flower at four weeks post-bloom and at harvest compared to insect pollination, wind pollination, and pollen exclusion treatment. Artificial pollination produced fruits of greater weight, size, and seed number compared to insect-pollinated flowers, and few fruits were produced in the pollen exclusion and wind pollination treatments. Kiwifruit producers experiencing similar conditions to ours should focus on artificially pollinating their crops rather than relying on managed or wild insects for kiwifruit pollination. Future research should evaluate other methods of artificial pollination to determine their effectiveness, efficiency, and economics in the pollination of kiwifruit grown within the United States.
▲红心猕猴桃▲
由于对美国境内种植的猕猴桃品种的授粉要求缺乏了解，农民同时采用多种授粉方法，例如租赁管理蜂种或人工授粉，以实现果实的高产。然而，实施多种授粉方法成本高昂，而且可能会导致资源利用效率低下。我们评估了两种管理蜜蜂（意大利蜜蜂和凤仙花熊蜂）对猕猴桃授粉的贡献，方法是：i) 确定每种蜂种采集者收集的猕猴桃花粉的相对丰度；ii) 比较昆虫和蜜蜂之间的坐果和果实质量。通过昆虫排除实验对花朵进行人工授粉。在西方蜜蜂和凤仙花的球核中携带的奇异果花粉的平均相对丰度之间存在显着差异，凤仙花携带的奇异果花粉平均比西方蜜蜂多 46%。与昆虫授粉、风授粉和花粉排斥处理相比，人工授粉的猕猴桃花在开花后四个星期和收获时每朵花的果实数量显着增加。与昆虫授粉的花朵相比，人工授粉产生的果实重量、大小和种子数量都更大，而在花粉排除和风授粉处理中产生的果实很少。与我们经历类似情况的猕猴桃生产者应该专注于人工授粉他们的作物，而不是依靠管理或野生昆虫为猕猴桃授粉。未来的研究应该评估其他人工授粉方法，以确定其在美国境内种植的猕猴桃授粉中的有效性、效率和经济性。
▲Kiwi pollination by honey bee
Introduction
Kiwifruit is a dioecious fruit-bearing plant with male and female flowers borne on separate plants. Male flowers produce viable pollen yet lack developed ovaries, ovules, and styles (Beutel 1990, Ferguson 1999), and female flowers, although perfect, lack viable pollen. Therefore, female flowers require a pollen vector to produce seed and fruit (Beutel 1990, Ferguson 1999). Kiwifruit size is dependent upon the number of viable pollen grains deposited on the stigma, and thus the number of seeds produced (Goodwin et al. 2013). To achieve high-quality marketable fruits (>100 g and >1,000 seeds), the stigma of a single female flower must receive between 2,000–3,000 viable pollen grains (Ferguson 1984, Hopping 1990, Testolin et al. 1991, Goodwin et al. 2013, Tacconi et al. 2016). In kiwifruit, inefficient pollination due to either pollen (inadequate quantity or quality of pollen) or pollinator (fewer pollinator visits or less pollen deposited per visit) limitation (Ashman et al. 2004) can result in low fruit set and unsatisfactory size, shape, and uniformity (Tacconi et al. 2016). For this reason, kiwifruit growers demand efficiently pollinated female flowers to ensure economic viability (Ferguson 1990, Costa 1999, Abbate et al. 2021).
▲阳光金果g3猕猴桃
介绍
猕猴桃是一种雌雄异株的果实植物，雄花和雌花分别开在不同的植株上。雄花产生有活力的花粉，但缺乏发育的子房、胚珠和花柱（Beutel 1990，Ferguson 1999），雌花虽然完美，但缺乏有活力的花粉。因此，雌花需要花粉载体来产生种子和果实（Beutel 1990，Ferguson 1999）。猕猴桃的大小取决于柱头上沉积的有活力的花粉粒的数量，以及产生的种子的数量（Goodwin 等人，2013）。为了获得高质量的适销水果（>100克和>1,000粒种子），一朵雌花的柱头必须接收2,000-3,000个有活力的花粉粒（Ferguson 1984，Hopping 1990，Testolin et al. 1991，Goodwin et al. 2017）。 2013，塔科尼等人，2016）。在猕猴桃中，由于花粉（花粉数量或质量不足）或传粉媒介（授粉媒介访问次数较少或每次访问沉积的花粉较少）限制而导致授粉效率低下（Ashman 等人，2004 年），可能会导致坐果率低以及大小、形状不理想。和均匀性（Tacconi 等人，2016）。因此，猕猴桃种植者需要有效授粉的雌花以确保经济可行性（Ferguson 1990，Costa 1999，Abbate et al. 2021）。
▲Kiwi pollination by honey bee
It is thought that kiwifruit pollination is mainly provided by insects, and to a lesser degree, wind (Testolin et al. 1991, Miñarro and Twizell 2015). Studies have documented wind pollinated kiwifruit flowers produce similar-sized fruits compared to insect-pollinated flowers (Bellini et al. 1989); however, most evidence suggests pollination via wind is ineffective in producing kiwifruits of marketable size (Donovan and Read 1991, Costa et al. 1993, Vaissière et al. 1996, Gonzalez et al. 1998, Morley-Bunker and Lyford 1999, Howpage et al. 2001, Pomeroy and Fisher 2002, Abbate et al. 2021). Honey bees and bumble bees are considered the most important pollinators of kiwifruit (Palmer-Jones and Clinch 1974, Matheson 1991a, 1991b, Vaissière et al. 1996, Howpage et al. 2001); however, their contribution to kiwifruit pollination has been debated (Clinch 1984, Read et al. 1989, Donovan and Read 1991, Costa et al. 1993, Pomeroy and Fisher 2002, Cnaani et al. 2006, Hanley et al. 2008, Miñarro and Twizell 2015, Somme et al. 2015, Abbate et al. 2021).
▲zespri redkiwi
人们认为猕猴桃授粉主要由昆虫提供，其次是风（Testolin et al. 1991，Miñarro and Twizell 2015）。研究表明，与昆虫授粉的花朵相比，风媒授粉的奇异果花产生的果实大小相似（Bellini 等，1989）；然而，大多数证据表明，风授粉对于生产适销规格的猕猴桃是无效的（Donovan and Read 1991, Costa et al. 1993, Vaissière et al. 1996, Gonzalez et al. 1998, Morley-Bunker and Lyford 1999, Howpage et al. . 2001，Pomeroy 和 Fisher 2002，Abbate 等人 2021）。蜜蜂和熊蜂被认为是猕猴桃最重要的传粉者（Palmer-Jones and Clinch 1974，Matheson 1991a，1991b，Vaissière et al. 1996，Howpage et al. 2001）；然而，它们对猕猴桃授粉的贡献一直存在争议（Clinch 1984，Read et al. 1989，Donovan and Read 1991，Costa et al. 1993，Pomeroy and Fisher 2002，Cnaani et al. 2006，Hanley et al. 2008，Miñarro and特威泽尔 2015，索姆等人 2015，阿巴特等人 2021）。
▲Extraction of pollen grain from anther
Studies argued honey bees are inefficient pollinators of kiwifruit due to a lack of attraction to the nectar-lacking blooms (Donovan and Read 1991, Costa et al. 1993, Pomeroy and Fisher 2002), and because they forage on concurrently flowering non-kiwifruit plants, often collecting as little as 2–4% kiwifruit pollen (Ford 1971, Blanchet et al. 1990). Similarly, bumble bees (Bombus impatiens and Bombus terrestris L. (Hymenoptera: Apidae)) have been evaluated as potential pollinators of kiwifruit (Read et al. 1989, Miñarro and Twizell 2015, Abbate et al. 2021), yet have been shown to be greatly influenced by accessible non-kiwifruit floral blooms collecting as little as 21% kiwifruit pollen (Pomeroy and Fisher 2002). Within the United States, no studies have evaluated the foraging preferences and contribution of A. mellifera and B. impatiens to kiwifruit pollination while stocked within a commercial kiwifruit orchard. Due to the varying conclusions concerning the contribution of wind and insects to kiwifruit pollination, several methods of artificial pollination, whereby pollen is applied manually or mechanically onto the pistil of a flower, have become common practice in commercial operations (Pinillos and Cuevas 2008). Artificial pollination is an important tool that supplements or replaces other forms of pollination when natural pollinators are not present or reliable (Pritchard and Edwards 2006). Like wind and insect pollination, artificial pollination of kiwifruit is known to have varying degrees of pollination success (Razeto et al. 2005).
▲Bee pollination
研究认为，蜜蜂是奇异果的低效传粉者，因为它们对缺乏花蜜的花朵缺乏吸引力（Donovan and Read 1991, Costa et al. 1993, Pomeroy and Fisher 2002），并且因为它们以同时开花的非奇异果植物为食，通常只收集 2-4% 的奇异果花粉（Ford 1971，Blanchet et al. 1990）。同样，熊蜂（Bombus impatiens 和 Bombus terrestris L.（膜翅目：蜜蜂科））已被评估为猕猴桃的潜在传粉者（Read et al. 1989，Miñarro and Twizell 2015，Abbate et al. 2021），但已被证明受到可获取的非猕猴桃花朵的极大影响，收集的猕猴桃花粉仅为 21%（Pomeroy 和 Fisher 2002）。在美国，尚无研究评估在商业猕猴桃果园中放养的蜜蜂和凤仙花的觅食偏好以及对猕猴桃授粉的贡献。由于关于风和昆虫对猕猴桃授粉的贡献的不同结论，几种人工授粉方法，即手动或机械地将花粉施用到花的雌蕊上，已成为商业操作中的常见做法（Pinillos 和 Cuevas 2008）。当自然授粉媒介不存在或不可靠时，人工授粉是补充或替代其他形式授粉的重要工具（Pritchard 和 Edwards 2006）。与风授粉和昆虫授粉一样，猕猴桃的人工授粉也具有不同程度的授粉成功率（Razeto 等，2005）。
▲Kiwi pollination by honey bee
Due to the varying results across kiwifruit pollination studies and the lack of knowledge concerning the pollination requirements of kiwifruit cultivars grown within the United States, orchard managers have turned to implement a mosaic of pollination methods to ensure high fruit yields. For example, orchard managers within the United States currently plant male and female kiwifruit plants near one another to facilitate cross-pollination via wind, introduce managed bees including honey bees and bumble bees to facilitate the movement of pollen from male to female flowers, and artificially pollinate their crops via hand pollination and pollen dusters (Brantley et al. 2019). Implementing multiple pollination methods is costly, time-consuming, and possibly an inefficient use of resources. In the southeastern United States, a successful golden fleshed female kiwifruit cultivar (A. chinensis var. chinensis ‘AU Gulf Coast Gold’) was developed and patented for commercial operations, yet its pollination requirements remain largely unknown (Dozier et al. 2018).
▲Kiwi pollination by honey bee
由于猕猴桃授粉研究的结果各不相同，并且缺乏对美国境内种植的猕猴桃品种的授粉要求的了解，果园管理者已转向实施多种授粉方法，以确保水果的高产。例如，美国的果园管理者目前将雄性和雌性猕猴桃植株种植在彼此靠近的地方，以促进通过风进行异花授粉，引入包括蜜蜂和大黄蜂在内的受管理蜜蜂，以促进花粉从雄花到雌花的移动，并人工人工授粉。通过手工授粉和花粉喷粉器为农作物授粉（Brantley et al. 2019）。实施多种授粉方法成本高昂、耗时，而且可能会导致资源利用效率低下。在美国东南部，一种成功的金肉雌性奇异果品种（A. chinensis var. chinensis ‘AU Gulf Coast Gold’）被开发出来并获得商业运营专利，但其授粉要求仍然很大程度上未知（Dozier 等人，2018）。
▲Male Kiwi fruit flower
Although commonly utilized for kiwifruit pollination, no studies in the United States have comparatively evaluated the foraging preferences of managed honey bees and bumble bees to determine their contribution to kiwifruit pollination. Therefore, the primary objectives of this study were to i) conduct a palynological study to compare the relative abundance of kiwifruit pollen contained within the corbicular pollen loads of both managed honey bees and bumble bees while stocked in a kiwifruit orchard typical of the southeastern United States, and ii) compare the effectiveness of common pollination methods (wind, insect, artificial pollination) on fruit set and fruit quality (fruit weight, fruit size, and seed counts) through an exclusion study while stocking honey bees and bumble bees within a kiwifruit orchard. We predicted that the amount of kiwifruit pollen collected by both managed honey bees and bumble bees would differ due to a difference in attraction between kiwifruit blooms and concurrently flowering non-kiwifruit plants (Pomeroy and Fisher 2002). We also predicted that artificial pollination would result in the greatest fruit set, fruit size, and seed counts compared to wind and insect pollination because more pollen grains would be deposited on the stigma of female flowers than the other pollination methods (Costa et al. 1993, Gonzalez et al. 1994, 1998, Abbate et al. 2021). The results of this research will assist orchard managers within the United States develop a science-backed pollination management plan for the kiwifruit industry.
▲Kiwi male and female flowers
尽管常用于猕猴桃授粉，但美国尚无研究对管理蜜蜂和熊蜂的觅食偏好进行比较评估，以确定它们对猕猴桃授粉的贡献。因此，本研究的主要目标是 i) 进行孢粉学研究，比较在美国东南部典型的猕猴桃果园中饲养的受管理蜜蜂和大黄蜂的球囊花粉负荷中所含猕猴桃花粉的相对丰度，和 ii) 通过排除研究比较常见授粉方法（风授粉、昆虫授粉、人工授粉）对坐果和果实质量（果实重量、果实大小和种子数量）的有效性，同时在猕猴桃内放养蜜蜂和熊蜂果园。我们预测，由于奇异果开花和同时开花的非奇异果植物之间的吸引力不同，管理的蜜蜂和大黄蜂收集的奇异果花粉量会有所不同（Pomeroy 和 Fisher 2002）。我们还预测，与风授粉和昆虫授粉相比，人工授粉将导致最大的坐果率、果实大小和种子数量，因为与其他授粉方法相比，更多的花粉粒会沉积在雌花的柱头上（Costa et al. 1993） ，冈萨雷斯等人，1994 年，1998 年，阿巴特等人，2021 年）。这项研究的结果将帮助美国果园管理者为猕猴桃产业制定科学支持的授粉管理计划。
▲干燥猕猴桃花粉
Methods
Experimental Setting
This work was performed during the 2020 kiwifruit growing season (April–October) within a 72.8 ha (180 acre) kiwifruit orchard located in central Alabama (Reeltown, Alabama; lat. 32°35ʹN, long. 85°47ʹW). All experiments were concentrated within a single 5.5 ha block which contained rows spaced 2.74 m. Each row alternated between one female golden-fleshed kiwifruit cultivar (Actinidia chinensis var. chinensis ‘AU Gulf Coast Gold’) and one of three male kiwifruit cultivars (A. chinensis var. chinensis ‘AU Golden Tiger’, ‘CK3’, and A. chinensis var. deliciosa ‘Chieftain’) to achieve a male to female plant ratio of 4:1. All plants chosen for the study were 5 years of age and were managed following the best management practices recommended for commercial kiwifruit production (Hasey 1994). Male pollinizers ‘AU Golden Tiger’, ‘CK3’, and ‘Chieftain’ were chosen because of their ploidy levels (hexaploid for both ‘AU Golden Tiger’ and ‘Chieftain’, and diploid for ‘CK3’), and because each male cultivar’s bloom period often overlaps with the bloom period of ‘AU Gulf Coast Gold’(Dozier et al. 2011, Spiers et al. 2018).
▲Female Kiwi fruit flower
方法
实验设置
这项工作是在 2020 年奇异果生长季节（4 月至 10 月）期间在位于阿拉巴马州中部（阿拉巴马州里尔敦；北纬 32°35，西经 85°47）的一个 72.8 公顷（180 英亩）奇异果园内进行的。所有实验都集中在一个 5.5 公顷的区块内，其中行间距为 2.74 m。每行交替种植一个雌性金肉猕猴桃品种（Actinidia chinensis var. chinensis 'AU Gulf Coast Gold'）和三个雄性猕猴桃品种之一（A. chinensis var. chinensis 'AU Golden Tiger'、'CK3' 和 A . chinensis deliciosa 'Chieftain') 的雌雄比例达到 4:1。研究中选择的所有植株均为 5 年龄，并按照商业奇异果生产推荐的最佳管理实践进行管理（Hasey 1994）。选择雄性授粉者“AU Golden Tiger”、“CK3”和“Chieftain”是因为它们的倍性水平（“AU Golden Tiger”和“Chieftain”均为六倍体，“CK3”为二倍体），并且因为每个雄性品种的花期经常与“AU Gulf Coast Gold”的花期重叠（Dozier et al. 2011，Spiers et al. 2018）。
▲Kiwi pollination▲
To ensure large numbers of blooms for each kiwifruit cultivar, all female kiwifruit plants were winter pruned between December 2019 and February 2020 (Strik 2005). Female kiwifruit plants were pruned by removing approximately 70% of the wood produced the previous season, replacement canes were tied to the trellis supports, and spurs on female plants were left intact as they are known to be very fruitful (Strik 2005). Winter pruning helps maintain a framework for the vines, a balance between vegetative growth and fruit production, and a canopy that utilizes available light efficiently (Strik 2005). Male plants were pruned at the end of the previous season’s bloom period by cutting canes back to a length between 15 and 30 cm with the goal of producing as many flowers as possible for pollination (Luh and Wang 1984, Strik 2005). Males were also trimmed in the winter by cutting back dead or weak canes, leaving canes between 1.0 and 1.5 m in length (Luh and Wang 1984, Strik 2005).
▲Pollen is packed into 200g jars – this amount of pollen comes from about 20kg of flowers
为了确保每个猕猴桃品种大量开花，所有雌性猕猴桃植株在 2019 年 12 月至 2020 年 2 月期间进行了冬季修剪（Strik 2005）。通过去除上一季节产生的约 70% 的木材来修剪雌性奇异果植株，将替换的藤蔓绑在棚架支撑上，并且雌性植株上的刺完好无损，因为众所周知它们非常结果子（Strik 2005）。冬季修剪有助于维持葡萄藤的框架、营养生长和果实生产之间的平衡，以及有效利用可用光线的树冠（Strik 2005）。在上一季节的花期结束时，通过将枝条剪短至 15 至 30 厘米的长度来修剪雄性植物，目的是产生尽可能多的花朵用于授粉（Luh 和 Wang 1984，Strik 2005）。雄性在冬季也会通过修剪枯死或弱的手杖进行修剪，使手杖长度在 1.0 至 1.5 m 之间（Luh 和 Wang 1984，Strik 2005）。
▲sungold g3 kiwi orchard
To ensure large numbers of blooms for each kiwifruit cultivar, all female kiwifruit plants were winter pruned between December 2019 and February 2020 (Strik 2005). Female kiwifruit plants were pruned by removing approximately 70% of the wood produced the previous season, replacement canes were tied to the trellis supports, and spurs on female plants were left intact as they are known to be very fruitful (Strik 2005). Winter pruning helps maintain a framework for the vines, a balance between vegetative growth and fruit production, and a canopy that utilizes available light efficiently (Strik 2005). Male plants were pruned at the end of the previous season’s bloom period by cutting canes back to a length between 15 and 30 cm with the goal of producing as many flowers as possible for pollination (Luh and Wang 1984, Strik 2005). Males were also trimmed in the winter by cutting back dead or weak canes, leaving canes between 1.0 and 1.5 m in length (Luh and Wang 1984, Strik 2005).
▲packaging kiwipollen
为了确保每个猕猴桃品种大量开花，所有雌性猕猴桃植株在 2019 年 12 月至 2020 年 2 月期间进行了冬季修剪（Strik 2005）。通过去除上一季节产生的约 70% 的木材来修剪雌性奇异果植株，将替换的藤蔓绑在棚架支撑上，并且雌性植株上的刺完好无损，因为众所周知它们非常结果子（Strik 2005）。冬季修剪有助于维持葡萄藤的框架、营养生长和果实生产之间的平衡，以及有效利用可用光线的树冠（Strik 2005）。在上一季节的花期结束时，通过将枝条剪短至 15 至 30 厘米的长度来修剪雄性植物，目的是产生尽可能多的花朵用于授粉（Luh 和 Wang 1984，Strik 2005）。雄性在冬季也会通过修剪枯死或弱的手杖进行修剪，使手杖长度在 1.0 至 1.5 m 之间（Luh 和 Wang 1984，Strik 2005）。
▲Kiwi pollination▲
In mid-April 2020, four bumble bee (Bombus impatiens) quads (16 colonies total; four colonies per quad) each weighing between 832 and 845 grams and containing approximately 200 workers were purchased (BioBest, Romulus, MI, USA) and 12 honey bee (Apis mellifera) colonies, each consisting of at least 10 frames of brood, 15 frames of adults (between 20,000 and 30,000 workers), and 5 frames of food obtained from our laboratory, were transferred to the kiwifruit orchard one day before the start of the A. chinensis var. chinensis ‘AU Gulf Coast Gold’ bloom. The bloom date of A. chinensis var. chinensis ‘AU Gulf Coast Gold’ was estimated from the previous season’s bloom date (Abbate et al. 2021) and because the female flowers were in the latter portion of the 7–10 day ‘popcorn’ stage (Kelley 2020, Oh et al. 2021).
▲picking kiwi flowers
2020 年 4 月中旬，购买了四只大黄蜂（Bombus impatiens）四群（总共 16 个蜂群；每个四群四个蜂群），每群重量在 832 至 845 克之间，包含约 200 名工蜂（BioBest，罗穆卢斯，密歇根州，美国）和 12 个蜂蜜蜜蜂 (Apis mellifera) 蜂群，每个蜂群至少由 10 框蜂巢、15 框成虫（20,000 至 30,000 名工蜂）和 5 框从我们实验室获得的食物组成，在开始前一天转移到猕猴桃果园中华猕猴桃变种chinensis ‘AU Gulf Coast Gold’ 开花。中华猕猴桃的花期chinensis“AU Gulf Coast Gold”是根据上一季节的开花日期估算的（Abbate et al. 2021），并且因为雌花处于 7-10 天“爆米花”阶段的后半部分（Kelley 2020，Oh et al. 2021）。 2021）。
▲Kiwi pollination by honey bee
To ensure numbers of colonies for both managed bee species reflected common stocking rates of commercial kiwifruit operations (Blanchet et al. 1990, Pomeroy and Fisher 2002), four circular 0.40 ha plots were established (61 m between each plot’s center point) across the entire length of the kiwifruit block (244 m) (Fig. 1). Within each 0.40 ha plot, one B. impatiens quad equating to a stocking rate of 10 colonies/ha was placed on a wooden pallet with a plastic weather covering, and three A. mellifera colonies equating to a stocking rate of 7 colonies/ha were placed on a separate wooden pallet spaced 9.1 m from the B. impatiens pallet. The stocking rates for each managed bee species are the currently used stocking rates for kiwifruit pollination in the southeastern United States. Because hail is not a major concern for kiwifruit growers in Alabama, hail nets were not employed during the study and therefore did not restrict pollen transfer via wind or insects.
▲猕猴桃对花授粉方法图片
为了确保两种管理蜂种的蜂群数量反映商业奇异果经营的常见放养率（Blanchet et al. 1990，Pomeroy and Fisher 2002），在整个区域建立了四个圆形 0.40 ha 地块（每个地块中心点之间 61 m）。猕猴桃块的长度（244 m）（图1）。在每个 0.40 公顷的地块内，将 1 个凤仙花菌落（相当于 10 个菌落/公顷的放养率）放置在带有塑料防雨罩的木托盘上，并在 3 个 A. mellifera 菌落（相当于 7 个菌落/公顷的放养率）上放置。放置在与凤仙花托盘间隔 9.1 m 的单独木托盘上。每个管理蜂种的放养率是美国东南部猕猴桃授粉目前使用的放养率。由于冰雹不是阿拉巴马州猕猴桃种植者的主要担忧，因此研究期间没有使用防雹网，因此不会限制花粉通过风或昆虫传播。
▲Kiwi pollination by honey bee
Fig. 1.Orientation of the four circular 0.40 ha experimental plots within the 5.5 ha kiwifruit block. The center of each plot was separated by 61 m from each other and spread out along a 244 m transect. Each 0.40 ha plot contained one bumble bee (Bombus impatiens) quad consisting of four colonies (10 colonies/ha), three honey bee (Apis mellifera) colonies (7 colonies/ha), and alternating female (always Actinidia chinensis var. chinensis ‘AU Gulf Coast Gold’) and male (one of A. chinensis var. chinensis ‘AU Golden Tiger’, ‘CK3,’ or A. chinensis var. deliciosa ‘Chieftain’) kiwifruit rows.
▲picking kiwi flowers
图 1. 5.5 公顷猕猴桃区块内 4 个圆形 0.40 公顷试验田的朝向。每个地块的中心彼此相距 61 m，并沿着 244 m 的横断面展开。每 0.40 公顷的地块包含 1 个大黄蜂（Bombus impatiens），由 4 个蜂群（10 个蜂群/公顷）组成，3 个蜜蜂（Apis mellifera）蜂群（7 个蜂群/公顷），以及交替的雌性蜜蜂（总是中华猕猴桃变种）。 AU Gulf Coast Gold'）和雄性（A. chinensis var. chinensis 'AU Golden Tiger'、'CK3' 或 A. chinensis var. deliciosa 'Chieftain' 之一）猕猴桃行。
▲Kiwi pollination by honey bee
Palynological Study
Pollen collection.
Sampling of A. mellifera and B. impatiens foragers was carried out from 9 April 2020 to 17 April 2020 to determine the flowering plants the workers were foraging on. We hand netted the first four A. mellifera and B. impatiens pollen foragers within each 0.4 ha plot as they returned to their colonies during three sampling periods morning [0800 am to 1100am], midday [1100 am to 1400 pm], and afternoon [1400 pm to 1800 pm]) (Azmi et al. 2015). Hand netting occurred when weather conditions were appropriate for sampling foraging bees (little to no wind, cloud coverage ≤50%, temperatures ≥15°C) (Krahner et al. 2021). From 9 April 2020 to 17 April 2020 air temperature and wind data were collected daily at half hour intervals from 08.00 to 18.00 from the closest weather station (EV Smith Research Center, Auburn University), which was approximately 20 km from the kiwifruit orchard. The minimum, maximum and average air temperatures, as well as wind speeds, during the study period were 15°C, 26°C, and 20°C, and 0 ms, 3.9 ms, and 1.9 ms, respectively.
▲picking kiwi flowers
孢粉学研究
花粉采集。
2020年4月9日至2020年4月17日期间，对蜜蜂采食者的蜜蜂和凤仙花进行了采样，以确定工蚁采食的开花植物。我们在上午 [上午 0800 点至上午 1100 点]、中午 [上午 1100 点至下午 1400 点] 和下午 [上午 0800 点至上午 1100 点]、中午 [上午 1100 点至下午 1400 点] 和下午 [下午 1400 点至下午 1800 点]）（Azmi 等人，2015 年）。当天气条件适合对觅食蜜蜂进行采样时（微风或无风、云覆盖率≤50%、温度≥15°C），就会使用手网（Krahner et al. 2021）。 2020年4月9日至2020年4月17日，每天08:00至18:00从最近的气象站（奥本大学EV史密斯研究中心）每隔半小时收集一次气温和风力数据，该气象站距离奇异果园约20公里。研究期间的最低、最高和平均气温以及风速分别为15°C、26°C和20°C，以及0毫秒、3.9毫秒和1.9毫秒。
▲Kiwi pollination by honey bee
Maximum air temperature and wind speeds generally occurred midday, whereas minimum air temperature and wind speeds generally occurred in the mornings and afternoons. The weather conditions during the study period were optimal for foraging bees and kiwifruit pollination via wind. Honey bee foraging behavior has been shown to be greatest at about 10°C (Tan et al. 2012), and wind speeds between 0 and 1.5 ms are suitable for kiwifruit pollination experiments (Costa et al. 1993). A total of 384 pollen foragers per bee species were netted over 8 days (16 foragers per species per sampling period per day). Netted A. mellifera and B. impatiens foragers containing corbicular pollen loads were temporarily placed in individual plastic 15 mL vials and stored in a cooler with an ice pack to anesthetize them. Once anesthetized, corbicular pollen loads were nonlethally removed using forceps and the bees were left to regain consciousness to fly back to their colonies unharmed (Rundlöf et al. 2022). To prevent the cross-contamination of pollen between bee species, sampling days, and sampling periods, designated nets for each bee species were used and washed daily in soapy water. Additionally, only new plastic 15 ml vials were used and forceps were washed with soapy water after each sampling period and day. For each day, corbicular pollen was pooled for each bee species at each sampling period before it was processed.
▲Kiwi Fruit Pollen
最高气温和风速一般出现在中午，最低气温和风速一般出现在上午和下午。研究期间的天气条件最适合蜜蜂觅食和猕猴桃通过风授粉。蜜蜂的觅食行为在 10°C 左右时表现最佳（Tan 等人，2012 年），0 到 1.5 毫秒之间的风速适合猕猴桃授粉实验（Costa 等人，1993 年）。在 8 天的时间里，每种蜂种总共捕获了 384 只花粉采集者（每个物种每天每个采样周期 16 只采集者）。将含有球状花粉负载的网状蜜蜂和凤仙花觅食者暂时放置在单独的 15 mL 塑料瓶中，并储存在带有冰袋的冷却器中以麻醉它们。麻醉后，用镊子非致命性地去除球状花粉负载，让蜜蜂恢复意识，毫发无伤地飞回蜂巢（Rundlöf et al. 2022）。为了防止蜂种、采样日和采样周期之间花粉的交叉污染，每个蜂种使用指定的网，并每天用肥皂水清洗。此外，仅使用新的 15 毫升塑料小瓶，并在每个采样周期和每天之后用肥皂水清洗镊子。每天，在处理之前，每个采样周期的每个蜂种的球状花粉都会被汇集起来。
▲Pollen Collection猕猴桃花采集
Palynological analyses.
Pollen samples were chemically processed by Global GeoLab Limited (Medicine Hat, Alberta, Canada) following standard palynological laboratory methods (Brown 2008), and quantified at the CENEX Laboratory at Louisiana State University (Baton Rouge, LA, USA). Pollen samples received by Global Geolab Limited were dehydrated and dissolved in glacial acetic acid, and the lipids, waxes, and cytoplasm were removed via an acetolysis chemical treatment to allow for easier identification of the pollen grains. After the acetolysis chemical treatment, pollen grains were rinsed with ethanol and water and then suspended in glycerine. The vial content was stirred thoroughly for one minute, then a small drop of the suspension was mounted on a 75 × 25 mm microscope slide and covered with an 18 × 18 mm #1-thickness glass coverslip. Coverslips were sealed with clear nail polish to prevent leakage and were sent to the CENEX Lab where microscope slides were examined at 600× and 1,000× magnification using light microscopy (Olympus BX41, Tokyo, Japan) to identify pollen types. A minimum of 300 identified pollen grains were counted for each sample using traverses that prevented duplicate counts of pollen; all slides were scanned for unique and rare types of pollen after the initial count was completed, and all pollen was identified to the lowest taxonomic group possible (i.e., family, genus, or species) (Ferguson et al. 2018). Due to the difficulty differentiating between male and female kiwifruit pollen grains, they were not quantified separately.
▲Pollen Collection猕猴桃花采集
孢粉学分析。
花粉样品由 Global GeoLab Limited（加拿大艾伯塔省麦迪辛哈特）按照标准孢粉学实验室方法（Brown 2008）进行化学处理，并在路易斯安那州立大学（美国路易斯安那州巴吞鲁日）的 CENEX 实验室进行定量。 Global Geolab Limited 收到的花粉样品经过脱水并溶解在冰醋酸中，并通过乙酰水解化学处理去除脂质、蜡质和细胞质，以便更容易识别花粉粒。丙酮水解化学处理后，用乙醇和水漂洗花粉粒，然后悬浮在甘油中。将小瓶内容物彻底搅拌一分钟，然后将一小滴悬浮液安装在 75 × 25 mm 显微镜载玻片上，并用 18 × 18 mm #1 厚度的玻璃盖玻片覆盖。盖玻片用透明指甲油密封以防止泄漏，并被送往 CENEX 实验室，在那里使用光学显微镜（奥林巴斯 BX41，日本东京）在 600 倍和 1,000 倍放大倍率下检查显微镜载玻片，以识别花粉类型。使用防止花粉重复计数的横移，对每个样本至少计数 300 个已识别的花粉粒；初始计数完成后，对所有载玻片进行扫描，寻找独特且稀有类型的花粉，并将所有花粉鉴定为可能的最低分类组（即科、属或种）（Ferguson 等人，2018）。由于难以区分雄性和雌性猕猴桃花粉粒，因此没有单独对它们进行定量。
▲Large packaged product kiwi pollen
Exclusion Study
Study design.
An exclusion study was performed to assess the contribution of managed A. mellifera and B. impatiens to A. chinensis var. chinensis ‘AU Gulf Coast Gold’ (hereby referred to as AU Gulf Coast Gold) pollination by quantifying fruit set among four pollination treatments: i) Insect Pollination, ii) Wind Pollination, iii) Artificial Pollination, and iv) Pollen Exclusion. The experiment was initiated in mid-April 2020, approximately two days before the bloom period of AU Gulf Coast Gold. Each pollination treatment was applied to each plant once and was replicated across 114 female kiwifruit plants.
▲Pollen Collection猕猴桃花采集
排除研究
学习设计。
进行了一项排除研究，以评估管理的意大利蜜蜂和凤仙花对中华蜜蜂的贡献。 chinensis ‘AU Gulf Coast Gold’（以下简称 AU Gulf Coast Gold）通过量化四种授粉处理中的坐果率进行授粉：i) 昆虫授粉，ii) 风授粉，iii) 人工授粉，以及 iv) 花粉排除。该实验于 2020 年 4 月中旬开始，大约是 AU Gulf Coast Gold 花期前两天。每个授粉处理对每株植物进行一次，并在 114 株雌性猕猴桃植株上重复进行。
▲kiwi blossoms that have completed pollination
To document the contribution of managed bees and wild pollinators to the pollination of AU Gulf Coast Gold, for the Insect Pollination treatment, a cluster of unopened female flower buds was tagged using flagging tape on each plant (Fig. 2a). During the bloom, managed A. mellifera and B. impatiens, as well as any wild pollinators, could freely visit the tagged floral cluster for pollen rewards. For the Wind Pollination treatment, we bagged one unopened cluster of female flower buds per plant using a 1,000 micron nylon monofilament mesh liquid filter bag (Cary Company part #NMO1000P1DS, Addison, IL, USA) to exclude all insects, but allow the passive passage of pollen through the mesh bag (Neal and Anderson 2004, McIver and Erickson 2012). The vine containing the cluster of flower buds was tagged with flagging tape and the number of flower buds present was recorded (Fig. 2b). For the Artificial Pollination treatment, we bagged one unopened cluster of female flower buds per plant with a 25-micron polyester felt liquid filter bag (Cary Company part #PES25P1DS, Addison, IL, USA) that prevented the passage of pollen via insects and wind from reaching the female kiwifruit flower buds during the bloom (Fig. 2c). To ensure kiwifruit pollen could not pass through the 25-micron bags, methods used by Neal and Anderson (2004) were followed to confirm kiwifruit pollen was incapable of passing through the material.
▲kiwi pollination
为了记录管理蜜蜂和野生传粉媒介对 AU Gulf Coast Gold 授粉的贡献，在昆虫授粉处理中，在每株植物上使用标记胶带标记一簇未开放的雌性花蕾（图 2a）。在开花期间，受管理的蜜蜂花和凤仙花以及任何野生传粉者都可以自由地访问标记的花簇以获得花粉奖励。对于风授粉处理，我们使用 1,000 微米尼龙单丝网状液体过滤袋（Cary Company 部件 #NMO1000P1DS，Addison，IL，USA）将每株植物的一簇未开放的雌性花蕾装袋，以排除所有昆虫，但允许被动通过通过网袋收集花粉（Neal 和 Anderson 2004，McIver 和 Erickson 2012）。用标记带标记含有花蕾簇的藤蔓，并记录存在的花蕾数量（图2b）。对于人工授粉处理，我们用 25 微米聚酯毡液体过滤袋（Cary Company 部件号 #PES25P1DS，Addison，IL，USA）将每株植物的一簇未开放的雌性花蕾装袋，以防止花粉通过昆虫和风传播在开花期间到达雌性猕猴桃花蕾（图2c）。为了确保奇异果花粉无法穿过 25 微米的袋子，遵循 Neal 和 Anderson (2004) 使用的方法来确认奇异果花粉无法穿过材料。
▲▲播宏猕猴桃花粉高品质、高倍体、高活性的猕猴桃花粉，可以满足30000亩果园的花粉需要。联系微信18030405084
Wooden clothespins were used to ensure a tight fit around the bag opening and the kiwifruit vine (Fig. 2c). Clothespins were necessary only for the 25-micron bags due to the material difference compared to the 1,000-micron bags used in the Wind Pollination treatment. Brantley et al. (2019) determined the effective pollination period of A. chinensis var. chinensis was approximately 4 days after anthesis (flower opening). For this reason, each bag within the Artificial Pollination treatment was opened every other day and checked for recently bloomed flowers. If open flowers were present, two puffs of ‘Chieftain’ pollen were applied with the use of a hand puffer (Antles Pollen Supplies, Inc., Modesto, CA, USA) to ensure adequate numbers of pollen grains were deposited (Dozier et al. 2018). Using methods from Kannely (2005), we determined that a single puff of pollen employed during our study expelled approximately 274,000 pollen grains. Pollen application occurred from the time of flower anthesis to flower senescence/petal drop (~6 days). Lastly, the same 25-micron polyester felt liquid filter bags were used for the Pollen Exclusion treatment to exclude all pollen via insects and wind. Unlike the Artificial Pollination treatment, artificial pollination was not applied to this treatment. For each treatment requiring an exclusion bag (Wind, Artificial Pollination, and Pollen Exclusion), bags remained on the cluster of flower buds until every flower within the bag opened and senesced, at which point the bags were removed and fruits were left to develop until the fall harvest. The number of flower buds present within each flower cluster was recorded for each pollination treatment on each plant.
▲picking kiwi flowers
使用木制衣夹确保袋子开口和猕猴桃藤周围紧密贴合（图 2c）。由于与风授粉处理中使用的 1,000 微米袋子相比，材料存在差异，因此仅 25 微米袋子需要使用衣夹。布兰特利等人。 (2019)测定了中华白藜芦醇的有效授粉期。 chinensis 大约在开花（开花）后 4 天。因此，人工授粉处理中的每个袋子每隔一天打开一次，检查最近盛开的花朵。如果存在开放的花朵，则使用手动喷枪（Antles Pollen Supplies, Inc., Modesto, CA, USA）喷洒两股“Chieftain”花粉，以确保沉积足够数量的花粉粒（Dozier 等，2015）。 2018）。使用 Kannely (2005) 的方法，我们确定在我们的研究中使用的一次花粉喷出大约 274,000 个花粉粒。花粉施用发生在从花开花期到花衰老/花瓣掉落期间（约6天）。最后，使用相同的 25 微米聚酯毡液体过滤袋进行花粉排除处理，以排除昆虫和风传播的所有花粉。与人工授粉处理不同，该处理没有应用人工授粉。对于需要排除袋的每种处理（风、人工授粉和花粉排除），袋子保留在花芽簇上，直到袋子内的每朵花都开放并衰老，此时将袋子移走，让果实发育直到秋天的收获。对于每株植物的每次授粉处理，记录每个花簇内存在的花芽的数量。
▲kiwi blossoms that have completed pollination
Fig. 2.Three clusters of Actinidia chinensis var. chinensis ‘AU Gulf Coast Gold’ representing A) Insect Pollinated flowers, with flagging tape tied to the kiwifruit vine, B) a kiwifruit vine with a 1,000-micron insect exclusion bag representing the Wind Pollinated treatment, and C) a kiwifruit vine with a fine-meshed 25-micron filter bag representing both the Pollen Exclusion and the Artificial Pollination treatments.
图2.三簇中华猕猴桃。 chinensis 'AU Gulf Coast Gold' 代表 A) 昆虫授粉的花朵，用旗带绑在奇异果藤上，B) 带有 1,000 微米昆虫排除袋的奇异果藤，代表风授粉处理，以及 C) 带有代表风授粉处理的奇异果藤细网状 25 微米过滤袋代表花粉排除和人工授粉处理。
▲kiwi blossoms that have completed pollination
Fruit quantity and quality assessments.
Fruit quantity assessments were conducted at four weeks post bloom (hereafter, fruit set; 11 May 2020) and at harvest (hereafter mature fruit; 15 September 2020). Fruit set was documented by recording the number of experimental flowers that produced fruit across the four treatments and 114 kiwifruit plants, and was defined as when petals abscised from the flower and fruit growth began (Richardson et al. 2011). Fruit set was quantified at this time in case kiwifruit plants or fruits became damaged, for example by storms or insect pests. This ensured that fruit set could be accurately estimated before harvest. The number of mature fruits was also quantified at harvest to estimate the total fruit production of each pollination treatment at harvest. All mature fruits were harvested once fruits reached two threshold parameters − a soluble solid content (SSC) of >6.5 °Brix (Burdon 2015) and a fruit hue angle of <110° (Minchin et al. 2003). To assess fruit quality at harvest, we randomly selected up to 20 fruits per pollination treatment and measured three parameters (fruit weight, fruit size, and seed counts). Fruit weights were taken to the nearest gram using a digital scale (VWR International, Randor, PA). To quantify fruit size, we calculated the fruit size index (FSI) of each fruit following the methods of Brantley et al. (2019) and Abbate et al. (2021). For this we quantified the number of seeds per fruit by manually pulverizing each fruit and forcing the fruit through a wire test sieve (1.00-mm mesh) (Newark Wire Cloth Company, Newark, NJ) with pressurized tap water, thus leaving all seeds behind.
▲Kiwi Pollen Collection & Sales
水果数量和质量评估。 果实数量评估在开花后四个星期（以下简称坐果；2020 年 5 月 11 日）和收获时（以下简称成熟果实；2020 年 9 月 15 日）进行。通过记录四种处理和 114 株猕猴桃植株中产生果实的实验花的数量来记录坐果，并定义为花瓣从花上脱落和果实开始生长的时间（Richardson 等，2011）。此时对坐果进行量化，以防猕猴桃植株或果实受到风暴或害虫等损害。这确保了在收获前可以准确估计坐果率。收获时成熟果实的数量也被量化，以估计收获时每次授粉处理的果实总产量。一旦果实达到两个阈值参数，即收获所有成熟的果实 - 可溶性固形物含量 (SSC) > 6.5 °Brix (Burdon 2015) 和果实色调角 < 110° (Minchin et al. 2003)。为了评估收获时的果实质量，我们在每次授粉处理中随机选择最多 20 个果实，并测量三个参数（果实重量、果实大小和种子数量）。使用数字秤（VWR International，Randor，PA）将果实重量精确到克。为了量化果实大小，我们按照 Brantley 等人的方法计算了每个果实的果实大小指数 (FSI)。 （2019）和阿巴特等人。 （2021）。为此，我们通过手动粉碎每个水果并用加压自来水迫使水果通过钢丝测试筛（1.00 毫米网孔）（新泽西州纽瓦克的纽瓦克钢丝布公司）来量化每个水果的种子数量，从而留下所有种子。
Statistical Analyses
A Wilcoxon Rank Sum test was used to test for differences in the relative abundance of kiwifruit pollen carried in the corbicula of A. mellifera and B. impatiens over the eight days. A Kruskal-Wallis one-way nonparametric analysis of variance was used to test for differences in the relative abundance of kiwifruit pollen collected by A. mellifera and B. impatiens among each sampling date and period (morning, midday, afternoon), and to finally test for differences in fruit set and mature fruit among the four pollination treatments (Insect Pollination, Wind Pollination, Artificial Pollination, and Pollen Exclusion) for A. chinensis var. chinensis AU Gulf Coast Gold at four weeks post-bloom and harvest, respectively. In cases with significant differences among treatments, a Dunn’s pairwise post hoc test was used to determine which treatment groups were significantly different from one another. Lastly, a Wilcoxon Rank Sum test was used to test for differences in fruit weight, FSI, and seed counts among the pollination treatments. Due to low numbers of fruits produced at harvest within the Wind Pollination (2 fruits) and Pollen Exclusion (1 fruit) treatments, they were excluded from the analysis. All statistical tests were performed using Statistix 9.0 Analytical Software, Tallahassee, FL, USA
▲Pollen Collection
统计分析
使用 Wilcoxon Rank Sum 检验来测试八天内，意大利蜜蜂和凤仙花的蚬中携带的猕猴桃花粉相对丰度的差异。采用Kruskal-Wallis单向非参数方差分析方法检验了猕猴桃和凤仙花采集的猕猴桃花粉在每个采样日期和时间段（上午、中午、下午）的相对丰度差异，最后得出测试四种授粉处理（昆虫授粉、风授粉、人工授粉和花粉排除）之间的中华猕猴桃坐果和成熟果实的差异。 chinensis AU Gulf Coast Gold 分别在开花后和收获后四个星期。如果治疗之间存在显着差异，则使用 Dunn 成对事后检验来确定哪些治疗组彼此之间存在显着差异。最后，使用 Wilcoxon Rank Sum 检验来测试授粉处理之间果实重量、FSI 和种子数的差异。由于风授粉（2 个果实）和花粉排除（1 个果实）处理中收获时产生的果实数量较少，因此将它们排除在分析之外。所有统计测试均使用 Statistix 9.0 分析软件（美国佛罗里达州塔拉哈西）进行
▲sungold g3 kiwifruit vine
Palynological Study
Overall, 15,616 pollen grains were counted (7,666 from A. mellifera and 7,950 from B. impatiens), representing a total of 26 distinct pollen taxa over the eight sampling days (Table 1). Nineteen pollen taxa were identified from the corbicular pollen of A. mellifera and 24 from the corbicular pollen of B. impatiens. Overall, A. mellifera collected the greatest relative abundances of pollen genera/species from Trifolium/Melilotus L./(L.) Lam. (Fabales: Fabaceae) (21.7%), followed by A. chinensis (21.3%), Rhus L. (Sapindales: Anacardiaceae) (15.6%), and Brassica L. (Brassicales: Brassicaceae) (12.1%) (Fig. 3). For B. impatiens, the greatest relative abundances of pollen genera/species collected were from A. chinensis (66.6%), Rhus (8.5%), Nyssa Gronov. ex L. (Cornales:Nyssaceae) (7.2%), and Trifolium/Melilotus (3.5%) (Fig. 3). For both A. mellifera and B. impatiens, pollen within the family Rosaceae Juss. accounted for the fourth greatest relative abundance of pollen collected (12.6% and 7.1%, respectively).
▲Pollen Collection
孢粉学研究
总体而言，对 15,616 个花粉粒进行了计数（其中 7,666 个来自蜜蜂花粉粒，7,950 个来自凤仙花花粉粒），代表 8 个采样日内总共 26 个不同的花粉类群（表 1）。从 A. mellifera 的球状花粉中鉴定出 19 个花粉类群，从 B. impatiens 的球状花粉中鉴定出 24 个花粉类群。总体而言，A. mellifera 从三叶草/草樨L./(L.) Lam 收集了相对丰度最高的花粉属/种。 （豆科：豆科）（21.7%），其次是A. chinensis（21.3%）、Rhus L.（无患子目：漆树科）（15.6%）和Brassica L.（芸苔目：十字花科）（12.1%）（图3） ）。对于凤仙花，收集的花粉属/种相对丰度最大的是中华凤仙花 (66.6%)、漆树 (8.5%)、Nyssa Gronov。 ex L.（玉米目：Nyssaceae）（7.2%）和三叶草/草樨属（3.5%）（图3）。对于 A. mellifera 和 B. impatiens 来说，都是蔷薇科 Juss 的花粉。占收集花粉相对丰度的第四位（分别为 12.6% 和 7.1%）。
▲Kiwi pollen
Table 1.Total number of pollen taxa quantified from the corbicula of honey bee (Apis mellifera) and bumble bee (Bombus impatiens) over an 8-day sampling period. Numerical values are expressed as mean percentages of pollen grains carried within the corbicula of each species over eight days. References refer to whether the flowering plant associated pollen taxa is a common nectar and/or pollen resource for insect pollinators
表 1.在 8 天的采样期内从蜜蜂 (Apis mellifera) 和大黄蜂 (Bombus impatiens) 的球蚴中量化的花粉类群总数。数值表示为八天内每个物种的球核内携带的花粉粒的平均百分比。参考文献涉及与开花植物相关的花粉类群是否是昆虫传粉媒介的常见花蜜和/或花粉资源
▲Drying kiwi pollen
Fig. 3.Corbicular pollen of A) honey bees (Apis mellifera) and B) bumble bees (Bombus impatiens) representing the top ten plant species visited. Relative abundances of corbicular pollen were averaged over the eight-day sampling period.
图 3.A) 蜜蜂 (Apis mellifera) 和 B) 熊蜂 (Bombus impatiens) 的球状花粉，代表了参观的前十种植物物种。球状花粉的相对丰度是八天采样期间的平均值。
▲刚采摘的猕猴桃公花，拿回去开始制花粉了
A significant difference was observed between the mean relative abundance of kiwifruit pollen carried in the corbicula of A. mellifera and B. impatiens over the eight days (z = 5.04, P < 0.0001), with B. impatiens carrying on average 46% more kiwifruit pollen than A. mellifera. The relative abundance of kiwifruit pollen collected by A. mellifera foragers on each sampling day (9–13 April 2020) was statistically different (χ2 = 14.98, df = 7, P = 0.008), yet results from Dunn’s test failed to identify differences by date (Fig. 4). For B. impatiens, the relative abundance of kiwifruit pollen collected between days 1 and 8 (9 and 13 April 2020, respectively) was statistically different (χ2 = 17.99, df = 7, P = 0.0003), but no other sampling dates were statistically different from one another (Fig. 4). Lastly, no significant differences were observed in the relative abundance of kiwifruit pollen collected between the three sampling periods (morning, midday, and afternoon) for both A. mellifera (χ2 = 1.71, df = 2, P = 0.44) and B. impatiens (χ2 = 1.30, df = 2, P = 0.54).
▲准备烘干猕猴桃鲜花药
八天内，意大利蜜蜂和凤仙花的蚬中携带的猕猴桃花粉的平均相对丰度存在显着差异（z = 5.04，P < 0.0001），其中凤仙花平均多携带 46% 的猕猴桃花粉花粉比蜜蜂花粉好。猕猴桃采集者在每个采样日（2020 年 4 月 9 日至 13 日）采集的猕猴桃花粉相对丰度存在统计差异（χ2 = 14.98，df = 7，P = 0.008），但 Dunn 检验的结果未能通过以下方法识别差异：日期（图 4）。对于凤仙花，第1天和第8天（分别为2020年4月9日和13日）收集的猕猴桃花粉的相对丰度存在统计差异（χ2 = 17.99，df = 7，P = 0.0003），但其他采样日期没有统计差异各不相同（图4）。最后，对于猕猴桃（χ2 = 1.71，df = 2，P = 0.44）和凤仙花来说，三个采样周期（上午、中午和下午）采集的猕猴桃花粉相对丰度没有显着差异。 （χ2 = 1.30，df = 2，P = 0.54）。
▲collecting kiwi anthers
Fig. 4.Percent relative abundance ± (s.e.m) of Actinidia chinensis var. chinensis ‘AU Gulf Coast Gold’ kiwifruit pollen per day averaged across each sampling period (morning, midday, and afternoon) for honey bee (Apis mellifera) and bumble bee (Bombus impatiens) during an 8-day sampling period in April of 2020. All pollen was quantified from corbicular pollen. A significant difference was observed between the mean relative abundance of kiwifruit pollen carried in the corbicula of A. mellifera and B. impatiens over the eight days (P < 0.0001). For each bee species, days with different letters (uppercase for B. impatiens, lowercase for A. mellifera) represent a significant difference in the relative abundance of kiwifruit pollen collected each day at P≤0.05.
图4 中华猕猴桃的相对丰度百分比±(s.e.m) 2020 年 4 月为期 8 天的采样期间，蜜蜂 (Apis mellifera) 和大黄蜂 (Bombus impatiens) 在每个采样周期（上午、中午和下午）的 chinensis 'AU Gulf Coast Gold' 奇异果花粉平均值。所有花粉均根据球状花粉进行定量。八天内，意大利蜜蜂和凤仙花的蚬中携带的猕猴桃花粉的平均相对丰度存在显着差异（P < 0.0001）。对于每种蜂种，不同字母的天数（凤仙花大写，意大利蜜蜂小写）代表每天采集的猕猴桃花粉相对丰度存在显着差异（P≤0.05）。
▲Collect kiwi blossoms
Exclusion Study
Overall, 1,674 kiwifruit flowers were included in the fruit quantity assessments, with 436, 442, 364, and 432 flowers utilized for the Insect Pollination, Wind Pollination, Artificial Pollination, and Pollen Exclusion treatments, respectively. Several kiwifruit vines were damaged during the growing season, thus a total of 114, 113, 106, and 109 kiwifruit plants were utilized for each pollination treatment (Insect Pollination, Wind Pollination, Artificial Pollination, and Pollen Exclusion, respectively). At four weeks post full bloom, the mean percentage of kiwifruit flowers that set fruit was statistically different among the four pollination treatments (χ2 = 338.2, df = 3, P < 0.0001). Artificial Pollination resulted in the greatest percent fruit set per flower (97.4%) with Insect Pollination (3.1%), Wind Pollination (0.4%), and Pollen Exclusion (0.4%) treatments not differing statistically from each other (P > 0.05, Dunn’s test) (Fig. 5). At harvest, the percentage of kiwifruit flowers that produced mature kiwifruits was significantly different among the four pollination treatments (χ2 = 297.5, df = 3, P < 0.0001). Artificial Pollination resulted in the greatest percentage of mature fruits per flower (76.0%), with Insect Pollination (2.8%), Wind Pollination (0.5%), and Pollen Exclusion (0.2%) treatments not being statistically different from one another (P > 0.05, Dunn’s test) (Fig. 6). Overall, 32 mature kiwifruits were included in the fruit quality assessments, with 12 and 20 fruits utilized for the Insect Pollination and Artificial Pollination treatments, respectively. Artificial Pollination resulted in the greatest fruit weights (z = 4.3, P < 0.0001), fruit size (FSI) (z = 4.3, P < 0.0001), and seed counts (z = 4.7, P < 0.0001) per fruit compared to the Insect Pollination treatment (Table 2).
▲Kiwi male flower
排除研究
总体而言，1674朵猕猴桃花被纳入果实数量评估，其中436、442、364和432朵花分别用于昆虫授粉、风授粉、人工授粉和花粉排除处理。在生长季节，几株猕猴桃藤蔓受损，因此每种授粉处理（分别为昆虫授粉、风授粉、人工授粉和花粉排除）共使用了114、113、106和109株猕猴桃植物。在盛花后四周，四种授粉处理的猕猴桃落果平均百分比存在统计学差异（χ2=338.2，df=3，P<0.0001）。人工授粉导致每朵花的坐果率最高（97.4%），昆虫授粉（3.1%）、风授粉（0.4%）和花粉排除（0.4%）处理在统计学上没有差异（P>0.05，Dunn检验）（图5）。收获时，四种授粉处理产生成熟猕猴桃的猕猴桃花百分比存在显著差异（χ2=297.5，df=3，P<0.0001）。人工授粉导致每朵花成熟果实的百分比最高（76.0%），昆虫授粉（2.8%）、风授粉（0.5%）和花粉排除（0.2%）处理彼此之间没有统计学差异（P>0.05，Dunn检验）（图6）。总体而言，32个成熟猕猴桃被纳入果实质量评估，其中12个和20个分别用于昆虫授粉和人工授粉处理。与昆虫授粉处理相比，人工授粉导致每个果实的最大果实重量（z=4.3，P<0.0001）、果实大小（FSI）（z=4.4，P<0.00001）和种子数量（z=4.7，P<0.0011）（表2）。
▲Collect kiwi blossoms
Table 2.Open in new tabSummary of kiwifruit quality variables for Actinidia chinensis var. chinensis ‘AU Gulf Coast Gold’
Variable Pollen Exclusion N Wind Pollination N Insect Pollination N Artificial Pollination N
Weight (g) 9.0 (NA) 1 31.5 (23.5) 2 21.2 (6.6)b 12 85.5 (4.7)a 20
Fruit size index (mm) 24.6 (NA) 1 35.5 (10.8) 2 29.6 (3.0)b 12 52.0(0.3)a 20
Seed count 3.0 (NA) 1 250.5 (217.5) 2 39.8 (19.4)b 12 573.9 (31.8)a 20
Mean numbers (±SE) for each variable followed by different letters (a or b) are significantly different at (P < 0.05) among Insect Pollination and Artificial Pollination treatments. The Pollen Exclusion treatment represented flowers that were completely isolated from pollen, the Wind Pollination treatment represented pollen vectored by wind only, Insect Pollination represented pollen vectored via insects and wind, and the Artificial Pollination treatment represented pollen vectored via a hand puffer only. Only the Insect Pollination and Artificial Pollination treatments were included in the statistical analysis due to small samples sizes observed in the Pollen Exclusion and Wind Pollination treatments (1 and 2, respectively).
▲picking kiwi flowers
表 2. 在新选项卡中打开猕猴桃品质变量摘要。 chinensis ‘AU 墨西哥湾黄金’
可变花粉排除 N 风授粉 N 昆虫授粉 N 人工授粉 N
重量 (g) 9.0 (NA) 1 31.5 (23.5) 2 21.2 (6.6)b 12 85.5 (4.7)a 20
果实大小指数 (mm) 24.6 (NA) 1 35.5 (10.8) 2 29.6 (3.0)b 12 52.0(0.3)a 20
种子数 3.0 (NA) 1 250.5 (217.5) 2 39.8 (19.4)b 12 573.9 (31.8)a 20
每个变量后跟不同字母（a 或 b）的平均数 (±SE) 在昆虫授粉和人工授粉处理之间显着不同 (P < 0.05)。花粉排除处理代表与花粉完全隔离的花朵，风授粉处理代表仅通过风传播的花粉，昆虫授粉代表通过昆虫和风传播的花粉，人工授粉处理代表仅通过手动吹管器传播的花粉。由于在花粉排除和风授粉处理（分别为 1 和 2）中观察到的样本量较小，因此仅将昆虫授粉和人工授粉处理纳入统计分析中。 Fig. 5.Mean percentage ± (s.e.m) of Actinidia chinensis var. chinensis ‘AU Gulf Coast Gold’ kiwifruit flowers that set fruit at four weeks post peak bloom (11 May 2020) for the four pollination treatments (Insect Pollination, Wind Pollination, Artificial Pollination, and Pollen Exclusion). Different letters above each column indicate a significant difference in treatment means at P = 0.05. 图5 中华猕猴桃的平均百分比±(s.e.m) chinensis ‘AU Gulf Coast Gold’奇异果花，在四种授粉处理（昆虫授粉、风授粉、人工授粉和花粉排除）的盛花期（2020 年 5 月 11 日）后四个星期结果。每列上方的不同字母表示处理平均值在 P = 0.05 时存在显着差异。
▲picking kiwi flowers
Fig. 6.Mean percentage ± (s.e.m) of Actinidia chinensis var. chinensis ‘AU Gulf Coast Gold’ kiwifruit flowers that produced mature kiwifruits per flower at harvest (September 2020) for the four pollination treatments (Insect Pollination, Wind Pollination, Artificial Pollination, and Pollen Exclusion). Different letters above each column indicate a significant difference in treatment means at P = 0.05. 图6 中华猕猴桃的平均百分比±(s.e.m) chinensis ‘AU Gulf Coast Gold’奇异果花，在收获时（2020 年 9 月）每朵花都结出成熟的奇异果，用于四种授粉处理（昆虫授粉、风授粉、人工授粉和花粉排除）。每列上方的不同字母表示处理平均值在 P = 0.05 时存在显着差异。
▲purifying kiwi pollen
Discussion Determining the contribution of managed bees to the pollination of kiwifruit grown within the southeastern United States is an important step in developing a pollination management strategy that will increase kiwifruit yields while decreasing pollination costs. Results from both our palynology and exclusion experiments support the idea that insect pollinators contributed minimally to the pollination of kiwifruit. Despite optimal orchard conditions (abundance of male and female blooms coinciding with one another, abundance of foraging bumble bees and honey bees, and favorable weather conditions) for the facilitation of cross-pollination, we still observed statistically lower percentages of fruit set, and fruit quality in insect-pollinated female kiwifruit flowers compared to flowers that were artificially pollinated. For this reason, we are confident that sufficient opportunities for pollination existed during the experiment and believe the low fruit set, and fruit quality associated with the Insect Pollination treatment was a result of pollinator limitation rather than pollen limitation. Furthermore, adequate levels of male pollen were available for cross-pollination during the ‘AU Gulf Coast Gold’ bloom, yet both managed bee species were drawn to competing non-kiwifruit blooms. As a result, orchard managers should put more emphasis on artificial pollination to achieve high fruit yields demanded by commercial kiwifruit operations. Kiwifruit exhibits a short bloom period (typically 1-2 weeks) and is, therefore, more susceptible to factors that could negatively impact pollination success such as pollen availability, pollinator abundance and activity, and adverse weather conditions during the bloom period (Clinch 1984, Testolin et al. 1991, Costa et al. 1993, Miñarro and Twizell 2015, Tacconi et al. 2016, Castro et al. 2021). To ensure adequate quantities of male kiwifruit pollen are available for cross-pollination, is it imperative that the bloom periods of at least one male kiwifruit pollinizer coincide with the female. For this reason, orchard managers in the southeastern United States plant multiple male pollinizers with staggered bloom periods alongside a single female cultivar to ensure the availability of pollen over a longer period. In April 2020, ‘AU Gulf Coast Gold’ reached peak bloom on 10 April and ‘CK3’ reached peak bloom approximately 3 days earlier. Although the peak bloom periods of ‘AU Gulf Coast Gold’ and ‘CK3’ were misaligned by several days, a high abundance of ‘CK3’ flowers, and thus male pollen, were present during the ‘AU Gulf Coast Gold’ bloom. ‘AU Golden Tigers’ peak bloom period often coincides with the ‘AU Gulf Coast Gold’ bloom, and for this reason, the cultivar has been referred to as the best male pollinizer for ‘AU Gulf Coast Gold’ (Dozier et al. 2011, Spiers et al. 2018). In April 2020, we observed the bloom periods of ‘AU Gulf Coast Gold’ and ‘AU Golden Tiger’ coinciding, thus providing an abundance of male kiwifruit pollen for cross-pollination. In our study orchard, the ‘Chieftain’ bloom period often lags ‘AU Gulf Coast Gold’ and ‘CK3’. In April 2020 ‘Chieftain’ began blooming in the last 2-3 days of the ‘AU Gulf Coast Gold’ bloom period, providing pollen in the latter portion of the ‘AU Gulf Coast Gold’ bloom. Due to the abundance of male kiwifruit flowers of several male pollinizers during the ‘AU Gulf Coast Gold’ bloom period, we believe an adequate quantity of male pollen was available within the orchard to facilitate cross-pollination via wind and/or insect pollinators. Because we observed low fruit set associated with the wind and insect-pollinated treatments when an abundance of male flowers was present during the ‘AU Gulf Coast Gold’ bloom, fruit set could have been influenced by other factors such as low pollinator abundance and activity or poor weather conditions during the bloom period.
▲Collect kiwi blossoms
讨论 确定管理蜜蜂对美国东南部种植的猕猴桃授粉的贡献是制定授粉管理策略的重要一步，该策略将提高猕猴桃产量，同时降低授粉成本。我们的孢粉学和排除实验的结果都支持昆虫传粉者对猕猴桃授粉贡献最小的观点。尽管有促进异花授粉的最佳果园条件（雄性和雌性花朵的丰富程度相互吻合，觅食的大黄蜂和蜜蜂的丰富程度，以及有利的天气条件），但我们仍然观察到，与人工授粉的花朵相比，昆虫授粉的雌性猕猴桃花朵的坐果率和果实质量在统计学上较低。因此，我们相信在实验期间存在足够的授粉机会，并认为与昆虫授粉处理相关的低坐果率和果实质量是传粉者限制的结果，而不是花粉限制的结果。此外，在“非盟墨西哥湾沿岸黄金”开花期间，有足够水平的雄性花粉可用于异花授粉，但两种受管理的蜜蜂都被竞争的非猕猴桃开花所吸引。因此，果园管理者应更加重视人工授粉，以实现商业猕猴桃运营所需的高水果产量。 猕猴桃的花期较短（通常为1-2周），因此更容易受到可能对授粉成功产生负面影响的因素的影响，如花粉可用性、传粉者丰度和活动，以及花期的不利天气条件（Clinch 1984，Testolin等人1991，Costa等人1993，Miñarro和Twizell 2015，Tacconi等人2016，Castro等人2021）。为了确保有足够数量的雄性猕猴桃花粉可用于异花授粉，至少一个雄性猕猴桃花粉器的花期必须与雌性猕猴桃花粉器重合吗。出于这个原因，美国东南部的果园管理者在一个雌性品种旁边种植了多个开花期交错的雄性花粉器，以确保花粉在更长时间内的可用性。2020年4月，“AU墨西哥湾沿岸黄金”在4月10日达到峰值，“CK3”在大约3天前达到峰值。尽管“非盟墨西哥湾沿岸黄金”和“CK3”的开花高峰期错开了几天，但在“非盟海湾海岸黄金”开花期间，出现了大量的“CK 3”花，从而产生了雄性花粉AU Golden Tigers的开花高峰期通常与“AU墨西哥湾沿岸黄金”的开花期相吻合，因此，该品种被称为“AU墨西哥海岸黄金”的最佳雄性授粉品种（Dozier等人，2011年；Spiers等人，2018年）。2020年4月，我们观察到“AU墨西哥湾沿岸黄金”和“AU金虎”的花期重合，从而为异花授粉提供了丰富的雄性猕猴桃花粉。在我们的研究果园中，“酋长”开花期往往落后于“澳大利亚墨西哥湾沿岸黄金”和“CK3”。2020年4月，“酋长”在“非盟墨西哥湾沿岸黄金”开花期的最后2-3天开始开花，为“非盟海湾海岸黄金”开花的后期提供花粉。由于在“澳大利亚墨西哥湾沿岸黄金”开花期，几种雄性授粉者的雄性猕猴桃花数量丰富，我们认为果园内有足够数量的雄性花粉，可以通过风媒和/或昆虫授粉者促进异花授粉。因为我们观察到，在“澳大利亚墨西哥湾沿岸黄金”开花期间，当存在大量雄花时，与风和昆虫授粉处理相关的低坐果率，坐果率可能受到其他因素的影响，如开花期间传粉者丰度和活动低或天气条件恶劣。
▲Kiwi insect pollination
High pollinator diversity and abundance are associated with fruit set and size equal to optimal pollination methods (artificial pollination) (Sharma et al. 2013, Miñarro and Twizell 2015), yet is dependent upon the pollinator community within any given orchard and surrounding habitat. A study conducted in 2019 and 2020 with our experimental kiwifruit orchard documented few pollinating insects visiting kiwifruit blooms (Abbate et al. 2021). Additionally, due to the low numbers of pollinators visiting the kiwifruit blooms, documenting the movement of pollinators between male and female flowers was not possible. Because of the low diversity and abundance of pollinating insects visiting kiwifruit blooms within our study orchard, the implementation of other forms of pollination including the stocking of managed bees must be considered to achieve high levels of pollination success (Castro et al. 2021). During the kiwifruit bloom period, weather conditions were optimal (average temperatures and wind speeds of 20°C, 1.9 ms, respectively) to facilitate cross-pollination via wind and insects, yet we still observed low fruit set in the wind pollination and insect pollination treatments. For the case of wind pollination, it could be that for the cultivar A. chinensis var. chinensis ‘AU Gulf Coast Gold’ pollination via wind is ineffective to produce fruits compared to other kiwifruit cultivars. In the case of insect pollination, the foraging behavior of managed bees while stocked for crop pollination has been shown to be influenced by poor weather conditions and competing blooms (Rogers et al. 2013, Miñarro and Twizell 2015, Castro et al. 2021). Apis mellifera workers are prone to inactivity during periods of cooler temperatures and periods of high winds and rain, whereas B. impatiens workers readily forage in adverse weather conditions (Rogers et al. 2013). In our study, each day except for 12 April 2020 (which experienced rain) had optimal weather conditions for foraging bees (Tan et al. 2012), and we observed both managed bee species to be highly active during the bloom period (frequently leaving and returning to their colonies). For this reason, and based upon our palynological results, we believe the foraging behaviors of both managed bee species were impacted by competing non-kiwifruit blooms rather than poor weather conditions. Apis mellifera and B. impatiens are both floral generalists (Ascher and Pickering 2020) and collected pollen from a diversity of flowering plant species growing within the kiwifruit orchard and surrounding areas. In the spring in central Alabama, many flowering plant species are in bloom and A. mellifera foragers were likely utilizing non-kiwifruit blooms for floral resources. Our palynological results showed five pollen types (A. chinensis, Brassica, Rhus, Rosaceae, and Trifolium/Melilotus) occurred in higher quantities (>10% mean relative abundance) in the corbicula of A. mellifera foragers than other pollen types. Except for A. chinensis, each of these plant taxa produces copious amounts of nectar and is often visited by honey bees (Free and Nuttall 1968, Crane 1975, Craig et al. 1988, Greco et al. 1996, Pomeroy and Fisher 2002, Abbate et al. 2021). We also visually observed A. mellifera foragers visiting the flowers of white clover (Trifolium repens L.) and wild radish (Raphanus raphanistrum L.) within the orchard during the ‘AU Gulf Coast Gold’ bloom period, supporting the idea that they were drawn to competing flowering plants. Lastly, while netting foragers, we also observed few (approximately 10%) A. mellifera foragers returning to their colonies with corbicular pollen loads, indicating they were likely visiting non-kiwifruit blooms for nectar rewards rather than pollen rewards. For the foragers that did contain corbicular pollen loads, only 21.3% of the pollen loads comprised kiwifruit pollen. Compare this figure to the relative abundance of almond pollen (74–96%) and blueberry pollen (57–80%) collected by A. mellifera foragers while stocked within commercial almond orchards and blueberry fields (Webster et al. 1985, Degrandi-Hoffman et al. 1992, Hoffman et al. 2018).
Bombus impatiens appeared to concentrate their foraging efforts on A. chinensis during the kiwifruit bloom period, but also visited three non-kiwifruit blooms (Nyssa, Rhus, and Rosaceae) to a greater degree than other flowering plants; all of which produce large quantities of nectar (Pellett 1920, Ramsay 1987, Maxwell and Knapp 2012). Although foragers of B. impatiens contained relatively high mean percentages of kiwifruit pollen within their corbicula compared to A. mellifera, the current recommended stocking rates of B. impatiens for kiwifruit pollination in the southeastern United States appear inadequate. These results aligned well with previous studies (Clinch 1984, Pomeroy and Fisher 2002, Lee et al. 2019). For example, Clinch (1984) determined honey bee foragers were the most abundant floral visitor to kiwifruit blooms in New Zealand yet were influenced by the presence of competing non-kiwifruit blooms. This same study also concluded that bumble bees were observed visiting kiwifruit blooms when male and female cultivars bloomed concurrently; however, their abundance was too low to contribute to kiwifruit pollination. In another study, Lee et al. 2019 investigated the foraging activity and pollination effects of A. mellifera and B. terrestris while stocked in net house units for kiwifruit pollination. Lee et al. 2019 observed B. terrestris workers returning to their hives had corbicular pollen loads that were approximately 3 times greater than that of A. mellifera. Despite this, fruit set and fruit quality were greater in A. mellifera pollinated kiwifruits, presumably due to the greater number of workers associated with A. mellifera colonies compared to B. terrestris. Similarly, Pomeroy and Fisher (2002) compared the pollination contribution of the same managed bee species (A. mellifera and B. terrestris) to kiwifruit within a New Zealand kiwifruit orchard (not within an enclosed space) and concluded that although B. terrestris displayed a greater fidelity toward the collection of kiwifruit pollen than A. mellifera, they were also influenced by the presence of competing non-kiwifruit flowering plants.
Despite stocking both managed bee species within the kiwifruit orchard at recommended rates, their combined contribution to kiwifruit was also minimal. The Artificial Pollination treatment yielded the greatest percentage of fruits per flower compared to all other pollination treatments (Insect Pollination, Wind Pollination, and Pollen Exclusion), and artificial pollination resulted in a 19-fold increase in fruit set at 4 weeks post-bloom and a 15-fold increase in mature fruit at harvest compared to the Insect Pollination treatment. Additionally, artificially pollinating ‘AU Gulf Coast Gold’ flowers resulted in statistically larger fruit weights and sizes, and contained a statistically greater number of seeds compared to insect pollinated flowers. Stocking B. impatiens colonies at higher rates within the kiwifruit orchard to increase fruit set and quality is not the optimal solution for pollination management in the southeastern United States, as stocking rates would need to be extremely high and would likely be too costly to match the fruit set, yields and fruit quality that artificial pollination provides (Herbertsson et al. 2016, Mallinger and Prasifka 2017, Lee et al. 2019). The potential economic losses associated with pollination limitation or failure justify the implementation of artificial pollination. Some studies have documented the combined benefits of implementing both insect pollination and artificial pollination as a pollination strategy to optimize fruit set and fruit quality (Lee et al. 2019, Castro et al. 2021), yet within our study orchard, implementing multiple pollination methods on a single crop to ensure high yields is costly, and may not be necessary. Although artificial pollination of kiwifruit is costly, it can offer many advantages over natural pollination (Castro et al. 2021, Wurz et al. 2021). Artificial pollination gives orchard managers more control of the timing and ensures adequate pollination of their crops (Oronje et al. 2012, Brantley et al. 2019). Both factors ensure consistent levels of yields and profits (Silveira et al. 2012), while simultaneously decreasing the number of aborted kiwifruits (Abbate et al. 2021). Artificial pollination, when implemented correctly, can provide the kiwifruit grower with pollination security when managed and wild insects cannot.
In conclusion, this study demonstrated that managed A. mellifera and B. impatiens foragers were inefficient pollinators of kiwifruit, most likely because they were drawn to competing non-kiwifruit blooms. Additionally, although B. impatiens collected greater mean relative abundances of kiwifruit pollen than A. mellifera, the small colony sizes associated with B. impatiens might explain why we observed low fruitset in the Insect Pollination treatment compared to the Artificial Pollination treatment. This also highlights that the current recommended stocking rates of B. impatiens are inadequate for kiwifruit pollination in the southeastern United States. Overall, artificial pollination provided the only commercially viable level of fruit set and yield. Therefore, based on our results, kiwifruit producers experiencing similar environmental conditions to us should put more emphasis on artificial pollination to pollinate their crops and increase profits. Due to the time and costs associated with hand pollination, future studies should comparatively evaluate the pollination efficiency and economics of other methods of artificial pollination (e.g., spraying dry pollen via boom sprayers) to hand pollination, as boom spraying requires less time, is less expensive for applying pollen, yet is often less efficient.
传粉媒介的多样性和丰度与最佳授粉方法（人工授粉）的坐果和大小有关（Sharma等人，2013年，Miñarro和Twizell，2015年），但取决于任何给定果园和周围栖息地内的传粉媒介群落。2019年和2020年，我们在实验猕猴桃园进行的一项研究记录了很少有授粉昆虫访问猕猴桃花（Abbate等人，2021）。此外，由于访问猕猴桃花朵的传粉者数量较少，无法记录传粉者在雄花和雌花之间的移动。由于我们研究果园内访问猕猴桃花的授粉昆虫的多样性和丰度较低，必须考虑实施其他形式的授粉，包括饲养管理蜜蜂，以实现高水平的授粉成功（Castro等人，2021）。在猕猴桃开花期间，天气条件最佳（平均温度和风速分别为20°C和1.9 ms），有助于通过风和昆虫进行异花授粉，但我们仍然观察到风授粉和昆虫授粉处理的坐果率较低。对于风媒传粉的情况，可能是因为与其他猕猴桃品种相比，通过风媒授粉的中华猕猴桃品种“AU墨西哥湾沿岸黄金”不能产生果实。在昆虫授粉的情况下，管理蜜蜂在为作物授粉而饲养时的觅食行为已被证明受到恶劣天气条件和竞争性花朵的影响（Rogers等人，2013年；Miñarro和Twizell，2015年；Castro等人，2021年）。意大利蜜蜂的工蜂在较冷的温度和大风大雨期间容易不活动，而B.凤仙的工蜂则容易在恶劣的天气条件下觅食（Rogers等人，2013）。在我们的研究中，除了2020年4月12日（经历了降雨）外，每天都有觅食蜜蜂的最佳天气条件（Tan等人，2012），我们观察到这两种受管理的蜜蜂在开花期都非常活跃（经常离开和返回它们的蜂群）。因此，根据我们的孢粉学结果，我们认为这两种受管理蜜蜂的觅食行为都受到了竞争性非猕猴桃开花的影响，而不是恶劣天气条件的影响。
意大利蜜蜂和凤仙花都是花卉多面手（Ascher和Pickering 2020），从猕猴桃园和周边地区生长的多种开花植物中收集花粉。在阿拉巴马州中部的春天，许多开花植物物种都在开花，意大利蜜蜂的觅食者可能会利用非猕猴桃的花朵作为花卉资源。我们的孢粉学结果表明，五种花粉类型（A.chinensis、Brassica、Rhus、Rosaceae和Trifolium/Melilotus）在A.mellifera觅食者的球茎中的数量（平均相对丰度>10%）高于其他花粉类型。除A.chinensis外，这些植物类群中的每一个都能产生大量的花蜜，经常被蜜蜂造访（Free和Nuttall 1968，Crane 1975，Craig等人1988，Greco等人1996，Pomeroy和Fisher 2002，Abbate等人2021）。我们还直观地观察到，在“澳大利亚墨西哥湾沿岸黄金”开花期间，意大利蜜蜂觅食者在果园里参观白三叶（白三叶）和野生萝卜（萝卜）的花朵，这支持了他们被竞争开花植物所吸引的观点。最后，在捕捉觅食者的同时，我们还观察到很少（约10%）的意大利蜜蜂觅食者带着球茎花粉回到它们的群落，这表明它们可能是为了花蜜奖励而不是花粉奖励而访问非猕猴桃花朵。对于确实含有球茎花粉负载的觅食者来说，只有21.3%的花粉负载包含猕猴桃花粉。将这一数字与意大利蜜蜂采集者在商业杏仁园和蓝莓田中采集的杏仁花粉（74-96%）和蓝莓花粉（57-80%）的相对丰度进行比较（Webster等人，1985年；Degrandi Hoffman等人，1992年；Hoffman等，2018年）。
在猕猴桃开花期间，不耐烦Bombus似乎将觅食精力集中在A.chinensis上，但也比其他开花植物更频繁地访问了三种非猕猴桃开花植物（Nyssa、Rhus和Rosaceae）；所有这些都会产生大量的花蜜（Pellett 1920，Ramsay 1987，Maxwell和Knapp 2012）。尽管与意大利蜜蜂相比，凤仙花的觅食者在其球茎中含有相对较高的猕猴桃花粉平均百分比，但目前美国东南部推荐的凤仙花猕猴桃授粉放养率似乎不足。这些结果与之前的研究一致（Clinch 1984，Pomeroy和Fisher 2002，Lee等人2019）。例如，Clinch（1984）确定，蜜蜂觅食者是新西兰猕猴桃花最丰富的花卉访客，但也受到竞争性非猕猴桃花的影响。这项研究还得出结论，当雄性和雌性品种同时开花时，观察到大黄蜂造访猕猴桃花朵；然而，它们的丰度太低，无法促进猕猴桃授粉。在另一项研究中，Lee等人在2019年调查了意大利蜜蜂和陆地蜜蜂在用于猕猴桃授粉的网房单元中的觅食活动和授粉效果。Lee等人在2019年观察到，回到蜂箱的B.terrestris工蜂的球茎花粉量大约是A.mellifera的3倍。尽管如此，A.mellifera授粉的猕猴桃的坐果率和果实质量更高，这可能是由于与B.terrestris相比，A.melifera群体的工人数量更多。同样，Pomeroy和Fisher（2002）比较了同一管理蜂种（A.melliferia和B.terrestri）在新西兰猕猴桃园（不是在封闭空间内）内对猕猴桃的授粉贡献，并得出结论，尽管B.terrestrris对猕猴桃花粉的收集表现出比A.melliferas更大的忠诚度，但它们也受到了竞争性非猕猴桃开花植物的影响。
尽管以推荐的速度在猕猴桃园中饲养了这两种受管理的蜜蜂，但它们对猕猴桃的贡献也很小。与所有其他授粉处理（昆虫授粉、风授粉和花粉排除）相比，人工授粉处理每朵花的果实百分比最高，人工授粉导致开花后4周的坐果率增加了19倍，收获时的成熟果实增加了15倍。此外，与昆虫授粉的花朵相比，人工授粉的“非盟墨西哥湾沿岸黄金”花朵在统计上导致了更大的果实重量和大小，并且含有更多的种子。在美国东南部，以更高的速度在猕猴桃园中储存B.impatius菌落以增加坐果率和质量并不是授粉管理的最佳解决方案，因为储存率需要非常高，而且可能成本过高，无法与人工授粉提供的坐果率、产量和果实质量相匹配（Herbertsson等人，2016年；Mallinger和Prasifka，2017年；Lee等人，2019年）。与授粉限制或失败相关的潜在经济损失证明了实施人工授粉的合理性。一些研究记录了将昆虫授粉和人工授粉作为优化坐果和果实质量的授粉策略的综合效益（Lee等人，2019年，Castro等人，2021年），但在我们的研究果园中，在单一作物上实施多种授粉方法以确保高产是昂贵的，可能不是必要的。尽管猕猴桃的人工授粉成本高昂，但与自然授粉相比，它可以提供许多优势（Castro等人，2021，Wurz等人，2021）。人工授粉使果园管理者能够更好地控制时间，并确保作物的充分授粉（Oronje等人，2012年；Brantley等人，2019年）。这两个因素确保了产量和利润的一致水平（Silveira等人，2012），同时减少了流产猕猴桃的数量（Abbate等人，2021）。人工授粉，如果实施得当，可以为猕猴桃种植者提供管理时的授粉安全，而野生昆虫则不能。
总之，这项研究表明，经过管理的意大利蜜蜂和凤仙花觅食者是猕猴桃的低效授粉者，很可能是因为它们被竞争的非猕猴桃花朵所吸引。此外，尽管B.凤仙花收集的猕猴桃花粉的平均相对丰度高于A.mellifera，但与B.凤仙花相关的小菌落大小可能解释了为什么我们在昆虫授粉处理中观察到的坐果率低于人工授粉处理。这也突显出，目前建议的B.impensis放养率不足以在美国东南部进行猕猴桃授粉。总体而言，人工授粉提供了唯一具有商业可行性的坐果率和产量。因此，根据我们的研究结果，经历与我们相似环境条件的猕猴桃生产商应该更加重视人工授粉，为作物授粉并增加利润。由于人工授粉的时间和成本，未来的研究应比较评估其他人工授粉方法（例如，通过臂式喷雾器喷洒干花粉）与人工授粉的授粉效率和经济性，因为臂式喷雾需要更少的时间，施用花粉的成本较低，但效率往往较低。
▲Bees pollinate kiwifruit
Acknowledgments
We thank Sophie Warny for all palynological quantifications, Jacob Kelley, Wesley Stone, and Katie Abbate for lab and field assistance, and Clint Wall from the Southeast Kiwifruit Farming Cooperative for the use of their kiwifruit orchard for this experiment. We thank Beth Redlin for their graphic design assistance.
Funding
This work was supported by the USDA National Institute of Food and Agriculture Multi-state Hatch project NC1173, the Alabama Agricultural Experiment Station, and a USDA Specialty Crop Block Grant Program (AL) – AM190100XXXXG055.
Author Contributions
AA: Conceptualization-Equal, Data curation-Lead, Formal analysis-Lead, Funding acquisition-Lead, Investigation-Lead, Methodology-Lead, Resources-Equal, Software-Equal, Supervision-Equal, Validation-Equal, Visualization-Equal, Writing – original draft-Lead, Writing – review & editing-Lead. JC: Conceptualization-Equal, Methodology-Equal, Supervision-Equal, Validation-Equal, Writing – review & editing-Supporting. GRW: Conceptualization-Equal, Funding acquisition-Equal, Supervision-Equal, Validation-Equal, Writing – review & editing-Equal.
Data Availability
The datasets generated during and/or analyzed during the current study are available from the corresponding author upon reasonable request.
致谢
我们感谢Sophie Warny提供的所有孢粉学定量，Jacob Kelley、Wesley Stone和Katie Abbate提供的实验室和现场协助，以及东南猕猴桃种植合作社的Clint Wall使用他们的猕猴桃园进行本次实验。我们感谢Beth Redlin的平面设计协助。
基金
这项工作得到了美国农业部国家食品和农业研究所多州孵化项目NC1173、阿拉巴马州农业实验站和美国农业部特种作物块拨款计划（AL）-AM190100XXXXG055的支持。
作者贡献
AA：概念化负责人、数据管理负责人、形式分析负责人、资金获取负责人、调查负责人、方法论负责人、资源负责人、软件负责人、监督负责人、验证负责人、可视化负责人、写作——初稿负责人、撰写——审查和编辑负责人。JC：概念平等、方法平等、监督平等、验证平等、写作-评论和编辑支持。GRW：概念化平等、资金获取平等、监督平等、验证平等、写作-评论和编辑平等。
数据可用性
在当前研究期间生成和/或分析的数据集可根据合理要求从相应作者处获得。
Journal of Economic Entomology 经济昆虫学期刊 第116卷第3期
2023年6月
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