對(duì)比昆蟲授粉 獼猴桃人工授粉效果更好

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. 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.

▲紅心獼猴桃▲

由于對(duì)美國(guó)境內(nèi)種植的獼猴桃品種的授粉要求缺乏了解，農(nóng)民同時(shí)采用多種授粉方法，例如租賃管理蜂種或人工授粉，以實(shí)現(xiàn)果實(shí)的高產(chǎn)。然而，實(shí)施多種授粉方法成本高昂，而且可能會(huì)導(dǎo)致資源利用效率低下。我們?cè)u(píng)估了兩種管理蜜蜂（意大利蜜蜂和鳳仙花熊蜂）對(duì)獼猴桃授粉的貢獻(xiàn)，方法是：i) 確定每種蜂種采集者收集的獼猴桃花粉的相對(duì)豐度；ii) 比較昆蟲和蜜蜂之間的坐果和果實(shí)質(zhì)量。通過昆蟲排除實(shí)驗(yàn)對(duì)花朵進(jìn)行人工授粉。在西方蜜蜂和鳳仙花的球核中攜帶的奇異果花粉的平均相對(duì)豐度之間存在顯著差異，鳳仙花攜帶的奇異果花粉平均比西方蜜蜂多 46%。與昆蟲授粉、風(fēng)授粉和花粉排斥處理相比，人工授粉的獼猴桃花在開花后四個(gè)星期和收獲時(shí)每朵花的果實(shí)數(shù)量顯著增加。與昆蟲授粉的花朵相比，人工授粉產(chǎn)生的果實(shí)重量、大小和種子數(shù)量都更大，而在花粉排除和風(fēng)授粉處理中產(chǎn)生的果實(shí)很少。與我們經(jīng)歷類似情況的獼猴桃生產(chǎn)者應(yīng)該專注于人工授粉他們的作物，而不是依靠管理或野生昆蟲為獼猴桃授粉。未來的研究應(yīng)該評(píng)估其他人工授粉方法，以確定其在美國(guó)境內(nèi)種植的獼猴桃授粉中的有效性、效率和經(jīng)濟(jì)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).

▲陽光金果g3獼猴桃

介紹
獼猴桃是一種雌雄異株的果實(shí)植物，雄花和雌花分別開在不同的植株上。雄花產(chǎn)生有活力的花粉，但缺乏發(fā)育的子房、胚珠和花柱（Beutel 1990，F(xiàn)erguson 1999），雌花雖然完美，但缺乏有活力的花粉。因此，雌花需要花粉載體來產(chǎn)生種子和果實(shí)（Beutel 1990，F(xiàn)erguson 1999）。獼猴桃的大小取決于柱頭上沉積的有活力的花粉粒的數(shù)量，以及產(chǎn)生的種子的數(shù)量（Goodwin 等人，2013）。為了獲得高質(zhì)量的適銷水果（>100克和>1,000粒種子），一朵雌花的柱頭必須接收2,000-3,000個(gè)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）。在獼猴桃中，由于花粉（花粉數(shù)量或質(zhì)量不足）或傳粉媒介（授粉媒介訪問次數(shù)較少或每次訪問沉積的花粉較少）限制而導(dǎo)致授粉效率低下（Ashman 等人，2004 年），可能會(huì)導(dǎo)致坐果率低以及大小、形狀不理想。和均勻性（Tacconi 等人，2016）。因此，獼猴桃種植者需要有效授粉的雌花以確保經(jīng)濟(jì)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, Mi?arro 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, Mi?arro and Twizell 2015, Somme et al. 2015, Abbate et al. 2021).

▲zespri redkiwi

人們認(rèn)為獼猴桃授粉主要由昆蟲提供，其次是風(fēng)（Testolin et al. 1991，Mi?arro and Twizell 2015）。研究表明，與昆蟲授粉的花朵相比，風(fēng)媒授粉的奇異果花產(chǎn)生的果實(shí)大小相似（Bellini 等，1989）；然而，大多數(shù)證據(jù)表明，風(fēng)授粉對(duì)于生產(chǎn)適銷規(guī)格的獼猴桃是無效的（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）。蜜蜂和熊蜂被認(rèn)為是獼猴桃最重要的傳粉者（Palmer-Jones and Clinch 1974，Matheson 1991a，1991b，Vaissière et al. 1996，Howpage et al. 2001）；然而，它們對(duì)獼猴桃授粉的貢獻(xiàn)一直存在爭(zhēng)議（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，Mi?arro and特威澤爾 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, Mi?arro 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

研究認(rèn)為，蜜蜂是奇異果的低效傳粉者，因?yàn)樗鼈儗?duì)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?yàn)樗鼈円酝瑫r(shí)開花的非奇異果植物為食，通常只收集 2-4% 的奇異果花粉（Ford 1971，Blanchet et al. 1990）。同樣，熊蜂（Bombus impatiens 和 Bombus terrestris L.（膜翅目：蜜蜂科））已被評(píng)估為獼猴桃的潛在傳粉者（Read et al. 1989，Mi?arro and Twizell 2015，Abbate et al. 2021），但已被證明受到可獲取的非獼猴桃花朵的極大影響，收集的獼猴桃花粉僅為 21%（Pomeroy 和 Fisher 2002）。在美國(guó)，尚無研究評(píng)估在商業(yè)獼猴桃果園中放養(yǎng)的蜜蜂和鳳仙花的覓食偏好以及對(duì)獼猴桃授粉的貢獻(xiàn)。由于關(guān)于風(fēng)和昆蟲對(duì)獼猴桃授粉的貢獻(xiàn)的不同結(jié)論，幾種人工授粉方法，即手動(dòng)或機(jī)械地將花粉施用到花的雌蕊上，已成為商業(yè)操作中的常見做法（Pinillos 和 Cuevas 2008）。當(dāng)自然授粉媒介不存在或不可靠時(shí)，人工授粉是補(bǔ)充或替代其他形式授粉的重要工具（Pritchard 和 Edwards 2006）。與風(fēng)授粉和昆蟲授粉一樣，獼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

由于獼猴桃授粉研究的結(jié)果各不相同，并且缺乏對(duì)美國(guó)境內(nèi)種植的獼猴桃品種的授粉要求的了解，果園管理者已轉(zhuǎn)向?qū)嵤┒喾N授粉方法，以確保水果的高產(chǎn)。例如，美國(guó)的果園管理者目前將雄性和雌性獼猴桃植株種植在彼此靠近的地方，以促進(jìn)通過風(fēng)進(jìn)行異花授粉，引入包括蜜蜂和大黃蜂在內(nèi)的受管理蜜蜂，以促進(jìn)花粉從雄花到雌花的移動(dòng)，并人工人工授粉。通過手工授粉和花粉噴粉器為農(nóng)作物授粉（Brantley et al. 2019）。實(shí)施多種授粉方法成本高昂、耗時(shí)，而且可能會(huì)導(dǎo)致資源利用效率低下。在美國(guó)東南部，一種成功的金肉雌性奇異果品種（A. chinensis var. chinensis ‘AU Gulf Coast Gold’）被開發(fā)出來并獲得商業(yè)運(yùn)營(yíng)專利，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

盡管常用于獼猴桃授粉，但美國(guó)尚無研究對(duì)管理蜜蜂和熊蜂的覓食偏好進(jìn)行比較評(píng)估，以確定它們對(duì)獼猴桃授粉的貢獻(xiàn)。因此，本研究的主要目標(biāo)是 i) 進(jìn)行孢粉學(xué)研究，比較在美國(guó)東南部典型的獼猴桃果園中飼養(yǎng)的受管理蜜蜂和大黃蜂的球囊花粉負(fù)荷中所含獼猴桃花粉的相對(duì)豐度，和 ii) 通過排除研究比較常見授粉方法（風(fēng)授粉、昆蟲授粉、人工授粉）對(duì)坐果和果實(shí)質(zhì)量（果實(shí)重量、果實(shí)大小和種子數(shù)量）的有效性，同時(shí)在獼猴桃內(nèi)放養(yǎng)蜜蜂和熊蜂果園。我們預(yù)測(cè)，由于奇異果開花和同時(shí)開花的非奇異果植物之間的吸引力不同，管理的蜜蜂和大黃蜂收集的奇異果花粉量會(huì)有所不同（Pomeroy 和 Fisher 2002）。我們還預(yù)測(cè)，與風(fēng)授粉和昆蟲授粉相比，人工授粉將導(dǎo)致最大的坐果率、果實(shí)大小和種子數(shù)量，因?yàn)榕c其他授粉方法相比，更多的花粉粒會(huì)沉積在雌花的柱頭上（Costa et al. 1993） ，岡薩雷斯等人，1994 年，1998 年，阿巴特等人，2021 年）。這項(xiàng)研究的結(jié)果將幫助美國(guó)果園管理者為獼猴桃產(chǎn)業(yè)制定科學(xué)支持的授粉管理計(jì)劃。

▲干燥獼猴桃花粉

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°35?N, long. 85°47?W). 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
實(shí)驗(yàn)設(shè)置
這項(xiàng)工作是在 2020 年奇異果生長(zhǎng)季節(jié)（4 月至 10 月）期間在位于阿拉巴馬州中部（阿拉巴馬州里爾敦；北緯 32°35，西經(jīng) 85°47）的一個(gè) 72.8 公頃（180 英畝）奇異果園內(nèi)進(jìn)行的。所有實(shí)驗(yàn)都集中在一個(gè) 5.5 公頃的區(qū)塊內(nèi)，其中行間距為 2.74 m。每行交替種植一個(gè)雌性金肉獼猴桃品種（Actinidia chinensis var. chinensis 'AU Gulf Coast Gold'）和三個(gè)雄性獼猴桃品種之一（A. chinensis var. chinensis 'AU Golden Tiger'、'CK3' 和 A . chinensis deliciosa 'Chieftain') 的雌雄比例達(dá)到 4:1。研究中選擇的所有植株均為 5 年齡，并按照商業(yè)奇異果生產(chǎn)推薦的最佳管理實(shí)踐進(jìn)行管理（Hasey 1994）。選擇雄性授粉者“AU Golden Tiger”、“CK3”和“Chieftain”是因?yàn)樗鼈兊谋缎运剑ā癆U Golden Tiger”和“Chieftain”均為六倍體，“CK3”為二倍體），并且因?yàn)槊總€(gè)雄性品種的花期經(jīng)常與“AU Gulf Coast Gold”的花期重疊（??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

為了確保每個(gè)獼猴桃品種大量開花，所有雌性獼猴桃植株在 2019 年 12 月至 2020 年 2 月期間進(jìn)行了冬季修剪（Strik 2005）。通過去除上一季節(jié)產(chǎn)生的約 70% 的木材來修剪雌性奇異果植株，將替換的藤蔓綁在棚架支撐上，并且雌性植株上的刺完好無損，因?yàn)楸娝苤鼈兎浅＝Y(jié)果子（Strik 2005）。冬季修剪有助于維持葡萄藤的框架、營(yíng)養(yǎng)生長(zhǎng)和果實(shí)生產(chǎn)之間的平衡，以及有效利用可用光線的樹冠（Strik 2005）。在上一季節(jié)的花期結(jié)束時(shí)，通過將枝條剪短至 15 至 30 厘米的長(zhǎng)度來修剪雄性植物，目的是產(chǎn)生盡可能多的花朵用于授粉（Luh 和 Wang 1984，Strik 2005）。雄性在冬季也會(huì)通過修剪枯死或弱的手杖進(jìn)行修剪，使手杖長(zhǎng)度在 1.0 至 1.5 m 之間（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

為了確保每個(gè)獼猴桃品種大量開花，所有雌性獼猴桃植株在 2019 年 12 月至 2020 年 2 月期間進(jìn)行了冬季修剪（Strik 2005）。通過去除上一季節(jié)產(chǎn)生的約 70% 的木材來修剪雌性奇異果植株，將替換的藤蔓綁在棚架支撐上，并且雌性植株上的刺完好無損，因?yàn)楸娝苤鼈兎浅＝Y(jié)果子（Strik 2005）。冬季修剪有助于維持葡萄藤的框架、營(yíng)養(yǎng)生長(zhǎng)和果實(shí)生產(chǎn)之間的平衡，以及有效利用可用光線的樹冠（Strik 2005）。在上一季節(jié)的花期結(jié)束時(shí)，通過將枝條剪短至 15 至 30 厘米的長(zhǎng)度來修剪雄性植物，目的是產(chǎn)生盡可能多的花朵用于授粉（Luh 和 Wang 1984，Strik 2005）。雄性在冬季也會(huì)通過修剪枯死或弱的手杖進(jìn)行修剪，使手杖長(zhǎng)度在 1.0 至 1.5 m 之間（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，購買了四只大黃蜂（Bombus impatiens）四群（總共 16 個(gè)蜂群；每個(gè)四群四個(gè)蜂群），每群重量在 832 至 845 克之間，包含約 200 名工蜂（BioBest，羅穆盧斯，密歇根州，美國(guó)）和 12 個(gè)蜂蜜蜜蜂 (Apis mellifera) 蜂群，每個(gè)蜂群至少由 10 框蜂巢、15 框成蟲（20,000 至 30,000 名工蜂）和 5 框從我們實(shí)驗(yàn)室獲得的食物組成，在開始前一天轉(zhuǎn)移到獼猴桃果園中華獼猴桃變種chinensis ‘AU Gulf Coast Gold’ 開花。中華獼猴桃的花期chinensis“AU Gulf Coast Gold”是根據(jù)上一季節(jié)的開花日期估算的（Abbate et al. 2021），并且因?yàn)榇苹ㄌ幱?7-10 天“爆米花”階段的后半部分（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.

▲獼猴桃對(duì)花授粉方法圖片

為了確保兩種管理蜂種的蜂群數(shù)量反映商業(yè)奇異果經(jīng)營(yíng)的常見放養(yǎng)率（Blanchet et al. 1990，Pomeroy and Fisher 2002），在整個(gè)區(qū)域建立了四個(gè)圓形 0.40 ha 地塊（每個(gè)地塊中心點(diǎn)之間 61 m）。獼猴桃塊的長(zhǎng)度（244 m）（圖1）。在每個(gè) 0.40 公頃的地塊內(nèi)，將 1 個(gè)鳳仙花菌落（相當(dāng)于 10 個(gè)菌落/公頃的放養(yǎng)率）放置在帶有塑料防雨罩的木托盤上，并在 3 個(gè) A. mellifera 菌落（相當(dāng)于 7 個(gè)菌落/公頃的放養(yǎng)率）上放置。放置在與鳳仙花托盤間隔 9.1 m 的單獨(dú)木托盤上。每個(gè)管理蜂種的放養(yǎng)率是美國(guó)東南部獼猴桃授粉目前使用的放養(yǎng)率。由于冰雹不是阿拉巴馬州獼猴桃種植者的主要擔(dān)憂，因此研究期間沒有使用防雹網(wǎng)，因此不會(huì)限制花粉通過風(fēng)或昆蟲傳播。

▲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

圖 1. 5.5 公頃獼猴桃區(qū)塊內(nèi) 4 個(gè)圓形 0.40 公頃試驗(yàn)田的朝向。每個(gè)地塊的中心彼此相距 61 m，并沿著 244 m 的橫斷面展開。每 0.40 公頃的地塊包含 1 個(gè)大黃蜂（Bombus impatiens），由 4 個(gè)蜂群（10 個(gè)蜂群/公頃）組成，3 個(gè)蜜蜂（Apis mellifera）蜂群（7 個(gè)蜂群/公頃），以及交替的雌性蜜蜂（總是中華獼猴桃變種）。 AU Gulf Coast Gold'）和雄性（A. chinensis var. chinensis 'AU Golden Tiger'、'CK3' 或 A. chinensis var. deliciosa 'Chieftain' 之一）獼猴桃行。

▲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

孢粉學(xué)研究
花粉采集。
2020年4月9日至2020年4月17日期間，對(duì)蜜蜂采食者的蜜蜂和鳳仙花進(jìn)行了采樣，以確定工蟻采食的開花植物。我們?cè)谏衔?[上午 0800 點(diǎn)至上午 1100 點(diǎn)]、中午 [上午 1100 點(diǎn)至下午 1400 點(diǎn)] 和下午 [上午 0800 點(diǎn)至上午 1100 點(diǎn)]、中午 [上午 1100 點(diǎn)至下午 1400 點(diǎn)] 和下午 [下午 1400 點(diǎn)至下午 1800 點(diǎn)]）（Azmi 等人，2015 年）。當(dāng)天氣條件適合對(duì)覓食蜜蜂進(jìn)行采樣時(shí)（微風(fēng)或無風(fēng)、云覆蓋率≤50%、溫度≥15°C），就會(huì)使用手網(wǎng)（Krahner et al. 2021）。 2020年4月9日至2020年4月17日，每天08:00至18:00從最近的氣象站（奧本大學(xué)EV史密斯研究中心）每隔半小時(shí)收集一次氣溫和風(fēng)力數(shù)據(jù)，該氣象站距離奇異果園約20公里。研究期間的最低、最高和平均氣溫以及風(fēng)速分別為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 (Rundl?f 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

最高氣溫和風(fēng)速一般出現(xiàn)在中午，最低氣溫和風(fēng)速一般出現(xiàn)在上午和下午。研究期間的天氣條件最適合蜜蜂覓食和獼猴桃通過風(fēng)授粉。蜜蜂的覓食行為在 10°C 左右時(shí)表現(xiàn)最佳（Tan 等人，2012 年），0 到 1.5 毫秒之間的風(fēng)速適合獼猴桃授粉實(shí)驗(yàn)（Costa 等人，1993 年）。在 8 天的時(shí)間里，每種蜂種總共捕獲了 384 只花粉采集者（每個(gè)物種每天每個(gè)采樣周期 16 只采集者）。將含有球狀花粉負(fù)載的網(wǎng)狀蜜蜂和鳳仙花覓食者暫時(shí)放置在單獨(dú)的 15 mL 塑料瓶中，并儲(chǔ)存在帶有冰袋的冷卻器中以麻醉它們。麻醉后，用鑷子非致命性地去除球狀花粉負(fù)載，讓蜜蜂恢復(fù)意識(shí)，毫發(fā)無傷地飛回蜂巢（Rundl?f et al. 2022）。為了防止蜂種、采樣日和采樣周期之間花粉的交叉污染，每個(gè)蜂種使用指定的網(wǎng)，并每天用肥皂水清洗。此外，僅使用新的 15 毫升塑料小瓶，并在每個(gè)采樣周期和每天之后用肥皂水清洗鑷子。每天，在處理之前，每個(gè)采樣周期的每個(gè)蜂種的球狀花粉都會(huì)被匯集起來。

▲Pollen Collection獼猴桃花采集

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獼猴桃花采集

孢粉學(xué)分析。
花粉樣品由 Global GeoLab Limited（加拿大艾伯塔省麥迪辛哈特）按照標(biāo)準(zhǔn)孢粉學(xué)實(shí)驗(yàn)室方法（Brown 2008）進(jìn)行化學(xué)處理，并在路易斯安那州立大學(xué)（美國(guó)路易斯安那州巴吞魯日）的 CENEX 實(shí)驗(yàn)室進(jìn)行定量。 Global Geolab Limited 收到的花粉樣品經(jīng)過脫水并溶解在冰醋酸中，并通過乙酰水解化學(xué)處理去除脂質(zhì)、蠟質(zhì)和細(xì)胞質(zhì)，以便更容易識(shí)別花粉粒。丙酮水解化學(xué)處理后，用乙醇和水漂洗花粉粒，然后懸浮在甘油中。將小瓶?jī)?nèi)容物徹底攪拌一分鐘，然后將一小滴懸浮液安裝在 75 × 25 mm 顯微鏡載玻片上，并用 18 × 18 mm #1 厚度的玻璃蓋玻片覆蓋。蓋玻片用透明指甲油密封以防止泄漏，并被送往 CENEX 實(shí)驗(yàn)室，在那里使用光學(xué)顯微鏡（奧林巴斯 BX41，日本東京）在 600 倍和 1,000 倍放大倍率下檢查顯微鏡載玻片，以識(shí)別花粉類型。使用防止花粉重復(fù)計(jì)數(shù)的橫移，對(duì)每個(gè)樣本至少計(jì)數(shù) 300 個(gè)已識(shí)別的花粉粒；初始計(jì)數(shù)完成后，對(duì)所有載玻片進(jìn)行掃描，尋找獨(dú)特且稀有類型的花粉，并將所有花粉鑒定為可能的最低分類組（即科、屬或種）（Ferguson 等人，2018）。由于難以區(qū)分雄性和雌性獼猴桃花粉粒，因此沒有單獨(dú)對(duì)它們進(jìn)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獼猴桃花采集

排除研究
學(xué)習(xí)設(shè)計(jì)。
進(jìn)行了一項(xiàng)排除研究，以評(píng)估管理的意大利蜜蜂和鳳仙花對(duì)中華蜜蜂的貢獻(xiàn)。 chinensis ‘AU Gulf Coast Gold’（以下簡(jiǎn)稱 AU Gulf Coast Gold）通過量化四種授粉處理中的坐果率進(jìn)行授粉：i) 昆蟲授粉，ii) 風(fēng)授粉，iii) 人工授粉，以及 iv) 花粉排除。該實(shí)驗(yàn)于 2020 年 4 月中旬開始，大約是 AU Gulf Coast Gold 花期前兩天。每個(gè)授粉處理對(duì)每株植物進(jìn)行一次，并在 114 株雌性獼猴桃植株上重復(fù)進(jìn)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

為了記錄管理蜜蜂和野生傳粉媒介對(duì) AU Gulf Coast Gold 授粉的貢獻(xiàn)，在昆蟲授粉處理中，在每株植物上使用標(biāo)記膠帶標(biāo)記一簇未開放的雌性花蕾（圖 2a）。在開花期間，受管理的蜜蜂花和鳳仙花以及任何野生傳粉者都可以自由地訪問標(biāo)記的花簇以獲得花粉獎(jiǎng)勵(lì)。對(duì)于風(fēng)授粉處理，我們使用 1,000 微米尼龍單絲網(wǎng)狀液體過濾袋（Cary Company 部件 #NMO1000P1DS，Addison，IL，USA）將每株植物的一簇未開放的雌性花蕾裝袋，以排除所有昆蟲，但允許被動(dòng)通過通過網(wǎng)袋收集花粉（Neal 和 Anderson 2004，McIver 和 Erickson 2012）。用標(biāo)記帶標(biāo)記含有花蕾簇的藤蔓，并記錄存在的花蕾數(shù)量（圖2b）。對(duì)于人工授粉處理，我們用 25 微米聚酯氈液體過濾袋（Cary Company 部件號(hào) #PES25P1DS，Addison，IL，USA）將每株植物的一簇未開放的雌性花蕾裝袋，以防止花粉通過昆蟲和風(fēng)傳播在開花期間到達(dá)雌性獼猴桃花蕾（圖2c）。為了確保奇異果花粉無法穿過 25 微米的袋子，遵循 Neal 和 Anderson (2004) 使用的方法來確認(rèn)奇異果花粉無法穿過材料。

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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

使用木制衣夾確保袋子開口和獼猴桃藤周圍緊密貼合（圖 2c）。由于與風(fēng)授粉處理中使用的 1,000 微米袋子相比，材料存在差異，因此僅 25 微米袋子需要使用衣夾。布蘭特利等人。 (2019)測(cè)定了中華白藜蘆醇的有效授粉期。 chinensis 大約在開花（開花）后 4 天。因此，人工授粉處理中的每個(gè)袋子每隔一天打開一次，檢查最近盛開的花朵。如果存在開放的花朵，則使用手動(dòng)噴槍（Antles Pollen Supplies, Inc., Modesto, CA, USA）噴灑兩股“Chieftain”花粉，以確保沉積足夠數(shù)量的花粉粒（Dozier 等，2015）。 2018）。使用 Kannely (2005) 的方法，我們確定在我們的研究中使用的一次花粉噴出大約 274,000 個(gè)花粉粒?；ǚ凼┯冒l(fā)生在從花開花期到花衰老/花瓣掉落期間（約6天）。最后，使用相同的 25 微米聚酯氈液體過濾袋進(jìn)行花粉排除處理，以排除昆蟲和風(fēng)傳播的所有花粉。與人工授粉處理不同，該處理沒有應(yīng)用人工授粉。對(duì)于需要排除袋的每種處理（風(fēng)、人工授粉和花粉排除），袋子保留在花芽簇上，直到袋子內(nèi)的每朵花都開放并衰老，此時(shí)將袋子移走，讓果實(shí)發(fā)育直到秋天的收獲。對(duì)于每株植物的每次授粉處理，記錄每個(gè)花簇內(nèi)存在的花芽的數(shù)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.

圖2.三簇中華獼猴桃。 chinensis 'AU Gulf Coast Gold' 代表 A) 昆蟲授粉的花朵，用旗帶綁在奇異果藤上，B) 帶有 1,000 微米昆蟲排除袋的奇異果藤，代表風(fēng)授粉處理，以及 C) 帶有代表風(fēng)授粉處理的奇異果藤細(xì)網(wǎng)狀 25 微米過濾袋代表花粉排除和人工授粉處理。

▲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

水果數(shù)量和質(zhì)量評(píng)估。 果實(shí)數(shù)量評(píng)估在開花后四個(gè)星期（以下簡(jiǎn)稱坐果；2020 年 5 月 11 日）和收獲時(shí)（以下簡(jiǎn)稱成熟果實(shí)；2020 年 9 月 15 日）進(jìn)行。通過記錄四種處理和 114 株獼猴桃植株中產(chǎn)生果實(shí)的實(shí)驗(yàn)花的數(shù)量來記錄坐果，并定義為花瓣從花上脫落和果實(shí)開始生長(zhǎng)的時(shí)間（Richardson 等，2011）。此時(shí)對(duì)坐果進(jìn)行量化，以防獼猴桃植株或果實(shí)受到風(fēng)暴或害蟲等損害。這確保了在收獲前可以準(zhǔn)確估計(jì)坐果率。收獲時(shí)成熟果實(shí)的數(shù)量也被量化，以估計(jì)收獲時(shí)每次授粉處理的果實(shí)總產(chǎn)量。一旦果實(shí)達(dá)到兩個(gè)閾值參數(shù)，即收獲所有成熟的果實(shí) - 可溶性固形物含量 (SSC) > 6.5 °Brix (Burdon 2015) 和果實(shí)色調(diào)角 < 110° (Minchin et al. 2003)。為了評(píng)估收獲時(shí)的果實(shí)質(zhì)量，我們?cè)诿看问诜厶幚碇须S機(jī)選擇最多 20 個(gè)果實(shí)，并測(cè)量三個(gè)參數(shù)（果實(shí)重量、果實(shí)大小和種子數(shù)量）。使用數(shù)字秤（VWR International，Randor，PA）將果實(shí)重量精確到克。為了量化果實(shí)大小，我們按照 Brantley 等人的方法計(jì)算了每個(gè)果實(shí)的果實(shí)大小指數(shù) (FSI)。 （2019）和阿巴特等人。 （2021）。為此，我們通過手動(dòng)粉碎每個(gè)水果并用加壓自來水迫使水果通過鋼絲測(cè)試篩（1.00 毫米網(wǎng)孔）（新澤西州紐瓦克的紐瓦克鋼絲布公司）來量化每個(gè)水果的種子數(shù)量，從而留下所有種子。

▲佳沛陽光金果

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

統(tǒng)計(jì)分析
使用 Wilcoxon Rank Sum 檢驗(yàn)來測(cè)試八天內(nèi)，意大利蜜蜂和鳳仙花的蜆中攜帶的獼猴桃花粉相對(duì)豐度的差異。采用Kruskal-Wallis單向非參數(shù)方差分析方法檢驗(yàn)了獼猴桃和鳳仙花采集的獼猴桃花粉在每個(gè)采樣日期和時(shí)間段（上午、中午、下午）的相對(duì)豐度差異，最后得出測(cè)試四種授粉處理（昆蟲授粉、風(fēng)授粉、人工授粉和花粉排除）之間的中華獼猴桃坐果和成熟果實(shí)的差異。 chinensis AU Gulf Coast Gold 分別在開花后和收獲后四個(gè)星期。如果治療之間存在顯著差異，則使用 Dunn 成對(duì)事后檢驗(yàn)來確定哪些治療組彼此之間存在顯著差異。最后，使用 Wilcoxon Rank Sum 檢驗(yàn)來測(cè)試授粉處理之間果實(shí)重量、FSI 和種子數(shù)的差異。由于風(fēng)授粉（2 個(gè)果實(shí)）和花粉排除（1 個(gè)果實(shí)）處理中收獲時(shí)產(chǎn)生的果實(shí)數(shù)量較少，因此將它們排除在分析之外。所有統(tǒng)計(jì)測(cè)試均使用 Statistix 9.0 分析軟件（美國(guó)佛羅里達(dá)州塔拉哈西）進(jìn)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

孢粉學(xué)研究
總體而言，對(duì) 15,616 個(gè)花粉粒進(jìn)行了計(jì)數(shù)（其中 7,666 個(gè)來自蜜蜂花粉粒，7,950 個(gè)來自鳳仙花花粉粒），代表 8 個(gè)采樣日內(nèi)總共 26 個(gè)不同的花粉類群（表 1）。從 A. mellifera 的球狀花粉中鑒定出 19 個(gè)花粉類群，從 B. impatiens 的球狀花粉中鑒定出 24 個(gè)花粉類群?？傮w而言，A. mellifera 從三葉草/草樨L./(L.) Lam 收集了相對(duì)豐度最高的花粉屬/種。 （豆科：豆科）（21.7%），其次是A. chinensis（21.3%）、Rhus L.（無患子目：漆樹科）（15.6%）和Brassica L.（蕓苔目：十字花科）（12.1%）（圖3） ）。對(duì)于鳳仙花，收集的花粉屬/種相對(duì)豐度最大的是中華鳳仙花 (66.6%)、漆樹 (8.5%)、Nyssa Gronov。 ex L.（玉米目：Nyssaceae）（7.2%）和三葉草/草樨屬（3.5%）（圖3）。對(duì)于 A. mellifera 和 B. impatiens 來說，都是薔薇科 Juss 的花粉。占收集花粉相對(duì)豐度的第四位（分別為 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 天的采樣期內(nèi)從蜜蜂 (Apis mellifera) 和大黃蜂 (Bombus impatiens) 的球蚴中量化的花粉類群總數(shù)。數(shù)值表示為八天內(nèi)每個(gè)物種的球核內(nèi)攜帶的花粉粒的平均百分比。參考文獻(xiàn)涉及與開花植物相關(guān)的花粉類群是否是昆蟲傳粉媒介的常見花蜜和/或花粉資源

▲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.

圖 3.A) 蜜蜂 (Apis mellifera) 和 B) 熊蜂 (Bombus impatiens) 的球狀花粉，代表了參觀的前十種植物物種。球狀花粉的相對(duì)豐度是八天采樣期間的平均值。

▲剛采摘的獼猴桃公花，拿回去開始制花粉了

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).

▲準(zhǔn)備烘干獼猴桃鮮花藥

八天內(nèi)，意大利蜜蜂和鳳仙花的蜆中攜帶的獼猴桃花粉的平均相對(duì)豐度存在顯著差異（z = 5.04，P < 0.0001），其中鳳仙花平均多攜帶 46% 的獼猴桃花粉花粉比蜜蜂花粉好。獼猴桃采集者在每個(gè)采樣日（2020 年 4 月 9 日至 13 日）采集的獼猴桃花粉相對(duì)豐度存在統(tǒng)計(jì)差異（χ2 = 14.98，df = 7，P = 0.008），但 Dunn 檢驗(yàn)的結(jié)果未能通過以下方法識(shí)別差異：日期（圖 4）。對(duì)于鳳仙花，第1天和第8天（分別為2020年4月9日和13日）收集的獼猴桃花粉的相對(duì)豐度存在統(tǒng)計(jì)差異（χ2 = 17.99，df = 7，P = 0.0003），但其他采樣日期沒有統(tǒng)計(jì)差異各不相同（圖4）。最后，對(duì)于獼猴桃（χ2 = 1.71，df = 2，P = 0.44）和鳳仙花來說，三個(gè)采樣周期（上午、中午和下午）采集的獼猴桃花粉相對(duì)豐度沒有顯著差異。 （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.

圖4 中華獼猴桃的相對(duì)豐度百分比±(s.e.m) 2020 年 4 月為期 8 天的采樣期間，蜜蜂 (Apis mellifera) 和大黃蜂 (Bombus impatiens) 在每個(gè)采樣周期（上午、中午和下午）的 chinensis 'AU Gulf Coast Gold' 奇異果花粉平均值。所有花粉均根據(jù)球狀花粉進(jìn)行定量。八天內(nèi)，意大利蜜蜂和鳳仙花的蜆中攜帶的獼猴桃花粉的平均相對(duì)豐度存在顯著差異（P < 0.0001）。對(duì)于每種蜂種，不同字母的天數(shù)（鳳仙花大寫，意大利蜜蜂小寫）代表每天采集的獼猴桃花粉相對(duì)豐度存在顯著差異（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
總體而言，1674朵獼猴桃花被納入果實(shí)數(shù)量評(píng)估，其中436、442、364和432朵花分別用于昆蟲授粉、風(fēng)授粉、人工授粉和花粉排除處理。在生長(zhǎng)季節(jié)，幾株獼猴桃藤蔓受損，因此每種授粉處理（分別為昆蟲授粉、風(fēng)授粉、人工授粉和花粉排除）共使用了114、113、106和109株獼猴桃植物。在盛花后四周，四種授粉處理的獼猴桃落果平均百分比存在統(tǒng)計(jì)學(xué)差異（χ2=338.2，df=3，P<0.0001）。人工授粉導(dǎo)致每朵花的坐果率最高（97.4%），昆蟲授粉（3.1%）、風(fēng)授粉（0.4%）和花粉排除（0.4%）處理在統(tǒng)計(jì)學(xué)上沒有差異（P>0.05，Dunn檢驗(yàn)）（圖5）。收獲時(shí)，四種授粉處理產(chǎn)生成熟獼猴桃的獼猴桃花百分比存在顯著差異（χ2=297.5，df=3，P<0.0001）。人工授粉導(dǎo)致每朵花成熟果實(shí)的百分比最高（76.0%），昆蟲授粉（2.8%）、風(fēng)授粉（0.5%）和花粉排除（0.2%）處理彼此之間沒有統(tǒng)計(jì)學(xué)差異（P>0.05，Dunn檢驗(yàn)）（圖6）?？傮w而言，32個(gè)成熟獼猴桃被納入果實(shí)質(zhì)量評(píng)估，其中12個(gè)和20個(gè)分別用于昆蟲授粉和人工授粉處理。與昆蟲授粉處理相比，人工授粉導(dǎo)致每個(gè)果實(shí)的最大果實(shí)重量（z=4.3，P<0.0001）、果實(shí)大?。‵SI）（z=4.4，P<0.00001）和種子數(shù)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. 在新選項(xiàng)卡中打開獼猴桃品質(zhì)變量摘要。 chinensis ‘AU 墨西哥灣黃金’
可變花粉排除 N 風(fēng)授粉 N 昆蟲授粉 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
果實(shí)大小指數(shù) (mm) 24.6 (NA) 1 35.5 (10.8) 2 29.6 (3.0)b 12 52.0(0.3)a 20
種子數(shù) 3.0 (NA) 1 250.5 (217.5) 2 39.8 (19.4)b 12 573.9 (31.8)a 20
每個(gè)變量后跟不同字母（a 或 b）的平均數(shù) (±SE) 在昆蟲授粉和人工授粉處理之間顯著不同 (P < 0.05)?；ǚ叟懦幚泶砼c花粉完全隔離的花朵，風(fēng)授粉處理代表僅通過風(fēng)傳播的花粉，昆蟲授粉代表通過昆蟲和風(fēng)傳播的花粉，人工授粉處理代表僅通過手動(dòng)吹管器傳播的花粉。由于在花粉排除和風(fēng)授粉處理（分別為 1 和 2）中觀察到的樣本量較小，因此僅將昆蟲授粉和人工授粉處理納入統(tǒng)計(jì)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. 圖5 中華獼猴桃的平均百分比±(s.e.m) chinensis ‘AU Gulf Coast Gold’奇異果花，在四種授粉處理（昆蟲授粉、風(fēng)授粉、人工授粉和花粉排除）的盛花期（2020 年 5 月 11 日）后四個(gè)星期結(jié)果。每列上方的不同字母表示處理平均值在 P = 0.05 時(shí)存在顯著差異。

▲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. 圖6 中華獼猴桃的平均百分比±(s.e.m) chinensis ‘AU Gulf Coast Gold’奇異果花，在收獲時(shí)（2020 年 9 月）每朵花都結(jié)出成熟的奇異果，用于四種授粉處理（昆蟲授粉、風(fēng)授粉、人工授粉和花粉排除）。每列上方的不同字母表示處理平均值在 P = 0.05 時(shí)存在顯著差異。

▲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, Mi?arro 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

討論 確定管理蜜蜂對(duì)美國(guó)東南部種植的獼猴桃授粉的貢獻(xiàn)是制定授粉管理策略的重要一步，該策略將提高獼猴桃產(chǎn)量，同時(shí)降低授粉成本。我們的孢粉學(xué)和排除實(shí)驗(yàn)的結(jié)果都支持昆蟲傳粉者對(duì)獼猴桃授粉貢獻(xiàn)最小的觀點(diǎn)。盡管有促進(jìn)異花授粉的最佳果園條件（雄性和雌性花朵的豐富程度相互吻合，覓食的大黃蜂和蜜蜂的豐富程度，以及有利的天氣條件），但我們?nèi)匀挥^察到，與人工授粉的花朵相比，昆蟲授粉的雌性獼猴桃花朵的坐果率和果實(shí)質(zhì)量在統(tǒng)計(jì)學(xué)上較低。因此，我們相信在實(shí)驗(yàn)期間存在足夠的授粉機(jī)會(huì)，并認(rèn)為與昆蟲授粉處理相關(guān)的低坐果率和果實(shí)質(zhì)量是傳粉者限制的結(jié)果，而不是花粉限制的結(jié)果。此外，在“非盟墨西哥灣沿岸黃金”開花期間，有足夠水平的雄性花粉可用于異花授粉，但兩種受管理的蜜蜂都被競(jìng)爭(zhēng)的非獼猴桃開花所吸引。因此，果園管理者應(yīng)更加重視人工授粉，以實(shí)現(xiàn)商業(yè)獼猴桃運(yùn)營(yíng)所需的高水果產(chǎn)量。 獼猴桃的花期較短（通常為1-2周），因此更容易受到可能對(duì)授粉成功產(chǎn)生負(fù)面影響的因素的影響，如花粉可用性、傳粉者豐度和活動(dòng)，以及花期的不利天氣條件（Clinch 1984，Testolin等人1991，Costa等人1993，Mi?arro和Twizell 2015，Tacconi等人2016，Castro等人2021）。為了確保有足夠數(shù)量的雄性獼猴桃花粉可用于異花授粉，至少一個(gè)雄性獼猴桃花粉器的花期必須與雌性獼猴桃花粉器重合嗎。出于這個(gè)原因，美國(guó)東南部的果園管理者在一個(gè)雌性品種旁邊種植了多個(gè)開花期交錯(cuò)的雄性花粉器，以確?；ǚ墼诟L(zhǎng)時(shí)間內(nèi)的可用性。2020年4月，“AU墨西哥灣沿岸黃金”在4月10日達(dá)到峰值，“CK3”在大約3天前達(dá)到峰值。盡管“非盟墨西哥灣沿岸黃金”和“CK3”的開花高峰期錯(cuò)開了幾天，但在“非盟海灣海岸黃金”開花期間，出現(xiàn)了大量的“CK 3”花，從而產(chǎn)生了雄性花粉AU Golden Tigers的開花高峰期通常與“AU墨西哥灣沿岸黃金”的開花期相吻合，因此，該品種被稱為“AU墨西哥海岸黃金”的最佳雄性授粉品種（Dozier等人，2011年；Spiers等人，2018年）。2020年4月，我們觀察到“AU墨西哥灣沿岸黃金”和“AU金虎”的花期重合，從而為異花授粉提供了豐富的雄性獼猴桃花粉。在我們的研究果園中，“酋長(zhǎng)”開花期往往落后于“澳大利亞墨西哥灣沿岸黃金”和“CK3”。2020年4月，“酋長(zhǎng)”在“非盟墨西哥灣沿岸黃金”開花期的最后2-3天開始開花，為“非盟海灣海岸黃金”開花的后期提供花粉。由于在“澳大利亞墨西哥灣沿岸黃金”開花期，幾種雄性授粉者的雄性獼猴桃花數(shù)量豐富，我們認(rèn)為果園內(nèi)有足夠數(shù)量的雄性花粉，可以通過風(fēng)媒和/或昆蟲授粉者促進(jìn)異花授粉。因?yàn)槲覀冇^察到，在“澳大利亞墨西哥灣沿岸黃金”開花期間，當(dāng)存在大量雄花時(shí)，與風(fēng)和昆蟲授粉處理相關(guān)的低坐果率，坐果率可能受到其他因素的影響，如開花期間傳粉者豐度和活動(dòng)低或天氣條件惡劣。 High pollinator diversity and abundance are associated with fruit set and size equal to optimal pollination methods (artificial pollination) (Sharma et al. 2013, Mi?arro 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, Mi?arro 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.

傳粉媒介的多樣性和豐度與最佳授粉方法（人工授粉）的坐果和大小有關(guān)（Sharma等人，2013年，Mi?arro和Twizell，2015年），但取決于任何給定果園和周圍棲息地內(nèi)的傳粉媒介群落。2019年和2020年，我們?cè)趯?shí)驗(yàn)獼猴桃園進(jìn)行的一項(xiàng)研究記錄了很少有授粉昆蟲訪問獼猴桃花（Abbate等人，2021）。此外，由于訪問獼猴桃花朵的傳粉者數(shù)量較少，無法記錄傳粉者在雄花和雌花之間的移動(dòng)。由于我們研究果園內(nèi)訪問獼猴桃花的授粉昆蟲的多樣性和豐度較低，必須考慮實(shí)施其他形式的授粉，包括飼養(yǎng)管理蜜蜂，以實(shí)現(xiàn)高水平的授粉成功（Castro等人，2021）。在獼猴桃開花期間，天氣條件最佳（平均溫度和風(fēng)速分別為20°C和1.9 ms），有助于通過風(fēng)和昆蟲進(jìn)行異花授粉，但我們?nèi)匀挥^察到風(fēng)授粉和昆蟲授粉處理的坐果率較低。對(duì)于風(fēng)媒傳粉的情況，可能是因?yàn)榕c其他獼猴桃品種相比，通過風(fēng)媒授粉的中華獼猴桃品種“AU墨西哥灣沿岸黃金”不能產(chǎn)生果實(shí)。在昆蟲授粉的情況下，管理蜜蜂在為作物授粉而飼養(yǎng)時(shí)的覓食行為已被證明受到惡劣天氣條件和競(jìng)爭(zhēng)性花朵的影響（Rogers等人，2013年；Mi?arro和Twizell，2015年；Castro等人，2021年）。意大利蜜蜂的工蜂在較冷的溫度和大風(fēng)大雨期間容易不活動(dòng)，而B.鳳仙的工蜂則容易在惡劣的天氣條件下覓食（Rogers等人，2013）。在我們的研究中，除了2020年4月12日（經(jīng)歷了降雨）外，每天都有覓食蜜蜂的最佳天氣條件（Tan等人，2012），我們觀察到這兩種受管理的蜜蜂在開花期都非常活躍（經(jīng)常離開和返回它們的蜂群）。因此，根據(jù)我們的孢粉學(xué)結(jié)果，我們認(rèn)為這兩種受管理蜜蜂的覓食行為都受到了競(jìng)爭(zhēng)性非獼猴桃開花的影響，而不是惡劣天氣條件的影響。
意大利蜜蜂和鳳仙花都是花卉多面手（Ascher和Pickering 2020），從獼猴桃園和周邊地區(qū)生長(zhǎng)的多種開花植物中收集花粉。在阿拉巴馬州中部的春天，許多開花植物物種都在開花，意大利蜜蜂的覓食者可能會(huì)利用非獼猴桃的花朵作為花卉資源。我們的孢粉學(xué)結(jié)果表明，五種花粉類型（A.chinensis、Brassica、Rhus、Rosaceae和Trifolium/Melilotus）在A.mellifera覓食者的球莖中的數(shù)量（平均相對(duì)豐度>10%）高于其他花粉類型。除A.chinensis外，這些植物類群中的每一個(gè)都能產(chǎn)生大量的花蜜，經(jīng)常被蜜蜂造訪（Free和Nuttall 1968，Crane 1975，Craig等人1988，Greco等人1996，Pomeroy和Fisher 2002，Abbate等人2021）。我們還直觀地觀察到，在“澳大利亞墨西哥灣沿岸黃金”開花期間，意大利蜜蜂覓食者在果園里參觀白三葉（白三葉）和野生蘿卜（蘿卜）的花朵，這支持了他們被競(jìng)爭(zhēng)開花植物所吸引的觀點(diǎn)。最后，在捕捉覓食者的同時(shí)，我們還觀察到很少（約10%）的意大利蜜蜂覓食者帶著球莖花粉回到它們的群落，這表明它們可能是為了花蜜獎(jiǎng)勵(lì)而不是花粉獎(jiǎng)勵(lì)而訪問非獼猴桃花朵。對(duì)于確實(shí)含有球莖花粉負(fù)載的覓食者來說，只有21.3%的花粉負(fù)載包含獼猴桃花粉。將這一數(shù)字與意大利蜜蜂采集者在商業(yè)杏仁園和藍(lán)莓田中采集的杏仁花粉（74-96%）和藍(lán)莓花粉（57-80%）的相對(duì)豐度進(jìn)行比較（Webster等人，1985年；Degrandi Hoffman等人，1992年；Hoffman等，2018年）。
在獼猴桃開花期間，不耐煩Bombus似乎將覓食精力集中在A.chinensis上，但也比其他開花植物更頻繁地訪問了三種非獼猴桃開花植物（Nyssa、Rhus和Rosaceae）；所有這些都會(huì)產(chǎn)生大量的花蜜（Pellett 1920，Ramsay 1987，Maxwell和Knapp 2012）。盡管與意大利蜜蜂相比，鳳仙花的覓食者在其球莖中含有相對(duì)較高的獼猴桃花粉平均百分比，但目前美國(guó)東南部推薦的鳳仙花獼猴桃授粉放養(yǎng)率似乎不足。這些結(jié)果與之前的研究一致（Clinch 1984，Pomeroy和Fisher 2002，Lee等人2019）。例如，Clinch（1984）確定，蜜蜂覓食者是新西蘭獼猴桃花最豐富的花卉訪客，但也受到競(jìng)爭(zhēng)性非獼猴桃花的影響。這項(xiàng)研究還得出結(jié)論，當(dāng)雄性和雌性品種同時(shí)開花時(shí)，觀察到大黃蜂造訪獼猴桃花朵；然而，它們的豐度太低，無法促進(jìn)獼猴桃授粉。在另一項(xiàng)研究中，Lee等人在2019年調(diào)查了意大利蜜蜂和陸地蜜蜂在用于獼猴桃授粉的網(wǎng)房單元中的覓食活動(dòng)和授粉效果。Lee等人在2019年觀察到，回到蜂箱的B.terrestris工蜂的球莖花粉量大約是A.mellifera的3倍。盡管如此，A.mellifera授粉的獼猴桃的坐果率和果實(shí)質(zhì)量更高，這可能是由于與B.terrestris相比，A.melifera群體的工人數(shù)量更多。同樣，Pomeroy和Fisher（2002）比較了同一管理蜂種（A.melliferia和B.terrestri）在新西蘭獼猴桃園（不是在封閉空間內(nèi)）內(nèi)對(duì)獼猴桃的授粉貢獻(xiàn)，并得出結(jié)論，盡管B.terrestrris對(duì)獼猴桃花粉的收集表現(xiàn)出比A.melliferas更大的忠誠度，但它們也受到了競(jìng)爭(zhēng)性非獼猴桃開花植物的影響。
盡管以推薦的速度在獼猴桃園中飼養(yǎng)了這兩種受管理的蜜蜂，但它們對(duì)獼猴桃的貢獻(xiàn)也很小。與所有其他授粉處理（昆蟲授粉、風(fēng)授粉和花粉排除）相比，人工授粉處理每朵花的果實(shí)百分比最高，人工授粉導(dǎo)致開花后4周的坐果率增加了19倍，收獲時(shí)的成熟果實(shí)增加了15倍。此外，與昆蟲授粉的花朵相比，人工授粉的“非盟墨西哥灣沿岸黃金”花朵在統(tǒng)計(jì)上導(dǎo)致了更大的果實(shí)重量和大小，并且含有更多的種子。在美國(guó)東南部，以更高的速度在獼猴桃園中儲(chǔ)存B.impatius菌落以增加坐果率和質(zhì)量并不是授粉管理的最佳解決方案，因?yàn)閮?chǔ)存率需要非常高，而且可能成本過高，無法與人工授粉提供的坐果率、產(chǎn)量和果實(shí)質(zhì)量相匹配（Herbertsson等人，2016年；Mallinger和Prasifka，2017年；Lee等人，2019年）。與授粉限制或失敗相關(guān)的潛在經(jīng)濟(jì)損失證明了實(shí)施人工授粉的合理性。一些研究記錄了將昆蟲授粉和人工授粉作為優(yōu)化坐果和果實(shí)質(zhì)量的授粉策略的綜合效益（Lee等人，2019年，Castro等人，2021年），但在我們的研究果園中，在單一作物上實(shí)施多種授粉方法以確保高產(chǎn)是昂貴的，可能不是必要的。盡管獼猴桃的人工授粉成本高昂，但與自然授粉相比，它可以提供許多優(yōu)勢(shì)（Castro等人，2021，Wurz等人，2021）。人工授粉使果園管理者能夠更好地控制時(shí)間，并確保作物的充分授粉（Oronje等人，2012年；Brantley等人，2019年）。這兩個(gè)因素確保了產(chǎn)量和利潤(rùn)的一致水平（Silveira等人，2012），同時(shí)減少了流產(chǎn)獼猴桃的數(shù)量（Abbate等人，2021）。人工授粉，如果實(shí)施得當(dāng)，可以為獼猴桃種植者提供管理時(shí)的授粉安全，而野生昆蟲則不能。
總之，這項(xiàng)研究表明，經(jīng)過管理的意大利蜜蜂和鳳仙花覓食者是獼猴桃的低效授粉者，很可能是因?yàn)樗鼈儽桓?jìng)爭(zhēng)的非獼猴桃花朵所吸引。此外，盡管B.鳳仙花收集的獼猴桃花粉的平均相對(duì)豐度高于A.mellifera，但與B.鳳仙花相關(guān)的小菌落大小可能解釋了為什么我們?cè)诶ハx授粉處理中觀察到的坐果率低于人工授粉處理。這也突顯出，目前建議的B.impensis放養(yǎng)率不足以在美國(guó)東南部進(jìn)行獼猴桃授粉?？傮w而言，人工授粉提供了唯一具有商業(yè)可行性的坐果率和產(chǎn)量。因此，根據(jù)我們的研究結(jié)果，經(jīng)歷與我們相似環(huán)境條件的獼猴桃生產(chǎn)商應(yīng)該更加重視人工授粉，為作物授粉并增加利潤(rùn)。由于人工授粉的時(shí)間和成本，未來的研究應(yīng)比較評(píng)估其他人工授粉方法（例如，通過臂式噴霧器噴灑干花粉）與人工授粉的授粉效率和經(jīng)濟(jì)性，因?yàn)楸凼絿婌F需要更少的時(shí)間，施用花粉的成本較低，但效率往往較低。

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.
致謝
我們感謝Sophie Warny提供的所有孢粉學(xué)定量，Jacob Kelley、Wesley Stone和Katie Abbate提供的實(shí)驗(yàn)室和現(xiàn)場(chǎng)協(xié)助，以及東南獼猴桃種植合作社的Clint Wall使用他們的獼猴桃園進(jìn)行本次實(shí)驗(yàn)。我們感謝Beth Redlin的平面設(shè)計(jì)協(xié)助。
基金
這項(xiàng)工作得到了美國(guó)農(nóng)業(yè)部國(guó)家食品和農(nóng)業(yè)研究所多州孵化項(xiàng)目NC1173、阿拉巴馬州農(nóng)業(yè)實(shí)驗(yàn)站和美國(guó)農(nóng)業(yè)部特種作物塊撥款計(jì)劃（AL）-AM190100XXXXG055的支持。
作者貢獻(xiàn)
AA：概念化負(fù)責(zé)人、數(shù)據(jù)管理負(fù)責(zé)人、形式分析負(fù)責(zé)人、資金獲取負(fù)責(zé)人、調(diào)查負(fù)責(zé)人、方法論負(fù)責(zé)人、資源負(fù)責(zé)人、軟件負(fù)責(zé)人、監(jiān)督負(fù)責(zé)人、驗(yàn)證負(fù)責(zé)人、可視化負(fù)責(zé)人、寫作——初稿負(fù)責(zé)人、撰寫——審查和編輯負(fù)責(zé)人。JC：概念平等、方法平等、監(jiān)督平等、驗(yàn)證平等、寫作-評(píng)論和編輯支持。GRW：概念化平等、資金獲取平等、監(jiān)督平等、驗(yàn)證平等、寫作-評(píng)論和編輯平等。
數(shù)據(jù)可用性
在當(dāng)前研究期間生成和/或分析的數(shù)據(jù)集可根據(jù)合理要求從相應(yīng)作者處獲得。

Journal of Economic Entomology 經(jīng)濟(jì)昆蟲學(xué)期刊 第116卷第3期
2023年6月