THE BOTANICAL REVIEW

VOL. 61 JANUARY-MARCH
1995 NO. 1

On Laboratory Bioassays in Allelopathy

INDERJIT 1

AND

K. i . M . DAKSHINI

Department of Botany, University of Delhi,

Delhi- l l O007, India

I. Abstract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29
II. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29
III. Bioassay Design . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30
IV. Bioassay Material . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30
V. Seed Germination and Growth Parameters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30
VI. Extraction of Allelochemicals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31
VII. Bioassay with Allelochemicals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32
VIII. Critical Age of AUelochemical Release . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33
IX. Microbes and Expression of Allelopathy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33
X. Active Concentration of Allelochemicals. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34
XI. Selection of Allelopathic Candidates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35
XII. Soil Texture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35
XIII. Chemical Characteristics of Amended Soils . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36
XIV. Life Cycle Pattern . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36
XV. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37
XVI. Acknowledgment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37
XVII. Literature Cited . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37

1. Present address: Department of Biology, Lakehead University, Thunder Bay, Ontario, Canada
P7B 5EI.

Copies of this issue [61(I)] may be purchased from the Scientific

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NY 10458-5125 USA. Please inquire as to prices.

The BotanicalReview 61(1): 28-44, January-March 1995

9 1995The New YorkBotanicalGarden 28
 ON LABORATORYBIOASSAYS IN ALLELOPATHY 29

I. Abstract

Allelopathy involves the complex chain of chemical communications among plants,

including microbes. Laboratory bioassays constitute a significant part of allelopathic
research, and various bioassays have been proposed to demonstrate allelopathy under
controlled lab conditions. However, many lab bioassays have little or no correspondence
to field interaction, which may be due to dissimilarity of the conditions of lab bioassay
to natural conditions, lack of standardized techniques, or absence of critical controls. Here
we discuss several lab bioassays presently used in allelopathic research for their suitability
to demonstrate allelopathy of ecological relevance. We recommend avoiding certain
practices, such as grinding plant material to evaluate allelopathic potential and isolation
of allelochemicals, using seed germination as the only criterion of growth response, using
sand, agar, or autoclaved soil, using organic solvents as extractants in allelopathic
bioassays, and eliminating microbial involvement. Care should be taken in the lab to
simulate natural conditions and attention should be given to habit, habitat, and life cycle
pattern of the allelopathic plants during designing of lab bioassays.

II. Introduction

Rice (1984) defined allelopathy as any direct or indirect (harmful or beneficial) effect
of a plant, including microbes, on another plant through release of chemicals that escape
into the environment. It involves a complex chain of chemical communication between
plant species (Harborne, 1987). The term, initially coined by Molisch in 1937 (Rice,
1984), includes both inhibitory and stimulatory effects (Rice, 1986). Allelopathic inter-
actions may play a key role in influencing the distribution of vegetation in nature, the
yield of various crop species, and weed interference (Muller, 1966; Del Moral & Muller,
1970; Putnam & Duke, 1978; Kaminsky, 1981; Aldrich, 1984; Rice, 1984; Horsley, 1991)
The significance of allelopathy to the ecological theory was highlighted by Muller (1966,
1969). Even low quantities of a chemical may significantly influence plant growth,
nutrient ion absorption, and, consequently, microclimate (Muller, 1966).
In spite of the large number of allelopathic studies, very few establish operation of
allelopathy under field conditions (Newman, 1982; Willis, 1985; Williamson, 1990).
Several mechanisms of interference may be operative in sequence or simultaneously under
field conditions, making it extraordinarily difficult to separate these mechanisms at the
field level (Putnam & Weston, 1986). Laboratory bioassays, on the other hand, allow
researchers to eliminate all possible alternative interferences through perfectly controlled
experimental designs and manipulation of nearly all parameters, so investigators can vary
complex field conditions one at a time to search for mechanistic interactions. Many
bioassays have been proposed to test allelopathy (Dekker et al., 1983; Einhellig et al.,
1985; Leather & Einhellig, 1985, 1988; Williamson & Richardson, 1988; Dornbos &
Spencer, 1990; Horsley, 1991 ; Grakhov et al., 1993), but most show little or no correspon-
dence to plant interactions in the field (Stowe, 1979; Leather & Einhellig, 1986), possibly
because it is difficult for lab bioassay experiments to simulate natural field conditions
(May & Ash, 1990). Chemicals are present in all plant parts, and their mere presence does
not establish allelopathy of ecological relevance (Heisey, 1990). For conclusive proof of
allelopathy, lab bioassays must demonstrate the effective release of allelochemicals into
the soil and the subsequent effect of those chemicals on associated plant species (Putnam
& Tang, 1986). In most of the studies null hypothesis appears to be less significant
30 THE BOTANICALREVIEW

(Williamson, 1990) and in many studies critical controls are absent. Williamson and
Richardson (1988) suggested that statistical measurement of treatments in comparison to
the control is required.

III. Bioassay Design

Allelopathic interactions are widely known in different groups of plants such as algae,
lichens, crops, and annual and perennial weeds (Rice, 1984; Putnam, 1985; Horsley, 1986,
1991 ; Lawre~,, 1993; Inderjit & Dakshini, 1994a, 1994b). Those designing an allelopathic
bioassay should keep in mind the group to which the suspected allelopathic plant belongs.
In this paper, we discuss bioassay for higher plants only. Operation of allelopathy in crops
and crop residues is different from that in weeds. Perennial weed allelopathy is different
from that of annual weeds because of perennials' evergreen nature. And biomass of annual
weeds' root systems is not enough to maintain an active concentration of allelochemicals,
while biomass does play a significant role in perennial weeds (e.g. Cyperus rotundus,
Pluchea lanceolata, lmperata cylindrica). Therefore, plant habit should be considered
when designing allelopathic bioassays.

IV. Bioassay Material

In many allelopathic studies, plant material is ground to assess allelopathic activity or

to identify and isolate allelochemicals (McCahon et al., 1973; Stowe, 1979; Rice, 1984;
Tanrisever et al., 1987; Heisey, 1990). This grinding results in the release of certain
enzymes, salts, amino acids, and nitrogen compounds, which may not be released under
natural circumstances (Chou & Muller, 1972). Allelopathic bioassays with ground mate-
rial are thus of little ecological relevance as the extraction procedure causes qualitative
and quantitative changes in the phytochemical profile. Further, relating allelopathy to such
extracts is a problem, because some compounds affecting plant growth may not be
naturally leached or exuded and therefore may not be physiologically significant even
though they may be significant in lab bioassays. Therefore, grinding of plant material in
allelopathic studies should be avoided (Putnam & Duke, 1978).

V. Seed Germination and Growth Parameters

Seed germination is a widely used parameter in allelopathic bioassays (Rice, 1984).

Williams and Hoagland (1982) suggested that seed germination may not be the primary
site for allelopathic interactions, and Stowe (1979) even advised against using seed
germination as a bioassay parameter. Weidenhamer et al. (1987) reported that alletopathic
results were influenced by seed number in germination bioassays, in relation to solution
volume. Furthermore, in comparison to seed germination, seedling growth showed more
response to certain categories of allelochemicals, such as phenolics (Rasmussen &
Einhellig, 1977; Einhellig & Rasmussen, 1978). However, some workers found oven dry
weight of radicle (Leather & Einhellig, 1985) and root length and root fresh weight to be
statistically more accurate (Cope, 1982; Pederson, 1986). Although seedling growth
response is widely used to assess allelopathic effects in lab bioassays, physiological and
biochemical characteristics will definitely help us understand the mechanism of action of
allelochemicals. Many physiological parameters as well were demonstrated to be affected
by the allelochemicals (Rice, 1984). Phenolic compounds have been shown to influence
 ON LABORATORYBIOASSAYS IN ALLELOPATHY 31

nutrient ion uptake (Glass & Bohm, 1971; Bergmark et al., 1992; Booker et al. 1992),
enzyme activities (Devi & Prasad, 1992), water relations (Barkosky & Einhellig, 1993),
and photosynthesis and respiration (Einhellig & Rasmussen, 1993; Hejl et al., 1993).
Inderjit and Dakshini (1992b) showed that water-soluble phenolics from the weed Pluchea
lanceolata influenced the chlorophyll content and net photosynthetic rate of leaves of
asparagus bean under greenhouse conditions. Therefore, we suggest studying the effect
of allelochemicals on more than growth parameters; investigators might look also at
seedling growth and the physiological and biochemical parameters of affected plant
species.

VI. Extraction of Allclochemicals

Reese (1979) defined an allelochemical as a "nonnutritional chemical produced by one

organism that affects the growth, health, behaviour or population biology of other
species." Allelochemicals belong to different categories of secondary compounds such as
phenols, benzoic and cinnamic acid derivates, flavonoids, coumarins, tannins, terpenoids,
alkaloids, and polyacetylenes (Evenari, 1949; Vancura, 1964; Whittaker & Feeny, 1971;
Rice, 1984). Allelochemicals are released into the environment through leaf leachates,
root exudates of living plants, and decomposed plant residues (Wilson & Rice, 1968;
Parenti & Rice, 1969; Putnam & Duke, 1978; Rice, 1984; Inderjit & Dakshini, 1991a,
1992b).
Extraction of allelochemicals is an integral part of allelopathic bioassays. One should
be logical in selecting extraction source and extractant. Extraction source could be either
vegetative parts of the suspected allelopathic plant or its associated soils. It is necessary
to establish not only the presence of these chemicals in biologically active concentrations
but also a persistence that would affect other plants in their vicinity (Putnam & Tang,
1986). Therefore, isolation and identification of chemicals from the environment is most
significant in establishing allelopathy. Stowe (1979) found that the classical bioassays
with whole plant extracts, foliar washings, and decomposed litter inhibited the germina-
tion and growth of plant species, although the plant may not be allelopathic under natural
conditions. Fisher (1979) stated that "it seems unlikely that the allelochemicals that may
be extracted from the plant material are actually those that reach the host plant, yet all our
information on allelopathic compounds is derived from extracts that never have been
exposed to soil." Cheng (1992) suggested that after chemicals enter the soil, a number of
processes take place--such as retention, transformation, and transport--which affect the
fate of these chemicals and hence their allelopathic potential. Therefore, it is important
to isolate allelochemicals from the environment/soils of a suspected allelopathic plant and
determine whether allelochemicats are present in an active concentration sufficient to
influence associated plant species.
Various workers extract phytochemicals in aqueous phase at room temperature
(Rice, 1984). However, to avoid interference of microbes, autoclaving is done for
higher quantity and wider range of phytochemicals, while extraction was done with
hot water and organic solvents such as methanol, chloroform, and NaOH (Guenzi
& McCalla, 1962; Lodhi & Nickell, 1973; Jackson & Willemson, 1976; Liebl &
Worsham, 1983; Ponder & Tadros, 1985; Choesin & Boerner, 1991; Harrison &
Peterson, 1991; Perez & Ormeno-Nunez, 1991). Organic solvents such as chloro-
form can extract chemicals from litter, soil organic matter, humic acid, and microbe
membranes (Schmidt, 1990). Extraction with organic solvents is thus of less eco-
32 THE BOTANICAL REVIEW

logical significance. The use of hot water or organic/inorganic solvents, therefore,

should be avoided, because there are qualitative and quantitative differences be-
tween the allelochemicals extracted in lab bioassays and those occurring in natural
conditions. Schmidt (1990) recommended against the use of organic solvents like
chloroform in extraction of allelochemicals from the soil. Whitehead et al. (1981)
suggested that the amounts of phenolic compounds can be best extracted with water
as an extractant in comparison to Ca(OH)2 or 2M NaOH. It is very likely that
allelopathic interference of ecological significance is due to water-soluble allelo-
chemicals which are available in free form. Further, in cultivated fields where
irrigation is frequent, water-soluble compounds are of greater ecological signifi-
cance (McPherson & Muller, 1969; Del Moral & Muller,1970). The phytochemicals
contributed to the soils are found in either free or bound form, and free or reversibly
bound forms are more accountable for allelopathic interactions in plants (Whitehead
et al., 1983; Dalton et al., 1989b).
Blum et al. (1994) suggested that neutral EDTA and water extraction of soils can be
used to estimate available (free and reversibly bound) phenolic acids in soils. Adsorption
of volatiles on the soil particles in Salvia leucophylla and their inhibitory activities on
herbs were also reported (Muller, 1966). Heisey and Delwiche (1985) found leaf extracts
of Trichostema lanceolatum to be very toxic in petri plate bioassays. But the same leaves,
when mixed with soil, had low allelopathic effects. They concluded, therefore, that T.
lanceolatum had low allelopathic potential under natural conditions. Lab bioassays may
establish whether a plant has allelopathic potential, but in order to confirm whether the
observed allelopathic potential is expressed under natural conditions, field studies are
necessary. Tang and Young (1982) suggested that the trapping system is more efficient
compared to the solvent extraction method and they found that it helps isolate and identify
phytochemicals from the rhizosphere of undisturbed roots.

VII. Bioassays with Allelochemicals

Many workers have tested the allelopathic potential of compounds by taking the
authentic compounds either in aqueous solution or in sterile soil (Dalton et al., 1983; Rice,
1984; Putnam & Tang, 1986). However, to test the allelopathic potential of chemicals, it
is most desirable either to add authentic compounds to the soils or to isolate compounds
from the soil associated with the allelopathic plant (Lodhi, 1975, 1976, 1978; Inderjit &
Dakshini, 1991b, 1992a). Hall et al. (1982) found that total phenolics equivalent to
chlorogenic acid inhibited the seed germination of Amaranthus retroflaxus but that
chlorogenic acid in its pure form did not inhibit the germination ofA. retroflaxus. Further,
many phenolics also become unavailable in soil due to either (1) their resistance to
microbial degradation because of their polymerization in the presence of microbes or (2)
their adsorption to the organic/inorganic soil particles (Martin et al., 1972; Martin &
Haider, 1976; Huang et al., 1977; Dalton et al., 1989a). Chang et al. (1969) found that red
clover soil sickness was due to various phenolic acids, which were the degraded products
of various isoflavonoids present in the red clover. Dornhos and Spencer (1990) suggested
modified agar bioassays, which are suitable for the lower quantity of water-soluble
compounds. However, they did not take soil into consideration, which is needed in order
to narrow the gap between lab bioassays and field interactions. Tanrisever et al. (1987)
identified ceratiolin from Ceratiola ericoides; however, it decomposes at room tempera-
ture to yield hydrocinnamic acid, which has higher inhibitory activity than ceratiolin
 ON LABORATORYBIOASSAYS IN ALLELOPATHY 33

against seed germination and seedling growth of Schizachyrium scoparium. Therefore,

the fate of allelochemicals in the soil should be given serious consideration (Williamson
& Weidenhamer, 1990).
In addition to testing the individual compounds, it is highly desirable to test also
the mixture of allelochemicals, as it is difficult to conclude and establish if one
compound is responsible for reduced growth (Liebl & Worsham, 1983). Allelopathic
interactions are the result more of synergistic activity of various allelochemicals
than of the activity of any single one (Williamson, 1990). Under field conditions,
additive or synergistic effects of allelochemicals may become influential even at
low concentrations (Rasmussen & Einhellig, 1977; Einhellig & Rasmussen, 1978;
Williams & Hoagland, 1982).

VIII. Critical Age of Allelochemical Release

Release of allelochemicals to the substratum starts only after the plant attains a definite
growth stage. Bell & Koeppe (1972) demonstrated that interference to the corn growth by
the annual weed Setariafeberi was brought about only after the weed attained significant
growth greatly in advance of the crop. Schumacher et al. (1983) reported that wild oat
becomes allelopathic at four-leaf stage to the growth of spring wheat. Maximum release
of allelochemicals from the roots of Parthenium hysterophorus was observed at the rosette
and flowering stages of the weed (Kanchan & Jayachandra, 1979). These observations
suggest the significance of the age at which plants begin to release allelochemicals. Walnut
trees expressed their allelopathic effects only after 15-25 years, when active concentra-
tions ofjuglone have built up in the soil (Ponder & Tadros, 1985). Kimber (1973) reported
that slightly green straw was more toxic than fully mature residues (Barnes & Putnam,
1983). In lab bioassays, then, particular attention should be paid to the plant growth stage
at which release of allelochemicals in active concentrations begins.

IX. Microbes and Expression of Ailelopathy

Soil microbes play an important role in qualitative and quantitative availability
of allelochemicals (Turner & Rice, 1975; Chapman & Lynch, 1983; Blum et al.,
1987; Nair et al., 1990; Chase et al., 1991b). Muller (1953; Muller & Muller, 1956)
found that the shrub Encelia farinosa contains water-soluble toxins that do not
contribute to its allelopathic activity under natural conditions. Such an inactivation
of toxins could be due to microbial activity or adsorption to soil colloids. Cutler
(1986) reported that herbicidal compounds from microorganisms can affect plant
growth. Many microbes utilize phenolic acids and flavonoids as a carbon source and
deplete phenolics from the medium (Westlake et al., 1959; Vaughan et al., 1983;
Blum et al., 1987). Fungi such as Aspergillusflavus and A. niger produce enzymes
when grown with rutin or quercetin, which degrade flavonols (Rice, 1984). Kamin-
sky (1981) suggested that the allelopathic potential of the shrub Adenostoma
fasciculatum may be due to its association with unidentified toxins produced by soil
organisms, which could account for allelopathic suppression of seed germination
and seedling growth of herbs. Many plant residues produce inhibitors only when
conditions become favourable for microbial growth (Cochran et al., 1977). Norstadt
and McCalla (1963) suggested that the toxins from plant residues and microorgan-
isms are jointly responsible for the inhibitory effect of the plant residues. Webb et
34 THE BOTANICALREVIEW

al. (1967) showed that the failure of regeneration of Grevillea robusta seedlings
was due to toxic factors associated with the living roots which may disturb the
microflora balance in its rhizosphere. In nature, plant leachates may stimulate the
activities of soil actinomycetes (Linderman & Gilbert, 1969; Katz et al., 1987).
DeFrank and Putnam (1985) reported that the soil-borne actinomycetes could
enhance allelopathic effects. Kaminsky (1981) reported that toxicity of soil associ-
ated with Adenostoma fasciculatum was due to soil microbes that produce inhibi-
tors. Various phytochemicals can affect plant growth by interfering with
mycorrhizae formation and by affecting survival of fungal symbionts (Persidsky et
al., 1965; Robinson, 1972; Del Moral et al., 1978; Kovacic et al., 1984). Patrick and
Koch (1963) found that the respiration of tobacco seedlings was inhibited by the
decomposing plant residues and not by aqueous extracts of fresh plant residues.
2(3H)-benzoxazolone (BOA) was transformed in soils into the more toxic substance
2,2'-oxo-l,l'azobenzene, and this soil transformation of BOA was mediated by the
microbe Actinotobactor calcoaceticus (Nair et al., 1990; Chase et al,, 1991a, 1991b),
Transformation of rye metabolite BOA into phytotoxic 2-amino-3H-phenoxazin-3-one
was reported in nonsterile soil, while BOA was recovered as such in sterile soil (Gagliardo
& Chilton, 1992). Rietveld et al. (1983) hypothesized a relationship among juglone
concentration in soil, juglone production by black walnut trees, and rate of breakdown by
aerobic heterotrophs. They suggested that better soil aeration could result in higher
aerobic metabolism by microbes that lower the concentrations ofjuglone. Therefore, the
role of microbes should not be overlooked, and, as rightly suggested by Schmidt (1990),
allelopathic experiments should be performed using nonsterile soils.
Further, many phenolic acids may influence microbial populations and result in
a shift of microbial ecology (Henderson, 1956; Olsen et al., 1971; Turner & Rice,
1975; Rose et at., 1983; Inderjit & Dakshini, 1991a; Shafer & Blum, 1991; Siqueira
et al., 1991). The significance of microbes in determining the allelopathic potential
is well documented (Sparling et al., 1981; Vaughan et al., 1983; Blum & Shafer,
1988); the role of microbes and their metabolites in phytotoxicity of decomposing
plant residues is also reported (Chapman & Lynch, 1983). Lovett and Duffield
(1981) observed that chemicals contributed by Camelina sativa were transformed
and liberated by its phyllosphere bacteria, which then induced allelopathic activity
(Lovett & Sagar, 1978).

X. Active Concentration of Allelochemicals

Data on active concentration of allelochemicals in soil is desirable to establish

allelopathy (Choesin & Boerner, 1991). The concentration is determined primarily
by rate of input to the environment (i.e., leaf leachate, root exudates, or decompo-
sition and leaching of plant debris), absorption and adsorption by seeds and roots,
fixation by soil component, and leaching and microbial degradation (Wang et al.,
1978; Huang et al., 1977; Rice, 1984; Dalton et al., 1983). It becomes very difficult,
therefore, to calculate the exact concentration of allelochemicals in the soil. Be-
cause of uneven distribution of phytotoxic substances (Liebl & Worsham, 1983) in
the soil and varied irrigation and agricultural practices, the inhibitory effects depend
on the probability of the exposure of roots of affected plants to the pockets of toxins.
Williamson and Weidenhamer (1990) suggested that toxicity was mainly a function
of existing concentration of toxins in the soil and their renewal rate by the
 ON LABORATORYBIOASSAYSIN ALLELOPATHY 35

allelopathic plant. Furthermore, concentration of the allelochemicals in the natural

fields depends on density and growth of allelopathic plant, habitat, pattern of
cultivation, and agricultural practices (Rice, 1984; Inderjit & Dakshini, 1994a). It
is therefore very difficult to collect data on exact concentrations of allelochemicals
under field conditions. In most of the lab bioassays, the allelopathic effect was
assessed after addition of vegetative parts or leachates to the soil (Rice, 1984). As
a result, amended soils have higher values for allelochemical pool. However, care
should be taken that higher content of allelochemicals in the amended soils should
be in the range of the allelochemical pool of natural soils associated with that
particular plant.

XI. Selection of Ailelopathic Candidates

Allelopathic effects are always mediated through chemicals that belong to dif-
ferent categories of secondary compounds such as phenolics, terpenoids, alkaloids,
polyacetylenes, fatty acids, peptides, etc. (Whittaker & Feeny, 1971; Rice, 1984).
However, it is very difficult to identify which group of secondary compound is
primarily responsible for allelopathic effects. Phenolics and terpenoids are the two
widely investigated groups of allelochemicals; however, phenolics are second only
to carbohydrates in abundance and are widely known for their allelopathic potential
(Muller, 1966; Levin, 1971; Rice, 1984). It is still not clear whether phenolics or
terpenoids should first be investigated as primary allelochemicals in a particular
plant. Phenolics are primary allelochemicals in temperate ecosystems, while ter-
penoids are of importance in arid and semi-arid environments (Horsley, 1991).
However, some workers preferred phenolics as primary allelochemicals because of
their easy identification and their ready availability due to their water-soluble nature
(Inderjit & Dakshini, 1990, 1992a, 1992b; Alsaadawi et al., 1985). Further, water-
soluble allelochemicals are of more significance where irrigation is frequent
(McPherson & Muller, 1969; Del Moral & Muller, 1970); however, a possible
transport mechanism of plant lipid in water, through micelle formation with natural
tensides such as sterols and fatty acids, has recently been suggested (Fischer, 1986).
Several sesquiterpenes were detected from Cyperus rotundus; however, growth
inhibitions in white clover and Rumex species were due to a mixture of phenolics
such as p-coumaric, ferulic, vanillic, p-hydrobenzoic, and protocatechuic acids
present in the purple nutsedge (Fischer, 1986).
Allelopathic interferences with phenolics should be more significant in soils of
low fertility or during periods of low fertility (Stowe & Osborn, 1980). Further, it
has been shown that plants growing under deficient nutrient conditions produce
more phenolics than plants under sufficient nutrient conditions (Del Moral &
Muller, 1970; Lehman & Rice, I972). Hence, collections of a species from many
habitats should be made in order to assess the conditions under which the plant best
exhibits its allelopathic potential.

XlI. Soil Texture

In many allelopathic studies, leaves, leachates, root exudates, or residues of

allelopathic plants are added to sand or agar instead of to soil of same textural class
as that of the plant's habitat (McPherson & Muller, 1969; Tinnin & Muller, 1971;
36 THE BOTANICALREVIEW

Chou & Muller, 1972; Patterson, 1981; El-Deck & Hess, 1986; Pederson, 1986;
Mersie & Singh, 1987; Anaya et al., 1990). Modification of allelopathic potential
by soil texture has been extensively worked out (Muller & Del Moral, 1966; Del
Moral & Muller, 1969, 1970; Del Moral & Cates, 1971; Oleszek & Jurzysta, 1987;
Inderjit & Dakshini, 1994c). Del Moral and Muller (1970) suggested that sandy soils
do not adsorb phenolics to the same extent as fine-textured soils. Additionally, they
reported that toxins are degraded more rapidly in soil under aerobic conditions and
that coarse soil has better aeration. Oleszek and Jurzysta (1987) reported that the
modification of saponin activity in soil was related to ability of medicagenic acid
glycosides to bind to sorption complex of soil. No sorption occurred in loose sand,
and sorption increased with heavier soils. Therefore, the highest degree of inhibition
was observed when alfalfa roots were amended with loose sand in comparison to
heavier soils. Ecologically relevant results in allelopathic bioassays, then, depend
also on soil selected for having texture similar to that of the plant's natural habitat.

XIII. Chemical Characteristics of Amended Soils

Generally, in allelopathic studies, leachates, extracts, residues, or plant parts are added
to the soil for assessing the allelopathic potential of the plant (Bieber & Hoveland, 1968;
Bhowmik & Doll, 1984; Mersie & Singh, 1987, 1988; Heisey, 1990; Inderjit & Dakshini,
199 la, 1992b). Since temperature, oxygen availability, osmotic potential, and light regime
all influence germination and growth (Reynolds, 1975; Duke et al., 1983; Anderson &
Louchs, 1986; Leather & Einhellig, 1986; Drew & Brockleurst, 1990; Willis & Groves,
1991), special care was taken to minimize the effect of these parameters in many
allelopathic bioassays. However, nutrients, which form a significant component of plant
parts and are known to contribute to the environment through natural leaching and
agricultural practices, are not given much attention in lab bioassays with amended soils
(Tamm, 1951; Tukey, 1966; Tukey & Morgan, 1963). Further, adding the leaves of a
suspected allelopathic plant may stimulate soil microbial population as discussed above,
which can cause immobilization of nutrients; distinguishing between allelopathy and the
nutrient effect, then, becomes difficult unless appropriate controls are used in such
experiments (Heisey, 1990). The alteration of physico-chemical characteristics of the soil
should affect the quantitative and qualitative availability of phytochemicals in the soil,
which, in turn, influences the allelopathic expression (Del Moral & Muller, 1970; White-
head et al., 1981, 1982; Dalton et al., 1983; Blum et al., 1987; Oleszek & Jurzysta, 1987;
Blum & Shafer, 1988). Inderjit and Dakshini (1994c) suggested that the gap between lab
bioassays and field interactions can be reduced by analysing amended soils for their
chemical characteristics to ensure that those characteristics are not significantly different
from those found in the residence of the allelopathic plant.

XIV. Life Cycle Pattern

Very few lab bioassays pay attention to the life cycle pattern of the allelopathic plants.
Though the problem is more significant in annual weeds, most studies treat annual and
perennial weeds similarly. It is very important to consider at what stage an annual weed
starts releasing atlelochemicals in active concentrations to the substratum and whether the
sensitive stage of a susceptible plant has passed prior to annual weeds' release of
allelochemicals. Crop losses by the weeds are largely related to the timing of crop and
 ON LABORATORY BIOASSAYS IN ALLELOPATHY 37

weed emergence (Forcella, 1993). It is very likely that monocarpic annuals such as
Polypogon, Cirsium, and Asphodelus may be allelopathic not to same-season crops but to
next-season crops, through accumulated residues. Therefore, in lab bioassays to test
annual weed allelopathy, associated susceptible plants should be selected, and life cycle
pattern should be carefully considered. Brede (1991) suggested the field apparatus used
to demonstrate allelopathy in annual bluegrass. Lab bioassays with soils amended with
weed parts, particularly monocarpic annuals, are thus of little significance.

XV. Conclusion

Laboratory bioassays in allelopathic research are of great significance. In nature many

factors are interacting (simultaneously and sequentially) and constantly changing, and
there are many co-linearities (e.g., debris increases, soil temperature decline, increased
water content of soil, and increased phenolic content). In laboratory bioassays, investiga-
tors can control these parameters and can vary one at a time to search for mechanistic
interactions.
The following measures can reduce the gap between lab bioassays and field interac-
tions in allelopathy:

1. Select a soil type similar to that of the allelopathic plant's residence.

2. Plant material should not be ground, the role of microbes should not be overlooked, and
extraction with organic solvents should be avoided.
3. Chemical analysis of amended soils should be performed in order to choose a soil whose
chemical characteristics most closely simulate those of the allelopathic plant's residence.
4. Careful consideration should be given to habit, habitat, and fife cycle pattern of allelopathic
plants, particularly weeds.

Protocols to establish allelopathy have been suggested (Putnam, 1985; Willis, 1985;
Putnam & Tang, 1986; Horsley, 1991; Zimdahl, 1993), but in order to gain definite insights
into allelopathy through lab bioassays, a holistic approach is required. It is important to
state that observing the above points will not prove that allelopathy alone is operative,
only that allelopathy offers the most reasonable explanation of the observed pattern. Close
consideration of the above points should, however, help in developing a model for lab
bioassays to monitor and establish allelopathy of ecological relevance.

XVI. Acknowledgment

We sincerely thank Professor Elroy L. Rice for his valuable suggestions and discussion
during his visit to our laboratory.

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