11

The Importance of Honey Bee (Apis mellifera carnica Poll.) in Pollination And Seed Yield of Alfalfa (Medicago sativa L.)

Goran Jevtić, Jasmina Radović . Institute of Forage Crops Kruševac, 37251 Globoder, Serbia

e-mail: goran.jevtic@ikbks.com

Abstract

Alfalfa is the most important forage crop and its seed yield potential exceeds 1000 kg ha-1. This high yield is very rarely achieved in practice, primarily due to poor pollination of alfalfa flowers which is a result of morphological traits of flower, low self fertilization and insufficient presence of adequate pollinators. Pollination of alfalfa and provision of pollinators are the most important moments in the seed production. Honey bee, due to its abundance, its range and mobility, although not the most effective pollinator in our agro-ecological conditions, is a significant factor in the production of alfalfa seed. Problems that arise during pollination of alfalfa by honey bee, led many authors to pay attention to the pollination of this crop. The importance of honey bees in the pollination of alfalfa, according to different authors, ranged from slight to very high impact. This paper presents the results obtained by use of honey bee colonies in alfalfa pollination, as well as additional measures to enhance pollination and have an impact on yield and yield components of alfalfa seeds. The number of other pollinators (solitary bees, bumblebees, etc.) was also observed in addition to the honey bee.

Key words: alfalfa, pollination, honey bees, seed yield components, seed yield

Importance of alfalfa

Perennial legumes have important role in providing cheap forages of high nutritive value and digestibility. Among them the most familiar and widespread in world agriculture is alfalfa. This species was cultivated before recorded history and it has had a long and rich history as a forage crop. Due to rich and variable genetic base it has good adaptability to different environment conditions and wide area of growing. Now it is cultivated in more than 80 countries on every continent on the area exceeding 35 million ha. It can grow between 55° northern and 50° southern latitude, and at 2500 m above sea level. It is cultivated without irrigation in dry regions with 200 mm annual precipitation as well as in humid region that receive 2500 mm precipitation. Deep and strongly developed root provides alfalfa with good tolerance to drought and high temperatures and even in years with very low precipitation it gives satisfactory yields (Radovic et al., 2009).

Alfalfa is the most important perennial forage crop in Serbia. It is the second most important forage crops after maize. Alfalfa is grown on over 200,000 ha in pure stand, which is about 4-5% of total agricultural area in Serbia (Đukić et al., 2007).

It is a remarkable crop in comparison with others. Alfalfa is used for livestock nutrition in different forms, most frequently as hay, but also dried/dehydrated in form of briquettes, as silage, haylage or for grazing. Alfalfa is harvested and stored primarily as hay or silage for use on the farm. It is recognized as the most widely adapted agronomic crops, important and the cheapest source of protein rich forages of excellent amino-acid composition and high digestibility, which is so valuable in economical animal husbandry. A number of inorganic elements are essential for normal growth and reproduction of animals, which is why mineral content of forages is important for animal feeding. Amount of N, P, Ca and K satisfy the needs of cattle and dairy cows, as well as in all microelements. Besides, rapid recovery

12

The Importance of Honey Bee (Apis mellifera carnica Poll.) in Pollination And Seed Yield of Alfalfa (Medicago sativa L.)

after cutting, longevity and tolerance to environmental stress is important for stabile yield during all periods of exploitation.

The practical value of alfalfa is not restricted to its fodder qualities alone. It also performs other important economic and biological functions: it enriches the soil with nitrogen, it is a good predecessor for many agricultural crops, it is a good green-manure and nectar producing crop which reduces effect of water and wind erosion by binding the soil. It is one of the few cultivated plants that can produce high level of biomass with minimum inputs. This is a reason why, besides its important role in conventional husbandry, alfalfa is getting great role in sustainable agriculture and organic production. All those characteristics confirm that alfalfa is really a “queen” of forages.

Apart from dry matter yield and quality of hay, seed yield of alfalfa varieties is also an important trait in determining the effective distribution of new cultivars to farmers. Alfalfa is a cross pollinating plant with complex flower structure. The alfalfa flower must be tripped and cross-pollinated by insects for maximum production of high-quality seed. Alfalfa seed production depends on many factors, but the most important factor is presence of pollinating bees. Pollination process includes a lot of factors which influence attendance and effectiveness of pollination bees, which is most important for high alfalfa seed production.

The aim of this paper was to present most of the problems that occur in the pollination of alfalfa (Medicago sativa L.) and all the methods and ways in which these problems can successfully be solved.

Pollination of alfalfa

The problems that are arising in the process of alfalfa pollination and fertilization have led many authors to pay special attention to this issue. Many authors claim that the honey bee is not very successful in the pollination of alfalfa (Benedek, 1976; Holm, 1984), primarily due to the anatomy of the flower. In addition, it was found that many other bee species are much more efficient pollinators of this important forage crops (Richards, 1984; Cane, 2002, Stanisavljević et al., 2003, etc.). However, those bee species have their specific drawbacks as they are highly dependent on meteorological conditions and anthropogenic factors. In addition, alfalfa is grown on large areas where there are no natural habitats for spontaneous pollinators. Tauber et al. (2004) continued to give primacy to honey bees in pollination, especially in the southern regions of the U.S., in California.

From the above stated, it can be concluded that the pollinating of alfalfa cannot be left to only one species, i.e. spontaneous pollinators, because of their low abundance and frequency and high dependence on various factors. To overcome this problem in pollination, the programs were started for selection of honey bee colonies for more successful pollination of forage crops. In the middle of the last century in the U.S. there were many studies on the introduction of honey bee as the pollinator of alfalfa. Mackensen and Nye (1969), and Nye and Mackensen (1970) analyzed a large number of honey bee colonies and determined differences in the amount of foraged pollen. In addition, it was found that the alfalfa pollen collecting is genetically conditioned honey bee trait. During the years of the breeding program, the colonies with better ability for collecting alfalfa pollen were obtained. In Serbia in the 1990s there were trials to breed honey bee lines that had better pollen foraging activity. It was found that the honey bee colonies had a pronounced variation in the pollen foraging and in the foraging of alfalfa pollen also (Džamić and Kulinčević, 1994; Jevtić 2007).

In addition to the honey bee selection programs, a comparative selection of alfalfa for more efficient pollination was simultaneously conducted. There were attempts to breed alfalfa lines with easily tripping flowers and the lines with the increased secretion of nectar.

Fertilization of alfalfa

To produce seed in the favourable years, alfalfa flowers must be tripped and cross-pollinated. Alfalfa is xenogame, entomophile plant and for its normal pollination it is necessary for pollinator to trip the flower, release the stylus and thus enable foreign pollen to set onto the stigma. Alfalfa pollen is moist,

13

sticky, heavy and coalescent into large lumps and cannot be transferred by wind, only by pollinating insects. There is self-pollination in alfalfa, however, as stated by Tysdal (1946), in field conditions less than one percent of flowers pollinated in such a way produce seed.

Analyzing the percentage of produced pods, Žarinov and Kljuj (1983) claim that only 0.3 to 0.7% of pods are produced without violent tripping of flowers and in most self-fertilized pods the seed is of poor quality. According to Zonjić et al. (1972), alfalfa is self-fertile to a lesser extent and its complete and high fertility is manifested in uncontrolled free pollination primarily as the xenogame plant.

Major role in the tripping of alfalfa flowers belongs to insects. Using isolated alfalfa plants, Gray (1925) showed that without the presence of insects the seed yield was reduced. In their study of pollination, Corbet et al. (1991) concluded that the commercial production of alfalfa seed was conditioned by insects due to xenogamy of alfalfa. The sequence of flower tripping was described by many authors (Martin, 1975; Mc Gregor, 1976).

The opening up of flowers (tripping) occurs when an insect enters in, and presses the top, large petal (vexillum). This pressure activates mechanism that opens lower, basic petal (naviculum), freeing up the reproductive system. Opening of the flower is rapid and frequently stylus strikes the honey bee and throws it away from flower or its proboscis gets stuck between anthers and the honey bee must spend a lot of effort to free its head out of the flower. Honey bees that collect nectar do not favour this “mechanism” and are able to quickly learn to access nectar by squeezing their proboscis between petals without tripping the flowers. This way, the bees take nectar but do not pollinate alfalfa flowers.

Alfalfa potential for seed production

Flowering alfalfa takes a relatively long time. Mc Gregor (1976) concludes that it takes a week to open up all the flowers in blooming. The unpollinated flower remains open for seven days, but very soon after pollination it wilts and falls off.

Due to its genetic characteristics, alfalfa is able to create a large number of flowers. Tysdal (1946) stated that in Nebraska 3.3 billion flowers per hectare were produced, but not all of these flowers produced seed.

Lesins (1950) has assumed that about 400 million flowers participate in pod producing, that each pod gives an average of 5 seeds and that 450,000 seeds weighs one kilogram and is consequently possible to achieve yield of 5,000 kg ha-1. In their research, Pedersen et al. (1956) showed that 46.7% of flowers may produce pods, so it is possible to get 2,000 kg ha-1 of seed. These high seed yields are never realized in the production practice and even cannot be achieved in trials. Stjepanović (1981) stated that the practical alfalfa seed yields are 20-30 times smaller than the calculated potential. Erić (1995) claims that in our agro-ecological conditions, average alfalfa seed yields range from 250-300 kg ha-1, and the highest recorded yields are 800-1000 kg ha-1.

Agro-metrological conditions for successful pollination and fertilization of alfalfa

Alfalfa grown for seed has significantly different requirements for its successful growth than that grown for fodder. It can be said that the production of alfalfa is often more conditioned by the influence of meteorological factors than by production technology (Erić, 1995; Marković, 1997). The temperature and humidity of the air and soil at the time of flowering, precipitation, light, wind speed, and other meteorological factors have impact on the seed yield. The medium daily mean temperatures ranging from 25 to 30°C are the most favourable for pollination of alfalfa (Erić, 1995). Relative humidity during flowering and pollination should be 30-50% (Žarinov and Kljuj, 1983). Increasing the relative humidity has a negative impact on the concentration of sugar in the nectar which reduces the activity of the honey bee (Ciurdaresku, 1974). The amount of precipitation affect the seed yield in such a way that higher yields of alfalfa seeds per plant are achieved in dry and hot summers, while in the humid and rainy year yields are lower (Hoopingarner and Waler, 1992). For alfalfa seed production, the most favourable years are the ones with an average rainfall of 600-650 lm-2 (Bošnjak and Stjepanović, 1978). Wind mainly affects the activity of the honey bees (Martin, 1975).

14

The Importance of Honey Bee (Apis mellifera carnica Poll.) in Pollination And Seed Yield of Alfalfa (Medicago sativa L.)

The study by Jevtić et al. (2006) observes that climatic conditions significantly affect the honey bee visits to the seed crops and thus the yield and yield components. In the dry and warm years, in the pollination of alfalfa honey bees were dominant (7:1). These conditions have been much better for seed production because twice as many seeds per area unit were received. In somewhat colder and more humid years there were considerably more spontaneous pollinators than honey bees (2:1). In addition, these weather conditions and poor honey bee visits are very detrimental for seed production.

Waler (1970), and Moffet and Caddel (1985) suggested that, in the conditions of the U.S., honey bee was much better pollinator in the southern, more arid areas, while in the areas in the northern and western regions, where the humidity is higher, solitary bees were primary pollinators. Sheesley et al. (1985) claimed that the visits of bees increased when the alfalfa field was a slightly moisturized, but reduced in the just moisturized or completely dry fields (Sheesley et al., 1985). Considering the previous statement, Sheesley and Atkins (1985) noted that the block irrigation of alfalfa was conducted in California. In addition, on every 200 m barrels for watering bees were placed.

Honey bee as the pollinator of alfalfa

Because of the significant problems that the honey bee has, when tripping the alfalfa flower, its role in the pollination of alfalfa for long was neglected. There were previous attempts to show its role in the proper light, so Megee and Keltty (1932), according to Mc Gregor (1976), shown in a limited way, by using the cages, that the honey bee is the pollinator of alfalfa. However, Hare and Vansell (1946), using pollen collectors, proved that honey bee collects large amounts of alfalfa pollen in favourable conditions. Using cages with insulation, Vansell and Todd (1946) showed that the honey bee has an essential role in the pollination of alfalfa. The flowers on plants that were placed in cages without bees had not even opened nor produced seed. In contrast, in the plants in cages with bees and in the open field there was an abundance of seed. More detailed results on the quantity of obtained seed were given by Pedersen et al. (1956), who reported that in cages without bees there was 13 kg ha-1 of seed, while in cages with bees it was up to 900 kg ha-1.

On the basis of the results obtained in the open field and in the cages, Bošnjak and Stjepanović (1978) stated that the tripping of flowers and pollination of alfalfa was 3-6 times better in the presence of honey bees than in their absence. While Zonjić (1953) claims that honey bees pollinate only 1-3% of visited flowers and the lead role in the pollination belongs to young honey bees. Schmit (1979) suggests that the honey bees are good pollinators only if there is no other nectar flora except alfalfa. Jevtić (2001) found that honey bees significantly affected alfalfa pollination.

In isolation without bees 23.3 kg ha-1 of seed were obtained, and in cages with bees 158.9-198 kg ha-1 depending on whether the cages contained one or two nuclei. In open pollination 304 kg ha-1 of seed were obtained, and the differences were statistically highly significant (Table 1).

Table 1. Yield and yield components of alfalfa seed depending on the way of improvement of pollination (Jevtić, 2001)

Treatment

TraitMechanical

improve.With one nucleus

With two nuclei

Without pollinators

With sugar syrup

Open pollination Average

LSD 0.050.01

Seed yield (kg ha-1) 427.4 156.9 198.0 23.3 449.0 304.1 259.5 71.67 97.41

% fert. infloresc. 75.42 46.85 52.97 38.05 71.72 67.18 58.70 10.8913.64

% fert. pods 49.28 24.99 27.45 16.31 51.35 39.44 27.19 10.4213.11

No. of seeds per pod 3.82 3.08 3.56 0.20 4.33 3.24 3.04 0.4270.580

15

Not all the honey bees in the same colony are equally active in the pollination of alfalfa. The largest contribution to the pollination is of the bees that collect pollen. For pollination of alfalfa it is important to note that the young nectar foragers in the beginning of their activities are able to successfully trip and pollinate flowers. They later “learn” to come up with nectar without tripping the flower and therefore do not take part in pollination. For this reason, it is very important that the colonies that pollinate alfalfa are very strong and have many young forager bees. Tucker (1956) pointed that honey bees which forage nectar trip only 0.3% of visited flowers. He gives great importance to young nectar foragers, citing the fact that they trip 70-85% of visited flowers during their “learning process”. Slightly higher percentage of flowers pollinated by honey bees than forage nectar was mentioned by Kropačova (1964) stating that 1.25-2.75% of all flowers these bees visited were also pollinated.

Ciurdaresku (1974) claimed that honey bee trips from 3.4 to 9.5% of visited flowers, which is much less efficient than some solitary bees which trip 45-100% of visited flowers. Pedersen (1962) found a positive correlation between the amount of nectar and the number of bees that visit alfalfa and, also, between bee visitation and the amount of obtained seed. Kropačova (1963) notes that alfalfa can produce large amounts of attractive nectar, stating the fact that alfalfa can secrete 466-2167 kg ha-1 of nectar. A great importance of nectar is recognized by Tauber et al. (1983) claiming that the quantity and quality of nectar are primary factors for pollinator activity, indicating a positive correlation between the amount of nectar and the number of seeds per pod. Kropačova (1964) found that the amount of nectar is significantly affected by humidity of air and soil.

It is undisputed that, for the successful pollination, a corresponding number of colonies per area unit are necessary. Unlike most agricultural crops which require up to 5 colonies per hectare for their pollination, alfalfa has specific requirements. Due to the problems honey bee has during alfalfa flower tripping and the unattractiveness of alfalfa pollen, the number of colonies companies for this crop is increased. Vansell (1951) states that use of 7 colonies per hectare, on a plot of 40 ha, received an average yield of 620 kg ha-1. In a similar experiment on 52 ha, using twice the number of colonies, more than 1,200 kg ha-1 was obtained. Jones (1958) recommended concentration of colonies in alfalfa field that will provide 2-7 nectar foragers per m2. Kropačova (1964) proved that there was a positive correlation between the number of honey bees visiting alfalfa and the number of tripped pollinated flowers in Northern Moravia (ex-Czechoslovakia). The highest seed production was achieved by using 9-18 honey bee colonies per hectare. Ponomariova (1976) published data that from 7 hectares average of 201 kg of seed was obtained by using 2 colonies per hectare and also 23.7 kg of honey was obtained per colony. On plots where honey bees were not used as supplemental pollinators, only 99 kg of alfalfa seed was obtained. In his book on pollination of plants, Free (1970) concludes that the best results were obtained by using 7-10 colonies per hectare, provided that there was no significant competitive pasture at a distance of 3 km, while Martin (1974) suggested 8-10 colonies. Hoopingarner and Waller (1992) suggested that this is not enough colonies for normal alfalfa pollination and that this number should be doubled. They supported their claim by the fact that the number of uncultivated (wild) colonies in the nature was greatly reduced, in particular due to diseases and parasites. These colonies have accounted for a significant pollination capacity, so due to the reduction of their number, the number of leased colonies must be increased to manage the pollination of certain area.

Using 10 colonies per hectare, Jevtić et al. (2004a) found that honey bee visited better and much pollinated plants better near the hive (at a distance of 50-100 m) compared to plants that are more distant from the hives (over 200 m). Hagler et al. (2011) determined that, in alfalfa field of 15.2 km2, the marked bees visited alfalfa flowers at the minimum of 45 m of distance from the hives and at maximum of 5,983 m from the hives.

Considering the number of colonies per hectare, the basic problem is that every beekeeper that provides bees for pollination also wants to have the surplus honey. However, this is difficult to achieve with alfalfa, especially if a large number of colonies was used per area unit. One strong honey bee colony may forage 22-45 kg of honey when there are two colonies per hectare (Mc Gregor, 1976). Using 4-6 colonies per hectare of alfalfa surplus honey can be achieved, and using ten or more colonies there is no surplus according to Hoopingarner and Waller (1992).

16

The Importance of Honey Bee (Apis mellifera carnica Poll.) in Pollination And Seed Yield of Alfalfa (Medicago sativa L.)

In order to attract honey bees to alfalfa flowers in large numbers, dressage may be used, i.e. feeding of the bees with sugar syrup in which the extracts of alfalfa flowers were mixed. This method was proposed by many Russian authors (Anciferova, 1979; Jakovljeva, 1981), who noted that the application of dressage may improve the pollination of alfalfa. In addition, alfalfa fields were sprayed with sugar syrup. Free (1965) proved that this measure does not affect fertilization positively because although the number of bees visiting alfalfa was increased the fertilization was reduced. However, this method held up, so Erić (1995) classifies it as the direct anthropogenic factor on the production of alfalfa seed. Spraying the alfalfa crop in bloom with 5-6% solution of sucrose has special significance in stimulating bees to pollinate alfalfa by making it more attractive to bees, because the other cultivated plants, especially sunflower, are more attractive than alfalfa itself. In addition to sugar syrup, alfalfa can be sprayed with chemicals that give off strong odours to attract bees (Free, 1962; Waler, 1970). Jevtić (2001) and Jevtić et al. (2007) found that by spraying seed crop with sugar syrup and mechanical improvement of pollination can significantly increase the seed yield (Table 1). Improving of pollination had shown significant differences in yield and yield components, but not in the quality of alfalfa seed (Jevtic et al., 2010).

Honey bees make a choice between the alfalfa clones based on floral aromas. When several alfalfa clone lines were offered to honey bees, they consistently showed a greater preference for certain lines (Loper and Waller, 1970). An attempt was made to identify compounds that are attractive to bees, so Loper et al. (1971) identified in the nectar of alfalfa aromatic compound ocimene. Loper (1972) has identified several terpenes in alfalfa varieties that were attractive to bees. Honey bee exhibits some differences visiting various coloured flowers of alfalfa (Clement, 1965).

Individual bees retain a tendency to forage nectar from the plants with the same coloured flowers. Steiner et al. (1992) devoted the attention to this issue and proved that, in addition to the affinity of bees to alfalfa plants based on flower colour, the location affect the frequency of visits to light-coloured flowers. According to Jevtic et al. (2004b) honey bees better visited light purple alfalfa flowers than purple and dark purple ones. Plants with light purple flowers had the highest percentage of fertilized flowers per plant, pods per inflorescence and seeds per pod.

The presence of competitive pasture at the time of alfalfa flowering had a large impact on the visiting of honey bees (Kirnu et al., 1997, Jevtić et al., 2007). Also, meteorological conditions (Jevtić et al., 2006), pollination improvement (Jevtić, 2001; Jevtić et al., 2005b), the time of day (Jevtić, 2007), (Table 2), the distance of the seed crop from the hive (Jevtić et al., 2004a), the alfalfa cultivar (Jevtić et al., 2005a) and other factors had significant impact on honey bee visits. Most of these factors affected the seed yield, and additionally the yield components.

Table 2. The number of honey bees and spontaneous pollinators per m2 of alfalfa in different periods of day and at different distances from hives (Jevtić, 2007).

Period of day 930-1000 1300-1330 1700-1730

Distance Honey bees Spont. pollin. Honey beesSpont. pollin. Honey bees

Spont. pollin.

50m 34.4 8.9 30.4 7.1 23.9 6.0100m 33.2 8.2 26.8 8.2 20.5 4.9200m 25.1 6.8 23.3 7.3 14.8 6.5

Average 30.90 7.97 26.83 7.53 19.73 5.8

The seed yield, in addition to pollinators, is influenced by: lot size (Lukić, 2000), cutting system (Karagić et al., 2007; Terzić, 2010), the position of the inflorescence on the stem (Kostić, 1996), cultivar (Lukić et al., 1993, Radović et al., 2006) and others. Growth stages that occur after the formation of flowers (fertilization, formation of the pods and seed) are also influenced by many factors, and the most by the weather, pests, mineral nutrition and others which may, during normal flowering and fertilization, to reduce seed yield to a minimum (Djukić et al., 1993; Erić, 1995; Vučković et al., 1997; Katić et al., 1998; Lukić, 2000; Terzić, 2010).

17

Other pollinators of alfalfa

Numerous authors cite the importance of a large number of wild bees for pollination of alfalfa. The greatest merit for our knowledge about these bees must be attributed to Bohart et al. (1960) and Hobbes (1973), who described in detail biology of these bees and their role in the pollination of alfalfa. Out of the several hundred species of solitary bees from several genera, there are two major types: alfalfa leafcutter (Megachilla rotundata L.) and alkali bees (Nomia melanderi L.). Bohart and Kmowlton (1960) showed that these two species can be manipulated, i.e. they can be transported in the alfalfa fields. He also found that alfalfa leafcutter has such traits that allow its domestication. Hobbes (1973), Richards (1984), and Krunić et al. (1995) reached the same conclusion.

As claimed by Mc Gregor (1976) alkali bees and alfalfa leafcutters are more efficient pollinators than honey bees when viewed as individuals. Their primary motive for visiting alfalfa flower is collecting pollen used to feed the brood. When honey bees visit alfalfa they rather collect nectar while searching for pollen in other plants. Cane (2002) states the fact that alfalfa leafcutters and alkali bees are much more successful in tripping and pollinating alfalfa flowers in comparison to honey bee (Fig 1). Results of Ivanova (1980) should be considered to understand the significance of solitary bees in pollination where from 7,384 insects counted on one hectare in the first cutting of alfalfa, 93.2% were solitary bees, 5.7% were wasps and bumblebees and 1.1% of total number was honey bees. Similar relationships were obtained in the second cut, but there was less insects in total (3979). Alfalfa leafcutter can increase seed yield if brought organized to the seed crop field, as was determined by Bosch and Kemp (2005).

Figure 1. Proportions of visited virgin alfalfa flowers that were tripped by visiting bee species and sexes flying in a greenhouse. Combinations of species and sex with different letters are significantly different by categorical analyses. The total number of flowers visited by each combination of species and sex is given in the right-hand column.

Using isolated cages, Cecen et al. (2008) found that the bumblebee (Bombus terrestris L.) is more successful pollinator of alfalfa compared to the honey bee. In cages with bumblebees, the average seed yield was 56.48 kg ha-1, while in cages with honey bees, 49.2 kg ha-1 and with free pollination 66.19 kg ha-1 was obtained.

Brunet and Stewart (2010) found that three species of the genus Bombus visited alfalfa flowers significantly more frequent and tripped them much better than the alfalfa leafcutter and honey bees. However, in conditions of Central Serbia, in the ten-year-long study (1997-2007) it was found that honey bees were dominant compared to all other pollinators (Table 3).

18

The Importance of Honey Bee (Apis mellifera carnica Poll.) in Pollination And Seed Yield of Alfalfa (Medicago sativa L.)

Table 3. Number of pollinators and their interactions on 1 m2 recorded during the half-hour observation and achieved seed yield in the experimental alfalfa plot

YearPollinators Pollinator interactions (%) Seed yield

(kg m-1)Honey bees Other pollinators Honey bees Other pollinators

1997 15.2 26.9 36.10 63.90 199.01998 45.5 6.6 87.52 12.48 409.22000 23.0 24.5 48.42 51.58 321.62001 28.1 11.0 71.87 28.13 430.92002 29.4 7.6 79.50 20.50 432.42003 26.0 4.2 86.10 13.90 398.22004 26.8 11.7 69.61 30.39 524.92005 19.9 4.8 83.97 16.03 182.52006 31.4 8.1 79.49 20.51 344.02007 25.4 9.3 73.20 26.80 548.2

Average 27.07 11.47 70.24 29.76 379.09

Selection of honey bee and alfalfa for pollination In early studies it was noted that the different races of the honey bee have had different affinity for

alfalfa flowers. Akerberg and Lesins (1949) noted that there is a difference in the behaviour between colonies of Italian honey bees (Apis mellifera ligustica L.) and the European dark honey bee (Apis mellifera mellifera L.). The first species did not visit alfalfa flowers when isolated with them, even when the colonies were supplemented with brood to increase the need for pollen. The colonies of European dark honey bees began to visit flowers and collect pollen on the third day after the isolation. Visiting continued throughout the entire period of flowering. Moffet and Caddel (1985) showed that honey bees pollinate alfalfa successfully in isolation by using them for pollination in order to create new lines of alfalfa. Bees visited flowers well and pollinated them successfully, so that in three years of work about thirty new alfalfa lines were created. The average number of seeds per plant in the best lines was up to 1550.

Given the fact that pollen foragers trip every flower that they visit Mackensen and Nye (1966, 1969), Nye and Mackensen (1968a, 1968b, 1970) created lines of bees with high and low alfalfa pollen foraging activity. They found that the tendency for alfalfa pollen foraging is hereditary. Selection program lasted for six generations and obtained lines differed significantly in the amount of collected pollen. The lines that foraged only 8% alfalfa pollen, but also the lines that forage over 80% of alfalfa pollen of the total amount of foraged pollen were obtained.

However, commercial production and use of bees that forage higher amounts of alfalfa pollen was not economically viable. Using honey bee colonies of different strength, Sheesley and Poduska (1969) determined that they forage an average of 17% of alfalfa pollen from the total amount of foraged pollen. From May 26 till August 6, while the experiment lasted, the amount of brood in colonies decreased by 69% and the normal decrease is about 10%, which shows that the colonies lost a significant amount of strength during the pollination of alfalfa. Special problems existed in the group of poor colonies companies, where 13 out of the 20 colonies either died off or were left with a very small amount of bees.

It was found that in the colonies from these regions there is high variation in alfalfa pollen foraging, which depending on the site ranged from 50 to 80% (Džamić and Kulinčević, 1994). In the research of Jevtić (2007), in the colonies sampled from six different regions of Serbia, it was found that there was variability in the all pollen foraging and foraging of alfalfa pollen, too. The average amount of pollen per colony in different years ranged from 10 to 30.9 g (Table 4), while the variation between individual colonies ranged from 0.8 to 71.3 g. The share of alfalfa pollen in the total amount of foraged pollen was 3.4-44.1%.

19

Table 4. Average amount of pollen per colony (g), average amount of alfalfa pollen (g) and the share of alfalfa pollen (%) in colonies from six regions of Serbia (Jevtić, 2007)

2002 2003 2004 2005 Average

Total pollen (g) 38.43 51.34 102.33 124.65 79.19

Alfalfa pollen (g) 3.73 10.07 34.84 25.73 18.59

Share of alfalfa pollen (%) 10.00 21.01 30.90 20.59 20.63

There were attempts of breeding alfalfa lines with easily trippable flowers. After many years of failure to create these lines due to the rapid degeneration, it was finally possible. Knapp et al. (1996) reported that in the United States four alfalfa germplasms were registered, two with easily trippable flowers and two with flowers more difficult to trip. As the starting material cultivars Saranac and CUF 101 were used. The ease of tripping was controlled by an electronic tripping meter opening. In addition to that, alfalfa lines with increased secretion of nectar were created (Tauber and Green, 1996). Honey bee visited those alfalfa lines much better, increasing pollination and seed production. Tested in the field, lines with increased secretion of nectar yielded significantly more pods and seeds per inflorescence compared to the lines with lower nectar secretion.

Apitechnical and agrotechnical practices in alfalfa pollination

The advantages of honey bees compared to the solitary bees as far as planned pollination are the following:

– The colonies of domestic bees can easily be transported to the alfalfa fields when it is most needed and in appropriate numbers;

– In numbers, because each honey bee colony daily produces up to 2,000 young bees which are good alfalfa pollinators in their first few days;

– The ability to manipulate the colonies and by that avoid chemical treatments that are damaging to the solitary bees.

To make the bees more efficient alfalfa pollinators, is a very important that the colonies are set in the field. The most effective setting is when colonies are grouped in 10 and that the distance between the groups is not higher than 200 meters. Pesenko (1980) lists two ways to secure alfalfa pollinators:

– Agrotechnical method involves increasing the abundance of the natural populations of bees and locating the alfalfa fields in the vicinity of nesting places of pollinators.

– Industrial method means artificial cultivation of specific species of insects that are the most efficient pollinators.

The two methods do not contradict each other and can complement each other. The first method can be fully achieved using existing agrotechnical resources and in the future of alfalfa pollination it should be seen only as agrotechnical measure. The second method involves the development of specific entomological industries, i.e. specialized branches of agriculture dealing with pollination.

Recently, due to the increased application of different agrotechnical practices, especially due to increased use of chemical agents, the number of solitary bees is decreased drastically and some species are even completely extinct.

20

The Importance of Honey Bee (Apis mellifera carnica Poll.) in Pollination And Seed Yield of Alfalfa (Medicago sativa L.)

Conclusions

– Alfalfa, as practically only xenogame species, depends on the activity of pollinators to achieve satisfactory seed yield.

– The honey bee is not as effective in pollination of alfalfa as are some solitary bees, but its advantage is in the number of colonies and the ability to manipulate them easily. In the pollination of alfalfa significantly greater importance is of pollen forager bees compared to the nectar foragers.

– In our agro-ecological conditions, the honey bee was dominant over other pollinators in 8 out of 10 studied years.

– The abundance of alfalfa pollinators is greatly influenced by weather conditions at the time of alfalfa flowering. The years with higher rainfall are more favourable to solitary bees, while the dry years are ideal for honey bees.

– Alfalfa pollinators such as honey bees are much more active in the morning and mid-day period, while their number decreases in the afternoon.

– The visits of honey bees to alfalfa are influenced by many factors, such as the number of hives per hectare, the distance of the crop from the hive, the amount of nectar, lot size, weather conditions, soil moisture, presence of competitive pasture, and many more.

– The selection of honey bees gave such colonies that are able to forage alfalfa pollen in the great extent. However, such a focused selection yielded some deficiencies in the productivity of the colonies.

– The selection of honey bees for pollination of alfalfa should be directed on improving the production traits in the colonies that were of the most successful in the pollination of alfalfa.

Acknowledgment

This research has been funded by Ministry of Education, Science and Technological Development of the Republic of Serbia (Project TR31057).

References

Akerberg A, Lesins K (1949). Insects pollinating alfalfa in central Sweden. Ann R Agricc Coll Sweden. 16: 630-643.

Анциферова TA (1979). Насекомые и люцерны. Пчеловодство No. 8: 12-13.Benedek P (1976). Insect pollination of crops: a review of studies in Hungary. International symposium

on melliferous flora, Budapest September 1976: 91-103. Bohart GE, Knowlton GF (1960). Managing alkali bees for alfalfa pollination. Logan, Utah State Univ.

Extension Services Leaflet 78: 1- 8 p.Bosch J, Kemp WP (2005). Alfalfa leafcutting bee population dynamics, flower availability, and

pollination rates in two Oregon alfalfa fields. Journal of Economic Entomology, vol. 98, no.4: 1077–1086.

Bošnjak D, Stepanović M (1978). Osnovni problemi u proizvodnji semena lucerke. Poljoprivredna sveska 258-259, Beograd: 61-62.

Brunet J, Stewart CM (2010). Impact of Bee Species and Plant Density on Alfalfa Pollination and Potential for Gene Flow. Hindawi Publishing Corporation, Psyche, Volume 2010, Article ID 201858, 7 pages.

Cane J H (2002). Pollinating bees (Hymenoptera: Apiformes) of U.S. alfalfa compared for rates of pod and seed set. Journal of Economic Entomology, vol. 95, no. 1: 22–27.

21

Cecen S, Gurel F, Karaca A (2008). Impact of honeybee and bumblebee pollination on alfalfa seed yield. Acta Agriculturae Scandinavica, Section B - Soil & Plant Science Volume 58, Issue 1: 77-81.

Ciurdaresku, G (1974). Polination as an important faktor for the yield alfalfa crops in Romania. Organizing comitete XII international grasland congress. Sectional Papers-plant introduction, Breeding and seed production - Moscow: 60-69.

Clement WMJr (1965). Flower colourt, a factor in attractiveness of alfalfa clones for honey bees. Crop Science. 5: 267-268.

Corbet S A, Williams I H, Osborne J L (1991). Bees and the pollination of crops and wild flowers in the european community. Bee world, vol. 72. 2: 47-59.

Đukić D, Katić, S, Mihajlović V (1993). Uticaj položaja cvasti na stabljici na komponente prinosa semena lucerke. Sav. poljoprivreda. Vol. 41 br. 3: 87-90.

Đukić D, Lugić Z, Vasiljević S, Radović J, Katić S, Stojanović I (2007): Domestic varieties of perennial legumes – breeding and quantitative traits. Proceedings of the Institute of Field and Vegetable Crops, Novi Sad, Vol. 44, no. 1, 1-7.

Džamić Z, Kulinčević J (1994). Varijabilnost društava kranjske medonosne pčele (Apis mellifera var. carnica) na sakupljanje polena. Jugoslovenski naučni skup za pčelarstvo sa međunarodnim učešćem, 01-03. jul, Sremski Karlovci, Zbornik izvoda: 30.

Erić P (1995). Tehnologija gajenja lucerke. Monografija Lucerka. Izd. Poljoprivredni Fakultet Novi sad: 43-86.

Free JB (1970). Insect Pollination of Crops. Academic Press, New York.Free JB (1965). Attempts to increase pollination by spraying crops with sugar syrup. J. Apic. Res. 4: 61-64.Gray HE (1925). Observations on tripping of alfalfa blossoms. Canad. Ent. 57: 236-237.Hagler JR, Mueller S, Teuber L R, Machtley S A, Van Deynze A (2011): Foraging range of honey bees,

Apis mellifera, in alfalfa seed production fields. Journal of Insect Science, Vol. 11 Article 144: 12.Hare QA, Vansell GH (1946). Pollen collection by honeybees in the delta, Utah, alfalfa seed-producing

area.Amer. Soc. Agronom Journal, 38: 462-469.Hobbs GA (1973). Alfalfa leafcutter bees for pollination alfalfa in Western Canada. Can. Dep. Agric.

Publ. 1945: 30. Holm SN (1984). Introduction and propagation of the leafcutter bee (M. rotundata) in Denmark.

Proc. int. Sym. on Poll., 27-30 IX 1983. Versailles, Tasei, Jnied: 455-460.Hoopingarner RA, Waller GD (1992). Crop Pollination. In:”The Hive and the Honey bee” 5th ed (J.N.

Graham ed.) Dadant and Sons, Hamilton, Illinois USA: 1043-1083.Иванов АИ (1980): Люцерна. Издателъство-Колос, Москва 1980: 348.Яковлева ЛП (1981): Медоносных пчел и люцерны. Пчеловодство Но. 7: 12-13.Jevtić G (2001). Uticaj medonosne pčele i ostalih oprašivača na prinos semena lucerke. Magistarska

teza, Poljoprivredni fakultet, Zemun, Beograd: 80.Jevtić G (2007). Varijabilnost ekotipova medonosne pčele (Apis mellifera carnica Poll.) i njihov značaj

u oprašivanju lucerke. Doktorska disertacija, Univerzitet u Beogradu, Poljoprivredni fakultet Beograd-Zemun: 139.

Jevtić G, Mladenović M, Nedić N, Radović J (2005a). Influence of cultivar on pollinator visiting, yield, yield components and seed quality components of lucerne. XXXIXth Apimondia International Apicultural Congres. Dublin, Ireland, 21th-26th August: 147.

Jevtić G, Radović J, Anđelković B, Štrbanović R, Milenković J, Anđelković S (2010). The influence of rainfall amount, pollinators and facilitatrion of pollination on the yield and alfalfa seed quality. Proccedings of the XII International Symposium on Forage Crops Of Republic of Serbia. Kruševac-Serbia 26-28 May, Book 2, 233-238.

Jevtić G, Radović J, Ignjatović S, Lugić Z, Nedić N. (2004b). Influence of alfalfa flower colour on pollinator visitation and seed yield components. Procc. 20th General Meeting of the EGF, Luzern, Switzerland, 21th-24th June: 451-453.

Jevtić G, Radović J, Lugić Z (2005b). Uticaj medonosne pčele (Apis mellifera carnica Poll.) i pospešivanja oprašivanja na prinos i kvalitet semena lucerke (Medicago sativa L.). Selekcija i semenarstvo Vol. 11, br. 1-4: 63-68.

22

The Importance of Honey Bee (Apis mellifera carnica Poll.) in Pollination And Seed Yield of Alfalfa (Medicago sativa L.)

Jevtić G, Radović J, Lugić Z, Ignjatović S (2004a). Uticaj rastojanja košnica od semenskog useva lucerke na posetu oprašivača, oprašivanje i prinos semena lucerke. Acta agriculturae Serbica. Vol. IX, 17 (2004) 9-657 (Special Issaue): 129-135.

Jevtić G, Radović J, Lugić Z, Sokolović D, Vasić T (2007). Uticaj medonosne pčele (Apis mellifera L.) i šećernog sirupa na prinos semena lucerke i crvene deteline. Institut za ratarstvo i povrtarstvo, Novi Sad, Zbornik radova, Vol. 44, No. I: 99-106.

Jevtić G, Radović J, Lugić Z, Vasić T (2006). The effect of climatic factors on the visit of pollinators and the seed yield of alfalfa. Proceedings of the 21st General Meeting of the European Grassland Federation Badajoz, Spain 3-6 April: 312-315.

Jones LG (1958). Recent studies on the role of honey bees in the cross-pollination of small-seeded legume crops. 10th Internat. Cong. Ent. Proc. Montreal: 967-970.

Karagić Đ, Katić S, Vasiljević S, Milić D (2007). Semenarstvo lucrke u Vojvodini. Institut za ratarstvo i povrtarstvo, Novi Sad, Zbornik radova, Vol. 44, No. I: 87-98.

Katić S, Mihajlović V, Pataki I (1998). Uticaj samooplodnje na zametanje mahuna i semena lucerke. Savremena poljoprivreda, Vol V br. 1-2: 91-95.

Кырну И, Барак И, Гросу Е (1977): Исследования по опылении люцерны (Medicago sativa L.). Апимондия ХХVI Международный Конгресс по Пчеловодству. Аделаида, Австралия, Издател’ство Апимондии, Бухарест Румыния: 395-397.

Knapp EE, Green WL, Tauber LR (1996). Registration of Four Alfalfa Germplasm Pools Selected for ease of Floret Tripping. Crop Sci. 36 (2): 475.

Kostić Ž (1996). Uticaj ekoloških uslova na prinos i kvalitet semena lucerke. Selekcija i semenarstvo. Vol. III br. 1-2: 84-86.

Kropačova S (1963). Nectar production of lucerne and the number of honeybees working on it . Sborn. Vys. Skoly Zemed. Brne, 1: 37-44.

Kropačova S (1964). A study of the function of honeybees as lucerne pollinators. Zool. Listy. Fol. Zool., 13(2): 143-154.

Krunić MD, Tesei JN, Pinzauti M (1995). Biology and menagement of Megachile rotundata Fab. under European conditions. Apocoltura (Italia), 10: 71-79.

Lesins K (1950). Investigations into seed setting of lucerne at ultuna Sweden. Ann. Roy. Agr. Col. (Uppsala: Sweden) 17: 441-483.

Loper GM, Waller GD (1970). Alfalfa flower aroma and flower selection by honeybees. Crop Sci. 10: 66-68.

Loper GM (1972). Medicago sativa and citrus depressa flower volatiles. Phytochemistry 11: 1865.Loper GM, Flath RA, Webster JL (1971). Identification of ocimene in alfalfa flower aroma by combined.

Crop Sci. 11: 61-63.Lukić D (2000). Lucerka. Izdavač Naučni institut za ratarstvo i povrtarstvo, Novi Sad, 1-458.Lukić D, Katić S, Mihajlović V (1993). Uticaj položaja cvasti na stabljici na komponente prinosa

semena lucerke. Sav. poljoprivreda. Vol. 41 br. 3: 87-90.Mackensen O, Nye WP (1966). Selecting and breeding honeybees for collecting alfalfa pollen. Jour.

Apic. Res. 5: 79-86.Mackensen O, Nye WP (1969). Selective breeding of honeybees for alfalfa pollen collecting: sixth

generation and outcrosses. Jour. Apic. Res. 8: 9-12.Marković D (1997). Produkcija i kvalitet sjemena lucerke u zavisnosti od izbora otkosa za sjeme na

području Doboja. Agroznanje, Godina I: 265-271.Martin EC (1975). Manegement of honey bees in agricultural crops pollination. In:”The Hive and the

Honey bee” 4th ed. (J.N. Grahmn, ed.) Dadant and Sons, Hamilton, Illinois USA.Mc Gregor SE (1976). Alfalfa in: Insect pollination of cultivated crop plans. Agricultural Handbook,

US Department of Agriculture No. 496: 64-75. Moffet OJ, Caddel JL (1985). Using honey bees to pollinate green house alfalfa. Apicultural research.

Dec. 1985: 835-837.Nye WP, Mackensen O (1968a). Selective breeding of honeybees for alfalfa pollen: 5th generation and

backcrosses. Jour. Apic Res. 7: 21-27.

23

Nye WP, Mackensen O (1968b). Breeding bees to the crop (alfalfa). Utah Sci. 29 (2): 46-48, 60.Nye WP, Mackensen O (1970). Selective breeding of honeybees for alfalfa pollen collection with testis

in high and low alfalfa pollen collection regions. Jour. Apic. Res 9: 61-64.Песенко ЮА (1980): Люцерновая пчела-листорез (Megachile rotundata) и её разведение для

опыления люцерны. Изд. “Наука” Ленингратское отделение: 1-136.Pedersen, MW (1962). An analysis of certain factors associated with pollination and seed production

in alfalfa. In 1st Inter. Symposium on pollination, Proc. Copenhagen, Aug. 1960. Commun. 7, Swedish Seed Growers, Assoc.: 64-77.

Pedersen MW, Petersen HL, Bohart GE, Levin MD (1956). A comparison of the effect of complete and partial cross-pollination of alfalfa on pod set, seeds per pod, and seed weight. Agron. Jour. 48: 177-180.

Ponomariova EG (1976): Efficiency of bee pollination when applying advanced agricultural techniques. International symposium. on melliferous flora, Budapest, IX 1976: 158-162.

Radović J, Lugić Z, Sokolović D, Jevtić G, Stanisavljević R (2006). Genetic variability for seed yield and seed yield components of alfalfa (Medicago sativa L.). Proceedings of 26th EUCARPIA Fodder Crops and Amenity Grasses Section Meeting and 16th EUCARPIA Medicago spp. Group Meeting., 3-7. September Perugia, Italy: 121-123.

Radović J, Sokolović D, Marković J (2009). Alfalfa-most important perennial forage legume in animal husbandry. Proceeding of 9th International Symposium, Modern Trend in Livestock Production. Belgrade, 7-9. October 2009: 465-476.

Richards KW (1984). Alfalfa leafcutter bee management in Western Canada. Publication 1495/E Agricultural Canada: Ottawa, Canada: 53.

Schmidt L (1979). Uloga pčela u oprašivanju lucerne i crvene deteline. IX kongres pčelara Jugoslavije Osijek 19-21 Aprila 1979. god. Zbornik radova: 41-42.

Sheesley B, Poduska B (1969). Honey bee pollination of alfalfa seed - improved by supplemental feeding. California agriculture, March, 1969: 11-13.

Sheesley MC, Curley RG, Brooks CR (1985). Alternate block irrigation management for alfalfa seed production. In Proc. California Alfalfa Seed Prod. Symp. Fresnoca. 12. March. University of California Cooperative Extension, Davis: 15.

Sheesly MC, Atkins EL (1985). Value of in-field honeybees watering sites in alfalfa seed pollination. In Proc. California Alfalfa Seed Prod. Symp. Fresnoca. 12. March. University of California Cooperative Extension, Davis: 13-14.

Stanisavljević Lj, Krunić M, Brajković M, Jevtić G (2003) Značaj medonosne i drugih vrsta pčela za oprašivanje gajenih biljaka. XI Naučno savetovanje sa međunarodnim učešćem, 6-9. februar, Poljoprivredni fakultet, Zemun, Zbornik radova: 82-88.

Steiner JJ, Beuselinck PR, Peaden RN, Kojis WP, Bingham ET (1992). Pollinator effects on crossing and genetic shift in a three-flower-colourt alfalfa population. Crop Science. 32: 73-77.

Stjepanović M (1981). Utjecaj ekoloških faktora na osjemenjavanje nekih sorata lucerne na području Istočne Slavonije. Doktorska disertacija, Poljoprivredni fakultet Osjek.

Tauber LR, Barnes DK, Rincker CM (1983). Effectivenes of Selection nectar Volume, Receptacle Diametar and Seed Yield Characteristics in Alfalfa. Crop Sci. 23 (2): 283-289.

Tauber LR, Green WL (1996). Registration of Six Alfalfa Germplasm Pools Selected for Nectar Volume. Crop Sci. 36 (2): 475-476.

Terzić D, (2010). Uticaj vremena kosidbe, đubrenja, mikroelementima i regulatorima rasta na pronos i kvalitet semena lucerke (Medicago sativa L.). Doktorska disertacija, Poljoprivredni fakultet Zemun.

Tucker KW (1956). Honey bee pollinators of alfalfa in Minnesota. 15th Alfalfa Imp. Conf. Rpt. St. Paul, Minn: 32-33.

Tysdal HM (1946). Influence of tripping soil moistoure, plant spacing and lodging on alfalfa seed producttion. Amer. Soc. Agron. Jour. 38: 515-535.

Vansell GH (1951). Use of honeybees in alfalfa seed production. U.S. Dept. Agr. Cir. 876: 11.Vansell GH, Todd FE (1946). Alfalfa tripping by insects. Amer. Soc Agron. Jour. 38: 470-478.

24

The Importance of Honey Bee (Apis mellifera carnica Poll.) in Pollination And Seed Yield of Alfalfa (Medicago sativa L.)

Vučković S, Pavišić-Popović J, Božić M, Zarić D, Prodanović S, Ćupina B, Radović J (1997). Uticaj nekih mikroelemenata na prinos semena lucerke. Agroznanje, Godina I: 273-277.

Waler GD (1970). Attracting honeybees to alfalfa citral geraniol and anis. Jour. Apic. Res. Vol. 9: 9-12.Žarinov VI, Kljuj VS (1983). Ljucerna. Izd. Urožaj. Kiev.Zonjić I (1953). Ispitivanje uticaja važnijih faktora kod zametanja semena lucerke (Medicago sativa).

Zbornik radova polj. fakulteta, Beograd, sveska 1: 1-41.Zonjić I, Krstić O, Tešić-Jovanović B, Miladinović M (1972). Proučavanje stepena autofertilnosti

populacija i inbridovanih linija lucerke. Zavod za Krmno bilje-Kruševac. Zbornik naučnih radova sveska IV: 149-155.