- Email: [email protected]
Potential uses of sicklepod (Cassia obtusifolia)
Industrial Crops and Products 8 (1998) 77 – 82
Potential uses of sicklepod (Cassia obtusifolia) T.P. Abbott a,*, S.F. Vaughn b, P.F. Dowd b, H. Mojtahedi c, R.F. Wilson d New Crops Research, National Center for Agricultural Utilization Research, Agricultural Research Ser6ice, USDA 1, 1815 N. Uni6ersity Street, Peoria, IL 61604, USA b Bioacti6e Agents Research, National Center for Agricultural Utilization Research, Agricultural Research Ser6ice, USDA, 1815 N. Uni6ersity Street, Peoria, IL 61604, USA c Department of Plant Pathology, Washington State Uni6ersity, 24106 N. Bunn Road, Prosser, Washington 99350 -9687, USA d Soybean and Nitrogen Fixation Research, Agricultural Research Ser6ice, USDA, North Carolina State Uni6ersity, Box 7620, Raleigh, NC 27695 -7620, USA a
Received 6 June 1997; accepted 2 October 1997
Abstract Sicklepod (Cassia obtusifolia) is a leguminous weed species that has become a severe problem in soybean production throughout the Southern United States. Economic incentives, such as premiums for low levels of foreign matter from cleaning soybeans prior to sale, could generate a large source of sicklepod seed in that area. This study was undertaken to evaluate C. obtusifolia seed for potential applications. As much as 41% of the seed was extractable. Some extracts were strong inhibitors of wheat, velvetleaf and sicklepod root growth, causing discoloration of the root meristems in a manner similar to that caused by naphthoquinones such as juglone and plumbagin. Some extracts increased weight gain in fall armyworm (Spodoptera frugiperda) causing them to grow to 50 – 100% larger than controls in a 7-day trial. Survival of Columbia root-knot nematode (Meloidogyne chitwoodi ) in the soil was inversely correlated to the amount of ground whole sicklepod amendment. No phytotoxic effects of the meal amendment on tomato plants or inhibition of germination for several crop seeds was observed at the levels tested. © 1998 Elsevier Science B.V. All rights reserved. Keywords: Sicklepod; Cassia obtusifolia; Gum; Nematode; Inhibitor; Fall armyworm; Allelochemical; Spodoptera frugiperda; Meloidogyne chitwoodi; Soil amendment
* Corresponding author. Tel: +1 309 6816533; fax: +1 309 6816524; e-mail: [email protected] 1 Names are necessary to report factually on available data; however, the USDA neither guarantees nor warrants the standard of the product, and the use of the name by USDA implies no approval of the product to the exclusion of others that may also be suitable.
1. Introduction Sicklepod (Cassia obtusifolia) is a leguminous weed species that has become a severe problem in soybean production areas throughout the Southern United States.
0926-6690/98/$19.00 © 1998 Elsevier Science B.V. All rights reserved. PII S 0 9 2 6 - 6 6 9 0 ( 9 7 ) 1 0 0 1 0 - 3
78
T.P. Abbott et al. / Industrial Crops and Products 8 (1998) 77–82
Cassia obtusifolia and C. tora may be the same species (Crawford et al., 1990) and are treated as the same in this paper. Effective control strategies for this noxious weed are limited by a lack of selective herbicides that discriminate among legumes and ‘no-till’ cultural practices which accentuate the spread of sicklepod seed. Sicklepod seed contains anthraquinones, such as obtusifolin, which produce toxic effects in livestock (McCormack and Neisler, 1980). When crushed with soybeans, these compounds also contribute to lower oil quality. Degradation products can cause undesirable colors in processed soybean oils that are difficult to remove with conventional refining technology. In 1993, soybeans harvested throughout the South contained an average 5% sicklepod seed (private communication). At that level of foreign matter, contract sales in North Carolina were discounted ca. $0.28/bushel. However, poor soybean quality plus high cost of disposal of sicklepod seed in an environmentally safe manner prompted Cargill, Inc. in North Carolina to offer a 1% per bushel contract premium for soybean containing 1% maximum foreign matter. The amended discount schedule became effective in October 1994. Economic incentives to clean soybeans prior to sale could generate a 50 million pound annual supply of sicklepod seed in North Carolina alone. Unless a market is found for these seeds, disposal on farm land could lead to greater infestations of this weed and further reduce soybean productivity. New uses for sicklepod could turn a problem into a resource. Research has shown that a large part of sicklepod seed composition is carbohydrate (68%), but the water-soluble gums content is low (7%) and sugars account for only 8% of the carbohydrates (Crawford et al., 1990). In addition, the seed contains 7% oil, 15 – 20% crude protein and less than 3% of methanol-extractable anthraquinone glycosides. Sicklepod has also been shown to contain antimicrobial components (Kitanaka and Takido, 1986). The polysaccharides in sicklepod are not yet well characterized, but the soluble gum has been analyzed (Varshney et al., 1973). Plant carbohydrates and gums in particular are sometimes insol-
uble in water because they contain ionic crosslinks or structure only solubilized at acid or basic pH (Ericson and Elbein, 1980). Ammonium oxalate for example, has been shown to solubilize gums with calcium crosslinked galactouronysyl units (Abbott et al., 1996). Meloidoggne chitwoodi (Columbia root-knot nematode) is a serious pest of potato in the Pacific Northwest (Mojtahedi et al., 1993). This nematode attacks tubers and causes brown spots and blemishes on the tubers. Blemished tubers are down-graded or even rejected by the processors. In the latter case, the entire crop is fed to cattle. Presently, soil fumigation with 1,3-dichloropropene or metham sodium is the only reliable method of control (Pinkerton et al., 1986). However, nematode control has been achieved using rapeseed or white mustard as a green manure, in combination with a contact organophosphate nematicide (Mojtahedi et al., 1993). If ground sicklepod or its water extract could be used on the farm as a soil amendment to inhibit weed germination, or decrease pest populations, fewer herbicides or pesticides might be required. Although sicklepod meal and extracts cause some toxic effects when digested, the toxic component would be expected to biodegrade in the soil readily because the sugar moiety in anthraquinone glycosides is easily attached and quinones are highly reactive. By comparison, the currently used fumigants and alternative organophosphothionates are highly toxic (Ali et al., 1986). This work was undertaken to assess sicklepod seed and extracts for valueadded products such as gums, allelochemicals and pesticides.
2. Experimental
2.1. Extracts of sicklepod seed Because water and traditional gum extraction techniques had failed to extract the carbohydrate component of sicklepod, a series of aqueous salt solutions with a range of pH, salt concentration, mono- and dibasic cations and anions was tested for solution of sicklepod gums. Cassia obtusifolia seeds, collected from U.S. Southeastern soybean
T.P. Abbott et al. / Industrial Crops and Products 8 (1998) 77–82
fields, were ground to pass a 20 mesh screen. A 10-g sample was weighed and water soluble components extracted with deionized water (400 ml) at 45°C for 4 h. The mixture was centrifuged and the supernatant removed, then dialyzed three times against 4, 12 and 12 l deionized water, and the retentate freeze-dried. Half of the permeate from the first dialysis was recovered and freeze-dried. The same procedure was followed but instead using 0.1 N or 0.5 N NaOH as the extraction solvent, and neutralizing the dialysis permeate with acid ion exchange resin to pH 7, before freeze-drying. Salt solutions (NaCl, NH4Cl, (NH4)2CO3, Na2CO3, Na2SO4, Ca(CH3COO)2) at 0.05, 0.1, 0.5 and 1.0 M concentration, 10 ml each, were added to 1.00 g of ground seed and held at 60°C for 2 h with occasional shaking. Solutions were cooled to room temperature and centrifuged to give a clear supernatant which was decanted and analyzed for total carbohydrate and percent carbohydrate greater than 10 000 molecular weight (Abbott et al., 1995). Additionally, the procedure of Varshney et al. (1973) was followed to determine the amount of soluble gum available by their procedure, which consists of extraction of gums with 1% acetic acid in water followed by precipitation of gums with ethanol.
2.2. Germination inhibition assays The water extract and the neutralized dialysis permeates from NaOH extracts of ground sicklepod seed were assayed for inhibitory effects against sicklepod, velvetleaf and wheat germination. Dried extracts (1 mg/ml) were tested in water agar (10 g/l) containing 0.1 g/l 2-(4-thiazolyl)benzimidazole (Aldrich, Milwaukee, WI) and 0.5 g/l chloramphenicol (Sigma, St. Louis, MO) as antifungal and antibacterial agents, respectively. Seeds were surface disinfected with 10% Chlorox for 15 min, rinsed with sterile ddH2O (twice) and incubated at 25°C for 24 – 48 h. Germinated seeds with protruding radicals (sicklepod and velvetleaf) or primary roots (wheat) of equal length were placed on the agar and incubated at 25°C in darkness on 45 degree slants until the control roots reached the edge of the plates.
79
Ground whole seed was mixed with a sandy soil (Bloomfield sand, Psammentic Hapludalf) at rates of 0.0, 0.1, 0.5, 1.0 and 5.0% (w/w). One hundred and fifty milliliters of the soil seed-meal mixtures were placed in 200-ml cups and 10 seeds of each bioassay species: wheat (Triticum aesti6um L. ‘Cardinal’), soybean (Glycine max (L.) Merr. ‘Williams’), corn (Zea mays L. ‘DeKalb IL 645786’) and hemp sesbania (Sesbania exaltata (Raf.) Rydb. ex A.W. Hill), were added and covered with approximately 1 cm of soil. The cups were watered to field capacity, placed in the growth chamber at a 16 h, 26°C day/8 h, 21°C night regime, and watered daily. A combination of fluorescent and metal halide lighting was used to give an illumination of approximately 1900 mE m − 1 s − 1 intensity.
2.3. Fall armyworm and nematicide assays The water and base extracts tested for inhibition of germinating seeds were also assayed for pesticidal activity against fall armyworms by previously described methods (Dowd, 1988). Dry extracts (1%) were added to the basic diet (5 ml liquified at 60°C) and mixed with a vortex mixer for 25 s. The diets were cooled, by standing at ambient temperature, dried and divided into pieces sufficient ( 0.25 g) to neonate larvae for 1 week ad libitum. Larvae and diet were placed individually in wells of a 24-well tissue culture plate and sealed with parafilm to prevent moisture loss. Ground whole seed was added at 10% of total diet weight. Controls were larvae fed the basic diet which had been treated similarly, but without added sicklepod fractions. Insect mortality was examined after 7 days. Twenty insects per treatment were tested. To investigate nematicidal activity, loamy sand greenhouse mix soil (80% sand, 15% silt, 5% clay, and 0.5% organic matter) was infested with tomato root pieces harboring Meloidogyne chitwoodi female, with attached egg masses. The infested soil was incubated for 4 weeks to allow the second stage juvenile to hatch. Infested soil samples (500 g, 8% moisture, five replicates each treatment) were then placed in plastic bags, and mixed with a contact organophosphate nematicide
T.P. Abbott et al. / Industrial Crops and Products 8 (1998) 77–82
80 Table 1 Sicklepod extracts Extractant
Water 0.1 N NaOH 0.5 N NaOH
Extract, % of seed weight
21.3 35.4 40.9
LMW, % of seed weight
14.2 10.9 12.4
HMW, % of seed weight
7.1 24.5 28.5
Analyses of HMW fraction
% gum
% crude protein
59.7 55.3 65.4
35.0 29.3 27.9
LMW, Low-molecular-weight (B10 000) dialysis permeate (neutralized) from the extract; HMW, High-molecular-weight (\10 000) dialysis retentate from the extract.
(13 kg ai/na, Ethoprop, MoCap, Rhone Poulenc), 0.5 – 4% sicklepod meal by weight, or left untreated as control. The bags were sealed and incubated for 1 week before the soil samples were placed in clay pots, and 3-week-old Rutger tomato seedlings were planted. The pots were randomized and maintained on a greenhouse bench (2396°C) for 3 weeks, before the roots were washed free from soil, stained with acid fuchsin and the number of invading nematodes were counted.
3. Results and discussion
3.1. Extractable components Using the procedure of Varshney et al. (1973), 2.82% soluble gum was extractable which is comparable to Varshney’s result. This is the lowest water-soluble gum content of any Cassia species as reported in previous gum surveys (Tookey and Jones, 1965). Water at 45°C solubilizes about 21% of the ground seed weight. There is a 2:1 ratio of dialysis permeate to retentate in the extract and analysis of the higher molecular weight retentate gives 60% carbohydrate gum and 35% protein (Table 1). As much as 41% of the C. obtusifolia seed can be extracted with basic solutions. Although a range of aqueous salt solutions at various pHs and concentrations were tried, only marginal changes in solubilization of C. obtusifolia gums occurred (Table 2). Only NaOH extraction, of those inves-
tigated, extracted significant amounts of gums from C. obtusifolia. Membrane separations are a relatively expensive industrial process compared to extraction, precipitation, centrifugation, drying and similar unit operations. Because, in our investigations, we take a view toward the most economical means for producing value-added products, we propose alternative, less-costly processes. The separation into low-molecular-weight constituents and gums plus protein fractions, for example, need not involve dialysis as detailed in the present experiment. The water extract dissolves all of the low molecular weight components, but the majority of the gum plus protein fraction is only soluble at higher pH. A sequential water, 0.1 N NaOH separation could be refined further by first extracting at a lower pH, where the protein would be even less soluble, followed by extraction at a higher pH and precipitation of the protein at its isoelectric point. Alternatively, the combined extract made at higher pH and then neutralized could potentially be spray-dried and tested as an encapsulated, slow-release preemergence herbicide or pesticide. Although gums are generally used as thickeners, rheological and chemical characterization of sicklepod gum properties have not yet been determined.
3.2. Seed germination inhibition Both of the permeates from the high pH extracts (after neutralization/deionization with acid ion exchange resin to pH 7) were strong inhibitors
T.P. Abbott et al. / Industrial Crops and Products 8 (1998) 77–82
81
Table 2 Salt solution (0.1 – 1.0 M) extraction of sicklepod Salt
NaCl NH4Cl (NH4)2CO3 Na2CO3 Na2SO4 Ca(OAc)2 1% HOAc/EtOH pptn.
pH Buffer
7 7 9–10 12–13 7 8 5
pH Extract
6 6 7– 10 12 7 7– 8 5
% of seed weight extracted as Sugars
Gums
6.1 – 8.2a 3.4 – 6.1a 6.1 – 8.6c 6.3 – 8.1b 6.7 – 8.6c 6.3 – 7.5b
5.0 – 6.8a 6.0 – 7.2 6.0 – 8.2a 5.9 – 7.9c 1.7 – 5.9b 3.0 – 4.3b 2.82
a
Increases with increasing salt concentration. Decreases with increasing salt concentration. c Peaks at mid salt concentration. b
of wheat, velvetleaf and sicklepod root growth, causing discoloration of the root meristems in a manner similar to that caused by naphthoquinones such as juglone and plumbagin. Previously published literature indicates that sicklepod seeds contain substituted anthraquinones including obtusifolin, obtusin, aurantio-obtusin and chryso-obtusin (Crawford et al., 1990) which may be at least partly responsible for the observed effects. In contrast, ground whole sicklepod seed (up to 5% in soil) did not inhibit germination of wheat, soybeans, corn or hemp. One explanation for the whole meal and water extract having different effects may be that the isolation by NaOH and neutralization with acid ion exchange resin changed the structures of the extracted materials from their structure in the plant. The exact cause of the difference requires further research.
Table 4 shows the effect of various levels of sicklepod seed meal on Meloidogyne chitwoodi nematodes. Survival of Columbia root-knot nematode in the soil was inversely correlated to the amounts of sicklepod amendment. No phytotoxic effects of the meal amendment on tomato plants was observed.
4. Conclusions It would appear that sicklepod has at least three opportunities for use. The first of these is Table 3 Seven-day weights of fall armyworm larvae on diets with sicklepod extracts Diet
3.3. Effect on fall armyworm and nematodes Table 3 shows an unusual effect of water and basic extracts of sicklepod on fall armyworm larvae. The hot water extract caused the larvae to consume more of the feed and grow to twice the size of larvae on the control diet. The water extract (ion-exchanged dialysis permeate) was most effective, although the base extracted materials showed a similar effect. Ground whole sicklepod seed had no effect on fall armyworm larvae when incorporated at 10% in the diet (Table 3, Experiment II).
Experiment I Control Control+1% sicklepod water extract Control+1% 0.1 N NaOH extract Control+1% 0.5 N NaOH extract Experiment II Control 10% cellulose powder (non-nutritive control) 10% sicklepod powder
7-Day weights (mg)
38.4 92.8 75.3 96.9a 61.5 94.3a 56.3 96.9a 25.5 93.1 23.2 93.2 27.6 93.4
Values are mean 9 standard error. a Weights are significantly higher than controls at PB0.05.
T.P. Abbott et al. / Industrial Crops and Products 8 (1998) 77–82
82
Table 4 Survival of Meloidogyne chitwoodi in soil amended with different amounts of sicklepod meal and bioassayed on Rutger tomato seedlings for 3 weeks Treatment
No. of infective nematodes
Control Mocap 10G Sicklepod meal Sicklepod meal Sicklepod meal Sicklepod meal
763a 0d 759a 374ab 220b 7c
0.5%a 1% 2% 4%
The values are means of five replicates. Means followed by the same letter do not differ at PB0.05 according to Duncan’s multiple range test. a % of soil by weight.
the potential for the base-extracted carbohydrate gum or gum-protein mixture to be tested in thickener or encapsulation applications, now that significant amounts have been solubilized in base. The results showing increased feeding by fall armyworm on a diet that included 1% sicklepod extract indicates that this extract may be useful for increasing the uptake of pesticide-laced bait by fall armyworm. Whether this is a palatability or feeding stimulant effect is not known, but the addition of 1% of the extract to a diet is unlikely to be an effect on feed efficiency because the protein, fat and carbohydrate in the sicklepod supplemented diet is 99% the same as in the control diet. Ground whole sicklepod seed meal had no adverse effects on tomato plants or several crop seeds when incorporated into soil at 5% but reduced nematode population. Therefore, its use as a nematicide may be possible. The next stage of testing for nematode control would be outdoor test plots with both the meal and water extracts.
References Abbott, T.P., Nelsen, T.C., Kleiman, R., 1995. Seed surface gums content using the Dubois carbohydrate analysis. Ind. Crops Prod. 4, 185 – 192. Abbott, T.P., Nelsen, T.C., Kleiman, R., 1996. Lesquerella gums. In: Princen, L.H., Rossi, C. (Eds.), Proceedings of the Third International Conference on New Industrial Crops and Products. Versa Press, Inc., East Peoria, IL, pp. 343 – 345. Ali, F.A.F., Chukwudebe, A.C., Fukuto, T.R., 1986. Toxicological properties of O,S,S, trialkyl phosphorodithioates. J. Agric. Food Chem. 34, 45 – 48. Crawford, L., McDonald, G.M., Friedman, M., 1990. Composition of sicklepod (Cassia obtusifolia) toxic weed seeds. J. Agric. Food Chem. 38, 2169 – 2175. Dowd, P.F., 1988. Toxicological and biochemical interactions of the fungal metabolites fusaric acid and kojic acid with xenobiotics in Heliothis zea (F.) and Spodoptera frugiperda (J.E. Smith). Pestic. Biochem. Physiol. 32, 123 – 134. Ericson, M.C., Elbein, A.D., 1980. Cell wall polysaccharides and glycoproteins. In: Preiss, J. (Ed.), The Biochemistry of Plants, Vol. 3. Carbohydrates, Structure and Function. Academic Press, New York, pp. 591 – 616. Kitanaka, S., Takido, M., 1986. Studies on the constituents in the roots of Cassia obtusifolia L. and the antimicrobial activities of constituents of the roots and the seeds. Yakugaku Zasshi 106, 302 – 306. McCormack, J.E., Neisler, W.E., 1980. Cassia obtusifolia toxicity in a dairy herd. Vet. Med. Small Anim. Clin. 75, 1849 – 1851. Mojtahedi, H., Santo, G.S., Wilson, T.H., Hang, A.N., 1993. Managing Meloidogyne chitwoodi on potato with rapeseed as green manure. Plant Dis. 77, 42 – 46. Pinkerton, J.N., Santo, G.S., Ponti, R.P., Wilson, J.H., 1986. Control of Meloidogyne chitwoodi in commercially grown Russet Burbank potatoes. Plant Dis. 70, 860 – 863. Tookey, H.L., Jones, Q., 1965. New sources of water-soluble seed gums. Econ. Bot. 19, 165 – 174. Varshney, S.C., Rizvi, S.A.I., Gupta, P.C., 1973. Structure of a polysaccharide from Cassia tora seeds. I. J. Agric. Food Chem. 21, 222 – 226.
Potential uses of sicklepod (Cassia obtusifolia) T.P. Abbott a,*, S.F. Vaughn b, P.F. Dowd b, H. Mojtahedi c, R.F. Wilson d New Crops Research, National Center for Agricultural Utilization Research, Agricultural Research Ser6ice, USDA 1, 1815 N. Uni6ersity Street, Peoria, IL 61604, USA b Bioacti6e Agents Research, National Center for Agricultural Utilization Research, Agricultural Research Ser6ice, USDA, 1815 N. Uni6ersity Street, Peoria, IL 61604, USA c Department of Plant Pathology, Washington State Uni6ersity, 24106 N. Bunn Road, Prosser, Washington 99350 -9687, USA d Soybean and Nitrogen Fixation Research, Agricultural Research Ser6ice, USDA, North Carolina State Uni6ersity, Box 7620, Raleigh, NC 27695 -7620, USA a
Received 6 June 1997; accepted 2 October 1997
Abstract Sicklepod (Cassia obtusifolia) is a leguminous weed species that has become a severe problem in soybean production throughout the Southern United States. Economic incentives, such as premiums for low levels of foreign matter from cleaning soybeans prior to sale, could generate a large source of sicklepod seed in that area. This study was undertaken to evaluate C. obtusifolia seed for potential applications. As much as 41% of the seed was extractable. Some extracts were strong inhibitors of wheat, velvetleaf and sicklepod root growth, causing discoloration of the root meristems in a manner similar to that caused by naphthoquinones such as juglone and plumbagin. Some extracts increased weight gain in fall armyworm (Spodoptera frugiperda) causing them to grow to 50 – 100% larger than controls in a 7-day trial. Survival of Columbia root-knot nematode (Meloidogyne chitwoodi ) in the soil was inversely correlated to the amount of ground whole sicklepod amendment. No phytotoxic effects of the meal amendment on tomato plants or inhibition of germination for several crop seeds was observed at the levels tested. © 1998 Elsevier Science B.V. All rights reserved. Keywords: Sicklepod; Cassia obtusifolia; Gum; Nematode; Inhibitor; Fall armyworm; Allelochemical; Spodoptera frugiperda; Meloidogyne chitwoodi; Soil amendment
* Corresponding author. Tel: +1 309 6816533; fax: +1 309 6816524; e-mail: [email protected] 1 Names are necessary to report factually on available data; however, the USDA neither guarantees nor warrants the standard of the product, and the use of the name by USDA implies no approval of the product to the exclusion of others that may also be suitable.
1. Introduction Sicklepod (Cassia obtusifolia) is a leguminous weed species that has become a severe problem in soybean production areas throughout the Southern United States.
0926-6690/98/$19.00 © 1998 Elsevier Science B.V. All rights reserved. PII S 0 9 2 6 - 6 6 9 0 ( 9 7 ) 1 0 0 1 0 - 3
78
T.P. Abbott et al. / Industrial Crops and Products 8 (1998) 77–82
Cassia obtusifolia and C. tora may be the same species (Crawford et al., 1990) and are treated as the same in this paper. Effective control strategies for this noxious weed are limited by a lack of selective herbicides that discriminate among legumes and ‘no-till’ cultural practices which accentuate the spread of sicklepod seed. Sicklepod seed contains anthraquinones, such as obtusifolin, which produce toxic effects in livestock (McCormack and Neisler, 1980). When crushed with soybeans, these compounds also contribute to lower oil quality. Degradation products can cause undesirable colors in processed soybean oils that are difficult to remove with conventional refining technology. In 1993, soybeans harvested throughout the South contained an average 5% sicklepod seed (private communication). At that level of foreign matter, contract sales in North Carolina were discounted ca. $0.28/bushel. However, poor soybean quality plus high cost of disposal of sicklepod seed in an environmentally safe manner prompted Cargill, Inc. in North Carolina to offer a 1% per bushel contract premium for soybean containing 1% maximum foreign matter. The amended discount schedule became effective in October 1994. Economic incentives to clean soybeans prior to sale could generate a 50 million pound annual supply of sicklepod seed in North Carolina alone. Unless a market is found for these seeds, disposal on farm land could lead to greater infestations of this weed and further reduce soybean productivity. New uses for sicklepod could turn a problem into a resource. Research has shown that a large part of sicklepod seed composition is carbohydrate (68%), but the water-soluble gums content is low (7%) and sugars account for only 8% of the carbohydrates (Crawford et al., 1990). In addition, the seed contains 7% oil, 15 – 20% crude protein and less than 3% of methanol-extractable anthraquinone glycosides. Sicklepod has also been shown to contain antimicrobial components (Kitanaka and Takido, 1986). The polysaccharides in sicklepod are not yet well characterized, but the soluble gum has been analyzed (Varshney et al., 1973). Plant carbohydrates and gums in particular are sometimes insol-
uble in water because they contain ionic crosslinks or structure only solubilized at acid or basic pH (Ericson and Elbein, 1980). Ammonium oxalate for example, has been shown to solubilize gums with calcium crosslinked galactouronysyl units (Abbott et al., 1996). Meloidoggne chitwoodi (Columbia root-knot nematode) is a serious pest of potato in the Pacific Northwest (Mojtahedi et al., 1993). This nematode attacks tubers and causes brown spots and blemishes on the tubers. Blemished tubers are down-graded or even rejected by the processors. In the latter case, the entire crop is fed to cattle. Presently, soil fumigation with 1,3-dichloropropene or metham sodium is the only reliable method of control (Pinkerton et al., 1986). However, nematode control has been achieved using rapeseed or white mustard as a green manure, in combination with a contact organophosphate nematicide (Mojtahedi et al., 1993). If ground sicklepod or its water extract could be used on the farm as a soil amendment to inhibit weed germination, or decrease pest populations, fewer herbicides or pesticides might be required. Although sicklepod meal and extracts cause some toxic effects when digested, the toxic component would be expected to biodegrade in the soil readily because the sugar moiety in anthraquinone glycosides is easily attached and quinones are highly reactive. By comparison, the currently used fumigants and alternative organophosphothionates are highly toxic (Ali et al., 1986). This work was undertaken to assess sicklepod seed and extracts for valueadded products such as gums, allelochemicals and pesticides.
2. Experimental
2.1. Extracts of sicklepod seed Because water and traditional gum extraction techniques had failed to extract the carbohydrate component of sicklepod, a series of aqueous salt solutions with a range of pH, salt concentration, mono- and dibasic cations and anions was tested for solution of sicklepod gums. Cassia obtusifolia seeds, collected from U.S. Southeastern soybean
T.P. Abbott et al. / Industrial Crops and Products 8 (1998) 77–82
fields, were ground to pass a 20 mesh screen. A 10-g sample was weighed and water soluble components extracted with deionized water (400 ml) at 45°C for 4 h. The mixture was centrifuged and the supernatant removed, then dialyzed three times against 4, 12 and 12 l deionized water, and the retentate freeze-dried. Half of the permeate from the first dialysis was recovered and freeze-dried. The same procedure was followed but instead using 0.1 N or 0.5 N NaOH as the extraction solvent, and neutralizing the dialysis permeate with acid ion exchange resin to pH 7, before freeze-drying. Salt solutions (NaCl, NH4Cl, (NH4)2CO3, Na2CO3, Na2SO4, Ca(CH3COO)2) at 0.05, 0.1, 0.5 and 1.0 M concentration, 10 ml each, were added to 1.00 g of ground seed and held at 60°C for 2 h with occasional shaking. Solutions were cooled to room temperature and centrifuged to give a clear supernatant which was decanted and analyzed for total carbohydrate and percent carbohydrate greater than 10 000 molecular weight (Abbott et al., 1995). Additionally, the procedure of Varshney et al. (1973) was followed to determine the amount of soluble gum available by their procedure, which consists of extraction of gums with 1% acetic acid in water followed by precipitation of gums with ethanol.
2.2. Germination inhibition assays The water extract and the neutralized dialysis permeates from NaOH extracts of ground sicklepod seed were assayed for inhibitory effects against sicklepod, velvetleaf and wheat germination. Dried extracts (1 mg/ml) were tested in water agar (10 g/l) containing 0.1 g/l 2-(4-thiazolyl)benzimidazole (Aldrich, Milwaukee, WI) and 0.5 g/l chloramphenicol (Sigma, St. Louis, MO) as antifungal and antibacterial agents, respectively. Seeds were surface disinfected with 10% Chlorox for 15 min, rinsed with sterile ddH2O (twice) and incubated at 25°C for 24 – 48 h. Germinated seeds with protruding radicals (sicklepod and velvetleaf) or primary roots (wheat) of equal length were placed on the agar and incubated at 25°C in darkness on 45 degree slants until the control roots reached the edge of the plates.
79
Ground whole seed was mixed with a sandy soil (Bloomfield sand, Psammentic Hapludalf) at rates of 0.0, 0.1, 0.5, 1.0 and 5.0% (w/w). One hundred and fifty milliliters of the soil seed-meal mixtures were placed in 200-ml cups and 10 seeds of each bioassay species: wheat (Triticum aesti6um L. ‘Cardinal’), soybean (Glycine max (L.) Merr. ‘Williams’), corn (Zea mays L. ‘DeKalb IL 645786’) and hemp sesbania (Sesbania exaltata (Raf.) Rydb. ex A.W. Hill), were added and covered with approximately 1 cm of soil. The cups were watered to field capacity, placed in the growth chamber at a 16 h, 26°C day/8 h, 21°C night regime, and watered daily. A combination of fluorescent and metal halide lighting was used to give an illumination of approximately 1900 mE m − 1 s − 1 intensity.
2.3. Fall armyworm and nematicide assays The water and base extracts tested for inhibition of germinating seeds were also assayed for pesticidal activity against fall armyworms by previously described methods (Dowd, 1988). Dry extracts (1%) were added to the basic diet (5 ml liquified at 60°C) and mixed with a vortex mixer for 25 s. The diets were cooled, by standing at ambient temperature, dried and divided into pieces sufficient ( 0.25 g) to neonate larvae for 1 week ad libitum. Larvae and diet were placed individually in wells of a 24-well tissue culture plate and sealed with parafilm to prevent moisture loss. Ground whole seed was added at 10% of total diet weight. Controls were larvae fed the basic diet which had been treated similarly, but without added sicklepod fractions. Insect mortality was examined after 7 days. Twenty insects per treatment were tested. To investigate nematicidal activity, loamy sand greenhouse mix soil (80% sand, 15% silt, 5% clay, and 0.5% organic matter) was infested with tomato root pieces harboring Meloidogyne chitwoodi female, with attached egg masses. The infested soil was incubated for 4 weeks to allow the second stage juvenile to hatch. Infested soil samples (500 g, 8% moisture, five replicates each treatment) were then placed in plastic bags, and mixed with a contact organophosphate nematicide
T.P. Abbott et al. / Industrial Crops and Products 8 (1998) 77–82
80 Table 1 Sicklepod extracts Extractant
Water 0.1 N NaOH 0.5 N NaOH
Extract, % of seed weight
21.3 35.4 40.9
LMW, % of seed weight
14.2 10.9 12.4
HMW, % of seed weight
7.1 24.5 28.5
Analyses of HMW fraction
% gum
% crude protein
59.7 55.3 65.4
35.0 29.3 27.9
LMW, Low-molecular-weight (B10 000) dialysis permeate (neutralized) from the extract; HMW, High-molecular-weight (\10 000) dialysis retentate from the extract.
(13 kg ai/na, Ethoprop, MoCap, Rhone Poulenc), 0.5 – 4% sicklepod meal by weight, or left untreated as control. The bags were sealed and incubated for 1 week before the soil samples were placed in clay pots, and 3-week-old Rutger tomato seedlings were planted. The pots were randomized and maintained on a greenhouse bench (2396°C) for 3 weeks, before the roots were washed free from soil, stained with acid fuchsin and the number of invading nematodes were counted.
3. Results and discussion
3.1. Extractable components Using the procedure of Varshney et al. (1973), 2.82% soluble gum was extractable which is comparable to Varshney’s result. This is the lowest water-soluble gum content of any Cassia species as reported in previous gum surveys (Tookey and Jones, 1965). Water at 45°C solubilizes about 21% of the ground seed weight. There is a 2:1 ratio of dialysis permeate to retentate in the extract and analysis of the higher molecular weight retentate gives 60% carbohydrate gum and 35% protein (Table 1). As much as 41% of the C. obtusifolia seed can be extracted with basic solutions. Although a range of aqueous salt solutions at various pHs and concentrations were tried, only marginal changes in solubilization of C. obtusifolia gums occurred (Table 2). Only NaOH extraction, of those inves-
tigated, extracted significant amounts of gums from C. obtusifolia. Membrane separations are a relatively expensive industrial process compared to extraction, precipitation, centrifugation, drying and similar unit operations. Because, in our investigations, we take a view toward the most economical means for producing value-added products, we propose alternative, less-costly processes. The separation into low-molecular-weight constituents and gums plus protein fractions, for example, need not involve dialysis as detailed in the present experiment. The water extract dissolves all of the low molecular weight components, but the majority of the gum plus protein fraction is only soluble at higher pH. A sequential water, 0.1 N NaOH separation could be refined further by first extracting at a lower pH, where the protein would be even less soluble, followed by extraction at a higher pH and precipitation of the protein at its isoelectric point. Alternatively, the combined extract made at higher pH and then neutralized could potentially be spray-dried and tested as an encapsulated, slow-release preemergence herbicide or pesticide. Although gums are generally used as thickeners, rheological and chemical characterization of sicklepod gum properties have not yet been determined.
3.2. Seed germination inhibition Both of the permeates from the high pH extracts (after neutralization/deionization with acid ion exchange resin to pH 7) were strong inhibitors
T.P. Abbott et al. / Industrial Crops and Products 8 (1998) 77–82
81
Table 2 Salt solution (0.1 – 1.0 M) extraction of sicklepod Salt
NaCl NH4Cl (NH4)2CO3 Na2CO3 Na2SO4 Ca(OAc)2 1% HOAc/EtOH pptn.
pH Buffer
7 7 9–10 12–13 7 8 5
pH Extract
6 6 7– 10 12 7 7– 8 5
% of seed weight extracted as Sugars
Gums
6.1 – 8.2a 3.4 – 6.1a 6.1 – 8.6c 6.3 – 8.1b 6.7 – 8.6c 6.3 – 7.5b
5.0 – 6.8a 6.0 – 7.2 6.0 – 8.2a 5.9 – 7.9c 1.7 – 5.9b 3.0 – 4.3b 2.82
a
Increases with increasing salt concentration. Decreases with increasing salt concentration. c Peaks at mid salt concentration. b
of wheat, velvetleaf and sicklepod root growth, causing discoloration of the root meristems in a manner similar to that caused by naphthoquinones such as juglone and plumbagin. Previously published literature indicates that sicklepod seeds contain substituted anthraquinones including obtusifolin, obtusin, aurantio-obtusin and chryso-obtusin (Crawford et al., 1990) which may be at least partly responsible for the observed effects. In contrast, ground whole sicklepod seed (up to 5% in soil) did not inhibit germination of wheat, soybeans, corn or hemp. One explanation for the whole meal and water extract having different effects may be that the isolation by NaOH and neutralization with acid ion exchange resin changed the structures of the extracted materials from their structure in the plant. The exact cause of the difference requires further research.
Table 4 shows the effect of various levels of sicklepod seed meal on Meloidogyne chitwoodi nematodes. Survival of Columbia root-knot nematode in the soil was inversely correlated to the amounts of sicklepod amendment. No phytotoxic effects of the meal amendment on tomato plants was observed.
4. Conclusions It would appear that sicklepod has at least three opportunities for use. The first of these is Table 3 Seven-day weights of fall armyworm larvae on diets with sicklepod extracts Diet
3.3. Effect on fall armyworm and nematodes Table 3 shows an unusual effect of water and basic extracts of sicklepod on fall armyworm larvae. The hot water extract caused the larvae to consume more of the feed and grow to twice the size of larvae on the control diet. The water extract (ion-exchanged dialysis permeate) was most effective, although the base extracted materials showed a similar effect. Ground whole sicklepod seed had no effect on fall armyworm larvae when incorporated at 10% in the diet (Table 3, Experiment II).
Experiment I Control Control+1% sicklepod water extract Control+1% 0.1 N NaOH extract Control+1% 0.5 N NaOH extract Experiment II Control 10% cellulose powder (non-nutritive control) 10% sicklepod powder
7-Day weights (mg)
38.4 92.8 75.3 96.9a 61.5 94.3a 56.3 96.9a 25.5 93.1 23.2 93.2 27.6 93.4
Values are mean 9 standard error. a Weights are significantly higher than controls at PB0.05.
T.P. Abbott et al. / Industrial Crops and Products 8 (1998) 77–82
82
Table 4 Survival of Meloidogyne chitwoodi in soil amended with different amounts of sicklepod meal and bioassayed on Rutger tomato seedlings for 3 weeks Treatment
No. of infective nematodes
Control Mocap 10G Sicklepod meal Sicklepod meal Sicklepod meal Sicklepod meal
763a 0d 759a 374ab 220b 7c
0.5%a 1% 2% 4%
The values are means of five replicates. Means followed by the same letter do not differ at PB0.05 according to Duncan’s multiple range test. a % of soil by weight.
the potential for the base-extracted carbohydrate gum or gum-protein mixture to be tested in thickener or encapsulation applications, now that significant amounts have been solubilized in base. The results showing increased feeding by fall armyworm on a diet that included 1% sicklepod extract indicates that this extract may be useful for increasing the uptake of pesticide-laced bait by fall armyworm. Whether this is a palatability or feeding stimulant effect is not known, but the addition of 1% of the extract to a diet is unlikely to be an effect on feed efficiency because the protein, fat and carbohydrate in the sicklepod supplemented diet is 99% the same as in the control diet. Ground whole sicklepod seed meal had no adverse effects on tomato plants or several crop seeds when incorporated into soil at 5% but reduced nematode population. Therefore, its use as a nematicide may be possible. The next stage of testing for nematode control would be outdoor test plots with both the meal and water extracts.
References Abbott, T.P., Nelsen, T.C., Kleiman, R., 1995. Seed surface gums content using the Dubois carbohydrate analysis. Ind. Crops Prod. 4, 185 – 192. Abbott, T.P., Nelsen, T.C., Kleiman, R., 1996. Lesquerella gums. In: Princen, L.H., Rossi, C. (Eds.), Proceedings of the Third International Conference on New Industrial Crops and Products. Versa Press, Inc., East Peoria, IL, pp. 343 – 345. Ali, F.A.F., Chukwudebe, A.C., Fukuto, T.R., 1986. Toxicological properties of O,S,S, trialkyl phosphorodithioates. J. Agric. Food Chem. 34, 45 – 48. Crawford, L., McDonald, G.M., Friedman, M., 1990. Composition of sicklepod (Cassia obtusifolia) toxic weed seeds. J. Agric. Food Chem. 38, 2169 – 2175. Dowd, P.F., 1988. Toxicological and biochemical interactions of the fungal metabolites fusaric acid and kojic acid with xenobiotics in Heliothis zea (F.) and Spodoptera frugiperda (J.E. Smith). Pestic. Biochem. Physiol. 32, 123 – 134. Ericson, M.C., Elbein, A.D., 1980. Cell wall polysaccharides and glycoproteins. In: Preiss, J. (Ed.), The Biochemistry of Plants, Vol. 3. Carbohydrates, Structure and Function. Academic Press, New York, pp. 591 – 616. Kitanaka, S., Takido, M., 1986. Studies on the constituents in the roots of Cassia obtusifolia L. and the antimicrobial activities of constituents of the roots and the seeds. Yakugaku Zasshi 106, 302 – 306. McCormack, J.E., Neisler, W.E., 1980. Cassia obtusifolia toxicity in a dairy herd. Vet. Med. Small Anim. Clin. 75, 1849 – 1851. Mojtahedi, H., Santo, G.S., Wilson, T.H., Hang, A.N., 1993. Managing Meloidogyne chitwoodi on potato with rapeseed as green manure. Plant Dis. 77, 42 – 46. Pinkerton, J.N., Santo, G.S., Ponti, R.P., Wilson, J.H., 1986. Control of Meloidogyne chitwoodi in commercially grown Russet Burbank potatoes. Plant Dis. 70, 860 – 863. Tookey, H.L., Jones, Q., 1965. New sources of water-soluble seed gums. Econ. Bot. 19, 165 – 174. Varshney, S.C., Rizvi, S.A.I., Gupta, P.C., 1973. Structure of a polysaccharide from Cassia tora seeds. I. J. Agric. Food Chem. 21, 222 – 226.