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Kenaf extract affects germination and post-germination development of weed, grass and vegetable seeds
INDUSTRIALCROPS AND PRODUCTS AN INTERNATIONAL
ELSEVIER
JOURNAL
Industrial Crops and Products 6 (1997) 59-69
Kenaf extract affects germination and post-germination development of weed, grass and vegetable seeds’ V.M. RUSSO”,“, C.L. Webber “USDepartment
III”, D.L. Myersb
of Agriculture, Agricultural Research Service, South Central Agricultural Research Laboratory, Lane, OK 74555, USA bChemistry Department, East Central University, Ada, OK 74820, USA
Received 14 August 1996; accepted 1 November 1996
Abstract Metabolites produced by one organism can affect development of other organisms. Kenaf (Hibiscus cannabinus L.) is used in products which put it in direct contact with other plants. This project was designed to determine whether kenaf plant extracts can affect germination and development of vegetable, grass and weed seeds. Frost-killed kenaf was chipped and either immediately frozen (weathered 0 months) or applied to the soil in mats in December and allowed to weather for 2 or 4 months. Kenaf samples, weathered from 0 to 4 months, were ground and soluble materials were extracted with distilled water. Seeds of cucumber (Cucumis sativus L.), green bean (Phaseolus vulgaris L.), tomato (Lycopersicon esculentum Mill.), redroot pigweed (Amaranthus retrojexus L.) and annual Italian ryegrass (Lo&m mult@orum Lam.) were exposed to 0, 16.7, 33.3, and 66.7 g/l of kenaf extract. Distilled water and three concentrations of polyethylene glycol (PEG) were included as controls. After 7 days, total germination and hypocotyl and radicle lengths were determined. Extracts of kenaf weathered up to 4 months, especially at the highest concentration, reduced germination in pigweed by 50-70%. Germination in tomato and ryegrass was reduced by 30% when exposed to the highest concentration of unweathered kenaf. As length of time of weathering of kenaf increased, germination and length of most plants increased. This suggests that, over time, the detrimental compounds in kenaf were leached or otherwise changed so that they had no effect or became beneficial. Non-weathered kenaf or its extracts, may be employed to suppress weeds. Alternatively, weathered kenaf tissue or extracts may stimulate germination and post-germination development of existing economic crops. Published by Elsevier Science B.V. Keywords:
Allelopathy;
Hypocotyl;
Radicle;
Kenaf;
Weed control;
Seed germination
* Corresponding author. Fax: + 1 405 8895783. ’ Mention of a trademark, vendor, or proprietary product does not constitute a guarantee or warranty of the product by the USDA and does not imply its approval to the exclusion of other products that may also be suitable. Published by Elsevier Science B.V. PII SO926-6690(96)00206-3
60
V.M. Russo et al. /Industrial
1. Introduction Chemical compounds produced by plants can escape into the environment and affect other plants (Rice, 1983). This occurs in plant communities (Gressel and Holm, 1964) and is a possible way that plants influence the development of competing plants (Bell and Koeppe, 1972; Muller, 1966). Kenaf (Hibiscus cannabinus L.) has been investigated as a source of fiber for paper products (Bagby et al., 1979; Clark et al., 1971; Nieschlag et al., 1960; White et al., 1970; Wilson et al., 1965) as an animal feed (Killinger, 1967; Phillips et al., 1989; Webber, 1993), as animal litter material (Tilmon et al., 1988) and as a bulking agent for sewage sludge (Webber, 1992). Kenaf has also received interest as a potting soil amendment (Williams et al., 1995) and for anti-erosion mats (Fisher, 1994). Using kenaf as either mulch or bed cover could expose the root to chemicals which may be detrimental to vegetable crops. These chemicals may or may not lose effectiveness as kenaf tissues are weathered. Russo et al. (1996) found that chipped kenaf used as a fall mulch restricted spring weed growth at a level comparable to that of black polyethylene plastic mulch. They attributed this to either physical conditions restricting weed development and/or possible allelopathic activity. In addition, some vegetables were adversely affected by planting into kenaf mulched beds. The objective of this study was to determine the effect of extracts from weathered and non-weathered kenaf on seed germination and subsequent plant development of selected vegetable, grass and weed species.
2. Materials
and methods
2.1. General Mature 193 day-old kenaf was harvested and chipped with a Gehl 2-row forage harvester on 1 December 1994, 2 days after a killing frost at Lane, OK. The plant material consisted mainly of stalks with the largest pieces being 6 cm long, 2
Crops and Products
6 (1997) 59-69
cm wide and 0.5 cm thick. Non-weathered kenaf (0 months) was frozen for several days. Prior to analysis, the tissue was oven-dried at 50°C for 72 h. Other chipped kenaf that was to be weathered was spread into a mat approximately 5 cm thick by 0.9 m wide and 4 m long on disked soil. Samples from the mat were collected in the first weeks of February and April 1995, after weathering for 2 and 4 months, respectively, and ovendried. Following drying, the kenaf was ground in a Wiley mill and passed through a 20-mesh screen. Dried kenaf at the full-strength concentration (66.7 g/l) was placed in a 2 1 beaker with 1 1 of distilled water and agitated on an orbital shaker for 12 h at room temperature (22°C). The extract was vacuum filtered through two layers of filter paper (Whatman No. 42). Three concentrations, of kenaf extract, made with distilled water, were used: full-strength, half-strength (33.3 g/l), and quarter-strength (16.7 g/l). Concentrations of PEG (15 000 MW) at 5, 8 and lo%, were included to account for the possible osmotic effects of the extracts (Smith, 1989; White et al., 1989). Distilled water controls (0 g/l) were also included. Osmotic potentials were determined using an Osmette A model No. 5002 automatic osmometer, which was calibrated using standards developed by Precision Systems (Natick, MA). A 2 ml aliquot was used for standardization and samples. Replicate determinations, using the standards, were reproducible to f 1 mOsm/kg of the specified osmolality. The plant extracts were filtered using a MicronSep cellulosic 0.1 micron filter (Micron Separations, Westboro, MA). Osmolality was determined by supercooling a sample below the normal freezing point, inducing freezing by seeding with mechanical agitation, i.e. with a stirring wire, evolving latent heat of fusion, and measuring the temperature rise of the sample. The temperature rise was reproducible for a given osmolality. When a sample freezes without mechanical agitation, it is termed to have preseeded, osmolality determinations become impossible because the amount of evolved heat is not uniform. Kenaf extracts from samples weathered for 2 and 4 months preseeded and their osmolalities were not measurable. The 33.3 and 66.7 g/l kenaf sam-
V.M. Russo et al. 1 Industrial
ples weathered 4 months required multiple filtrations over a period of days using the 0.1 micron filter. All other plant extract samples required only one filtration and in the case of the fresh extract, a 0.45 micron filter was sufficient. No difference in osmolality was observed when either the 0.1 or the 0.45 micron filter was used. For osmotic measurement, a 2 ml aliquot was transferred with an Eppendorf pipette into a sample tube placed in the refrigeration well of the osmometer. Osmotic determinations were also performed on all PEG concentrations, and on the deionized water used for the sample preparations. The pH of the 66.7 g/l extract from each harvest time was determined. Dilutions, PEG treatments and distilled water were adjusted to the pH of the 66.7 g/l extract solution at each collection time. The experimental design was a randomized complete block with five replications and was repeated twice. Percentile data were transformed to radians. Data were analyzed with the general linear methods procedures in SAS Institute, 1988. 2.2. Aqueous
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6 (1997) 59-69
61
sterilized, placed on petri plates, moistened and incubated as described earlier. Seed were observed daily. When the radicle emerged, ten seeds were transferred to Petri plates containing distilled water or concentrations of the kenaf extract. After 7 days, hypocotyl and radicle length, percent of radicle discolored and the number of secondary roots formed were determined.
3. Results 3.1. PEG concentration: germination post-germination development
and
PEG concentration affected germination, hypocotyl, radicle, and total length of some seedlings (Fig. lA-D). Only germination of green bean decreased at the higher PEG concentrations. The hypocotyl, radicle and total length of green bean, tomato and pigweed decreased, whereas the other plants were unaffected as PEG concentration increased.
extract 3.2. Kenaf extract:
2.2.1. Germination Seeds of cucumber (Cucumis sativus L.), green bean (Phaseolus vulgaris L.), tomato (Lycopersicon esculentum Mill.), redroot pigweed (Amarnnthus refroflexus L.) and annual Italian ryegrass (Lo&m mult@7orum Lam.) were surface sterilized prior to treatment for 1 min in a 50% commercial hypochlorite solution, rinsed with running tap water for 10 min and air dried. Twenty-five seeds of each species were placed in separate plastic Petri plates fitted with 2 layers of 9 cm filter paper (Whatman No. 2) and moistened with 10 ml of extract, PEG or distilled water. The covered Petri plates were placed in a non-illuminated incubator at 27°C. Percent seed gepination was recorded after 7 days. Seeds were considered to be germinated if radicle length was equal to seed width of each species. 2.2.2. Post-germination development Twenty-five seeds of each species were surface
germination
Germination of all species was, in part or whole, affected by extract concentration and amount of time of weathering. The interaction of amount of time that of weathering by concentration of kenaf extract affected germination of all species except green bean (Table 1). With non-weathered kenaf (0 months), germination of tomato and ryegrass decreased, green bean were not affected and pigweed germination decreased steadily as concentration of the kenaf extract increased to 66.7 g/l, cucumber increased (Fig. 2A). As kenaf extract concentration weathered up to 2 months was increased to 16.7 g/l germination of pigweed decreased, and was unchanged thereafter, whereas other plants were not affected (Fig. 2B). When exposed to extract from kenaf weathered for 4 months, germination of pigweed decreased as concentration was increased to 66.7 g/l, but other crops were not affected (Fig. 2C).
V.M. Russo et al. / Industrial Crops and Products 6 (1997) 59-69
62
100
A
I
I ----me__._._
..e.-w..
.-rR---------&-
---
--
‘I
80
I
60 ___..----
40
*20 0
Fig. 1. Osmolality (- 0 -) of distilled water (0% PEG) and PEG concentration effect on: A, germination; B, hypocotyl length; C, radicle length; and D, total seedling length of 7 day old cucumber, green bean, tomato, pigweed and ryegrass seedlings. The vertical bars are S.E.
V.M. Russo et al. 1 industrial Crops and Products 6 (1997) 59-69
63
Kenaf Weathered 0 Months -----____
loo 60
~-___._____~___.__----~---------i
60
I- -*_.
--..
--e
___..-.---t
40
t ----.__
--__
20
J
0
Kenaf Weathered 4 Months 100
ir
100
11
------__.__
_____-.---
60
b B
40
%
’
1
l-
80-
--60
k ____--.----.+----._.____
--40
20
0
--
1
-
.
0
I
20
l. TO
16.7
33.3
66.7
Kcnaf Extracf (@IL) -----Greenbran -----.RFW=
-cucumk --.-pi-
----Tcmnab --o--0MloQlity
Fig. 2. Effect of kenaf extract concentration and distilled water (0 g/l) on germination pigweed, and ryegrass seedlings, and osmolality (- c -) if kenaf was: A, non-weathered; 4 months. The vertical bars are S.E.
I
of 7 day old cucumber, green bean, tomato, or B, weathered 2 months; or C, weathered
V.M. Russo et al. /Industrial Crops and Products 6 (1997) 59-69
64
Table 1 Germination and post-germination development of 7 day old seedlings of vegetables, weed and grass exposed to varying concentrations of extract from kenaf weathered on soil Treatment
Green bean Months weathered Concentration (C) Interaction: C x M Cucumber Months weathered Concentration (C) Interaction: C x M Tomato Months weathered Concentration (C) Interaction: C x M Pigweed Months weathered Concentration (C) Interaction: C x M Ryegrass Months weathered Concentration (C) Interaction: C x M
Germination (%)
Length (mm)
Number of secondary roots
Hypocotyl
Radicle
Total
(M)
** NS NS
NS ** *
** ** **
** ** **
(M)
** ** *
NS ** **
* ** **
** ** **
(M)
** ** *
* ** **
** ** **
** ** **
(M)
** ** *
* * **
** ** **
** ** **
(M)
** ** *
* * **
** ** **
** ** **
NS, non-significant and *,**, significant at PcO.05
** * **
NS NS NS
or P
3.3. Aqueous extract: post-germination development 3.3.1. General Post-germination development of all species was, in part or whole, affected by extract concentration, weathering length and their interaction (Table 1). Tissue discoloration occurred only on green bean and pigweed radicles. The amount of discoloration of green bean radicles (average, 25%) was not affected by either weathering or concentration of kenaf extract. Pigweed radicles exposed to non-weathered kenaf (0 months) extract showed O-55.2% discoloration as extract concentration was increased from control to 66.7 g/l (data not shown). Only green bean and pigweed produced secondary roots. Secondary root number (average two per radicle) on pigweed was not affected by treatment. However, secondary root number on green bean radicles was affected by months of weathering, concentration of extract and their
interactions (Table 1). As time of weathering and concentration of kenaf extract increased, the number of secondary roots increased (data not shown). Hypocotyl, radicle and total seedling length responded differently to extracts from kenaf with different lengths of time of weathering, extract concentrations and their interactions (Table 1). Exceptions were for cucumber and green bean where hypocotyl length was not affected by weathering of kenaf. 3.3.2. Green bean For each length of time of weathering, hypocotyl, radicle and total seedling lengths generally increased as the concentration of extract increased above that of the controls (Figs. 3 and 4). Exceptions were for radicle and total seedling lengths exposed to non-weathered kenaf (0 months) where length increased as extract concentration increased to 33.3 g/l and then decreased as concentration was increased to 66.7 g/l. In neither
V.M. Russo et al. 1 Industrial Crops and Products 6 (1997) 59-69
65
Kenaf Weathered 0 Months T ‘O”
Radicles
160~D
Kenaf Weathered 2 Months 150 125 100 75 50 25 0
Kenaf Weathered 4 Months
0
16.7
33.3
66.7
KenatMract (g/L) -----GmenbePn
-c-
---Tenrate
__....Qqmaa
-..-p@#j
--o--osmoh(i
0
18.7
33.3
66.7
Fig. 3. Effect of kenaf extract concentration and distilled water (0 g/l) on hypocotyl or radicle length of 7 day old cucumber, green bean, tomato, pigweed seedlings, and ryegrass and osmolality (- 0 -) if kenaf was: A and D, non-weathered; or B and E, weathered 2 months; or C and F, weathered 4 months. The vertical bars are S.E.
V.M. Russo et al. /Industrial Crops and Products 6 (1997) 59-69
66
Total Length 25Q-
Kenaf Weathered
0 Months
Kenaf Weathered
2 Months
A
50 --
l-
__-..-
+ _-__._.__._
_._m_
+_______.____l L
*
0
CO
Kenaf Weathered
=
b
4 Months
&&xj
_Ii
I;
--loo
__--
___---
_I_____-
_*-.-
t
$---
.-_._
-- B
____I
50
A
7
0
16.7
+0 66.7
33.3 Kenaf Extmcf (gL)
-----Gmanbenn
-Cucumber -.__p-
-‘---‘R)agnrr,
Fig. 4. Effect of kenaf extract
concentration
and distilled
tomato, pigweed and ryegrass seedlings and osmolality weathered 4 months. The vertical bars are S.E.
---1onmta -0Muly
water (0 g/l) on total seedling length of 7 day old cucumber, green bean, or B, weathered 2 months; or C, (- 0 -) if kenaf was: A, non-weathered;
V.M. Russo et al. /Industrial Crops and Products 6 (1997) 59-69
case was radicle or total length exposed to 66.7 g/l strength extract shorter than those exposed to controls. 3.3.3. Cucumber Hypocotyl, radicle and total seedling length generally increased as extract concentration increased regardless of length of time of weathering of kenaf (Figs. 3 and 4). Exceptions were for radicle and total seedling lengths for seedlings exposed to non-weathered kenaf (0 months), which first increased and then decreased, as concentration increased from controls to 16.7 g/l extract, and then decreased as extract concentration increased to 66.7 g/l. 3.3.4. Tomato Seedling hypocotyls exposed to concentrations of kenaf extract weathered for up 2 months first increased and then decreased as extract concentration increased. For seedlings exposed to kenaf extract weathered 4 months, hypocotyl length increased as concentration increased to 16.7 g/l and was unchanged thereafter. For seedlings exposed to extract of kenaf weathered for up to 2 months, radicle length decreased as concentration increased. When exposed to kenaf extract weathered 4 months, radicle length increased and then decreased as concentration of kenaf in extract increased. Seedling total length first increased and then decreased as concentration of extract from kenaf weathered up to 4 months increased. Total length of seedlings exposed to distilled water was longer than for those exposed to 66.7 g/l extract from non-weathered kenaf. When exposed to kenaf weathered for 2 months, the total length was similar for seedlings exposed to distilled water and 66.7 g/l extract. When exposed to kenaf weathered for 4 months, total length for seedlings exposed to distilled water was shorter than those exposed to 66.7 g/l extract. 3.3.X Pigweed Hypocotyl length of seedlings exposed to nonweathered kenaf (0 months) decreased as concentration increased to 66.7 g/l (Fig. 3A). Hypocotyls were longer when exposed to 66.7 g/l extract weathered for 2 months than when exposed to
61
controls (Fig. 3B). Length of hypocotyls exposed to extract of kenaf weathered for 4 months increased and then remained unchanged as concentration increased (Fig. 3C). Seedling radicle length decreased from that of the distilled water control when exposed to all concentrations of non-weathered kenaf (0 months) (Fig. 3D). Radicles exposed to kenaf weathered for 2 or 4 months were unchanged as extract concentration increased (Fig. 3E, F). Total seedling length decreased as concentration of non-weathered kenaf (0 months) increased (Fig. 4A). Total length was unchanged for seedlings exposed to all concentrations of kenaf weathered for 2 months (Fig. 4B), and was increased as concentration of kenaf weathered for 4 months increased (Fig. 4C). 3.3.6. Ryegrass Length of hypocotyls exposed to non-weathered kenaf (0 months) increased from those for controls to 16.7 g/l, and then decreased (Fig. 3A). Lengths of hypocotyls exposed to extract from kenaf weathered for 2 or 4 months increased as extract strength increased to 16.7 g/l and was unchanged thereafter (Fig. 3B, C). Radicles exposed to non-weathered kenaf (0 months) were shorter than those exposed to controls (Fig. 3D). Radicles exposed to kenaf weathered for 2 months were unchanged with increasing kenaf concentration (Fig. 3E). Radicle length for seedlings exposed to kenaf extract weathered for 4 months increased as concentration increased to 16.7 g/l and were unchanged thereafter (Fig. 3F). Total length of seedlings exposed to non-weathered kenaf extract (0 months) remained unchanged as concentration increased (Fig. 4A). When exposed to kenaf extract weathered for 2 or 4 months, total seedling length increased as extract concentration increased to 16.7 g/l and remained unchanged thereafter (Fig. 3B).
4. Discussion Extracts from weathered and non-weathered frost-killed kenaf appear to have properties that reduced germination of pigweed, and to a lesser degree that of ryegrass and tomato. As kenaf
68
V.M. Russo et al. 1 Industrial Crops and Products 6 (1997) 59-69
weathered, it appeared that compounds in tissues were leached, or otherwise changed, so that there was no effect or caused improved length of hypocotyls and/or radicles. The osmolality data suggest that with longer weathering, chemical changes occur in the kenaf tissues that may bind materials that have apparent allelopathic qualities. It was difficult to obtain osmolality determinations for some samples. In these cases the sample preseeded, possibly due to suspended solids. The material causing these difficulties was small enough to pass the 0.1 micron filters. The biological significance of these components is not yet known. This suggests that osmolality data need to be carefully evaluated for these types of studies. Russo et al. (1996) reported that spring weed population for the untreated bed was higher than those beds mulched with kenaf the previous fall. The reduced weed population was observed after several months of weathering of the chipped kenaf. Possibly the spring allelopathic compounds present in freshly chipped, material is absent in the kenaf tissue that had been weathered. This suggests that the allelopathic components may have leached from the tissue over the winter and were deposited in the germination region of the soil where there may still have some residual potency (Muller, 1966; Rice, 1983; White et al., 1989). Weathering of biological material is not confined to abiotic degradation. Fungi and bacteria may also be involved in this degradation. These biological entities could be responsible for the apparent allelopathic effect. However, detrimental effects were associated with extracts from non-weathered kenaf tissue. Since the non-weathered samples were immediately frozen, there was insufficient time for extensive biological colonization to occur during processing. Thus, allelopathic effects can be attributed to the extract from the kenaf tissue rather than from any other biological entity. In addition, since the seed were surface sterilized, and there was no indication of fungal or bacterial induced effects on germination and postgermination development, the results should be attributed to the extract obtained from kenaf.
For cucumber, green bean and ryegrass, PEG concentration did not affect germination and post-germination development. However, the time of weathering and concentration of kenaf, or both affected seed germination and/or post-germination development. This suggests that factors associated with kenaf and not the physical osmotic effect caused reduced germination and changes in length of hypocotyls and/or radicals. Green bean had decreased germination and green bean, tomato and pigweed had shorter hypocotyls or radicles when exposed to PEG. When treated with non-weathered kenaf extracts, green bean germination was unaffected but germination of tomato and ryegrass was reduced. The germination of pigweed was reduced to a larger extent with the kenaf extract than PEG. This suggests that the suppression or promotion of germination and/or growth of these plants were due to the biochemical component (kenaf) with the effect of osmotic potential being of less importance. Further examination of non-weathered kenaf tissue might yield metabolites that exhibit properties that can be used to suppress weeds. Alternatively this material, after weathering, may produce metabolites, or supply nutrients, that could be beneficial in the early development of some vegetable crops.
References Bagby, M.O., Cunningham, R.L., Touzinsky, G.F., Hamerstrand, G.E., Curtis, E.L. and Hofreiter, B.T., 1979. Kenaf thermomechanical pulp in newsprint. TAPPI/NPFP Committee Progress Report 10. Atlanta, GA. Bell, D.T. and Koeppe, D.E., 1972. Noncompetitive effects of giant foxtail on the growth of corn. Agron. J., 64: 321l 325. Clark, T.F., Cunningham, R.L. and Wolff, I.A., 1971. A search for new fiber crops. Tappi, 54: 63-65. Fisher, G., 1994. Manufacturing non-woven kenaf products. Int. Kenaf Assoc. Conf. Proc., New Orleans, LA, 6: 5456. Gressel, J.B. and Helm, L.G., 1964. Chemical inhibition of crop germination by weed seed and the nature of the inhibition by Abunlon theophrasii. Weed Res., 4: 44-53. Killinger, G.B., 1967. Potential uses of kenaf (Hibiscus cannabinus L.). Florida Soil Crop Sci. Sot. Proc., 27: 4- 11.
V.M. Russo et al. /Industrial Muller, C.H., 1966. The role of chemical inhibition (allelopathy) in vegetation composition. Bull. Torrey Bot. Club., 93: 332-351. Nieschlag, H.J., Nelson, G.H., Wolff, I.A. and Perdue, R.E.. Jr., 1960. A search for new fiber crops. Tappi, 43: 193201. Phillips, W.A., Rao, S. and Dao, T., 1989. Nutritive value of immature whole plant kenaf and mature tops for growing ruminants. Prcic. Assoc. Advancement of Industrial Crops, pp. 17-22. Rice, E.L., 1983. Pest control with nature’s chemicals: allelochemics and pheromones in gardening and agriculture. University Oklahoma Press, Norman, OK, 224~. Russo, V.M., Cartwright, B. and Webber, C., III, 1996. Mulching: effects on erosion of soil beds, and on yield of autumn and spring-sown vegetables. Biol. Agric. Hort., 14: in press. SAS Institute. 1988. SASSTAT User’s Guide, Release 6.03 Edition. SAS Institute, Cary, NC, 1028~. Smith, A.E., 1989. The potential allelopathic characteristics of Bitter Sneezeweed (Helenium amarum). Weed Sci., 17: 6655669. Tilmon, H.D., Taylor, R. and Malone, G., 1988. Kenaf: An alternative crop for Delaware. In: J. Janick and Simon,
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J.E. (Eds.), Advances in New Crops. Timber Press, Portland, OR, pp. 301-302. Webber, C.L., III., 1992. Kenaf (Hibiscus cannabinus L.) yield components as affected by sewage sludge applications. Proc. TAPPI Pulping Conf. pp. 57-62. Webber, CL., III., 1993. Crude protein and yield components of six kenaf cultivars as affected by crop maturity. Ind. Crops Prod. 2: 27-31. White, F.D., Cummins, D.G., Whiteley, E.L., Fike, W.T., Greig, J.K., Martin, J.A., Killinger, G.B., Higgins, J.J. and Clark, T.F., 1970. Cultural and harvesting methods for kenaf. USDA. Production Research Rept. 113. Washington, DC, 38~. White, R.H., Worsham, A.D. and Blum, U., 1989. Allelopathic potential of legume debris and aqueous extracts. Weed Sci. 37: 614-619. Williams, R.D., Baldwin, B.S. and Reichert, N.A., 1995. Kenaf core as the major component in greenhouse potting media. Seventh Annual International Kenaf Association Conference Proceedings, Irving, TX. pp. 25-32. Wilson, F.D., Summers, T.E., Joyner, J.F., Fisher, D.W. and Seale, C.C., 1965. ‘Everglades 41’ and ‘Everglades 71’, two new varieties of kenaf (Hibiscus cannabinus L.) for the fiber and seed. Fla. Agr. Exp. Cir. p. 168.
ELSEVIER
JOURNAL
Industrial Crops and Products 6 (1997) 59-69
Kenaf extract affects germination and post-germination development of weed, grass and vegetable seeds’ V.M. RUSSO”,“, C.L. Webber “USDepartment
III”, D.L. Myersb
of Agriculture, Agricultural Research Service, South Central Agricultural Research Laboratory, Lane, OK 74555, USA bChemistry Department, East Central University, Ada, OK 74820, USA
Received 14 August 1996; accepted 1 November 1996
Abstract Metabolites produced by one organism can affect development of other organisms. Kenaf (Hibiscus cannabinus L.) is used in products which put it in direct contact with other plants. This project was designed to determine whether kenaf plant extracts can affect germination and development of vegetable, grass and weed seeds. Frost-killed kenaf was chipped and either immediately frozen (weathered 0 months) or applied to the soil in mats in December and allowed to weather for 2 or 4 months. Kenaf samples, weathered from 0 to 4 months, were ground and soluble materials were extracted with distilled water. Seeds of cucumber (Cucumis sativus L.), green bean (Phaseolus vulgaris L.), tomato (Lycopersicon esculentum Mill.), redroot pigweed (Amaranthus retrojexus L.) and annual Italian ryegrass (Lo&m mult@orum Lam.) were exposed to 0, 16.7, 33.3, and 66.7 g/l of kenaf extract. Distilled water and three concentrations of polyethylene glycol (PEG) were included as controls. After 7 days, total germination and hypocotyl and radicle lengths were determined. Extracts of kenaf weathered up to 4 months, especially at the highest concentration, reduced germination in pigweed by 50-70%. Germination in tomato and ryegrass was reduced by 30% when exposed to the highest concentration of unweathered kenaf. As length of time of weathering of kenaf increased, germination and length of most plants increased. This suggests that, over time, the detrimental compounds in kenaf were leached or otherwise changed so that they had no effect or became beneficial. Non-weathered kenaf or its extracts, may be employed to suppress weeds. Alternatively, weathered kenaf tissue or extracts may stimulate germination and post-germination development of existing economic crops. Published by Elsevier Science B.V. Keywords:
Allelopathy;
Hypocotyl;
Radicle;
Kenaf;
Weed control;
Seed germination
* Corresponding author. Fax: + 1 405 8895783. ’ Mention of a trademark, vendor, or proprietary product does not constitute a guarantee or warranty of the product by the USDA and does not imply its approval to the exclusion of other products that may also be suitable. Published by Elsevier Science B.V. PII SO926-6690(96)00206-3
60
V.M. Russo et al. /Industrial
1. Introduction Chemical compounds produced by plants can escape into the environment and affect other plants (Rice, 1983). This occurs in plant communities (Gressel and Holm, 1964) and is a possible way that plants influence the development of competing plants (Bell and Koeppe, 1972; Muller, 1966). Kenaf (Hibiscus cannabinus L.) has been investigated as a source of fiber for paper products (Bagby et al., 1979; Clark et al., 1971; Nieschlag et al., 1960; White et al., 1970; Wilson et al., 1965) as an animal feed (Killinger, 1967; Phillips et al., 1989; Webber, 1993), as animal litter material (Tilmon et al., 1988) and as a bulking agent for sewage sludge (Webber, 1992). Kenaf has also received interest as a potting soil amendment (Williams et al., 1995) and for anti-erosion mats (Fisher, 1994). Using kenaf as either mulch or bed cover could expose the root to chemicals which may be detrimental to vegetable crops. These chemicals may or may not lose effectiveness as kenaf tissues are weathered. Russo et al. (1996) found that chipped kenaf used as a fall mulch restricted spring weed growth at a level comparable to that of black polyethylene plastic mulch. They attributed this to either physical conditions restricting weed development and/or possible allelopathic activity. In addition, some vegetables were adversely affected by planting into kenaf mulched beds. The objective of this study was to determine the effect of extracts from weathered and non-weathered kenaf on seed germination and subsequent plant development of selected vegetable, grass and weed species.
2. Materials
and methods
2.1. General Mature 193 day-old kenaf was harvested and chipped with a Gehl 2-row forage harvester on 1 December 1994, 2 days after a killing frost at Lane, OK. The plant material consisted mainly of stalks with the largest pieces being 6 cm long, 2
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cm wide and 0.5 cm thick. Non-weathered kenaf (0 months) was frozen for several days. Prior to analysis, the tissue was oven-dried at 50°C for 72 h. Other chipped kenaf that was to be weathered was spread into a mat approximately 5 cm thick by 0.9 m wide and 4 m long on disked soil. Samples from the mat were collected in the first weeks of February and April 1995, after weathering for 2 and 4 months, respectively, and ovendried. Following drying, the kenaf was ground in a Wiley mill and passed through a 20-mesh screen. Dried kenaf at the full-strength concentration (66.7 g/l) was placed in a 2 1 beaker with 1 1 of distilled water and agitated on an orbital shaker for 12 h at room temperature (22°C). The extract was vacuum filtered through two layers of filter paper (Whatman No. 42). Three concentrations, of kenaf extract, made with distilled water, were used: full-strength, half-strength (33.3 g/l), and quarter-strength (16.7 g/l). Concentrations of PEG (15 000 MW) at 5, 8 and lo%, were included to account for the possible osmotic effects of the extracts (Smith, 1989; White et al., 1989). Distilled water controls (0 g/l) were also included. Osmotic potentials were determined using an Osmette A model No. 5002 automatic osmometer, which was calibrated using standards developed by Precision Systems (Natick, MA). A 2 ml aliquot was used for standardization and samples. Replicate determinations, using the standards, were reproducible to f 1 mOsm/kg of the specified osmolality. The plant extracts were filtered using a MicronSep cellulosic 0.1 micron filter (Micron Separations, Westboro, MA). Osmolality was determined by supercooling a sample below the normal freezing point, inducing freezing by seeding with mechanical agitation, i.e. with a stirring wire, evolving latent heat of fusion, and measuring the temperature rise of the sample. The temperature rise was reproducible for a given osmolality. When a sample freezes without mechanical agitation, it is termed to have preseeded, osmolality determinations become impossible because the amount of evolved heat is not uniform. Kenaf extracts from samples weathered for 2 and 4 months preseeded and their osmolalities were not measurable. The 33.3 and 66.7 g/l kenaf sam-
V.M. Russo et al. 1 Industrial
ples weathered 4 months required multiple filtrations over a period of days using the 0.1 micron filter. All other plant extract samples required only one filtration and in the case of the fresh extract, a 0.45 micron filter was sufficient. No difference in osmolality was observed when either the 0.1 or the 0.45 micron filter was used. For osmotic measurement, a 2 ml aliquot was transferred with an Eppendorf pipette into a sample tube placed in the refrigeration well of the osmometer. Osmotic determinations were also performed on all PEG concentrations, and on the deionized water used for the sample preparations. The pH of the 66.7 g/l extract from each harvest time was determined. Dilutions, PEG treatments and distilled water were adjusted to the pH of the 66.7 g/l extract solution at each collection time. The experimental design was a randomized complete block with five replications and was repeated twice. Percentile data were transformed to radians. Data were analyzed with the general linear methods procedures in SAS Institute, 1988. 2.2. Aqueous
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61
sterilized, placed on petri plates, moistened and incubated as described earlier. Seed were observed daily. When the radicle emerged, ten seeds were transferred to Petri plates containing distilled water or concentrations of the kenaf extract. After 7 days, hypocotyl and radicle length, percent of radicle discolored and the number of secondary roots formed were determined.
3. Results 3.1. PEG concentration: germination post-germination development
and
PEG concentration affected germination, hypocotyl, radicle, and total length of some seedlings (Fig. lA-D). Only germination of green bean decreased at the higher PEG concentrations. The hypocotyl, radicle and total length of green bean, tomato and pigweed decreased, whereas the other plants were unaffected as PEG concentration increased.
extract 3.2. Kenaf extract:
2.2.1. Germination Seeds of cucumber (Cucumis sativus L.), green bean (Phaseolus vulgaris L.), tomato (Lycopersicon esculentum Mill.), redroot pigweed (Amarnnthus refroflexus L.) and annual Italian ryegrass (Lo&m mult@7orum Lam.) were surface sterilized prior to treatment for 1 min in a 50% commercial hypochlorite solution, rinsed with running tap water for 10 min and air dried. Twenty-five seeds of each species were placed in separate plastic Petri plates fitted with 2 layers of 9 cm filter paper (Whatman No. 2) and moistened with 10 ml of extract, PEG or distilled water. The covered Petri plates were placed in a non-illuminated incubator at 27°C. Percent seed gepination was recorded after 7 days. Seeds were considered to be germinated if radicle length was equal to seed width of each species. 2.2.2. Post-germination development Twenty-five seeds of each species were surface
germination
Germination of all species was, in part or whole, affected by extract concentration and amount of time of weathering. The interaction of amount of time that of weathering by concentration of kenaf extract affected germination of all species except green bean (Table 1). With non-weathered kenaf (0 months), germination of tomato and ryegrass decreased, green bean were not affected and pigweed germination decreased steadily as concentration of the kenaf extract increased to 66.7 g/l, cucumber increased (Fig. 2A). As kenaf extract concentration weathered up to 2 months was increased to 16.7 g/l germination of pigweed decreased, and was unchanged thereafter, whereas other plants were not affected (Fig. 2B). When exposed to extract from kenaf weathered for 4 months, germination of pigweed decreased as concentration was increased to 66.7 g/l, but other crops were not affected (Fig. 2C).
V.M. Russo et al. / Industrial Crops and Products 6 (1997) 59-69
62
100
A
I
I ----me__._._
..e.-w..
.-rR---------&-
---
--
‘I
80
I
60 ___..----
40
*20 0
Fig. 1. Osmolality (- 0 -) of distilled water (0% PEG) and PEG concentration effect on: A, germination; B, hypocotyl length; C, radicle length; and D, total seedling length of 7 day old cucumber, green bean, tomato, pigweed and ryegrass seedlings. The vertical bars are S.E.
V.M. Russo et al. 1 industrial Crops and Products 6 (1997) 59-69
63
Kenaf Weathered 0 Months -----____
loo 60
~-___._____~___.__----~---------i
60
I- -*_.
--..
--e
___..-.---t
40
t ----.__
--__
20
J
0
Kenaf Weathered 4 Months 100
ir
100
11
------__.__
_____-.---
60
b B
40
%
’
1
l-
80-
--60
k ____--.----.+----._.____
--40
20
0
--
1
-
.
0
I
20
l. TO
16.7
33.3
66.7
Kcnaf Extracf (@IL) -----Greenbran -----.RFW=
-cucumk --.-pi-
----Tcmnab --o--0MloQlity
Fig. 2. Effect of kenaf extract concentration and distilled water (0 g/l) on germination pigweed, and ryegrass seedlings, and osmolality (- c -) if kenaf was: A, non-weathered; 4 months. The vertical bars are S.E.
I
of 7 day old cucumber, green bean, tomato, or B, weathered 2 months; or C, weathered
V.M. Russo et al. /Industrial Crops and Products 6 (1997) 59-69
64
Table 1 Germination and post-germination development of 7 day old seedlings of vegetables, weed and grass exposed to varying concentrations of extract from kenaf weathered on soil Treatment
Green bean Months weathered Concentration (C) Interaction: C x M Cucumber Months weathered Concentration (C) Interaction: C x M Tomato Months weathered Concentration (C) Interaction: C x M Pigweed Months weathered Concentration (C) Interaction: C x M Ryegrass Months weathered Concentration (C) Interaction: C x M
Germination (%)
Length (mm)
Number of secondary roots
Hypocotyl
Radicle
Total
(M)
** NS NS
NS ** *
** ** **
** ** **
(M)
** ** *
NS ** **
* ** **
** ** **
(M)
** ** *
* ** **
** ** **
** ** **
(M)
** ** *
* * **
** ** **
** ** **
(M)
** ** *
* * **
** ** **
** ** **
NS, non-significant and *,**, significant at PcO.05
** * **
NS NS NS
or P
3.3. Aqueous extract: post-germination development 3.3.1. General Post-germination development of all species was, in part or whole, affected by extract concentration, weathering length and their interaction (Table 1). Tissue discoloration occurred only on green bean and pigweed radicles. The amount of discoloration of green bean radicles (average, 25%) was not affected by either weathering or concentration of kenaf extract. Pigweed radicles exposed to non-weathered kenaf (0 months) extract showed O-55.2% discoloration as extract concentration was increased from control to 66.7 g/l (data not shown). Only green bean and pigweed produced secondary roots. Secondary root number (average two per radicle) on pigweed was not affected by treatment. However, secondary root number on green bean radicles was affected by months of weathering, concentration of extract and their
interactions (Table 1). As time of weathering and concentration of kenaf extract increased, the number of secondary roots increased (data not shown). Hypocotyl, radicle and total seedling length responded differently to extracts from kenaf with different lengths of time of weathering, extract concentrations and their interactions (Table 1). Exceptions were for cucumber and green bean where hypocotyl length was not affected by weathering of kenaf. 3.3.2. Green bean For each length of time of weathering, hypocotyl, radicle and total seedling lengths generally increased as the concentration of extract increased above that of the controls (Figs. 3 and 4). Exceptions were for radicle and total seedling lengths exposed to non-weathered kenaf (0 months) where length increased as extract concentration increased to 33.3 g/l and then decreased as concentration was increased to 66.7 g/l. In neither
V.M. Russo et al. 1 Industrial Crops and Products 6 (1997) 59-69
65
Kenaf Weathered 0 Months T ‘O”
Radicles
160~D
Kenaf Weathered 2 Months 150 125 100 75 50 25 0
Kenaf Weathered 4 Months
0
16.7
33.3
66.7
KenatMract (g/L) -----GmenbePn
-c-
---Tenrate
__....Qqmaa
-..-p@#j
--o--osmoh(i
0
18.7
33.3
66.7
Fig. 3. Effect of kenaf extract concentration and distilled water (0 g/l) on hypocotyl or radicle length of 7 day old cucumber, green bean, tomato, pigweed seedlings, and ryegrass and osmolality (- 0 -) if kenaf was: A and D, non-weathered; or B and E, weathered 2 months; or C and F, weathered 4 months. The vertical bars are S.E.
V.M. Russo et al. /Industrial Crops and Products 6 (1997) 59-69
66
Total Length 25Q-
Kenaf Weathered
0 Months
Kenaf Weathered
2 Months
A
50 --
l-
__-..-
+ _-__._.__._
_._m_
+_______.____l L
*
0
CO
Kenaf Weathered
=
b
4 Months
&&xj
_Ii
I;
--loo
__--
___---
_I_____-
_*-.-
t
$---
.-_._
-- B
____I
50
A
7
0
16.7
+0 66.7
33.3 Kenaf Extmcf (gL)
-----Gmanbenn
-Cucumber -.__p-
-‘---‘R)agnrr,
Fig. 4. Effect of kenaf extract
concentration
and distilled
tomato, pigweed and ryegrass seedlings and osmolality weathered 4 months. The vertical bars are S.E.
---1onmta -0Muly
water (0 g/l) on total seedling length of 7 day old cucumber, green bean, or B, weathered 2 months; or C, (- 0 -) if kenaf was: A, non-weathered;
V.M. Russo et al. /Industrial Crops and Products 6 (1997) 59-69
case was radicle or total length exposed to 66.7 g/l strength extract shorter than those exposed to controls. 3.3.3. Cucumber Hypocotyl, radicle and total seedling length generally increased as extract concentration increased regardless of length of time of weathering of kenaf (Figs. 3 and 4). Exceptions were for radicle and total seedling lengths for seedlings exposed to non-weathered kenaf (0 months), which first increased and then decreased, as concentration increased from controls to 16.7 g/l extract, and then decreased as extract concentration increased to 66.7 g/l. 3.3.4. Tomato Seedling hypocotyls exposed to concentrations of kenaf extract weathered for up 2 months first increased and then decreased as extract concentration increased. For seedlings exposed to kenaf extract weathered 4 months, hypocotyl length increased as concentration increased to 16.7 g/l and was unchanged thereafter. For seedlings exposed to extract of kenaf weathered for up to 2 months, radicle length decreased as concentration increased. When exposed to kenaf extract weathered 4 months, radicle length increased and then decreased as concentration of kenaf in extract increased. Seedling total length first increased and then decreased as concentration of extract from kenaf weathered up to 4 months increased. Total length of seedlings exposed to distilled water was longer than for those exposed to 66.7 g/l extract from non-weathered kenaf. When exposed to kenaf weathered for 2 months, the total length was similar for seedlings exposed to distilled water and 66.7 g/l extract. When exposed to kenaf weathered for 4 months, total length for seedlings exposed to distilled water was shorter than those exposed to 66.7 g/l extract. 3.3.X Pigweed Hypocotyl length of seedlings exposed to nonweathered kenaf (0 months) decreased as concentration increased to 66.7 g/l (Fig. 3A). Hypocotyls were longer when exposed to 66.7 g/l extract weathered for 2 months than when exposed to
61
controls (Fig. 3B). Length of hypocotyls exposed to extract of kenaf weathered for 4 months increased and then remained unchanged as concentration increased (Fig. 3C). Seedling radicle length decreased from that of the distilled water control when exposed to all concentrations of non-weathered kenaf (0 months) (Fig. 3D). Radicles exposed to kenaf weathered for 2 or 4 months were unchanged as extract concentration increased (Fig. 3E, F). Total seedling length decreased as concentration of non-weathered kenaf (0 months) increased (Fig. 4A). Total length was unchanged for seedlings exposed to all concentrations of kenaf weathered for 2 months (Fig. 4B), and was increased as concentration of kenaf weathered for 4 months increased (Fig. 4C). 3.3.6. Ryegrass Length of hypocotyls exposed to non-weathered kenaf (0 months) increased from those for controls to 16.7 g/l, and then decreased (Fig. 3A). Lengths of hypocotyls exposed to extract from kenaf weathered for 2 or 4 months increased as extract strength increased to 16.7 g/l and was unchanged thereafter (Fig. 3B, C). Radicles exposed to non-weathered kenaf (0 months) were shorter than those exposed to controls (Fig. 3D). Radicles exposed to kenaf weathered for 2 months were unchanged with increasing kenaf concentration (Fig. 3E). Radicle length for seedlings exposed to kenaf extract weathered for 4 months increased as concentration increased to 16.7 g/l and were unchanged thereafter (Fig. 3F). Total length of seedlings exposed to non-weathered kenaf extract (0 months) remained unchanged as concentration increased (Fig. 4A). When exposed to kenaf extract weathered for 2 or 4 months, total seedling length increased as extract concentration increased to 16.7 g/l and remained unchanged thereafter (Fig. 3B).
4. Discussion Extracts from weathered and non-weathered frost-killed kenaf appear to have properties that reduced germination of pigweed, and to a lesser degree that of ryegrass and tomato. As kenaf
68
V.M. Russo et al. 1 Industrial Crops and Products 6 (1997) 59-69
weathered, it appeared that compounds in tissues were leached, or otherwise changed, so that there was no effect or caused improved length of hypocotyls and/or radicles. The osmolality data suggest that with longer weathering, chemical changes occur in the kenaf tissues that may bind materials that have apparent allelopathic qualities. It was difficult to obtain osmolality determinations for some samples. In these cases the sample preseeded, possibly due to suspended solids. The material causing these difficulties was small enough to pass the 0.1 micron filters. The biological significance of these components is not yet known. This suggests that osmolality data need to be carefully evaluated for these types of studies. Russo et al. (1996) reported that spring weed population for the untreated bed was higher than those beds mulched with kenaf the previous fall. The reduced weed population was observed after several months of weathering of the chipped kenaf. Possibly the spring allelopathic compounds present in freshly chipped, material is absent in the kenaf tissue that had been weathered. This suggests that the allelopathic components may have leached from the tissue over the winter and were deposited in the germination region of the soil where there may still have some residual potency (Muller, 1966; Rice, 1983; White et al., 1989). Weathering of biological material is not confined to abiotic degradation. Fungi and bacteria may also be involved in this degradation. These biological entities could be responsible for the apparent allelopathic effect. However, detrimental effects were associated with extracts from non-weathered kenaf tissue. Since the non-weathered samples were immediately frozen, there was insufficient time for extensive biological colonization to occur during processing. Thus, allelopathic effects can be attributed to the extract from the kenaf tissue rather than from any other biological entity. In addition, since the seed were surface sterilized, and there was no indication of fungal or bacterial induced effects on germination and postgermination development, the results should be attributed to the extract obtained from kenaf.
For cucumber, green bean and ryegrass, PEG concentration did not affect germination and post-germination development. However, the time of weathering and concentration of kenaf, or both affected seed germination and/or post-germination development. This suggests that factors associated with kenaf and not the physical osmotic effect caused reduced germination and changes in length of hypocotyls and/or radicals. Green bean had decreased germination and green bean, tomato and pigweed had shorter hypocotyls or radicles when exposed to PEG. When treated with non-weathered kenaf extracts, green bean germination was unaffected but germination of tomato and ryegrass was reduced. The germination of pigweed was reduced to a larger extent with the kenaf extract than PEG. This suggests that the suppression or promotion of germination and/or growth of these plants were due to the biochemical component (kenaf) with the effect of osmotic potential being of less importance. Further examination of non-weathered kenaf tissue might yield metabolites that exhibit properties that can be used to suppress weeds. Alternatively this material, after weathering, may produce metabolites, or supply nutrients, that could be beneficial in the early development of some vegetable crops.
References Bagby, M.O., Cunningham, R.L., Touzinsky, G.F., Hamerstrand, G.E., Curtis, E.L. and Hofreiter, B.T., 1979. Kenaf thermomechanical pulp in newsprint. TAPPI/NPFP Committee Progress Report 10. Atlanta, GA. Bell, D.T. and Koeppe, D.E., 1972. Noncompetitive effects of giant foxtail on the growth of corn. Agron. J., 64: 321l 325. Clark, T.F., Cunningham, R.L. and Wolff, I.A., 1971. A search for new fiber crops. Tappi, 54: 63-65. Fisher, G., 1994. Manufacturing non-woven kenaf products. Int. Kenaf Assoc. Conf. Proc., New Orleans, LA, 6: 5456. Gressel, J.B. and Helm, L.G., 1964. Chemical inhibition of crop germination by weed seed and the nature of the inhibition by Abunlon theophrasii. Weed Res., 4: 44-53. Killinger, G.B., 1967. Potential uses of kenaf (Hibiscus cannabinus L.). Florida Soil Crop Sci. Sot. Proc., 27: 4- 11.
V.M. Russo et al. /Industrial Muller, C.H., 1966. The role of chemical inhibition (allelopathy) in vegetation composition. Bull. Torrey Bot. Club., 93: 332-351. Nieschlag, H.J., Nelson, G.H., Wolff, I.A. and Perdue, R.E.. Jr., 1960. A search for new fiber crops. Tappi, 43: 193201. Phillips, W.A., Rao, S. and Dao, T., 1989. Nutritive value of immature whole plant kenaf and mature tops for growing ruminants. Prcic. Assoc. Advancement of Industrial Crops, pp. 17-22. Rice, E.L., 1983. Pest control with nature’s chemicals: allelochemics and pheromones in gardening and agriculture. University Oklahoma Press, Norman, OK, 224~. Russo, V.M., Cartwright, B. and Webber, C., III, 1996. Mulching: effects on erosion of soil beds, and on yield of autumn and spring-sown vegetables. Biol. Agric. Hort., 14: in press. SAS Institute. 1988. SASSTAT User’s Guide, Release 6.03 Edition. SAS Institute, Cary, NC, 1028~. Smith, A.E., 1989. The potential allelopathic characteristics of Bitter Sneezeweed (Helenium amarum). Weed Sci., 17: 6655669. Tilmon, H.D., Taylor, R. and Malone, G., 1988. Kenaf: An alternative crop for Delaware. In: J. Janick and Simon,
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J.E. (Eds.), Advances in New Crops. Timber Press, Portland, OR, pp. 301-302. Webber, C.L., III., 1992. Kenaf (Hibiscus cannabinus L.) yield components as affected by sewage sludge applications. Proc. TAPPI Pulping Conf. pp. 57-62. Webber, CL., III., 1993. Crude protein and yield components of six kenaf cultivars as affected by crop maturity. Ind. Crops Prod. 2: 27-31. White, F.D., Cummins, D.G., Whiteley, E.L., Fike, W.T., Greig, J.K., Martin, J.A., Killinger, G.B., Higgins, J.J. and Clark, T.F., 1970. Cultural and harvesting methods for kenaf. USDA. Production Research Rept. 113. Washington, DC, 38~. White, R.H., Worsham, A.D. and Blum, U., 1989. Allelopathic potential of legume debris and aqueous extracts. Weed Sci. 37: 614-619. Williams, R.D., Baldwin, B.S. and Reichert, N.A., 1995. Kenaf core as the major component in greenhouse potting media. Seventh Annual International Kenaf Association Conference Proceedings, Irving, TX. pp. 25-32. Wilson, F.D., Summers, T.E., Joyner, J.F., Fisher, D.W. and Seale, C.C., 1965. ‘Everglades 41’ and ‘Everglades 71’, two new varieties of kenaf (Hibiscus cannabinus L.) for the fiber and seed. Fla. Agr. Exp. Cir. p. 168.