HORTICULTURAL ENTOMOLOGY
Flies (Diptera: Muscidae: Calliphoridae) Are Efficient Pollinators of Allium ampeloprasum L. (Alliaceae) in Field Cages STEPHEN L. CLEMENT,1,2 BARBARA C. HELLIER,1 LESLIE R. ELBERSON,1 RUSSELL T. STASKA,1 AND MARC A. EVANS3
J. Econ. Entomol. 100(1): 131Ð135 (2007)
ABSTRACT In conjunction with efforts to identify efÞcient insect pollinators for seed multiplication of cross-pollinated plant species stored and maintained by USDAÐARS Western Regional Plant Introduction Station (WRPIS), experiments were conducted to assess and compare the efÞciency of the house ßy, Musca domestica L. (Diptera: Muscidae), and Calliphora vicina Robineau-Desvoidy (Diptera: Calliphoridae), and different densities of each ßy species, to pollinate leek, Allium ampeloprasum L., plant inventory (PI) accessions in Þeld cages for seed yield maximization and high germination. Cages with ßowering plants were exposed to 0 ßies or stocked with 100, 250, and 500 M. domestica or C. vicina pupae per week for 6 (2002) and 7 (2004) wk. Seed yield (weight per cage) increased linearly as ßy densities (C. vicina or M. domestica) increased from 0 to 500 pupae per week, with 500 ßy cages averaging 340.7 g (C. vicina) and 70.5 g (M. domestica) of seed in 2002 (PI 368343) and 615.3 g (PI 168977) and 357.5 g (PI 368343) in 2004 when only the C. vicina was used. For 0, 100, and 250 ßy cages, seed yields averaged between 2.3 and 175.3 g in 2002 and 10.7 and 273.1 g in 2004. Mean 100-seed weights between treatments ranged narrowly between 0.4 and 0.5 g in 2002 and 0.3 and 0.4 g in 2004, and germination rates of seed lots from “ßy cages” were mostly ⱖ80% in both years. The C. vicina is an efÞcient and cost-effective pollinator ($388.97 for pupae and shipping, compared with $2,400 for honey bee, Apis mellifera L. [Hymenoptera: Apidae], nuclei) for caged leek accessions, with 250 and 500 C. vicina pupae per week required to produce sufÞcient seed (130 g) to Þll an accession storage bag in the WRPIS gene bank. KEY WORDS leek, Calliphora vicina, Musca domestica, germplasm regeneration, insect pollination
Regeneration of seed-propagated plant species is one of the most important tasks for gene bank curators and managers. This activity, which normally involves Þeld nurseries with multiple plant accessions, is necessary to replenish seed stocks in cold storage that are low in viability and supply. However, there is a risk of unintended hybridization among accessions of allogamous (outcrossing) entomophilous plant species if accessions are clustered in Þeld nurseries. To prevent insect-meditated cross-pollination between clustered accessions in open-Þeld nurseries, gene bank curators use spatial isolation of accessions or Þeld cages over plants with suitable insect pollinators (Clark et al. 1997, Schittenhelm et al. 1997). The USDAÐARS Western Regional Plant Introduction Station (WRPIS), Pullman, WA, is a major gene bank for plant collections (358 plant genera, 2,194 Mention of a trademark or a product does not constitute a guarantee or warranty by the USDA and does not imply its approval over other suitable products. 1 USDAÐARS, Plant Germplasm Introduction and Testing Research Unit, 59 Johnson, Hall, Washington State University, Pullman, WA 99164-6402. 2 Corresponding author, e-mail:
[email protected]. 3 Department of Statistics, Washington State University, Pullman, WA 99164-3144.
plant species, and a seed lot inventory of ⬇72,500 accessions) in the U.S. National Plant Germplasm System. A sizeable percentage of WRPIS germplasm is cross-pollinated species, including species in the genus Allium (Alliaceae). Among the 105 species and 978 accessions of Allium in WRPIS collections are 204 leek accessions, A. ampeloprasum L. [sometimes classiÞed as A. porrum (L.)]. Leek is a vegetable belonging to the onion family and is used as an ingredient in soups and salads. There are three other horticultural groups in A. ampeloprasum (Kurrat, Great-headed Garlic, and Pearl Onion), which are mainly cultivated in Europe and the Middle East, but on a smaller scale than leeks. Leeks are an important crop in Europe. Wild A. ampeloprasum originates from Portugal in the west through the Mediterranean countries to Iran and elsewhere in the Middle East (van der Meer and Hanelt 1990, Brewster 1994). Leeks, which do not normally bulb like onions, are grown from seed. A leek seed stalk can approach 1.2 m in height and is terminated by a single umbel containing hundreds of bell-shaped ßowers with light purple petals. These protandrous ßowers facilitate out-crossing. However, protandry only offers a partial barrier to self-pollination because pollen from ßowers that typically open over 2Ð 4 wk can fertilize receptive stigma of more
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advanced ßowers in the same umbel (McGregor 1976, Brewster 1994). This potential for self-pollination complicates seed regeneration because leeks are subject to severe inbreeding depression, which can lead to severe loss of vigor and reduced survival in seedlings derived from self-pollination (Currah 1986, Brewster 1994, Silvertand et al. 1995). Therefore, leek accessions must be regenerated without risk of contamination by pollen from neighboring accessions, yet under conditions that encourage high rates of cross-pollination. These requirements can be accomplished by coupling a suitable insect pollinator with caged plants of a leek accession. The honey bee, ßies in the genera Calliphora, Lucilia (Diptera: Calliphoridae), and Eristalis (Diptera: Syrphidae), and the solitary bee Osmia rufa L. (Hymenoptera: Megachilidae) have been used to pollinate caged onions, A. cepa L. (Moffett 1965; Bohart et al. 1970; Free 1970; Schittenhelm et al. 1997; Currah and Ockendon 1983, 1984). Although some of these investigators found that ßies and honey bees were equivalent in their suitability as pollinators for caged onions (Currah and Ockendon 1983, 1984), others reported that calliphorid and Eristalis ßies were more suitable than honey bees for cross-pollinating onions in cages (Bohart et al. 1970, Free 1970). It is well known that onion ßowers are unattractive to the honey bee (Bohart et al. 1970, McGregor 1976, Silva et al. 2003), so it is not surprising that some ßy species might be better pollinators for caged onions, as well as other alliums. Schittenhelm et al. (1997) did not recommend the solitary bee O. rufa as a pollinator for caged onions because it was repelled by the plants. Without published accounts on the efÞciency of different insects for pollinating A. ampeloprasum in cages, but armed with the results of the aforementioned reports on A. cepa pollination, we chose to evaluate two ßy species for their efÞciency to pollinate leek accessions in cages. In addition, ßy pupae were available from commercial suppliers, whereas honey bees, Apis mellifera L. (Hymenoptera: Apidae), were unavailable at the start of this study. The research objective of this multiyear study was to assess and compare the efÞciency of the house ßy, Musca domestica L. (Diptera: Muscidae), and Calliphora vicina Robineau-Desvoidy (Diptera: Calliphoridae), and variable densities of each species, to pollinate caged leek accessions in the Þeld for seed yield maximization and high germination. Using our results, we identify the best ßy pollinator for the WRPIS leek regeneration program. Last, we compare the economics of ßies versus honey bees for pollination of caged leek accessions. Materials and Methods Sources of Plants and Flies. The plants selected for this study were grown from seed of two plant inventory (PI 368343, from the former Yugoslavia; PI 168977, from Turkey) accessions of A. ampeloprasum obtained from the WRPIS gene bank. In 2002, ßy pupae were purchased from Rincon-Vitova, Ventura,
Vol. 100, no. 1
CA (M. domestica) and Forked Tree Ranch, Porthill, ID (C. vicina). Once a week between 8 July and 12 August 2002, these vendors express-mailed 5,000Ð15,000 pupae of their respective ßy species to Pullman, WA. In 2004, only C. vicina pupae were required for this study, and 15,000 pupae were purchased from Forked Tree Ranch and shipped weekly, 6 July to 16 August. Caged Field Experiments. The experiments were conducted, 2001Ð2002 and 2003Ð2004, at a major WRPIS seed regeneration site (25 ha) bordering the Snake River and near Central Ferry in southeastern Washington (46⬚ 40⬘ 13⬙ N, 117⬚ 45⬘ 8⬙ W). Plots were established using 4-wk-old seedlings grown from seed in a glasshouse and transplanted to the Þeld on 26 and 27 September 2001 and 15Ð17 October 2003. The seedlings were transplanted into black plastic ground cover for weed suppression and watered with drip irrigation as needed. Nitrogen was applied before transplanting at 45 kg 䡠 ha⫺1. No pesticides were applied and weed control was by cultivation before transplantation and hand hoeing thereafter. A plot (4 by 7 m) consisted of four 6-m-long rows on 0.3-m centers, with each row containing 18 Ð20 evenly spaced transplants for a total of 75Ð 80 plants per plot. The spacing between plots was 1.5 m. Each plot was thinned to 53 and 49 bolting plants on 8 May 2002 and 16 June 2004, respectively. In June 2002 and 2004, before plants ßowered, each plot was covered with a screen Þeld cage (4 by 7 by 2 m) (Synthetic Industries, Gainesville, GA) and randomly assigned a ßy density treatment: 100, 250, and 500 M. domestica or C. vicina pupae, and one 0 ßy cage (no pupae added) in 2002; and 0, 100, 250, and 500 C. vicina pupae in 2004. These assignments resulted in seven treatment cages per block in 2002 (one leek accession [PI 368343] ⫻ two ßy species ⫻ 3 ßy rates ⫹ one 0 ßy cage) and eight treatment cages per block in 2004 (two leek accessions [PI 368343, PI 168977] ⫻ one ßy species ⫻ 4 ßy rates). Each experiment was replicated four times in randomized complete blocks for a total of 28 and 32 cages in 2002 and 2004, respectively. Cages were secured to the ground by using metal stakes and bottom edges along each side were buried in soil. Fly pupae were placed in cages at the onset of ßowering and until ßowering was complete. There were six weekly release dates (8 JulyÐ12 August) in 2002 and seven (6 JulyÐ16 August) in 2004. Pupae were placed in a plastic pot (15 cm in diameter) positioned on its side on the soil surface of each cage. The pot was held in place by a few grams of top soil. This technique sheltered pupae from wind and direct sunlight and allowed us to easily monitor adult emergence. Flies began emerging from pupae within one day and usually continued for another 2Ð3 d. A plastic pan (29 by 35 cm) Þlled with 15 cm of water-saturated vermiculite (water added as needed) was placed in a corner of each cage to provide a constant supply of moisture for ßies during periods of ambient temperature extremes (10.2Ð 41.8⬚C, 2002; 12.5Ð39.7⬚C, 2004). We thinned to 47 primary umbels per cage (one per plant) on 22 August 2002 and 16 September 2004. The mature umbels were hand-harvested during the last
February 2007 Table 1.
CLEMENT ET AL.: FLY POLLINATION OF A. ampeloprasum
133
Partial ANOVA results for the effects of fly pollinator species and pollinator density on seed yield of A. ampeloprasum, 2002
Source
df
Fly species (F) Fly density (D) F⫻D Linear trend for D Nonparallel regression for F Error
1 2 2 1 1 18
100-seed wt/cage
Seed germination
F
Seed wt/cage P
F
P
F
P
292.13 109.64 44.95 219.15 88.82
⬍0.0001 ⬍0.0001 ⬍0.0001 ⬍0.0001 ⬍0.0001
8.34 2.21 0.23 4.07 0.13
0.0098 0.1385 0.7951 0.0589 0.7268
0.00 0.47 0.20 0.03 0.29
0.9750 0.0346 0.8201 0.8620 0.5945
week of October 2002 and 2004, after which they were hand-threshed with a Vogel thresher (Bills Welding, Pullman, WA) and cleaned of nonseed material by using an in-house built laboratory air screen cleaner. Cleaned and dried seed lots were stored at room temperature (⬇21⬚C) before weighing seed in November and December 2002 and 2004. Data were recorded for total seed weight per cage (47 umbels) and for the weight of a random sample of 100 seeds per cage. In addition, a representative sample of 100 seeds from each cage was germinated between sections of moist blotter paper in a plastic germination box (11.5 by 11.5 cm) (50 seeds per box). Seed germination at the “loop stage” (Brewster 1994) was recorded after 21 d at 20⬚C (photoperiod of 8:16 [L:D] h) in a controlled temperature incubator (Hoffman Manufacturing Inc., Albany, OR). This germination procedure is in accordance with that established by the Association of OfÞcial Seed Analysts (AOSA 1998). Statistical Analysis. Data (seed weights [total, 100 seed sample] and germination rates [%] per cage) from each year were analyzed separately using PROC GLM based on a randomized complete block design with a two-way treatment structure (SAS Institute 2003). Analysis of 2002 data assessed the main effects for ßy species (M. domestica and C. vicina) and density (0, 100, 250, and 500 pupae) per ßy species, and the interactions, on seed yield and germination of PI 368343. Analysis of 2004 data (seed yield and germination comparisons, and the interactions) involved PI 368343 and PI 168977 and different C. vicina densities (0, 100, 250, and 500 pupae). Orthogonal polynomial contrasts were used to assess a linear trend in seed yield response due to ßy density and to determine whether the linear trends were parallel for two ßy species (2002) and two leek accessions (2004). Mean comparisons were made using Fisher least signiÞcant difference (LSD) (␣ ⫽ 0.05 for all comparisons). Regression analysis was performed to using the PROC REG procedure to assess the linear relationship between seed yield and ßy density (SAS Institute 2003).
species on seed yield per cage were signiÞcant (Table 1). Seed yield (weight per cage) increased linearly (P ⬍ 0.0001) as densities increased for C. vicina (seed yield ⫽ 0.982 ⫹ 0.682 䡠 ßy density; r2 ⫽ 0.97) and M. domestica (seed yield ⫽ ⫺2.680 ⫹ 0.135 䡠 ßy density; r2 ⫽ 0.72). Except for the main effect of ßy species, no other signiÞcant P values were detected for the 100seed weights (Table 1). This main effect is illustrated by mean ⫾ SEM 100-seed weights (grams) of 0.44 ⫾ 0.01 (0 ßy cages), 0.44 ⫾ 0.01 (M. domestica cages), and 0.41 ⫾ 0.01 (C. vicina cages) (LSD ⫽ 0.02). Overall, mean 100-seed weights ranged narrowly between 0.4 and 0.5 g (Table 2). The seed yield data in Table 2 illustrate the positive effect of high ßy densities on pollination and seed production of caged leek plants. In 2002, signiÞcantly more seed (x ⫽ 340.7 g) was produced by caged plants that received 500 C. vicina pupae per week compared with 0, 100, and 250 M. domestica and C. vicina pupae per week (averages of 2.3Ð175.3 g). Additionally, the results for seed weight per cage clearly show that C. vicina was superior to M. domestica for cage pollination and seed production (Table 2). The superior performance of C. vicina in 2002 led to its exclusive use in 2004 and for pollination of two leek accessions. Analysis of the 2004 data revealed significant effects of accession, ßy density, and the accession ⫻ ßy density interaction on seed yield per cage. In addition, this analysis generated a signiÞcant linear trend for ßy density and nonparallel regressions for accessions (Table 3). Seed yield (weight per cage) increased linearly (P ⬍ 0.0001) for PI 368343 (seed yield ⫽ 22.52 ⫹ 0.716 䡠 ßy density; r2 ⫽ 0.89) and PI Table 2. Yields and germination rates of seed produced by caged leek plants (PI 368343) exposed to different fly pollinators and densities, 2002 Mean ⫾ SEM Diptera species
Fly density (no. pupae/wk)
M. domestica
100 250 500 100 250 500 0
Results and Discussion For 2002, the effects of ßy species and ßy density on seed yield per cage of PI 368343 were signiÞcant. Also, there was a signiÞcant ßy ⫻ ßy density interaction on seed yield per cage, indicating a differential effect of ßy density on the pollination efÞciency of M. domestica and C. vicina. As well, the linear trend for ßy density and nonparallel regressions for the effect of ßy
C. vicina No ßies
Seed wt/ cage (g) 11.8 ⫾ 3.6a 19.7 ⫾ 3.9a 70.5 ⫾ 15.5b 65.2 ⫾ 3.9b 175.3 ⫾ 13.9c 340.7 ⫾ 19.1d 2.3 ⫾ 0.9a
100-seed Seed wt/cage germination (g) (%) 0.5 ⫾ 0.0 0.4 ⫾ 0.0 0.4 ⫾ 0.0 0.4 ⫾ 0.0 0.4 ⫾ 0.0 0.4 ⫾ 0.0 0.4 ⫾ 0.0
87.8 ⫾ 2.4 83.5 ⫾ 4.1 86.0 ⫾ 1.5 85.5 ⫾ 0.9 84.5 ⫾ 2.9 87.5 ⫾ 2.9 78.3 ⫾ 5.7
Means (n ⫽ 4) in the one column followed by a common letter are not signiÞcantly different (P ⫽ 0.05; LSD). Mean comparisons were not computed for the values in the other columns because ßy species ⫻ ßy density interactions were not signiÞcant.
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JOURNAL OF ECONOMIC ENTOMOLOGY
Table 3. 2004
Partial ANOVA results for the effects of fly pollinator density on seed yield of two leek accessions (PI 168977 and PI368343),
Source
df
Accession (A) Fly density (D) A⫻D Linear trend for D Nonparallel regression for A Error
1 3 3 1 1 21
100-seed wt/cage
Seed germination
F
Seed wt/cage P
F
P
F
P
17.50 111.87 8.99 334.82 20.98
⬍0.0004 ⬍0.0001 ⬍0.0005 ⬍0.0001 ⬍0.0002
1.02 6.87 2.06 19.80 3.20
0.3236 0.0021 0.1359 0.0002 0.0881
19.15 6.73 0.57 19.80 1.58
0.0003 0.0023 0.6402 0.0002 0.2231
168977 (seed yield ⫽ 3.169 ⫹ 1.194 䡠 ßy density; r2 ⫽ 0.94) because C. vicina densities for each of these accessions increased from 0 to 500 pupae per week. Our analysis of 100-seed weight data detected significance for ßy density and a linear trend for ßy density (Table 3). The signiÞcance for ßy density (across the two accessions) is illustrated by mean ⫾ SEM 100-seed weights (grams) of 0.37 ⫾ 0.01 (0 ßy cages), 0.36 ⫾ 0.01 (100 C. vicina), 0.34 ⫾ 0.01 (250 C. vicina), and 0.33 ⫾ 0.01 (500 C. vicina) (LSD ⫽ 0.02). The differences between mean 100-seed weights across all 2004 treatments were not pronounced (0.3Ð 0.4 g) (Table 4); however, these means are generally lower than the 2002 values (Table 2) for reasons not evident to us. Caged leeks stocked weekly with 500 C. vicina pupae produced the highest seed yields in 2004 (Table 4), a pattern also recorded in 2002 (Table 2). Also, more seed, on average, was produced by PI 168977 than by PI 368343 in 2004, irrespective of C. vicina density (Table 4). Interestingly, mean seed weights from 2002 and 2004 caged plants of PI 368343, across all ßy densities, were roughly equivalent (Tables 2 and 4). Main and interaction effects for seed germination rates in 2002 were not statistically signiÞcant (Table 1), with mean values from 78.3% (0 ßy cages) to 87.8% (100 M. domestica) (Table 2). By contrast, mean germination rates differed signiÞcantly (P ⬍ 0.05) among 2004 treatments (Table 4). Regarding these 2004 germination rates, there was a signiÞcant accession effect, Table 4. Yield and germination rates of seed produced by caged plants of two leek accessions (PI 168977 and PI 368343) exposed to different densities of the fly pollinator C. vicina, 2004 Mean ⫾ SEM Fly density Accession (no. pupae/wk) PI 368343
PI 168977
Vol. 100, no. 1
0 100 250 500 0 100 250 500
Seed wt/ cage (g) 10.7 ⫾ 2.1a 75.5 ⫾ 9.0ad 255.1 ⫾ 37.1b 357.5 ⫾ 17.0c 20.2 ⫾ 1.1a 119.3 ⫾ 10.1d 273.1 ⫾ 31.5b 615.3 ⫾ 52.9e
100-seed Seed wt/cage germination (g) (%) 0.4 ⫾ 0.0 0.3 ⫾ 0.0 0.3 ⫾ 0.0 0.3 ⫾ 0.0 0.3 ⫾ 0.0 0.4 ⫾ 0.0 0.3 ⫾ 0.0 0.3 ⫾ 0.0
69.8 ⫾ 1.4a 73.3 ⫾ 2.7ab 78.3 ⫾ 1.0bc 83.3 ⫾ 2.3cd 80.0 ⫾ 1.5bc 83.0 ⫾ 3.0cd 84.5 ⫾ 2.3cd 88.0 ⫾ 3.7d
Means (n ⫽ 4) in the two columns followed by a common letter are not signiÞcantly different (P ⫽ 0.05; LSD). Mean comparisons were not computed for the values in the middle column because the accession ⫻ ßy density interaction was not signiÞcant.
as revealed by the generally higher mean values for PI 168977, and an overall linear trend for ßy density (Tables 3 and 4). The analysis also revealed a significant effect for ßy density on the germination rates of each accession (Table 3), which can be discerned from the results in Table 4. Three general conclusions can be gathered from our overall results. First, insect pollination is essential for high seed yields of leek accessions in Þeld cages. Second, ßies possess desirable attributes for insect pollination of leek accessions, with C. vicina superior to M. domestica for pollination and high seed production of caged plants. The 250 and 500 C. vicina treatments produced the highest seed yields (weight per cage) in 2002 and 2004. By contrast, mean 100-seed weights among treatments were relatively constant each year. Finally, there was a trend for higher germination rates of seed lots from “ßy cages” compared with “0 ßy cages.” A minimally successful WRPIS Allium seedÐregeneration cycle requires the production of 10,000 seeds per accession, which equates to ⬇36 g of A. ampeloprasum seed. However, a much larger amount of seed (130 g) is required to Þll an accession storage bag in the WRPIS gene bank. Our results show that 250 and 500 C. vicina pupae per week were required for 47 caged plants to produce at least 130 g of seed. Moreover, we posit that these high C. vicina densities effected high rates of cross-pollination. Good crosspollination can lead to a high degree of heterozygosity and the production of high quality seed in a leek accession. It also can negate the inßuence of deleterious alleles responsible for inbreeding depression and other genetic defects (Currah 1986, Brewster 1994). Expenses for M. domestica and C. vicina (pupae and shipping) were $298.20 and $178.83, respectively, in 2002 and $210.14 for C. vicina in 2004. Had honey bees been available for this study, the cost for a four-frame nucleus of A. mellifera would have been $50.00. Thus, the expense of using 24 honey bee nuclei in each year of this study would have totaled at least $2,400, an amount that far exceeds the amount paid for the ßy pupae. In conclusion, our results show that C. vicina (250 Ð500 ßies per week per cage) is an efÞcient and cost-effective pollinator for seed production of leek accessions at the WRPIS gene bank.
February 2007
CLEMENT ET AL.: FLY POLLINATION OF A. ampeloprasum Acknowledgments
We are grateful to Kurt Tetrick and Scott McGee for expert help and assistance with establishing and maintaining the Þeld plots, and to Jacqueline Cruver for seed cleaning operations. Rich Hannan provided valuable input on an early draft of this manuscript.
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