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Effect of Soaking Time and Cooking Time on Qualities of Red Kidney Bean Flour

Kamolwan Jangchud and Nongsuda Bunnag

ABSTRACT Soaking time and cooking time of red kidney bean affected the reduction of raffinose and stachyose contents in its flour. Red kidney beans were soaked in distilled water for 6 and 12 h, and then cooked by steaming (30, 60, and 90 min) or boiled in pressure cooker (10 and 15 min). After extraction of raffinose and stachyose contents from flour made from soak-cooked beans, the results from HPLC showed an important decrease in oligosaccharides of soak-cooked beans. The highest decrease in raffinose and stachyose was to soak beans for 12 h and then boil in pressure cooker for 15 min which was 47% and 44%, respectively. Key words: red kidney bean, oligosaccharides, raffinose, stachyose, HPLC INTRODUCTION Red kidney beans (Phaseolus vulgaris) are the whole grain consumed in the greatest quantity in the world (Carpenter, 1981). They are an important economical source of protein in the diet of many developed and developing countries (Nielsen, 1991) ; however, Thailand has exported most of the red kidney beans (Tharatthapan, 1996). Red kidney beans are rich in B-complex vitamins and minerals (Koehler et al., 1987; Guzman-Maldonado and Paredes-Lopez, 1998). Moreover, red kidney beans are a very low in sodium, cholesterol, and saturated fatty acids but rich in unsaturated fatty acids such as linoleic acid (Barampama and Simard, 1994). They are not only a good source of both soluble and insoluble dietary fiber but also health benefits, including reduced risk of heart disease and colon cancer (Hughes, 1991; Guzman-Maldonado and Paredes-Lopez, 1998). Although they are advantages, several factors detract from their full nutritional potential such as the presence of antinutritional factors that cause flatulence (Olson et al., 1981) and low protein digestibility which may limit amino acid availability (Nielsen, 1991). Raffinose oligosaccharides (e.g. raffinose, stachyose, and verbascose) have been called antinutritional factors that contribute to flatulence production (Reddy et al., 1989; Guzman-Maldonado and Paredes-Lopez, 1998). They were not digested due to the lack ­1,6-galactosidase activity in the mammalian intestinal mucosa and cause intestinal microflora to produce flatus (Olson et al., 1981; Sathe and Salunkhe, 1989). To improve nutritional quality of red kidney beans, soaking, cooking, germination, fermentation or irradiation treatments may be used to develop protein nutrition value (Sathe and Salunkhe, 1989; Guzman-Maldonado and Paredes-Lopez, 1998). Jood et al. (1985) and Barampama and Simard (1994) studied the effect of processing on oligosaccharides in legumes and found that soaking, cooking, germination or fermentation can be use to reduce antinutritional factors.

The objective of this research was to evaluate the effect of soaking and cooking (steaming and pressure cooker) at different length of time on the oligosaccharide contents in red kidney beans. MATERIALS AND METHODS 1. Materials Red kidney beans. Red kidney beans were purchased from local market and stored at room temperature (30°C). Reagents and standards. The raffinose and stachyose standards were obtained from Fluga (Buchs, Switzerland). Acetonitrile and methanol were obtained from J.T.Baker (Philipsberg, New Jersey). All standards and solvents were HPLC grade. 2. Experimental design A 2x3 factorial design with two levels (6 and 12 h) of soaking and three levels (30, 60, and 90 min) of steaming and a 2X2 factorial design with

two levels (6 and 12 h) of soaking and two levels (10 and 15 min) of boiling in pressure cooker were investigated. 3. Sample preparation Raw red kidney bean flour. Red kidney beans were washed thoroughly with three times of distilled water at room temperature and dried using tray dryer (Model HA-40, BWS Trading, Ltd., Thailand) at 60°C for 1 h. After drying, they were reduced particle size by food processor and finally ground with a Hammer Mill (Model AP-S, Hosokawa Micron Corp., Japan) equipped with a 0.5 mm screen. Raw kidney bean flour was placed into plastic container and stored at 4°C during the period of analysis. Cooked red kidney bean flour. The method of preparation as given in Figure 1 was followed in this investigation. Red kidney beans were washed throughly with three times of distilled water at room temperature, and then were soaked in distilled water at a ratio of 1:10 for 6-h and 12-h at room temperature.

Red kidney beans soaked

6h 12 h

cooked steamed (at 100 oC)

30 min 60 min 90 min

boiled in pressure cooker (at 15 psi, 121 oC)

10 min 15 min

reduced particle size by food processor dried at 60 oC, 4 1/2 hour. passed through a 0.5 mm screen Cooked red kidney bean flours

Figure 1 Preparation steps of cooked red kidney bean flours.

The soaked samples were cooked by steaming at 100°C for 30 min, 60 min, and 90 min or boiled in pressure cooker at 15 psi for 10 and 15 min (1:1.5 red kidney beans to water ratio). After the processed samples were reduced particle size by food processor, they were dried at 60°C for 4 1/2 h and finally ground with a Hammer Mill equipped with a 0.5 mm screen. All the soak-cooked bean flours were placed into plastic containers and stored at 4° C during the period of analysis. Concentration of oligosaccharides were calculated in a dry basis. 4. Raffinose and stachyose oligosaccharide analysis Raffinose and stachyose standard solution. Stock standard mixture solution was prepared by accurately weighed raffinose and stachyose standard and dissolved in deionized water. The other four concentrations were prepared from stock standard mixture solution diluted with deionized water. The resulting peak areas were plotted against concentrations for the calibration curve by using the external standard method. Extraction. Based on a modification procedure of Barampama and Simard (1994), sample was weighed about 1.5 g in a 20-ml test tube, added 10 ml of 80% ethanol : water (v/v) for first extraction. The sample was throughly mixed using a vortex mixer, and then was shaken in a boiling water bath at 80°C for 15 min. The mixture was centrifuged for 5 min at 2,800 rpm using a centrifuge (Model Universal 16R, Andreas Hettich GmbH&Co., Germany), and the supernatant was transferred to another 50-ml test tube. For the sample residue in the first centrifuge tube, added 5 ml of 80% ethanol : water (v/v) and repeated extraction as in the previous described. Finally, 10 ml of 80% ethanol : water (v/v) was added to the residue and the extraction repeated as in previous step. The three collected supernatants were combined in a 5-ml polyethylene centrifuge tube, then added 2 ml of 10% lead acetate : water (w/v)) to deproteinize the solution. The mixture was shaken using a vortex

mixer until it was homogeneous, and then centrifuged for 20 min at 2,800 rpm. The supernatant was transferred to another centrifuge tube, then added 0.5 ml of 10% oxalic acid : water (w/v). The supernatant was centrifuged for 20 min at 2,800 rpm to remove the excess lead acetate in the sample, the clear extract was quantitatively transferred to a 25-ml volumetric flask and made up volume with deionized water. It was stored immediately at ­20° C for further analysis. All experiments were conducted in duplicate. The analytical method for sample cleanup was modified by the procedure of Barampama and Simard (1994) and Sánchez-Mata et al. (1998). The prepared extract was purified by filtered through a Sep-Pak C18 cartridge (Waters Associates, Milford, MA), which was prewetted with 5 ml of methanol followed by 5 ml of deionized water. Before injection, the sample was filtered through a 0.45 µm cellulose acetate filter (Sartorius, AG). The eluent was collected in a 4-ml clear shell viol for raffinose and stachyose analysis by HPLC. HPLC Quantification. Aliquots of filtered sample (20 µl) was injected to the reverse phase HPLC system with Waters Associates Liquid Chromatograph (Waters, Milford, MA), equipped with a Water 600 controller, Waters 600 pump and a Waters 410 differential refractometer. The separation of oligosaccharides was achieved by a 4 × 250 mm, 5 mm (Merk, Darmstadt, Germany) LiChrosphere NH 2 column, connected to a µBondapak NH2 guard column (Waters, Milford, MA) using a 10-µl microsyringe (Model MS-NR 10, Ito Corp., Fuji). The isocratic mobile phase contained acetonitrile : water (70:30) at 0.8 ml/min flow rate. The mobile phase was filtered using a 0.45 mm filter membrane. The column temperature was controlled at 30°C and the temperature of RI detector was set at 35°C with high sensitivity (512). A completed analysis was carried out about 13 min. Data were collected and processed by Millenium version 2.10 program on a PC 130-486 DX2 computer connected to HPLC apparatus. The

concentrations of raffinose and stachyose in the samples were calculated using the average peak areas compared with the standard, and expressed as g/100g of dry beans. 5. Statistical analysis Analysis of variance (ANOVA) was done to determine and compare differences in raffinose and stachyose contents. Duncan's Multiple Range Test was performed for post hoc multiple comparisons. Significant differences were establish at = 0.05. RESULTS AND DISCUSSION The chromatograms of raffinose and stachyose in mature red kidney bean separated by LiChrosphere NH2 Column are shown in Figure 2. According to the chromatogram, the retention time of raffinose and stachyose are 8.25 and 11.50 min, respectively. The effect of various soaking times and steaming times on raffinose and stachyose contents are shown in Figure 3 and 4, respectively. The increase in soaking time and steaming time significantly (P 0.05) decreased raffinose. The extent of loss was increased as the time of soaking

increased from 6 to 12 h or as the time of steaming increased from 30 to 90 min. After being soaked for 6 h, stachyose contents were decreased significantly (P 0.05) as the steaming time increased from 30 to 60 min. However, at the 60-min and 90-min steaming, stachyose content was not significantly different (P>0.05) for 6-h soaking. At 12-h soaking, the increase in 60-min to 90-min steaming was significantly (P 0.05) affected the reduction of stachyose from 2.22 to 1.90 g/100 g of dry beans. Soaking for 12 h followed by steaming for 90 min represented the lowest raffinose and stachyose contents with 0.63 and 1.90 g/100 g of dry beans, respectively. Therefore, soaking time and cooking time affected the reduction of raffinose oligosaccharides in red kidney beans. The result was agreed with the study of Jood et al. (1985), and Barampama and Simard (1994) who found on that soaking and cooking reduced oligosaccharides in legumes. The effect of soaking and boiling in pressure cooker on raffinose and stachyose in red kidney bean flour are showed in Figure 5 and 6, respectively. Raffinose and stachyose were decreased significantly (P 0.05) after soaking time increased.

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Figure 2 HPLC chromatogram of raffinose and stachyose in raw red kidney bean.

The increased in cooking time was not significantly (P>0.05) affected raffinose content ; however, the significant (P 0.05) reduction was found in stachyose contents. It was indicated that the 12-h soaking followed by boiling in pressure cooker for 15 min present the highest decrease of raffinose and stachyose by 0.53 and 1.60 g/100 g of dry beans, respectively.

The results of raffinose and stachyose content and the percent loss under different treatments are summarized in Table 1. Stachyose content was higher than that of raffinose in raw red kidney beans which was supported by Salunkhe et al. (1989) who studied the oligosaccharide compositions of raw red kidney bean and found that raffinose and stachyose contents were 0.93% and 2.44%,

1.5 6-h soaking 12-h soaking

Stachyose content (mg/100 g of beans)

3.5 3 2.5 2 1.5 1 0.5 a 6-h soaking ab b b 12-h soaking b

Raffinose content (mg/100 g of beans)

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0 30 60 Cooking time (min) 90

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Figure 3 Raffinose content (g/100 g of dry beans) of red kidney bean flour after soaking and steaming. abcde Means with different letters are significantly different (P 0.05)

Figure 4 Stachyose content (g/100 g of dry beans) of red kidney bean flour after soaking and steaming. abc Means with different letters are significantly different (P 0.05)

1.5 6-h soaking

(mg/100 g of beans) Raffinose content

12-h soaking

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Stachyose content (mg/100 g of beans)

2.5 2 1.5 1 0.5 0

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12-h soaking b c

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0 10 Boiling time (min) 15

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Figure 5 Raffinose content (g/100 g of dry beans) of red kidney bean flour after soaking and boiling in pressure cooker. ab Means with different letters are significantly different (P 0.05)

Figure 6 Stachyose content (g/100 g of dry beans) of red kidney bean flour after soaking and boiling in pressure cooker. abc Means with different letters are significantly different (P<0.05)

Table 1 Raffinose and stachyose content (g/100 g of dry beans) in red kidney beans and flour subjected to various treatments. Treatments 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. Raw red kidney beans 6-h soaking and boiling in pressure cooker for 10 min 6-h soaking and boiling in pressure cooker for 15 min 12-h soaking and boiling in pressure cooker for 10 min 12-h soaking and boiling in pressure cooker for 15 min 6-h soaking and steaming for 30 min 6-h soaking and steaming for 60 min 6-h soaking and steaming for 90 min 12-h soaking and steaming for 30 min 12-h soaking and steaming for 60 min 12-h soaking and steaming for 90 min

in parentheses represent variation (%) in reference compared to raw beans.

Raffinose 1.00±0.03 0.60±0.02 (-40)a 0.56±0.00 (-44) 0.55±0.00 (-45) 0.53±0.03 (-47) 0.80±0.01 (-20) 0.77±0.00 (-23) 0.76±0.01 (-24) 0.71±0.02 (-29) 0.67±0.02 (-33) 0.63±0.01 (-37)

Stachyose 2.87±0.19 1.98±0.00 (-30) 1.79±0.02 (-37) 1.78±0.01 (-38) 1.60±0.00 (-44) 2.31±0.03 (-20) 2.18±0.02 (-24) 2.14±0.00 (-25) 2.22±0.01 (-23) 2.17±0.03 (-24) 1.90±0.09 (-34)

aValues

respectively. Soaking and cooking reduced raffinose and stachyose in all treatments. For steaming process, the highest reduction of raffinose (37%) and stachyose (34%) were conducted by soaking for 12 h followed by steaming for 90 min. For boiling in pressure cooker, soaking for 12 h followed by cooking for 15 min was the highest decreased in raffinose and stachyose by 47% and 44%, respectively. Salunkhe et al. (1989) also reported that Great Northern, kidney, and pinto beans which were soaked in either distilled water or a mixed salt solution for 18 h at 22°C reduced raffinose oligosaccharides within the range of 32.77 ­ 51.02%. The combination of soaking and cooking reduced raffinose oligosaccharides which causes flatulence. The reduction was caused by a loss of sugar to water during diffusion (Barampama and Simard, 1994) ; therefore, the soaking water should be discarded prior to cooking. However, the nutritional quality such as some vitamins and minerals may also be lost in the soaking water along with flatulence-causing oligosaccharides (Deshpande et al., 1989). In addition, prolonged cooking time may even reduce the nutritive quality

of beans; however, quick-cooking beans such as pressure cooking seems to offer not only a decrease in cooking time and fuel requirement, but also appear to have as good nutritional qualities as the standard cooked beans (Salunkha et al, 1989). CONCLUTION Soaking and cooking is an appropriate combination processing to improve nutritional quality of red kidney bean flour by reducing the amounts of flatus-producing oligosaccharides. The red kidney bean flour that was processed by soaking raw red kidney beans for 12 h and boiling in pressure cooker for 15 min could decrease the raffinose and stachyose contents by 47% and 44%, respectively. ACKNOWLEDGEMENT The anthors would like to acknowledge Kasetsart University Research and Development Institute (KURDI) for a grant to support this study.

LITERATURE CITED Barapama, Z. and R.E. Simard. 1994. Oligosaccharide, antinutritional factors, and protein digestibility of dry beans as affected by processing. J. Food Sci. 59 : 833-838. Carpenter, K.J. 1981. The Nutritional contribution of dry beans (Phaseolus vulgaris) in perspective. Food Technol. 35 : 77. Deshpande, S.S., S.S. Kadam, and D.K. Salunkhe. 1989. Soaking, p. 135. In D.K. Salunkhe and S.S. Kadam (eds.). CRC Handbook of World Food Legumes : Nutritional Chemistry, Processing Technology, and Utilization Volume III. CRC Press, Florida. Guzman-Maldonado, S.H. and O. ParedesLopez.1998. Functional products of plants indigenous to Latin America: Amaranth, Quinoa, Common Beans, and Botanicals, pp. 308-312. In G. Mazza (ed.). Functional Foods : Biochemical and Processing Aspects. Technomic Publishing, Pennsylvania. Hughes, J.S. 1991. Potential contribution of dry bean dietary fiber to health. Food Technol. 45 : 122-126. Jood, S., U. Mehta, R. Singh, and C.M. Bhat. 1985. Effect of processing on flatus-producing factors in legumes. J. Agric. Food. Chem. 33 : 268271. Koehler, H.H., C. Chang, G. Scheier, and D.W. Burke. 1987. Nutrient composition, protein quality and sensory properties of thirty-six cultivars of dry beans (Phaseolus vulgaris L.). J. Food Sci. 52 : 1335-1340. Nielsen, S.S. 1991. Digestibility of legume proteins. Food Technol. 45 : 112. Olson, A.C., G.M. Gray, M.R. Gumbmann, C.R.

Sell, and J.R. Wagner. 1981. Flatus causing factors in legumes, pp.277-294. In R.L. Ory (ed.). Antinutrients and Natural Toxicants in Foods. Food and Nutrition Press, Connecticut. Reddy, N.R., S.K. Sathe, and D.K. Salunkhe.1989. Carbohydrates, p. 64. In D.K. Salunkhe and S.S. Kadam (eds). CRC Handbook of World Food Legumes : Nutritional Chemistry, Processing Technology, and Utilization Volume I. CRC Press, Florida. Salunkhe, D.K., S.K. Sathe, and S.S. Deshpande. 1989. French Bean, pp. 49-50. In D.K. Salunkhe and S.S. Kadam (eds.). CRC Handbook of World Food Legumes : Nutritional Chemistry, Processing Technology, and Utilization Volume II. CRC Press, Florida. Sánchez-Mata, M.C., M.J. Peñuela-Teruel, M. Cámara-Hurtado, C. DÌez-Marqués, and M. E. Toriia-Isasa. 1998. Determination of mono-, di-, and oligosaccharides in legumes by HighPerformance Liquid Chromatography using an amino- bonded silica column. J. Agric. Food Chem. 46 : 3648-3652. Sathe, S.K. and D.K. Salunkhe. 1989. Technology of removal of unwanted components of dry legumes, pp. 249, 251. In D.K. Salunkhe and S.S. Kadam (eds.). CRC Handbook of World Food Legumes : Nutritional Chemistry, Processing Technology, and Utilization Volume III. CRC Press, Florida. Tharatthapan, C. 1996. Development of Planting and Red Kidney Bean Production. Department of Agriculture, Bangkok. 35 p.

Received date Accepted date

: :

04/10/01 28/12/01

Cotton Seed Treatment in Lao PDR

Somnuck Thirasack

ABSTRACT In 1992 and 1993 insecticide, trials were carried out in two locations in Lao PDR to compare the effectiveness of four insecticides (imidachloprid, benfuracarb, carbosulfan, furathiocarb) applied in seed treatment before sowing , and three modes of application of imidachloprid (powder, coating, pelting) in the control of early cotton pests. The results showed that such seed treatment could accelerate the plant emergence and reduced the rate of damping-off. All the insecticides showed an effect against cotton aphid, leafhopper, whitefly, scale insect and grasshopper when compared to an untreated check, but variations in the level of efficiency were registered according to the active ingredients and the pests. Overall, imidachloprid was the most effective, with the longer residual effect. Key words: cotton, seed treatment, pest, Integrated Pest Management, Lao INTRODUCTION In Lao PDR, at the beginning of growing season, cotton seedlings and young plants are attacked by several insect pests. The most important species are the leafhopper Amrasca biguttula Ishida (Homoptera, Cicadellidae) and the aphid Aphis gossypii Glover (Homoptera, Aphididae) which can affect the production (Castella et al., 1993). Several other species are also observed feeding on the plants during the vegetative phase of development, but in most cases the incidences remain low and they are not of economic importance. Among them the most common are the scale insect Ferrisia virgata Cockerell (Homoptera, Pseudococidae), the whitefly Bemisia tabaci (Gennadius) (Homoptera, Aleyrodidae), several unidentified species of thrips (Thysanoptera), and locusts and grasshoppers (Orthoptera). A. biguttula is considered as the main cotton pest recorded from Southeast Asia (Matthews, 1994). Nymphs and adults injure cotton leaves through injection of toxic saliva while feeding inside veins. The first symptom is a yellowish turning to reddish coloration of the margin of leaves followed by dryness. Depending on the plant stage, the infestation level, and the duration of infestation, leafhopper attacks can reduce plant growth, cause the abortion of the first fruiting branch and increase shedding of squares and young bolls by affecting the photosynthesis. In case of early attacks the vegetative development remains limited and the plant becomes bush-like due to shortening of the internodes. Due to limitations of favourable cultural practices, disadvantages of chemical sprays, and lack of suitable biological control agents, breeding resistant cultivars could be the most practical and cheapest way to control leafhoppers, and therefore pubescence is a major source of resistance (Parnell et al., 1949; Renou et al., 1998). Nevertheless, during almost two weeks after germination, before appearance of hairiness, cotton plants are susceptible

Ministry of Agriculture and Forestry, Cabinet Office, P.O. Box 811, Vientiane, Lao PDR.

to leafhopper attacks and can be seriously affected by this insect (Renou et al., 1998). A. gossypii is a sap-sucking pest widely distributed in the world. This is a very polyphagous insect which has been recorded from almost 900 host plants. On cotton, it develops on the lower surface of the leaves constituting colonies. It is responsible of two kinds of damage. On the one hand, the penetration of the stylets in the vegetal induces characteristic deformation of the leaves which become crumpled and curl down. On the other hand, it excretes droplets of honeydew deposited on open bolls when infestation occurs at the end of the season. The sugary exudate soils the fibre, producing sticky cotton, and is an organic substrate used by fungus to grow. Moreover A. gossypii is a vector of a virus disease, called cotton blue disease or leaf roll, presented in Lao PDR (Cauquil and Vaissayre, 1971). Attacks of these pests at the beginning of the growing season often make it necessary to control the population in order to avoid an outbreak. Therefore, in most cases the farmers make one or two insecticide sprayings with knapsack sprayer equipped with a single lance. The active ingredient commonly used is dimethoate implemented at the rate of almost 300 g ai/ha. Besides the fact that this broad spectrum insecticide is noxious for the user, it is not environmently friendly and , among other undesirable effects, it eliminates some predators of the pests (Wilson et al., 1998). One solution to avoid such a consequence, consists of treating the seeds with a systemic insecticide before planting (Graham, 1998). The objective of this research work was to evaluate the efficacy of insecticides used for seed treatment in the control of early cotton pests in Lao PDR, and to compare methods used to treat the seed. MATERIALS AND METHODS The trials to study insecticide efficiency

were performed in 1992 during the wet season at Napok Research Center (sowing date July 27) and in a farmer's field at Ban Hai (sowing date July 20). The comparison of modalities of treatment was carried out in 1993 during the dry season at Napok Center (sowing date December 2). These two localities are located in the vicinity of Vientiane Municipality (102.6°E ­ 18.0°N), at an altitude of 170 m above sea level, with an annual rainfall of 1400 to 1900 mm. To evaluate insecticides efficiency, the seeds were slightly humidified, and then treated with powder insecticides in plastic bags. In 1993, this mode of seed treatment was compared to two other ones called caoting and pelting. The former consisted of covering the seeds with a thin layer of clay, and then spraying them with the insecticide diluted in water. The later consisted of mixing the slightly humidified seeds with insecticide and then covering each of them with a thin layer of clay. Randomized Complete Block Design with 4 replications at Ban Hai in 1992 and Napok in 1993 and 6 replications at Napok in 1992 was employed. The elementary plot dimension was 54 m2 (3 rows with 18 m long) in 1992 and 60 m2 (3 rows with 20 m long) in 1993. Planting materials used were varieties of Gossypium hirsutum L.: P 288 and KK I at Napok in 1992 and 1993, respectively, and S 295 at Ban Hai in 1992. The modalities of studies in different trials are shown in Table 1. The counting of plant emergence and insects started 6 days after sowing. Registered data are given in Table 2. After recording data on MULTIPLAN software, all variables have been analysed using STATITCF software. Sometimes it was necessary to transform certain variables to improve further parameters of analysis: indices of normality, absence of residues and equality of deviation of residues. In this case, means mentioned in the table were taken into account of transformation. It was noted that two contrasts were employed for the trials from Napok and Ban Hai in 1992, to complete the interpretations: contrast 1

Table 1 Modalities of the different trials carried out at Napok Station and Ban Hai in 1992 and 1993. Active Ingredients No treatment Benfuracarb Carbosulfan Furathiocarb Imidachloprid Imidachloprid Imidachloprid Types of treatment powder powder powder powder coating pelting Doses Napok 1992 X X X X X Ban Hai 1992 X X X X X Napok 1993 X X X

0.80 % 0.80 % 0.80 % 0.35 % 0.35 % 0.35 %

Table 2 Data recorded according to the localities and the types of observation. Observations Emergence Jassids Aphids Scale insects Grasshoppers Whitefly Thrips Damping-off Production of squares Production of flowers Production of bolls Height Yield Napok 1992 X X X X X X X X X X Ban Hai 1992 X X X X X X X X Napok 1993 X X X X X

opposed the check one (no treatment) to different seed treatments and contrast 2 was to compare the effect of imidachloprid to carbamates (Table 3). This method permitted one to compare one treatment (or several) with several other ones taken together. A contrast is defined as a linear combination between the p averages compared: C = i x i where is a coefficient and varies from 1 to p with i = 0. The coefficients used are mentioned in Table 3. The level of significance was obtained by comparing the ratio of the sum of the squares of the standard

deviations of the contrasts and the residual standard deviation using an F-test (Gouet, 1974). Table 3 Coefficients used to test the contrasts. Objects No treatment Imidachloprid Carbosulfan Benfuracarb Furathiocarb Contrast 1 +4 -1 -1 -1 -1 Contrast 2 0 +3 -1 -1 -1

RESULTS AND DICUSSION 1) Comparison of active ingredients 1-a) Emergence No significant difference were revealed,

either at Ban Hai or at Napok, concerning emergence (Tables 4, 5). In both localities, plant densities were normal and no insecticide phytotoxicity was detected. Nevertheless, at Ban Hai, the rate of 50 % of emergence was reached 8.1 days after sowing

Table 4 Percentages of emergence at different dates after sowing and dates of 50 % emergence at Ban Hai in 1992. (DAS = days after sowing). Objects 6 DAS No treatment Benfuracarb Carbosulfan Furathiocarb Imidachloprid F-test (treatment) Sign. Prob. CV (%) Transformation Contrast 1 Sign. Pro. Contrast 2 Sign. Pro. 40.7 41.0 40.4 40.7 42.9 0.23 ns 9.9 ns ns Emergence of plant (%) 9 DAS 41.5 41.2 41.5 42.9 46.1 1.25 ns 8.5 ns 0.1 12 DAS 61.4 56.5 56.5 55.2 61.7 1.25 ns 9.4 arcsin r. (x) ns 0.1 Date 50 % emergence (DAS) 9.5 9.2 8.6 9.2 8.1 0.72 ns 19.0 ns ns

Table 5 Percentages of emergence at different dates after sowing and dates of 50 % emergence at Napok in 1992. (DAS = days after sowing). Objects 6 DAS No treatment Benfuracarb Carbosulfan Furathiocarb Imidachloprid F-test (treatment) Sign. Prob. CV (%) Transformation Contrast 1 Sign. Pro. Contrast 2 Sign. Pro. 75.5 73.7 79.6 76.3 79.7 1.12 ns 7.9 ns ns Emergence of plant (%) 9 DAS 79.3 78.2 80.1 77.8 81.7 0.53 ns 6.7 ns ns 12 DAS 79.8 78.3 80.9 77.8 82.3 0.74 ns 6.5 arcsin r.(x) ns ns 15 DAS 77.9 78.4 80.9 80.4 82.3 0.92 ns 5.8 ns ns 45 DAS 80.3 82.3 80.0 79.8 80.8 0.90 ns 3.1 ns ns

with imidachloprid while this percentage was obtained in average after 9 days with the other products, and after 9.5 days in untreated plots. Even if no significant difference have been pointed out, this result could originate, at least in part, from the effect of insecticide. Goddard and Leser (1997) mentioned that the presence of systemic insecticides tended to increase final stands over the untreated check, and Stringer and Mitchell (1997) concluded that with imidachloprid treated seed, seedling stand densities were slightly higher than those with furadan/disulfoton treated seed. Thus, an action of active ingredient on the emergence of plant and vigour of seedling could be envisaged.

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1-b) Plant growth Great difference was noted according to locality. The better growth was registered at Ban Hai where the plants reached 70 to 80 cm high 60 days after sowing. At Napok after the same period of time, in spite of the speeding-up of growth from 45 days after sowing, the plants were not taller than 25 cm (Figures 1, 2). This variation of one place to the other, was mostly the consequences of the influence of environmental conditions, like rainfall and soil fertility, or the plant physiology out was not due to pests incidence. However the data the recorded at Ban Hai, showed that with imidachloprid there was a plant tendency to be higher than when treated

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Figure 1 Growth of the plants at Ban Hai (1992).

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Days after sowing

Figure 2 Growth of the plants at Napok (1992).

with other products as well as when untreated. This result could be connected to the previous observation concerning the rate of emergence and the plant vigor. Moreover, Van Duyn et al. (1998) reported that seedlings emerging from seeds treated with imidachloprid and aldicarb were significantly larger than those from the untreated plants. Stringer and Mitchell (1997) also mentioned that carbamate insecticides could result in a physiological effect manifested by more rapid plant development. 1-c) Effect on damping-off Cotton seed and plant can be infested with various disease organisms, fungus and bacteria, which may cause it to decay in the soil, injure and kill seedlings, or have harmful effect on the plants. According to Angladette (1948) in Lao PDR, the most important disease is the cotton wilt caused by Fusarium oxysporum f. vasinfectum Atk. which can affect the seedlings a few days after emergence. The penetration of the fungus inside the plant can occur through wounds produced in the root during nematode penetration. Although no fungicide were used, a difference was noted in Ban Hai between treated and untreated seeds, 6 days after sowing

(contrast 1, Table 6). Even if no significant difference was noted between the active ingredients (contrast 2, Table 6), the reduction of damping-off was particularly marked with furathiocarb and imidachloprid. One explanation could be a nematicide effect of the insecticides. Nevertheless, if such an effect has been reported with aldicarb, it does not occur with furathiocarb and imidachloprid (Burmester et al., 1997). 1-d) Control of pests Seed treatment allows control of different early pests which attack cotton plants at the beginning of the crop season. Nevertheless, the results demonstrated that the level of efficiency depended on the active ingredient used, and on the insect species (Tables 7, 8). Concerning aphids, the best control was obtained with imidachloprid. The control level with the three carbamates was generally lower, particularly at Ban Hai in 1992, where the untreated check was the most affected by this pest (Table 7). With regard to leafhopper, all the products had an effect on this insect. Nevertheless, the data recorded at Ban Hai in 1992, showed that the residual effect with carbamates was shorter than

Table 6 Incidences of damping-off at Ban Hai in 1992. (DAS = days after sowing). Objects 6 DAS No treatment Benfuracarb Carbosulfan Furathiocarb Imidachloprid F-test (treatment) Sign. Prob. CV (%) Transformation Contrast 1 Sign. Pro. Contrast 2 Sign. Pro. 6.8 b 4.5 ab 4.5 ab 2.8 a 3.0 a 7.1 0.05 27.9 0.01 ns Percentages of plants affected by damping-off 9 DAS 1.3 1.8 1.8 1.8 2.0 0.1 ns 85.9 ns ns 12 DAS 0.0 0.5 0.3 0.5 0.5 0.8 ns 147.5 ns ns total 2.2 2.0 1.9 1.8 1.9 1.4 ns 13.7 log (x+1) 0.05 ns

that with imidachloprid (Table 7). The former gave a satisfactory protection during the period of almost two weeks after planting, whereas the insecticide effect of the later was registered throughout the first

month of the crop. Starting from about 45 days after sowing the population of leafhoppers decreased at Ban Hai, and it was not possible to detect longer effect of imidachloprid. But, trials carried out in

Table 7 Infestation by aphid and jassids at Ban Hai in 1992. (DAS = days after sowing). Objects % leaves with aphids 15 DAS No treatment Benfuracarb Carbosulfan Furathiocarb Imidachloprid F-test (treatment) Sign. Prob. CV (%) Transformation Contrast 1 Sign. Pro. Contrast 2 Sign. Pro. 4.1 2.9 10.9 4.1 4.1 1.9 ns 87.0 arc sin (root (x)) ns ns 30 DAS 22.0 c 12.8 b 12.9 b 14.0 b 8.1 a 23.8 0.01 14.7 0.01 0.01 15 DAS 6.0 c 4.2 b 4.0 b 4.2 b 3.3 a 81.1 0.01 6.9 0.01 0.01 No. jassids/1000 leaves 30 DAS 9.0 b 9.2 b 7.9 b 8.2 b 3.9 a 29.9 0.01 10,9 log (x+1) 0.01 0.01 45 DAS 0.9 1.0 1.6 2.4 0.9 1.1 ns 89.1 log (x+1) ns ns 60 DAS 7.5 10.5 12.0 12.0 4.0 2.5 ns 47.4 ns 0.01

Means followed by the same letter in the column are not significantly different as determined by DMRT at P>0.05.

Table 8 Efficiency of seed treatment against each insect pest / 10 plants at Napok in 1992. Objects Thrips No treatment Benfuracarb Carbosulfan Furathiocarb Imidachloprid F-test (treatment) Sign. Prob. CV (%)39.1 Transformation Contrast 1 Sign. Pro. Contrast 2 Sign. Pro. 63.5 b 29.3 a 43.5 ab 48.2 ab 31.8 a 3.9 0.05 49.4 ns ns Jassids 20.7 b 8.5 a 7.2 a 10.2 a 6.8 a 7.1 0.01 124.0 0.01 ns Number of pests/10 plants Aphids 15.3 8.5 7.7 5.2 4.2 1.2 ns 109.0 0.1 ns Whitefly 1.1 0.2 0.1 0.2 0.0 b a a a a Scale insects Grasshoppers 8.2 0.2 0.0 0.1 0.6 b a a a a 1.5 0.7 1.0 0.7 0.0 b ab ab ab a

8.6 0.01 157.1 log (x+1) 0.01 ns

3.2 0.05 82.8 log (x+1) 0.01 ns

4.3 0.05 log (x+1) 0.01 0.05

Means followed by the same letter in the column are not significantly different as determined by DMRT at P>0.05.

Thailand, showed that the persistence effect of this insecticide could be longer than 45 days after sowing (Genay, 1994). Attacks of thrips were observed only at Napok in 1992 (Table 8). In this case, two active ingredients, imidachloprid and benfuracab, rank first. Carbosulfan and furathiocarb were less efficient, and were not significantly different from the untreated check. Early appearance of scale insect was observed at Napok in 1992 (Table 8). In this experiment, the difference was noted between the level of infestation which occurred in the treated and the untreated plots, but no difference was pointed out regarding the four insecticides. Finally, in spite of low pest populations, the results acquired at Napok in 1992, showed that the different active ingredients tested had effect against whitefly and grasshoppers. In this case, imidachloprid was the most efficient insecticide in control of the later (Table 8). 1-e) Flowering and yield No significant difference was found between the treated and untreated plots (Table 9). Only at Ban Hai the results indicated a superiority of imidachloprid in comparison with other insecticides (P< 0.05) (contrast 2) regarding yields, but no difference was noted with untreated check. Trials performed in the U.S.A. indicate that, when pest incidence is high enough, seed treatment with imidachloprid and aldicarb can increase the production over the untreated check by 30% and even 60% (Van Duyn et al., 1998). Accordingly, the fact that no difference has been revealed could be the consequence of the small size of the experimental plots (Michel Bruno, pers. comm.) combined or not combined with too low pests injury at the beginning of crop season. To try to determine the profitability of seed treatment, further investigations should be performed. Actually, price of seed treatment per ha with carbamate and with imidachloprid are approrimately 10 and 20 US $ respectively, while seed cotton is sold by farmers at 22 cents per kg. That means that systemic insecticide

must increase the production of at least 45 kg/ha for carbamates and 90 kg/ha for imidachloprid to be profitable. 2) Comparison of modes of application No difference was noted between the three modes of application of imidachloprid (Table 10). They were equivalent in relation to pest control and yield. A similar study performed in Thailand led to the same conclusion (Genay, 1994). Consequently more sophisticated seed treatment methods like coating and pelting are notneeded; applying insecticide on moistened seeds would be sufficient. CONCLUSION In Lao PDR the most important early pests of cotton crop are leafhoppers (A. biguttula) and aphids (A. gossypii). The remaining species, scale insects, grasshoppers, whitefly (B. tabaci) and thrips, have a lower incidence on the crop. Field studies carried out in 1992 in the vicinity of Vientiane, showed that seed treatment with benfuracarb, carbosulfan, furathiocarb, and imidachloprid employing whatever the mode of application, allowed one to control early cotton pests. As a general rule, the best protection as well as the longer residual effect was obtained with imidachloprid resulting in the most efficient control of leafhoppers, aphids and thrips. Concerning the later, a good control was also obtained with benfuracarb. Even if control level depended on the active ingredient, seed treatment represented a valuable and more environmentally friendly alternative to insecticide sprayings regarding the control of pests. However, no effect of seed treatment on the production was observed, and it should be of great interest to perform the same study throughout several cropping seasons. That should permit one to test insecticide effectiveness under different conditions of pest incidence, and to assess the economic interest of seed treatment to implement an integrated pest management strategy in Lao PDR.

Table 9 Production of flowers and yields at Ban Hai (1992) (DAS = days after sowing). Objects Number of squares/plant 45 DAS No treatment Benfuracarb Carbosulfan Furathiocarb Imidachloprid F-test (treatment) Sign. Prob. CV (%) Transformation Contrast 1 Sign. Pro. Contrast 2 Sign. Pro. 13.4 10.5 9.2 9.6 12.6 0.7 ns 40.9 ns ns 60 DAS 11.9 10.8 9.2 9.6 12.8 0.7 ns 33.0 ns ns Number of flowers/plant 45 DAS 0.1 0.0 0.2 0.0 0.2 1.7 ns 172.3 ns 0.1 60 DAS 0.8 0.5 0.4 0.4 0.7 1.3 ns 50.8 0.1 ns 2043 1673 1565 1782 2262 1.8 ns 20.4 ns 0.05 Yield (Kg/ha of seed cotton)

Table 10 Comparison of the efficiency of the three modes of application of imidachloprid (Napok, 1992). Objects % leaves infested by aphids 19.4 19.1 18.1 % leaves infested by jassids 28.9 24.7 19.6 No. of bolls/plant at harvest 15.1 11.1 11.0 Yield (kg/ha of seed-cotton) 2020 1768 2106

Imida. powder Imida. coating Imida pelting F-test not analysed Sign. Prob. C.V. (%) Transformation

3.2 ns 23.0 -

2.7 ns 15.1 -

4.6 ns 17.9 -

0.3 ns 30.7 -

LITERATURE CITED Angladette, A. 1948. Note sur la culture du cotonnier en Indochine. Coton et Fibres tropicales 3 (34) : 73-111. Burmester, C.B., W. Gazaway, D.J. Potter, D. Derrick, and E. Ingram. 1997. Impact of atplant and post-plant nematicides on cotton

production in reniform nematode infested fields, pp. 6-10. In Proceedings of the Beltwide Cotton Conferences. National Cotton Council of America. January 6-10, New Orleans, Louisiana, U.S.A. Castella, J.C., B. Chantharat, S. Thirasack, and G. Trébuil. 1993. Le cotonnier au Laos. Les enseignements d'une expérience de Recherche-

Développement-Formation. Coopération agricole bilatérale française/CNRA-MAF RDP Laos. 88 p. Cauquil, J. 1977. Etudes sur une maladie d' origine virale du cotonnier: la maladie bleue. Coton et Fibres tropicales 32 (3) : 259-278 Cauquil, J. 1993. Maladies et ravageurs du cotonnier en Afrique au sud du Sahara. CIRAD., France. 92 p. Cauquil, J. and M. Vaissayre. 1971. La maladie bleue du cotonnier en Afrique: transmission de cotonnier à cotonnier par Aphis gossypii Glover. Coton et Fibres tropicales. 26 (4) : 463-466. Christides, B.G., and G.J. Harrison. 1955. Cotton Growing Problems. McGraw-Hill, Londo, U.K. 633 p. Genay, J.P. 1994. Trois années d'épérimentation phytosanitaire sur cotonnier en Thaïlande (1991-1993): bilan et perspectives. Series Documents de travail. CIRAD-CA, Montpellier, France. 53 p. Goddard, G.F., and J.F. Leser. 1997. Effects of soil systemic insecticides and cotton seed vigor on cotton stands and yields, pp. 1165-1168 In Proceedings of the Beltwide Cotton Conferences. National Cotton Council of America. January 6-10, New Orleans, Louisiana, U.S.A. Gouet, J.P. 1974. Les comparaisons de moyennes et de variances. Application à l'agronomie. ITCF ed., Paris, France. 55 p. Graham, C.T. 1998. Performance of Gaucho seed treatment across the mid-South and Southeast, pp. 1187-1188. In Proceedings of the Beltwide Cotton Conferences. National Cotton Council of America. January 5-9, San Diego, California, U.S.A. Guillemat, J. 1947 La flétrissure ou "wilt" du cotonnier due à Fusarium vasindectum Atk. Coton et Fibres tropicale 2 (1) : 17-27. Hussain, M.A., and K.B. Lal. 1940. The Bionomics of Empoasca devastans Dist. On some varieties of cotton in Punjab. Indian Journal of

Entomology 2: 123-136. Inaizumi, M. 1980. Studies on the life-cycle and polymorphism of Aphis gossypii Glover (Homoptera, Aphididae). Special Bulletin of the College of Agriculture, Utsunamiya University, Japan. 37. 132 p. Kirkpatrick, T.L. 1998. Interactions of nematodes and fungal wilt pathogens, pp. 123-125. In Proceedings of the Beltwide Cotton Conferences. National Cotton Council of America. January 5-9, San Diego, California, U.S.A. Leclant, F. and J.P. Deuine. 1994. Aphids (Hemiptera: Aphididae), pp. 285-323. In G.A. Matthews and J.P. Tunstall (eds.). Insect Pests of Cotton. CAB International, Oxon, U.K. : 285-323. Matthews, G.A. 1994. Jassids (Hemiptera: Cicadellidae), pp 353-357. In G.A. Matthews and J. P. Tunstall (eds.). Insect Pests of Cotton, CAB International, Oxon, U.K. Parnell, F.R., H.E. King, and D.F. Ruston. 1949. Insect resistance and hairiness of the cotton plant. Bulletin of Entomological Research 39 : 539-575. Renou, A. 1996. Studies about jassid injuries and their incidence on fruiting production and survival, pp 49-52. In Report. Development Oriented Research on Agrarian Systems Project, Kasetsart University, Bangkok. Renou, A., E. Weerawan, and W. Chograttanameteekul. 1998. Importance of jassid egg deposition affected by leaf age and cultivar in relation to jassids resistance in cotton, pp.1-8. In Annual Report. Development Oriented Research on Agrarian Systems Project, Kasetsart University, Bangkok. Scott, W.P., J.W. Smith, and G.L. Snodgrass. 1986. Impact of early season use of selected in secticides on cotton arthropod population and yield. Journal of Economic Entomology 73 : 797-804. Stringer, S.J., and H.R. Mitchell. 1997. Effect of

Furadan/Disulfoton on cotton growth and development, pp. 1171-1176. In Proceedings of the Beltwide Cotton Conferences. National Cotton Council of America. January 6-10, New Orleans, Louisiana, U.S.A. Turnipseed, S.G., and M.J. Sullivan. 1998. Consequences of early-season foliar insecticides in cotton in South Carolona, pp. 1050-1051. In Proceedings of the Beltwide Cotton Conferences. National Cotton Council of America. January 5-9, San Diego, California, U.S.A. Uthamasamy, S. 1994. Host resistance to the leafhopper, Amrasca devastans (Distant) in cotton, Gossypium spp. Challenging the future; pp. 494-498. In G.A. Constable and N. W. Forrester (eds.).Proceedings of the World Cotton Research Conference. CSIRO, February 14-17, 1994. Brisban, Melbourne, Australia. Van Duyn, J., J.R. Bradley, A.L. Lambert, C.P.C. Suh, and J. Faircloth. 1998. Thrips management with Gaucho seed treatment in North Carolina

cotton, pp. 1183-1187. In Proceedings of the Beltwide Cotton Conferences. National Cotton Council of America. January 5-9, San Diego, California, U.S.A. Wilson, L.J., L.R. Bauer, and D.A. Lally. 1998. Effect of early insecticide use on predators and outbreaks of spider mites (Acari: Tetranychidae) in cotton. Bulletin of Entomological Research 88 : 477-488. Xia, J.Y. 1994. An integrated cotton insect pest management system for cotton-wheat intercropping in North China. Challenging the future; pp. 511-517 In G.A. Constable and N. W. Forrester (eds.). Proceedings of the World Cotton Research Conference, CSIRO, February 14-17, 1994, Brisban, Melbourne, Australia: 517.

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