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Effects of climatic and edaphic factors on soybean flowers and on the subsequent attractiveness of the plants to honey bees
Field Crops Research, 6 (1983) 267--278
267
Elsevier Science Publishers B.V., Amsterdam -- Printed in The Netherlands
EFFECTS OF CLIMATIC A N D E D A P ~ I C FACTORS ON SOYBEAN FLOWERS A N D ON THE SUBSEQUENT ATTRACTIVENESS OF THE PLANTS TO HONEY BEES
D.C. ROBACKER, P.K. FLO2"rUM, D. SAMMATARO and E.H. ERICKSON
Department of Entomology, University of Wisconsin-Madison, and U.S. Department of Agriculture, Agricultural Research Service, Bee Research Unit, Madison, WI 53706 (U.S.A.) (Accepted 9 November 1982)
ABSTRACT Robacker, D.C., Flottum, P.K., Sammataro, D. and Erickson, E.H., 1983. Effects of climatic and edaphic factors on soybean flowers and on the subsequent attractiveness of the plants to honey bees. Field Crops Res., 6: 267--278. Soybean (Glycine max) plants were grown at various day and night air temperatures, soil temperatures and soil concentrations of N, P and K, to investigate effects of environmental conditions on flower characteristics, including flower production, color intensity, openness, size, nectar secretion and aroma emanation and on attractiveness of the plants to honey bees. Most flower characteristics increased as day air temperatures at which plants were grown increased from 20 to 24 ° C and reached maximum values at 28 ° C before plateauing or declining at 32 ° C, although flower size and nectar secretion continued to increase as growing temperature increased to 32 ° C. Of two flower aroma components, emanation of one c o m p o n e n t increased while the other decreased with increases in growing temperatures. The hypothesis suggested is that the two aroma chemicals may communicate flower-readiness information to pollinators. Flower production and flower openness responded linearly to night air temperature at which plants were grown, attaining highest values at higher (22, 26 ° C) vs. lower (14, 18 ° C) temperatures. Flower production also responded linearly to soil temperature, attaining highest values at higher (28--32 ° C) vs. lower (16--20 ° C) temperatures. Of two levels each of N (75 and 175 ppm) and P (15 and 30 ppm) tested, the higher level of N stimulated greater flower production, flower size and nectar secretion while the higher level of P decreased the same three flower characteristics. Conversely, lower N and higher P promoted flower openness. Honey bee attractiveness of plants varied positively with flower characteristics such that plants grown at a day air temperature of 28 ° C, night air temperatures of 22 and 26 ° C, the higher level of N of the lower level of P were the most attractive to honey bees.
INTRODUCTION
Although soybean (Glycine max [L.] Merr.) is generally considered a selfpollinating species, yield increases of about 10--40% have been demonstrated Mention of a proprietary product in this paper does not constitute an endorsement by the USDA. 0378-4290/83/$03.00
© 1983 Elsevier Science Publishers B.V.
268 when comparing honey bee (Apis mellifera L.) pollinated vs. self-pollinated plants (Erickson, 1975a; Juliano, 1977; Erickson et al., 1978). Further, soybean flowers possess many entomophilous characteristics (Erickson and Garment, 1979) and honey bees produce substantial honey crops when they visit soybeans (Abrams et al., 1978; Erickson and Robins, 1979). However, soybean flowers are not always attractive to honey bees, leading to the hypothesis that environmental conditions during growth and flowering of plants affect the development of entomophilous flower characteristics. The objective of this work is to evaluate the effects of environmental conditions during plant growth on flowers and on attractiveness of soybean plants to honey bees. To accomplish this objective, three experiments were conducted. The first experiment investigated effects of day and night temperatures, the second effects of soil temperature at 2-day air temperatures, and the third examined effects of N, P and K nutrition. Various flower characteristics assumed to be involved in honey bee attraction were measured and actual honey bee attractiveness tests were conducted in all three experiments. Effects of environment on plant characteristics were not investigated in this work since this has been reported by others (Caldwell, 1973; Norman, 1978). METHODS
General The following conditions were constant in all three experiments. Soybean (var. Mitchell, Group IV) plants were grown in controlled environment rooms in the University of Wisconsin Biotron. Seeds were surface sterilized with 10% hypochlorite solution and sown individually in 20-cm pots containing sand. Photophase was 16 h, average light flux at plant level was about 600 pE m-2 s-1 (fluorescent and incandescent lights) and relative humidity was 70%. Plants were irrigated with nutrient solution often enough to prevent apparent stress. Light intensity and temperature were changed incrementally during the first and last hours of photophase to simulate natural day/night changes.
Experiments Experiment 1: Day and night air temperature This experiment tested effects of 16 combinations of day and night air temperatures in the Biotron's crossed-gradients room which is capable of maintaining four distinct temperature regions at any one time. The four regions were programmed with the following day/night temperature combinations: 20/14, 24/18, 28/22 and 32/26 ° C. During the first and last hours of photophase, groups of plants were moved among the four temperature regions to achieve the full factorial complement of 16 treatments. For example, during the day one region held the treatments 20/14, 20/18, 20/22 and 20/26 ° C, during the last hour of photophase, plants of the latter three treatments were
269 moved to their night positions in the 24/18, 28/22 and 32/26°C regions, respectively. During the first hour of photophase, these plants were returned to their day positions. Other temperature regions were handled similarily. There were three replications of each treatment. Treatments were necessarily confounded with position since temperature regions could n o t be randomized within the room. Plants were irrigated with half-strength Hoagland solution (N--105 ppm, P--15 ppm, K--128 ppm). Experiment 2: Soil temr~erature at two air temperatures Plants were grown in the crossed-gradients room and were irrigated with half-strength Hoagland solution. Day air temperature was 28 ° C and night air temperature was 20--24 ° C during a 2-week germination period. Plants were then divided into 2-day/night air temperature groups of 24/18 and 32/22 ° C, each with 16 plants. Within each group, day soil temperatures were adjusted to values ranging from 16 to 32°C using heat exchangers (Robacker et al., 1982). Soil temperatures decreased at night as a result of the decrease in air temperature such that plants receiving higher day soft temperatures also received higher night soil temperatures. In general, soil temperatures decreased by 4--8 ° C (greater decreases for higher day values) at night. Therefore, the integrity of the overall soil temperature treatments was maintained during both the day and night. As before, air temperature treatments were necessarily confounded with position. Experiment 3: NPK experiment Plants were grown in a large plant room where they received day/night air temperatures of 28/20 ° C. Plants were irrigated with nutrient solution similar to half strength Hoagland solution except for the concentrations of N, P and K. Levels of N, P and K were: N--75 and 175 p p m as urea; P--15 and 30 ppm as monobasic/dibasic sodium phosphate in a 2.8:1 molar ratio; K--75 and 175 p p m as potassium chloride. These ranges in nutrients are generally within acceptable limits for Mitchell soybeans although the effects of each are influenced by levels of the other t w o (Caldwell, 1973). The two levels each of N, P and K were administered in factorial combination of eight treatments with four replications per treatment. Flower measurements Flower production was measured as the number of fresh (< 1 day old) flowers at 5--9 h after the onset of the photophase, 7 days after the day of first flower for each plant. Flower color intensity was measured from color photographs of one typical flower per plant selected 5--8 h after the onset of the photophase, 2--4 days after first flower. Intensity of red and blue was rated from 1--12 (12: most intense) b y comparing photographs with a two-dimensional color chart (Maerz and Paul, 1950) on which intensity of red increased along one axis and blue
270
along the other. Overall color intensity was assigned by adding the values for red and blue. Flower size was also determined from the photographs, since flowers were photographed beside a m m scale. Flower size was calculated by measuring the base diameter at the point most proximal to the pedicel where the petals emerge from the calyx. Flower openness was determined as the mean of the degree of openness of all flowers per plant. Flowers were rated on a scale of 1--5 (5: fully open), 5--7 h after the onset of the photophase, 7 days after first flower. Nectar secretion was determined as the mean of nectar secretion of four flowers per plant. Flowers were sampled with 1 p L D r u m m o n d Microcaps @, 2.5--4.5 h after the onset of the photophase, 5--8 days after first flower. Nectary development was determined from the mean of three flowers per plant for the soil temperature experiment and from one flower per plant for the NPK experiment, but was not determined for the day and night air temperature experiment. Flowers were collected during the peak flowering period of each plant at 3--5 h after the onset of the photophase, fixed in 2.5% glutaraldehyde /1.0 M phosphate buffer, rinsed 3 times for 20 min each with 0.1 M phosphate buffer, and dehydrated through a graded series of ethanol (50, 70, 90, 100%) for 20 min at each dilution. Transverse sections were cut at about 2 mm above the flower-pedicel junction and examined under a light microscope. Nectary wall thickness was measured with a microscope eyepiece micrometer and used as the indicator of nectary development. Flower aroma was measured from 10 to 15 flowers collected from 5--6 h after the onset of the photophase, 9 days after first flower for each plant from the day and night air temperature experiment. The flowers were placed into a 20 ml glass syringe and the plunger inserted to the 5 ml line. After a 1 h equilibration at 28 ° C, the 5 ml of air were injected onto a column packed with 5% SE 30 on 100/120 Gas Chrom Q, at 140°C (Varian 3700 Gas Chromatograph with FID detector). Quantities of volatile components of flower aroma were determined by measuring peak heights.
Honey bee attractiveness index A honey bee bioassay r o o m was set up in the Biotron. The room was maintained at full light flux (about 600 uE m -2 s-l), day/night temperatures of 28/20 ° C, relative h u m i d i t y of 70% and a 12 h photophase which began synchronously with the 16 h photophase used in the other rooms. One colony of about 3000 bees was placed in the r o o m 2 weeks before the beginning of assays and fed a pollen/sugar cake once each week. The room also contained three " s t a n d a r d " soybean plants (var. Mitchell) which were grown at day/ night temperatures of 28/20 ° C and other conditions the same as in the day/ night air temperature experiment. The three plants were replaced weekly with three fresh plants to keep the standards near peak flowering condition. For each bioassay, the test plant was placed in the bioassay room, watered
271 to drip point, and allowed to equilibrate, with bee visitation, for 0.5 h. During the next 0.5 h, the number of bees foraging at the test plant was recorded during seven predetermined 1-min observation periods. Also during this 0.5 h, the number of bees foraging at the three standards was recorded during six predetermined 1-min observation periods. Attractiveness index was calculated b y dividing the total number of bees observed foraging at the test plant by the total number observed at the three standard plants. Bioassays were conducted from 3--8 h after the onset of the photophase since bees most actively visited soybean flowers during this time. Whenever possible, plants from the same treatment were tested at different times of the day to prevent confounding of treatments with time of day. All 48 plants from the day and night air temperature experiment, 16 plants representing highest and lowest soil temperatures at each air temperature from the soil temperature experiment, and t w o plants from each of the eight treatments from the NPK experiment were bioassayed individually, 7 days after first flower for each plant.
Statistical analyses Completely randomized designs were assumed for all analyses despite confounding of treatments with positions in both the day/night air temperature experiment and the soil temperature experiment, and of treatments with day on which attractiveness bioassays were conducted in all three experiments. Position bias seemed unimportant since the only apparent position difference -- light flux -- had no significant effects on dependent variables when analyzed as an independent variable. Effects of bioassay date were ruled o u t since mean visitation to "standard" plants did not change over days. Regression analyses were used to calculate main and interaction effects of treatments on flower characteristics and on h o n e y bee attractiveness indices. RESULTS AND DISCUSSION
Effects of day air temperature Main effects of day air temperature are reported in Table I. The data show that plants grown at a day air temperature of 20°C were inferior to those grown at other temperatures. That is, they produced the second fewest flowers which individually were the smallest, least intensely colored, least open and secreted the lowest amounts of nectar. In general, plants grown at higher day air temperatures were much improved over those grown at 20 ° C, b u t effects on individual flower characteristics varied. Flower size and nectar secretion increased linearly with temperature from 20 to 32°C (P < 0.01). Flower openness and flower color intensity increased linearly (P < 0.01) with temperature from 20 to 24 and 20 to 28 ° C, respectively. Both remained unchanged with further increases to 32 ° C, resulting in quadratic regression components (P <
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0.01). Flower production showed a highly significant (P < 0.01) quadratic curve with peak production at 24--28°C and lower production of flowers at 20 and 32 ° C. Gas chromatographic analyses of flower aroma uncovered 2 peaks (Table I). The peak of lower retention time, component 1, varied inversely (r = -0.74, P < 0.01) with the peak of higher retention time, component 2. Data in Table I show that aroma component 2 responded to _growingtemperatures similarily to most other flower characteristics. Thus, component 2 increased with growing temperature from 20 to 24 ° C and remained unchanged with further temperature increases, resulting in significant (P < 0.01) linear and quadratic regression components. Component 1, however, decreased with growing temperature from 20 to 28°C and remained unchanged with further temperature increase, also resulting in significant (P < 0.01) linear and quadratic regression coefficients, but of opposite sign to those for component 2. Presently, the hypothesis that aroma components 1 and 2 communicate flower readiness for visitation and/or pollination to pollinators, is under investigation. Within limits, increased air temperatures generally increase nectar production in most species of plants investigated (Shuel, 1967). For soybeans, Jaycox (1970) reported nectar secretion to be favored by day air temperatures near 27°C for field grown plants and Erickson (1975b) reported 21°C as a lower limit for both nectar secretion and flower opening in many soybean cultivars (Mitchell not included). Our results agree with the literature and extend the effects of day air temperature on soybean flowers to include flower production, flower color intensity, flower size and flower aroma. Honey bee attractiveness index, which is theoretically dependent on the quality of flower characteristics, increased dramatically as growing temperatures of test plants increased from 20 to 24 ° C, then remained unchanged with further increases to 32 ° C. Linear and quadratic regression components were highly significant (P < 0.01). Thus honey bee attractiveness index and all flower characteristics, except aroma component 1, produced essentially the same curve in response to day air temperatures. These results suggest that honey bee attractiveness index is in fact varying in response to changes in flower characteristics.
Effects of night air temperature Main effects of night air temperature are reported in Table I. The data show that, compared to day air temperatures, night air temperatures at which plants were grown had little impact on flower characteristics. However, both flower production (P < 0.05) and flower openness (P < 0.01) showed linear increases with night temperature increases from 14 to 26 ° C. Nectar secretion responded similarily, but the result was not significant. Honey bee attractiveness index also increased linearly (P < 0.01) with increases in night temperature, possibly in response to changes in flower production, flower openness and nectar secretion.
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275 The authors are aware of no studies reporting effects of night air temperatures on soybean flower characteristics. Related studies on the effects of night air temperature have demonstrated no significant changes in nectar secretion by red clover (Shuel, 1952), increased sucrose/reducing-sugar ratios at low temperatures for alfalfa nectar (Walker et al., 1974), increased flower openness during the day following warmer nights for lima bean (Fisher and Weaver, 1974) and increased flower production with higher night temperatures for sugarcane (Berding, 1981).
Interactions o f day and night air temperatures Only flower color intensity showed a significant interaction effect (P < 0.01). This occurred because increases in night air temperature at a day air temperature of 20 ° C caused a linear increase in color intensity whereas night air temperature had no effects at other day air temperatures. Flower color intensity for plants grown at day air temperatures of 20 ° C and night air temperatures of 14, 18 and 22°C were significantly lower than for plants grown at any other day/night air temperature combinations (P ~ 0.01L
Effects o f soil temt~erature Main effects of soil temperature are shown in Table II. Note that the temperatures in Table II are day soil temperatures. Corresponding night soil temperatures were 4--8 ° C lower, respectively, for day soil temperatures of 16-32°C. The data show that flower production increased linearly (P < 0.01) with increasing soil temperature at which plants were grown. This effect was consistent at b o t h the 24 and 32°C day air temperatures, therefore the data in Table II are the combined results of the t w o day air temperatures. No other flower characteristics were significantly affected by soil temperature in our study. In a similar study, however, Shuel and Shivas (1953) reported that nectar sugar production of snapdragons subjected to soil temperatures of 15.5, 21 and 27 ° C at the beginning of the flowering stage increased with increasing soil temperature. Because so few flower characteristics were affected by soil temperature, honey bee attractiveness index also was not significantly affected. However, the value of h o n e y bee attractiveness index for higher soil temperature plants was slightly higher than for lower soil temperature plants (26 vs 22 ° C), possibly due to higher flower production by plants grown at higher soil temperatures. This effect was also consistent at both day air temperatures.
Effects o f N, P and K The main effects of N, P and K are reported in Table III. The data show that N and P had major effects on flower characteristics, while K had no sig-
276 TABLE III Main effects o f N, P and K on soybean flower characteristics and honey bee attractiveness index Measurement
N (ppm) 75
Flower production (flowers/plant) Flower color intensity a Flower size (mm) Flower openness a Nectar secretion (nl/flower) Neetary wall development (#) Honey bee attractiveness index b
P (ppm) 175
15
K (ppm) 30
75
175
18"*
27 c**
32**
15"*
22
25
14 1.9"* 4.6** 25"
15 2.2** 4.2** 40*
15 2.2** 4.2** 45"*
14 2.0** 4.6** 20"*
15 2.0 4.4 28
14 2.2 4.4 35
140 0.4*
160 0.9*
150 0.8*
150 0.4*
140 0.5
160 0.8
aArbitrary units. Color intensity: 1--24, 24: most intense. Flower openness: 1--5, 5: most open. bValues are ratios of number of foragers at test plants divided by number of foragers at standard plants during bioassays. c . and ** indicate 5 and 1% significance levels, respectively.
nificant effects. Flower production (P < 0.01), flower size (P ~ 0.01) and nectar secretion (P < 0.05) were all higher for plants grown at the higher (175 ppm) vs. the lower (75 ppm) concentrations of N. However, the same three flower characteristics were lower (P < 0.01) for plants grown at the higher (30 ppm) vs. the lower (15 ppm) concentrations of P. Conversely, flowers grown at either the lower level of N or the higher level of P were more-fully open (P ~ 0.01) than those grown under the opposite conditions. This result, in which flower openness increased and decreased while other flower characteristics decreased and increased, respectively, contrasts with results of the day and night air temperature experiment in which all flower characteristics including flower openness generally varied together. Flower color intensity and nectary wall development were not affected at the levels of N and P used in our experiments. Flower production, nectary wall development and flower size were affected by NP interactions (P ~ 0.05). Flowers grown at the lower level of N in combination with the higher level of P were dramatically smaller and had smaller nectary wails than flowers grown at the other three NP combinations, which were approximately equal to each other in flower size and nectary wall development. Plants grown at the higher level of N in combination with the lower level of P produced about 2 × as many flowers as plants grown at the other three NP combinations, which again were similar to each other in their flower production. These results suggest that the N/P ratio may be an important de-
277 terminant of many flower characteristics, probably as it relates to N uptake (Meyer et al., 1973). Effects of N, P and K nutrition on flower characteristics of soybeans have received little attention from researchers. In other plant species, most experiments have indicated decreased nectar secretion associated with high soil N concentration (Ryle, 1954a; Shuel, 1955, 1957). However, Kaziev (1967) reported increased nectar secretion in cotton when N was increased at adequate levels of P and K, suggesting the importance of N/P and N/K ratios. Experiments have also shown that P and K may promote or inhibit nectar secretion in some plants (Ryle, 1954a, b; Shuel, 1957; Kaziev, 1967). Honey bee attractiveness index was significantly higher (P < 0.05) for plants grown at either the higher level of N or the lower level of P, and was not affected by K. The greater attractiveness of higher-N and lower-P plants appears to be related to flower characteristics, as the more attractive plants had greater flower production, flower size and nectar secretion. CONCLUSIONS
Climatic and edaphic conditions during growth of soybean plants affected the number of flowers produced and various characteristics of the individual flowers. The most powerful factors were day air temperature and levels of N and P applied in nutrient solution. Effects of night air temperature and soil temperature were relatively less, and effects of K were not evident, possibly due to the low range in concentration used in this study. In general, higher levels of these factors, except P for which opposite results were obtained, resulted in more and better soybean flowers. With a few exceptions, environmental conditions which promoted greater flower production, larger flower size, more intensely colored flowers, more fully-open flowers and higher nectar secretion were precisely the conditions which promoted greater honey bee attractiveness of plants grown under those conditions. Thus, environmental factors altered attractiveness of plants to honey bees through effects on flower characteristics. Eventually, application of this kind of information may lead to increased yields of both soybeans and soybean honey through increased visitation by honey bees. ACKNOWLEDGEMENTS This research was supported by the College of Agricultural and Life Sciences, University of Wisconsin, Madison, WI. The authors thank Lowell Zirbel and Ellen Garvens for technical assistance, and the University of Wisconsin Biotron staff for its indispensible assistance in setup and maintenance of our experimental conditions.
278 REFERENCES Abrams, R.I., Edwards, C.R. and Harris, T., 1978. Yields and crosspollination of soybeans as affected by honey bees and alfalfaleafcutting bees. Am. Bee J., 118: 555, 556, 558, 560. Berding, N., 1981. Improved flowering and pollen fertility in sugarcane under increased night temperatures. Crop Sci., 21: 863--867. Caldwell, B.E., 1973. Soybean: Improvement, Production and Uses. American Society of Agronomy, Madison, WI, 681 pp. Erickson. E.H., 1975a. Effect of honey bees on yield of three soybean cultivars. Crop Sci., 15: 84--86. Erickson. E.H., 1975b. Variability of floral characteristics influences honey bee visitation to soybean blossoms. Crop Sci., 15: 767--771. Erickson. E.H. and Garment, M.B., 1979. Soya-bean flowers: Nectary ultrastructure, nectar guides, and orientation on the flower by foraging honey bees. J. Apic. Res., 18: 3--11. Erickson. E.H. and Robins, J.M., 1979. Honey from soybeans: The influence of soil conditions. Am. Bee J., 119: 444, 445, 448--450. Erickson. E.H., Berger, G.A., Shannon, J.G. and Robins, J.M., 1978. Honey bee pollination increases soybean yields in the Mississippi Delta region of Arkansas and Missouri. J. Econ. Entomol., 71: 601--603. Fisher, V.J. and Weaver, C.K., 1974. Flowering, pod set and pod retention of lima bean in response to night temperature, humidity and soil moisture. J. Am. Soc. Hortic. Sci., 99: 448--450. Jaycox, E.R., 1970. Ecological relationships between honey bees and soybeans. Am. Bee J., 110: 306--307,343--345, 383--385. Juliano, J.C., 1977. Polinizacao entomofila na soja. In: Anais do 4 ° Congresso Brasileiro de Apicultura, 1976, Curitiba, PR, Brazil, pp. 235--239. Kaziev, T.I., 1967. Some agrotechnical measures expediting increased nectar productivity of cotton. In: J.I. Hambleton, G.F. Townsend and V.D. Harnaj (Editors), Proc. XXI Int. Apic. Cong., 1967, University of Maryland, College Park, MD. Apimondia Publishing House, Bucharest, Romania, pp. 452--454. Maerz, A. and Paul, M.R., 1950. A Dictionary of Color. McGraw-Hill, New York, NY, 208 pp. Meyer, B.S., Anderson, D.B., Bohning, R.H. and Fratianne, D.G., 1973. Introduction to Plant Physiology. D. van Nostrand, New York, NY, 565 pp. Norman, A.G., 1978. Soybean Physiology, Agronomy, and Utilization. Academic Press, New York, NY, 249 pp. Robacker, D.C., Flottum, P.K. and Erickson, E.H., 1982. A heat exchanger for soil temperature control of potted plants. Agron. J., 74: 147--148. Ryle, M., 1954a. The influence of nitrogen, phosphate and potash on the secretion of nectar. Part I. J. Agric. Sci., 44: 400--407. Ryle, M., 1954b. The influence of nitrogen, phosphate and potash on the secretion of nectar. Part II. J. A~ric. Sci., 44: 408--419. Shuel, R.W., 1952. Some factors affecting nectar secretion in red clover. Plant Physiol., 27: 95--110. Shuel, R.W., 1955. Nectar secretion in relation to nitrogen supply, nutritional status, and growth of the plant. Can. J. Agric. Sci., 35: 124--138. Shuel, R.W., 1957. Some aspects of the relation between nectar secretion and nitrogen, phosphorus and potassium nutrition. Can. J. Plant Sci., 37: 220--236. Shuel, R.W., 1967. The influence of external factors on nectar production. Am. Bee J., 107: 54--56. Shuel, R.W. and Shivas, J.A., 1953. The influence of soil physical condition during the flowering period on nectar production in snapdragon. Plant Physiol., 28: 645--651. Walker, A.K., Barnes, D.K. and Furgala, B., 1974. Genetic and environmental effects on quantity and quality of alfalfa nectar. Crop Sci., 14: 235--238.
267
Elsevier Science Publishers B.V., Amsterdam -- Printed in The Netherlands
EFFECTS OF CLIMATIC A N D E D A P ~ I C FACTORS ON SOYBEAN FLOWERS A N D ON THE SUBSEQUENT ATTRACTIVENESS OF THE PLANTS TO HONEY BEES
D.C. ROBACKER, P.K. FLO2"rUM, D. SAMMATARO and E.H. ERICKSON
Department of Entomology, University of Wisconsin-Madison, and U.S. Department of Agriculture, Agricultural Research Service, Bee Research Unit, Madison, WI 53706 (U.S.A.) (Accepted 9 November 1982)
ABSTRACT Robacker, D.C., Flottum, P.K., Sammataro, D. and Erickson, E.H., 1983. Effects of climatic and edaphic factors on soybean flowers and on the subsequent attractiveness of the plants to honey bees. Field Crops Res., 6: 267--278. Soybean (Glycine max) plants were grown at various day and night air temperatures, soil temperatures and soil concentrations of N, P and K, to investigate effects of environmental conditions on flower characteristics, including flower production, color intensity, openness, size, nectar secretion and aroma emanation and on attractiveness of the plants to honey bees. Most flower characteristics increased as day air temperatures at which plants were grown increased from 20 to 24 ° C and reached maximum values at 28 ° C before plateauing or declining at 32 ° C, although flower size and nectar secretion continued to increase as growing temperature increased to 32 ° C. Of two flower aroma components, emanation of one c o m p o n e n t increased while the other decreased with increases in growing temperatures. The hypothesis suggested is that the two aroma chemicals may communicate flower-readiness information to pollinators. Flower production and flower openness responded linearly to night air temperature at which plants were grown, attaining highest values at higher (22, 26 ° C) vs. lower (14, 18 ° C) temperatures. Flower production also responded linearly to soil temperature, attaining highest values at higher (28--32 ° C) vs. lower (16--20 ° C) temperatures. Of two levels each of N (75 and 175 ppm) and P (15 and 30 ppm) tested, the higher level of N stimulated greater flower production, flower size and nectar secretion while the higher level of P decreased the same three flower characteristics. Conversely, lower N and higher P promoted flower openness. Honey bee attractiveness of plants varied positively with flower characteristics such that plants grown at a day air temperature of 28 ° C, night air temperatures of 22 and 26 ° C, the higher level of N of the lower level of P were the most attractive to honey bees.
INTRODUCTION
Although soybean (Glycine max [L.] Merr.) is generally considered a selfpollinating species, yield increases of about 10--40% have been demonstrated Mention of a proprietary product in this paper does not constitute an endorsement by the USDA. 0378-4290/83/$03.00
© 1983 Elsevier Science Publishers B.V.
268 when comparing honey bee (Apis mellifera L.) pollinated vs. self-pollinated plants (Erickson, 1975a; Juliano, 1977; Erickson et al., 1978). Further, soybean flowers possess many entomophilous characteristics (Erickson and Garment, 1979) and honey bees produce substantial honey crops when they visit soybeans (Abrams et al., 1978; Erickson and Robins, 1979). However, soybean flowers are not always attractive to honey bees, leading to the hypothesis that environmental conditions during growth and flowering of plants affect the development of entomophilous flower characteristics. The objective of this work is to evaluate the effects of environmental conditions during plant growth on flowers and on attractiveness of soybean plants to honey bees. To accomplish this objective, three experiments were conducted. The first experiment investigated effects of day and night temperatures, the second effects of soil temperature at 2-day air temperatures, and the third examined effects of N, P and K nutrition. Various flower characteristics assumed to be involved in honey bee attraction were measured and actual honey bee attractiveness tests were conducted in all three experiments. Effects of environment on plant characteristics were not investigated in this work since this has been reported by others (Caldwell, 1973; Norman, 1978). METHODS
General The following conditions were constant in all three experiments. Soybean (var. Mitchell, Group IV) plants were grown in controlled environment rooms in the University of Wisconsin Biotron. Seeds were surface sterilized with 10% hypochlorite solution and sown individually in 20-cm pots containing sand. Photophase was 16 h, average light flux at plant level was about 600 pE m-2 s-1 (fluorescent and incandescent lights) and relative humidity was 70%. Plants were irrigated with nutrient solution often enough to prevent apparent stress. Light intensity and temperature were changed incrementally during the first and last hours of photophase to simulate natural day/night changes.
Experiments Experiment 1: Day and night air temperature This experiment tested effects of 16 combinations of day and night air temperatures in the Biotron's crossed-gradients room which is capable of maintaining four distinct temperature regions at any one time. The four regions were programmed with the following day/night temperature combinations: 20/14, 24/18, 28/22 and 32/26 ° C. During the first and last hours of photophase, groups of plants were moved among the four temperature regions to achieve the full factorial complement of 16 treatments. For example, during the day one region held the treatments 20/14, 20/18, 20/22 and 20/26 ° C, during the last hour of photophase, plants of the latter three treatments were
269 moved to their night positions in the 24/18, 28/22 and 32/26°C regions, respectively. During the first hour of photophase, these plants were returned to their day positions. Other temperature regions were handled similarily. There were three replications of each treatment. Treatments were necessarily confounded with position since temperature regions could n o t be randomized within the room. Plants were irrigated with half-strength Hoagland solution (N--105 ppm, P--15 ppm, K--128 ppm). Experiment 2: Soil temr~erature at two air temperatures Plants were grown in the crossed-gradients room and were irrigated with half-strength Hoagland solution. Day air temperature was 28 ° C and night air temperature was 20--24 ° C during a 2-week germination period. Plants were then divided into 2-day/night air temperature groups of 24/18 and 32/22 ° C, each with 16 plants. Within each group, day soil temperatures were adjusted to values ranging from 16 to 32°C using heat exchangers (Robacker et al., 1982). Soil temperatures decreased at night as a result of the decrease in air temperature such that plants receiving higher day soft temperatures also received higher night soil temperatures. In general, soil temperatures decreased by 4--8 ° C (greater decreases for higher day values) at night. Therefore, the integrity of the overall soil temperature treatments was maintained during both the day and night. As before, air temperature treatments were necessarily confounded with position. Experiment 3: NPK experiment Plants were grown in a large plant room where they received day/night air temperatures of 28/20 ° C. Plants were irrigated with nutrient solution similar to half strength Hoagland solution except for the concentrations of N, P and K. Levels of N, P and K were: N--75 and 175 p p m as urea; P--15 and 30 ppm as monobasic/dibasic sodium phosphate in a 2.8:1 molar ratio; K--75 and 175 p p m as potassium chloride. These ranges in nutrients are generally within acceptable limits for Mitchell soybeans although the effects of each are influenced by levels of the other t w o (Caldwell, 1973). The two levels each of N, P and K were administered in factorial combination of eight treatments with four replications per treatment. Flower measurements Flower production was measured as the number of fresh (< 1 day old) flowers at 5--9 h after the onset of the photophase, 7 days after the day of first flower for each plant. Flower color intensity was measured from color photographs of one typical flower per plant selected 5--8 h after the onset of the photophase, 2--4 days after first flower. Intensity of red and blue was rated from 1--12 (12: most intense) b y comparing photographs with a two-dimensional color chart (Maerz and Paul, 1950) on which intensity of red increased along one axis and blue
270
along the other. Overall color intensity was assigned by adding the values for red and blue. Flower size was also determined from the photographs, since flowers were photographed beside a m m scale. Flower size was calculated by measuring the base diameter at the point most proximal to the pedicel where the petals emerge from the calyx. Flower openness was determined as the mean of the degree of openness of all flowers per plant. Flowers were rated on a scale of 1--5 (5: fully open), 5--7 h after the onset of the photophase, 7 days after first flower. Nectar secretion was determined as the mean of nectar secretion of four flowers per plant. Flowers were sampled with 1 p L D r u m m o n d Microcaps @, 2.5--4.5 h after the onset of the photophase, 5--8 days after first flower. Nectary development was determined from the mean of three flowers per plant for the soil temperature experiment and from one flower per plant for the NPK experiment, but was not determined for the day and night air temperature experiment. Flowers were collected during the peak flowering period of each plant at 3--5 h after the onset of the photophase, fixed in 2.5% glutaraldehyde /1.0 M phosphate buffer, rinsed 3 times for 20 min each with 0.1 M phosphate buffer, and dehydrated through a graded series of ethanol (50, 70, 90, 100%) for 20 min at each dilution. Transverse sections were cut at about 2 mm above the flower-pedicel junction and examined under a light microscope. Nectary wall thickness was measured with a microscope eyepiece micrometer and used as the indicator of nectary development. Flower aroma was measured from 10 to 15 flowers collected from 5--6 h after the onset of the photophase, 9 days after first flower for each plant from the day and night air temperature experiment. The flowers were placed into a 20 ml glass syringe and the plunger inserted to the 5 ml line. After a 1 h equilibration at 28 ° C, the 5 ml of air were injected onto a column packed with 5% SE 30 on 100/120 Gas Chrom Q, at 140°C (Varian 3700 Gas Chromatograph with FID detector). Quantities of volatile components of flower aroma were determined by measuring peak heights.
Honey bee attractiveness index A honey bee bioassay r o o m was set up in the Biotron. The room was maintained at full light flux (about 600 uE m -2 s-l), day/night temperatures of 28/20 ° C, relative h u m i d i t y of 70% and a 12 h photophase which began synchronously with the 16 h photophase used in the other rooms. One colony of about 3000 bees was placed in the r o o m 2 weeks before the beginning of assays and fed a pollen/sugar cake once each week. The room also contained three " s t a n d a r d " soybean plants (var. Mitchell) which were grown at day/ night temperatures of 28/20 ° C and other conditions the same as in the day/ night air temperature experiment. The three plants were replaced weekly with three fresh plants to keep the standards near peak flowering condition. For each bioassay, the test plant was placed in the bioassay room, watered
271 to drip point, and allowed to equilibrate, with bee visitation, for 0.5 h. During the next 0.5 h, the number of bees foraging at the test plant was recorded during seven predetermined 1-min observation periods. Also during this 0.5 h, the number of bees foraging at the three standards was recorded during six predetermined 1-min observation periods. Attractiveness index was calculated b y dividing the total number of bees observed foraging at the test plant by the total number observed at the three standard plants. Bioassays were conducted from 3--8 h after the onset of the photophase since bees most actively visited soybean flowers during this time. Whenever possible, plants from the same treatment were tested at different times of the day to prevent confounding of treatments with time of day. All 48 plants from the day and night air temperature experiment, 16 plants representing highest and lowest soil temperatures at each air temperature from the soil temperature experiment, and t w o plants from each of the eight treatments from the NPK experiment were bioassayed individually, 7 days after first flower for each plant.
Statistical analyses Completely randomized designs were assumed for all analyses despite confounding of treatments with positions in both the day/night air temperature experiment and the soil temperature experiment, and of treatments with day on which attractiveness bioassays were conducted in all three experiments. Position bias seemed unimportant since the only apparent position difference -- light flux -- had no significant effects on dependent variables when analyzed as an independent variable. Effects of bioassay date were ruled o u t since mean visitation to "standard" plants did not change over days. Regression analyses were used to calculate main and interaction effects of treatments on flower characteristics and on h o n e y bee attractiveness indices. RESULTS AND DISCUSSION
Effects of day air temperature Main effects of day air temperature are reported in Table I. The data show that plants grown at a day air temperature of 20°C were inferior to those grown at other temperatures. That is, they produced the second fewest flowers which individually were the smallest, least intensely colored, least open and secreted the lowest amounts of nectar. In general, plants grown at higher day air temperatures were much improved over those grown at 20 ° C, b u t effects on individual flower characteristics varied. Flower size and nectar secretion increased linearly with temperature from 20 to 32°C (P < 0.01). Flower openness and flower color intensity increased linearly (P < 0.01) with temperature from 20 to 24 and 20 to 28 ° C, respectively. Both remained unchanged with further increases to 32 ° C, resulting in quadratic regression components (P <
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0.01). Flower production showed a highly significant (P < 0.01) quadratic curve with peak production at 24--28°C and lower production of flowers at 20 and 32 ° C. Gas chromatographic analyses of flower aroma uncovered 2 peaks (Table I). The peak of lower retention time, component 1, varied inversely (r = -0.74, P < 0.01) with the peak of higher retention time, component 2. Data in Table I show that aroma component 2 responded to _growingtemperatures similarily to most other flower characteristics. Thus, component 2 increased with growing temperature from 20 to 24 ° C and remained unchanged with further temperature increases, resulting in significant (P < 0.01) linear and quadratic regression components. Component 1, however, decreased with growing temperature from 20 to 28°C and remained unchanged with further temperature increase, also resulting in significant (P < 0.01) linear and quadratic regression coefficients, but of opposite sign to those for component 2. Presently, the hypothesis that aroma components 1 and 2 communicate flower readiness for visitation and/or pollination to pollinators, is under investigation. Within limits, increased air temperatures generally increase nectar production in most species of plants investigated (Shuel, 1967). For soybeans, Jaycox (1970) reported nectar secretion to be favored by day air temperatures near 27°C for field grown plants and Erickson (1975b) reported 21°C as a lower limit for both nectar secretion and flower opening in many soybean cultivars (Mitchell not included). Our results agree with the literature and extend the effects of day air temperature on soybean flowers to include flower production, flower color intensity, flower size and flower aroma. Honey bee attractiveness index, which is theoretically dependent on the quality of flower characteristics, increased dramatically as growing temperatures of test plants increased from 20 to 24 ° C, then remained unchanged with further increases to 32 ° C. Linear and quadratic regression components were highly significant (P < 0.01). Thus honey bee attractiveness index and all flower characteristics, except aroma component 1, produced essentially the same curve in response to day air temperatures. These results suggest that honey bee attractiveness index is in fact varying in response to changes in flower characteristics.
Effects of night air temperature Main effects of night air temperature are reported in Table I. The data show that, compared to day air temperatures, night air temperatures at which plants were grown had little impact on flower characteristics. However, both flower production (P < 0.05) and flower openness (P < 0.01) showed linear increases with night temperature increases from 14 to 26 ° C. Nectar secretion responded similarily, but the result was not significant. Honey bee attractiveness index also increased linearly (P < 0.01) with increases in night temperature, possibly in response to changes in flower production, flower openness and nectar secretion.
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275 The authors are aware of no studies reporting effects of night air temperatures on soybean flower characteristics. Related studies on the effects of night air temperature have demonstrated no significant changes in nectar secretion by red clover (Shuel, 1952), increased sucrose/reducing-sugar ratios at low temperatures for alfalfa nectar (Walker et al., 1974), increased flower openness during the day following warmer nights for lima bean (Fisher and Weaver, 1974) and increased flower production with higher night temperatures for sugarcane (Berding, 1981).
Interactions o f day and night air temperatures Only flower color intensity showed a significant interaction effect (P < 0.01). This occurred because increases in night air temperature at a day air temperature of 20 ° C caused a linear increase in color intensity whereas night air temperature had no effects at other day air temperatures. Flower color intensity for plants grown at day air temperatures of 20 ° C and night air temperatures of 14, 18 and 22°C were significantly lower than for plants grown at any other day/night air temperature combinations (P ~ 0.01L
Effects o f soil temt~erature Main effects of soil temperature are shown in Table II. Note that the temperatures in Table II are day soil temperatures. Corresponding night soil temperatures were 4--8 ° C lower, respectively, for day soil temperatures of 16-32°C. The data show that flower production increased linearly (P < 0.01) with increasing soil temperature at which plants were grown. This effect was consistent at b o t h the 24 and 32°C day air temperatures, therefore the data in Table II are the combined results of the t w o day air temperatures. No other flower characteristics were significantly affected by soil temperature in our study. In a similar study, however, Shuel and Shivas (1953) reported that nectar sugar production of snapdragons subjected to soil temperatures of 15.5, 21 and 27 ° C at the beginning of the flowering stage increased with increasing soil temperature. Because so few flower characteristics were affected by soil temperature, honey bee attractiveness index also was not significantly affected. However, the value of h o n e y bee attractiveness index for higher soil temperature plants was slightly higher than for lower soil temperature plants (26 vs 22 ° C), possibly due to higher flower production by plants grown at higher soil temperatures. This effect was also consistent at both day air temperatures.
Effects o f N, P and K The main effects of N, P and K are reported in Table III. The data show that N and P had major effects on flower characteristics, while K had no sig-
276 TABLE III Main effects o f N, P and K on soybean flower characteristics and honey bee attractiveness index Measurement
N (ppm) 75
Flower production (flowers/plant) Flower color intensity a Flower size (mm) Flower openness a Nectar secretion (nl/flower) Neetary wall development (#) Honey bee attractiveness index b
P (ppm) 175
15
K (ppm) 30
75
175
18"*
27 c**
32**
15"*
22
25
14 1.9"* 4.6** 25"
15 2.2** 4.2** 40*
15 2.2** 4.2** 45"*
14 2.0** 4.6** 20"*
15 2.0 4.4 28
14 2.2 4.4 35
140 0.4*
160 0.9*
150 0.8*
150 0.4*
140 0.5
160 0.8
aArbitrary units. Color intensity: 1--24, 24: most intense. Flower openness: 1--5, 5: most open. bValues are ratios of number of foragers at test plants divided by number of foragers at standard plants during bioassays. c . and ** indicate 5 and 1% significance levels, respectively.
nificant effects. Flower production (P < 0.01), flower size (P ~ 0.01) and nectar secretion (P < 0.05) were all higher for plants grown at the higher (175 ppm) vs. the lower (75 ppm) concentrations of N. However, the same three flower characteristics were lower (P < 0.01) for plants grown at the higher (30 ppm) vs. the lower (15 ppm) concentrations of P. Conversely, flowers grown at either the lower level of N or the higher level of P were more-fully open (P ~ 0.01) than those grown under the opposite conditions. This result, in which flower openness increased and decreased while other flower characteristics decreased and increased, respectively, contrasts with results of the day and night air temperature experiment in which all flower characteristics including flower openness generally varied together. Flower color intensity and nectary wall development were not affected at the levels of N and P used in our experiments. Flower production, nectary wall development and flower size were affected by NP interactions (P ~ 0.05). Flowers grown at the lower level of N in combination with the higher level of P were dramatically smaller and had smaller nectary wails than flowers grown at the other three NP combinations, which were approximately equal to each other in flower size and nectary wall development. Plants grown at the higher level of N in combination with the lower level of P produced about 2 × as many flowers as plants grown at the other three NP combinations, which again were similar to each other in their flower production. These results suggest that the N/P ratio may be an important de-
277 terminant of many flower characteristics, probably as it relates to N uptake (Meyer et al., 1973). Effects of N, P and K nutrition on flower characteristics of soybeans have received little attention from researchers. In other plant species, most experiments have indicated decreased nectar secretion associated with high soil N concentration (Ryle, 1954a; Shuel, 1955, 1957). However, Kaziev (1967) reported increased nectar secretion in cotton when N was increased at adequate levels of P and K, suggesting the importance of N/P and N/K ratios. Experiments have also shown that P and K may promote or inhibit nectar secretion in some plants (Ryle, 1954a, b; Shuel, 1957; Kaziev, 1967). Honey bee attractiveness index was significantly higher (P < 0.05) for plants grown at either the higher level of N or the lower level of P, and was not affected by K. The greater attractiveness of higher-N and lower-P plants appears to be related to flower characteristics, as the more attractive plants had greater flower production, flower size and nectar secretion. CONCLUSIONS
Climatic and edaphic conditions during growth of soybean plants affected the number of flowers produced and various characteristics of the individual flowers. The most powerful factors were day air temperature and levels of N and P applied in nutrient solution. Effects of night air temperature and soil temperature were relatively less, and effects of K were not evident, possibly due to the low range in concentration used in this study. In general, higher levels of these factors, except P for which opposite results were obtained, resulted in more and better soybean flowers. With a few exceptions, environmental conditions which promoted greater flower production, larger flower size, more intensely colored flowers, more fully-open flowers and higher nectar secretion were precisely the conditions which promoted greater honey bee attractiveness of plants grown under those conditions. Thus, environmental factors altered attractiveness of plants to honey bees through effects on flower characteristics. Eventually, application of this kind of information may lead to increased yields of both soybeans and soybean honey through increased visitation by honey bees. ACKNOWLEDGEMENTS This research was supported by the College of Agricultural and Life Sciences, University of Wisconsin, Madison, WI. The authors thank Lowell Zirbel and Ellen Garvens for technical assistance, and the University of Wisconsin Biotron staff for its indispensible assistance in setup and maintenance of our experimental conditions.
278 REFERENCES Abrams, R.I., Edwards, C.R. and Harris, T., 1978. Yields and crosspollination of soybeans as affected by honey bees and alfalfaleafcutting bees. Am. Bee J., 118: 555, 556, 558, 560. Berding, N., 1981. Improved flowering and pollen fertility in sugarcane under increased night temperatures. Crop Sci., 21: 863--867. Caldwell, B.E., 1973. Soybean: Improvement, Production and Uses. American Society of Agronomy, Madison, WI, 681 pp. Erickson. E.H., 1975a. Effect of honey bees on yield of three soybean cultivars. Crop Sci., 15: 84--86. Erickson. E.H., 1975b. Variability of floral characteristics influences honey bee visitation to soybean blossoms. Crop Sci., 15: 767--771. Erickson. E.H. and Garment, M.B., 1979. Soya-bean flowers: Nectary ultrastructure, nectar guides, and orientation on the flower by foraging honey bees. J. Apic. Res., 18: 3--11. Erickson. E.H. and Robins, J.M., 1979. Honey from soybeans: The influence of soil conditions. Am. Bee J., 119: 444, 445, 448--450. Erickson. E.H., Berger, G.A., Shannon, J.G. and Robins, J.M., 1978. Honey bee pollination increases soybean yields in the Mississippi Delta region of Arkansas and Missouri. J. Econ. Entomol., 71: 601--603. Fisher, V.J. and Weaver, C.K., 1974. Flowering, pod set and pod retention of lima bean in response to night temperature, humidity and soil moisture. J. Am. Soc. Hortic. Sci., 99: 448--450. Jaycox, E.R., 1970. Ecological relationships between honey bees and soybeans. Am. Bee J., 110: 306--307,343--345, 383--385. Juliano, J.C., 1977. Polinizacao entomofila na soja. In: Anais do 4 ° Congresso Brasileiro de Apicultura, 1976, Curitiba, PR, Brazil, pp. 235--239. Kaziev, T.I., 1967. Some agrotechnical measures expediting increased nectar productivity of cotton. In: J.I. Hambleton, G.F. Townsend and V.D. Harnaj (Editors), Proc. XXI Int. Apic. Cong., 1967, University of Maryland, College Park, MD. Apimondia Publishing House, Bucharest, Romania, pp. 452--454. Maerz, A. and Paul, M.R., 1950. A Dictionary of Color. McGraw-Hill, New York, NY, 208 pp. Meyer, B.S., Anderson, D.B., Bohning, R.H. and Fratianne, D.G., 1973. Introduction to Plant Physiology. D. van Nostrand, New York, NY, 565 pp. Norman, A.G., 1978. Soybean Physiology, Agronomy, and Utilization. Academic Press, New York, NY, 249 pp. Robacker, D.C., Flottum, P.K. and Erickson, E.H., 1982. A heat exchanger for soil temperature control of potted plants. Agron. J., 74: 147--148. Ryle, M., 1954a. The influence of nitrogen, phosphate and potash on the secretion of nectar. Part I. J. Agric. Sci., 44: 400--407. Ryle, M., 1954b. The influence of nitrogen, phosphate and potash on the secretion of nectar. Part II. J. A~ric. Sci., 44: 408--419. Shuel, R.W., 1952. Some factors affecting nectar secretion in red clover. Plant Physiol., 27: 95--110. Shuel, R.W., 1955. Nectar secretion in relation to nitrogen supply, nutritional status, and growth of the plant. Can. J. Agric. Sci., 35: 124--138. Shuel, R.W., 1957. Some aspects of the relation between nectar secretion and nitrogen, phosphorus and potassium nutrition. Can. J. Plant Sci., 37: 220--236. Shuel, R.W., 1967. The influence of external factors on nectar production. Am. Bee J., 107: 54--56. Shuel, R.W. and Shivas, J.A., 1953. The influence of soil physical condition during the flowering period on nectar production in snapdragon. Plant Physiol., 28: 645--651. Walker, A.K., Barnes, D.K. and Furgala, B., 1974. Genetic and environmental effects on quantity and quality of alfalfa nectar. Crop Sci., 14: 235--238.