Screening methods for the evaluation of crop allelopathic potential

THE VOL. 67

BOTANICAL

REVIEW

JULY-SEPTEMBER 2001

NO. 3

Screening Methods for the Evaluation of Crop Allelopathic Potential H. W U 1, J. PRATLEY 1, D . LEMERLE 2, T. HAIG 1, AND M . A N 3

z Farrer Centre for Conservation Farming and s Environmental and Analytical Laboratories Charles Sturt University Locked Bag 588 Wagga Wagga, NSW 2678, Australia e NSW Agriculture Wagga Wagga Agricultural Institute Wagga Wagga, NSW 2650, Australia

I. Abstract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . II. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . III. Key Issues in Designing Screening Bioassays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. General Requirements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Separation of Allelopathy from Competition . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Bioassay Species of Interest . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D. Pregermination of Bioassay Species . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E. Densities of Donor and Receiver Species . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . F. Simulation of Natural Release of Allelochemieals . . . . . . . . . . . . . . . . . . . . . . . . IV. Aqueous Extract Screening Bioassays under Laboratory Conditions . . . . . . . . . . . . V. Seedling Screening Bioassays under Controlled Environments . . . . . . . . . . . . . . . . . A. Pot Screening . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Plant Box Method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Relay Seeding Technique . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D. Equal Compartment Agar Method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VI. Field Screening . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VII. Chemical Screening . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VIII. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . IX. Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . X. Literature Cited . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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I. Abstract There is increasing interest in the d e v e l o p m e n t o f allelopathic crop varieties for w e e d suppression. Allelopathie varieties are likely to be able to suppress w e e d s by natural exudation o f

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THE BOTANICAL REVIEW

bioactive allelochemicals, thereby reducing dependence upon synthetic herbicides. Screening bioassays are essential tools in identifying crop accessions with allelopathic potential. A number of crops have been screened for this allelopathic trait, and key issues in selecting and designing screening bioassays are reviewed. It is recommended that a combination of different bioassays be used in the evaluation of crop allelopathic potential. Laboratory bioassays, field testing, and chemical screening are important steps, and none of them can be precluded if conclusive evidence of crop allelopathy is to be established. More concerted efforts are needed in screening crop germplasm before the development of allelopathic varieties occurs.

II. Introduction Allelopathy is a process whereby plants provide themselves with a competitive advantage by putting phytotoxins into the near environment (Pratley, 1996). The increasing interest in allelopathy has been driven by the recognition that agroecological applications of allelopathy may provide alternatives to synthetic herbicides for weed control (Romeo & Weidenhamer, 1999), and it is well documented that allelopathy has potential for weed management (Rice, 1984; An et al., 1998). Recent achievements in the genetic control of allelopathy and in the development of allelopathic varieties for weed management have been reviewed by Wu et al. (1999). During the past several decades, screening for allelopathic genotypes has been conducted in a number of field crops, including, cucumber (Cucumber sativus L.) (Putnam & Duke, 1974) and wheat (Triticum aestivum L.) (Wu et al., 1998, 2000a, 2000b). Some crop accessions have been shown to possess strong allelopathic potential against the growth of a number of weed species. A plant with allelopathic potential is referred to as the "donor plant," while the plant in the vicinity affected by the allelopathic compounds from the donor plant is referred to as the "receiver plant." Donor and receiver plants can affect each other through allelopathy and competition. The combined effect of these two interactions has been termed "interference" (Muller, 1969). In allelopathic interactions, some phytotoxic substances are released by donor plants into the environment to affect the growth of receiver plants, whereas in competitive interactions, a growth resource is removed from the environment by one plant so that the growth resources available to other plants are reduced (Wu et al., 2000a). Laboratory bioassay is the first step used to investigate the possible involvement of allelopathy (Foy, 1999). Many bioassays have been designed to identify the role of allelopathy in donor-receiver interactions (Pederson, 1986; Shilling & Yoshikawa, 1987; Weidenhamer et al., 1989; Liu & Lovett, 1993; Haugland & Brandsaeter, 1996). Limited efforts have also been made to develop screening bioassays for identifying crop accessions with allelopathic potential for weed suppression. These bioassays can basically be categorized as aqueous extract screening, seedling screening, field screening, and chemical screening (Table I). The objectives of this review are to discuss some of the key methodological obstacles in designing and selecting an appropriate screening bioassay and some of the most commonly used screening bioassays for assessing allelopathic potential in crop germplasm.

III. Key Issues in Designing Screening Bioassays A. GENERAL REQUIREMENTS The single most important factor in the search for allelopathic crop accessions is the convenience and reliability of a screening bioassay. Screening bioassays must be inexpensive,

EVALUATION OF CROP ALLELOPATHIC POTENTIAL

Table

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I

Screening methods in the evaluation of crop allelopathic potential Test species Screening method

Donor

Receiver/Chemical

References

Extract screening

Triticumaestivum

Triticum aestivum

Ipomaea batatas

Lolium rigidum Cyperus esculentus

Guenzi et al., 1967; Kimber, 1967 Wu et al., 1998 Harrison & Peterson, 1986 Harrison & Peterson, 1986 Peters & Mohammed Zam, 1981 Peters & Mohammed Zam, 1981 Kim & Shin, 1998; Kim et al., 1999

Medicago sativa Festuca arundinacea

Lotus corniculatus Trifolium pratense

Oryza sativa Seedling screening Pot screening method Cucumber sativus Plant box method

Avena spp. Oryza sativa

Echinochloa crus-galli

Brassica hirta Panicum miliaceum Brassica kaber Lactuca sativa Echinochloa crus-galli lndigofera hirsuta

Triticum speltoides

Avena fatua Sisymbrium orientate

Relay seeding technique

Oryza sativa

Echinochloa crus-galli

Equal compartment agar method Field screening

Triticum aestium

Lolium rigidum

Oryza sativa

Hetheranthera limosa Ammannia coccinea Cyperus difformis Echinochloa crus-galli

Chemical screening

Triticum aestivum

DIMBOA ~

Triticum aestivum

Phenolic acids

Hordeum spp.

Graminea

Avena spp. Sorghum bicolor

Hordenine~ Scopoletina Sorgoleonea

Putnam & Duke, 1974 Putnam & Duke, 1974 Fay & Duke, 1977 Fujii, 1992; Maneechote & Krasaesindhu, 1996 Maneechote & Krasaesindhu, 1996 Maneechote & Krasaesindhu, 1996 Hashem and Adkins, 1998 Hashem and Adkins, 1998 Navarez & Olofsdotter, 1996 Olofsdotter & Navarez, 1996 Wu et al., 2000a, 2000b Dilday et al., 1994, 1998 Dilday et al., 1994, 1998 Hassan et al., 1998 Olofsdotter & Navarez, 1996 Hassan et al., 1998 Kim & Shin, 1998; Kim et al., 1999 Niemeyer, 1988 Copaja et al., 1991 Nicol et al., 1992 Wu, 1999 Wu et al., 2000c, 2000d, 2001 Hanson et al., 1981; Lovett & Hoult, 1992 Lovett et al., 1994 Fay & Duke, 1977 Nimbal et al., 1996

a D I M B O A = 2,4-dihydroxy-7-methoxy-1,4-benzoxazin-3-one; gramine = N,N-dimethyl-3-aminomethylindole; hordenine = 4-(2-dimethylaminoethyl)phenol; scopoletin = 6-methoxy-7hydroxycoumarin; sorgoleone = 2-hydroxy-5-methoxy-3-[(8"Z,11 "Z)-8",l 1 ",14"-pentadecatrienyl]-pbenzoquinone.

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rapid, and easy to operate, have broad application to numerous target species, be reproducible and statistically valid, and require a limited amount of time and space. Bioassays should also be sensitive to detect the differences of allelopathic activity between accessions. No single bioassay meets all of these requirements, so a combination of different bioassays is, undoubtedly, the best approach to screening. However, financial constraints usually force the choice of one or only a few. B. SEPARATION OF ALLELOPATHYFROM COMPETITION It may not matter what is causing the suppression of weeds in the field, but it is very important to be able to distinguish allelopathy from competition in order to manipulate the allelopathic potential of a crop species. Allelopathy and competition occur simultaneously in the field where crop plants often grow together with the weeds, and it is extraordinarily difficult, if not impossible, to separate these mechanisms of interference at the field level (Inderjit & Del Moral, 1997). The complexity of allelopathic interactions complicates the choice of an appropriate bioassay (Romeo & Weidenhamer, 1999); as a result, few bioassays have adequately addressed the separation of allelopathic interactions from other plant-plant interactions (Elakovich, 1999). Progress is needed in bioassay methodology to distinguish allelopathy from other interference mechanisms (Fuerst & Putnam, 1983; Weidenhamer, 1996; Romeo & Weidenhamer, 1999). Experiments can be conducted under controlled conditions to understand some particular aspect(s) of allelopathy (Foy, 1999), and several bioassays have been developed to separate allelopathy from competition (Liu & Lovett, 1993; Wu et al., 2000a) C. BIOASSAY SPECIES OF INTEREST Many species are used in bioassays to indicate allelopathic activity (Shilling & Yoshikawa, 1987). Some standard indicator species, such as lettuce (Lactuca sativa), radish (Raphanus sativa), and duckweed (Lemna minor), have been recommended for the preliminary testing of allelopathic activity because of their availability and high sensitivity to allelopathic actions (Putnam et al., 1983; Leather & Einhellig, 1986; Fujii, 1992). Romeo and Weidenhamer (1999), however, argued that these indicator species are not involved in the actual allelopathic association and are of no agronomic importance as weeds. Crop allelopathic activity measured on these indicator species may not truly represent the response of a particular weed species. It has been demonstrated that test species and biotypes within species differ significantly in their susceptibility to an allelopathic source (An et al., 1997). If the purpose of a bioassay is to identify ecologically meaningful interactions, then use of artificially sensitive species should be avoided (Inderjit & Dakshini, 1995; Foy, 1999). Bioassays should ideally be conducted with plants species naturally occurring or cultivated in association with allelopathic plants. The weeds of interest should be included as the receiver species (Wu et al., 2000a). Selection of several test species, at least in laboratory bioassays, should give more meaningful results (Foy, 1999). D. PREGERMINATIONOF BIOASSAY SPECIES The use of pregerminated seeds is preferred to the use of nongerminated seeds due to the reduction in experimental error (Wardle et al., 1993; Ben-Hammouda et al., 1995; Romeo & Weidenhamer, 1999; Wu et al., 2000a). The measurement of radicle elongation is commonly

EVALUATION OF CROP ALLELOPATH1CPOTENTIAL

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used to determine allelopathic activity; however, interpretation of such data requires some caution, because it may be confounded by delays in germination. Also, seed dormancy of many wild species can result in varied speed of germination (Romeo & Weidenhamer, 1999). Olofsdotter et al. (1995) argued that weed seeds germinated unevenly, resulting in a large variation of radicle elongation measurements. Wardle et al. (1993) found that pregermination of the seeds allows selection of uniformly germinated seeds for more sensitive elongation measurements. The sowing of pregerminated seeds also guarantees constant densities of both donor and receiver seedlings required by the experimental design and data analysis. Although crop seeds usually germinate more uniformly than do weed seeds, the pregermination of crop seeds is also recommended (Wu et al., 2000a). E. DENSITIESOF DONORAND RECEIVERSPECIES Manipulating seedling densities can result in changes in concentration effects. Weidenhamer et al. (1987, 1989) and Thijs et al. (1994) found that phytotoxic effects in both laboratory bioassays and greenhouse growth studies depend on both receiver density and concentration. They observed that phytotoxicity of known toxins decreased as the plant density of receiver species increased. They attributed this decline to a reduced availability (i.e., dilution effect) of toxins to individual plants as the density of receiver species increased. On the other hand, the density of donor species is another aspect to be considered. Wu et al. (2000a) found that wheat-seedling allelopathy on the inhibition of root growth of annual ryegrass (receiver) was dependent upon seedling density of donor wheat plants. The inhibition was enhanced as wheat-seedling density increased. The response of root length of ryegrass as a function of wheat seedling density was fitted into a logistic regression model (Y = 59.00/[ 1 + EXP {-2"(-1.0496 - 3.2789*log(X/10))} ] + 6.57, r 2= 0.998). As wheat-seedling density increased, the active concentration ofallelochemicals exuded by wheat seedlings was presumed to increase accordingly, resulting in greater inhibition on the root growth of ryegrass. It is therefore of importance that the density of both donor and receiver species be taken into account when designing bioassays. Densities of bioassay species similar to field situations should be given preference. F. SIMULATIONOF NATURALRELEASEOF ALLELOCHEMICALS Screening bioassays should simulate the natural release of allelochemicals from the living donor plants into the growth medium (Wu et al., 2000a) and simulate field conditions as much as possible (Williamson, 1990; Foy, 1999). Unlike herbicides, which are applied at a given rate and subsequently decrease in concentration with time, allelochemicals are likely continually or sporadically being released by living plants and can be removed from the soil solution as a result of plant uptake, microbial degradation, and adsorption (Weidenhamer, 1996). Thus it is likely that the toxicity of allelochemicals is a function of both concentration (static availability at a given point in time) and flux rate (dynamic availability based on the total amount of chemicals moving in and out of the system over a period of time) (Williamson & Weidenhamer, 1990). Chronic exposure to a low dose of an allelopathic agent may have deleterious effects on plant growth (Blum & Rebbeck, 1989). It is essential to ensure the realistic cogrowtb of two bioassay species during the experiment period. The removal of donor plants followed by the planting of receiver species should be avoided, although the competition and allelopathy can be separated in such a manner. The removal of donor plants stops the constant release of allelochemicals, thereby underestimating the allelopathic effects of the donor species.

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The advantages and disadvantages of some of the commonly used screening bioassays (see Table I) are discussed below.

IV. Aqueous Extract Screening Bioassays under Laboratory Conditions Aqueous extract bioassays have been widely employed to evaluate residue allelopathy of a suspected donor species. In general, extract bioassays are conducted in petri dishes by placing seeds of receiver species on substrata (often filter paper) moistened with aqueous plant extracts of donor species (Pederson, 1986; Ben-Hammouda et al., 1995; Wu et al., 1998). Dishes receiving distilled water are included as control. The petri dishes are placed in an incubator under light or dark conditions for a given period of time (usually two to seven days), and germination and radicle elongation are measured. Extract bioassays are simple, rapid, and straightforward and can be used preliminarily to determine residue allelopathy for weed control or impacts of weed residues on crops. Many replications can be undertaken to satisfy statistical analysis. The varietal difference in residue allelopathy can be easily determined (Wu et al., 1998). However, extract bioassays have received strong criticism (Chou & Muller, 1972; Stowe, 1979; Inderjit & Dakshini, 1995). The process involved in the preparation of aqueous extracts could result in the release of certain enzymes, salts, amino acids, and nitrogen compounds, all of which may not be released under natural circumstances (Chou & Muller, 1972). Bioassays using ground plant material are thus of little ecological relevance, for the extraction procedure causes qualitative and quantitative changes in the phytochemical profile (Inderjit & Dakshini, 1995). In addition, plant-extract bioassays with filter paper often give inconsistent results due to nonuniform wetting or localized swelling of the filter paper (Pederson, 1986). Extract-agar bioassays were developed to overcome these problems (Pederson, 1986; Ben-Hammouda et al., 1995). It is therefore essential that extract bioassays be followed by carefully designed bioassays using crop residues under greenhouse and field conditions. Excellent reviews are available on such crop-residuesoil-amending bioassays (Blum, 1999; Foy, 1999; Inderjit & Dakshini, 1999).

V. Seedling Screening Bioassays under Controlled Environments Because of the criticisms of aqueous extract bioassays (Chou & Muller, 1972; Stowe, 1979), screening bioassays using intact plant seedlings have been developed. Crop plants at the seedling stage have been used in allelopathy studies (Wu et al., 2000a). The interaction between crop and weed is critical at the seedling stage. If a weed species can be allelopathically suppressed by crop plants during the seedling-establishment period, crop plants will gain an advantage over the weeds. A number of screening bioassays have been designed and employed to assess crop-seedling allelopathy. A. POT SCREENING Pot screening was used to evaluate 526 accessions of cucumber (Cucumbersativus) for the allelopathic potential against a broad-leaved weed, white mustard (Brassica hirta), and a narrow-leaved weed, proso millet (Panicum miliaceum) (Putnam & Duke, 1974). This pot screening included three stages.' Stage 1 was the preliminary and unreplicated screening, where 4 seeds of each cucumber accession (donor) and 10 seeds of each receiver weed species were planted together in 7.6-cm pots containing 270 g of quartz sand in a controlledenvironment chamber. Each weed was also grown in the absence of cucumbers as a control. Each pot received an initial watering of 80 ml of half-strength Hoagland's solution, which


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was supplied again when necessary. Weed numbers and shoot fresh weights were recorded after 10 days of planting. Stage 2 was where potential accessions from the initial experiments were selected and confirmed in replicated experiments. At stage 3, strongly allelopathic and nonallelopathic accessions were selected to demonstrate allelopathy on receiver weeds in the absence of cucumbers. Under sterile growth conditions, pots received 80 ml of nutrient solution initially and were leached with an additional 120 ml of solution on days 4, 6 and 8. Tenmilliliter portions of the leachates collected on each day were applied to eight replications of receiver weeds (10 seeds per lot) planted in 30 cm3 of quartz sand in compartmentalized plastic trays. Weed plants receiving nutrient solution at the same time intervals were used as controls. Weed-shoot flesh weights were recorded after 10 days of planting. This screening method, with three stages in sequence, has several advantages. A large number of crop accessions can be screened in stage 1 with unreplicated experiments, which would greatly reduce the time and space for a large screening project. Reliable results are obtained in the replicated experiments in stage 2. The collection of root leachates in stage 3 is an important step in order to substantiate donor-seedling allelopathy without the influences of competition and microbial involvement. However, seeds of bioassay species are not pregerminated in this method, and this could result in varied densities of both donor and receiver species across accessions and pots. It has been found that allelopathic activity is density dependant (Weidenhamer et al., 1987, 1989; Thijs et al., 1994; Wu et al., 2000a). Pregerminated seeds are preferred in allelopathy studies (Wu et al., 2000a). B. PLANTBOX METHOD Fuj ii (1992) developed a "plant box method" (PBM) to assess seedling allelopathy of 189 rice accessions on lettuce. The PBM requires one to two months to nurse the rice seedlings in sand culture with nutrient solution. Rice seedlings are transplanted into a cellulose dialysis tube (CDT) filled with 0.5% water agar medium and placed in the center of one side of a sterilized square plastic box filled with 11 of 0.5% water agar. The CDT is held by a specially engineered holding frame. Surface-sterilized seeds of receiver species are planted onto the agar surface in rows concentric to the CDT. The plant box is then wrapped with a transparent film to reduce evaporation and contamination. Radicle length of receiver seedlings is recorded after a given period of growth. This bioassay was also employed to assess upland and wild rice against Echinochloa crus-galli, Indigofera hirsuta, and Lactuca sativa (Maneechote & Krasaesindhu, 1996) and Triticum spp against wild oat (Avenafatua) and Indian hedge mustard (Sisymbrium orientate) (Hashem & Adkins, 1998). This PBM bioassay can clearly observe the inhibitory effect of the donor species on the root growth of the receiver species over time. The allelochemicals produced by the donor seedlings are able to diffuse through the CDT into the agar medium to affect the growth of receiver species (Hashem & Adkins, 1998). The inhibition on receiver plants is gradually reduced when the distance between donor and receiver plants increases. However, a large box is required with this bioassay, which is a problem when a large screening program is to be conducted. The bioassay is also time consuming, requiring the pregrowth of donor seedlings up to two months. This bioassay is therefore not suitable for a mass screening program (Navarez & Olofsdotter, 1996). C. RELAY SEEDINGTECHNIQUE To overcome the problems associated with the PBM, Navarez and Olofsdotter (1996) developed the "relay seeding technique" (RST) for the evaluation of rice-seedling allelopathy.

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The RST involves seeding receiver plant species with 7-day-old donor seedlings and growing them together for 10 days in a well-watered petri dish enclosed in a transparent growth box containing distilled water. The petri dish is lined with one layer of circular filter paper and also connected to a bridge filter paper strip for supplying water from the growth box. Donor seeds are sown in rows and covered with perlite to prevent donor-plant roots from arching. The growth box is covered with a transparent plastic lid, and distilled water is added when necessary. Shoot and root lengths of receiver plants are recorded after 10 days of planting. The disadvantages of the RST are that roots of bioassay species may adhere to the filter paper and break when detached, resulting in errors in the measurement of root length / dry matter (Haugland & Brandsaeter, 1996; Wu et al., 2000a). Microbial contamination is also a problem. Although RST successfully eliminates competition for water and nutrients, competition for light is still present (Navarez & Olofsdotter, 1996). Furthermore, the trimming of the excessive sho'ot growth of donor seedlings may trigger the reallocation and release of the allelochemicals (Wu et al., 2000a), which is a characteristic response when plants are stressed (Rice, 1984). D. EQUAL COMPARTMENT AGAR METHOD Recently, a laboratory screening bioassay, the "equal compartment agar method" (ECAM), was developed and employed to assess wheat-seedling allelopathy on annual ryegrass (Lolium rigidum) (Wu et al., 2000a, 2000b). Briefly, pregerminated donor seeds are uniformly selected and aseptically sown on an agar surface in three rows in one-half of a glass beaker prefilled with water agar (nutrient free). The beaker is wrapped with a piece of parafilm and placed in a controlled-growth cabinet. After the growth of wheat seedlings for seven days, pregerminated seeds of receiver-weed species are then sown on the other half of the agar surface in three rows. A piece of pre-autoclaved white paperboard is inserted across the center and down the middle of the beaker, with the lower edge of the paperboard kept I cm above the agar surface. The entire beaker is thereby divided into two equal compartments occupied separately by donor and receiver seedlings. The beaker is again wrapped with parafilm and placed back in the growth cabinet for continuous growth of 10 more days. After 10 days of cogrowth of weed with crop seedlings in the growth cabinet, both wheat and weed seedlings are harvested for the measurements of growth parameters, such as root and shoot length. The ECAM is derived from PBM and RST, with the combined advantages of these two techniques and other additional benefits. ECAM provides a rapid, simple, and inexpensive procedure for the initial screening of the allelopathic potential of a crop against a target weed species under laboratory conditions (Wu et al., 2000a). The paperboard used in the bioassay successfully precludes resource competition between donor and receiver seedlings. Roots of donor plants were distributed throughout the bottom of the beaker and fully interacted with receiver plants. Allelochemicals released from the living donor roots were diffused into the agar medium to affect the root growth of the receiver plants. The continuous growth of donor seedlings ensured a constant release and accumulation of allelochemicals into the agar medium, which simulated the continuous growth in nature. The setup of this bioassay is aseptically managed so that microbial involvement is also avoided. The screening results obtained could therefore be attributed solely to the allelopathic effect of donor seedlings (Wu et al., 2000a). Intact receiver seedlings can be easily removed from the agar for measurements and further chemical analysis. By such a design, the water agar can also be collected for further chemical component analysis. Responsible allelochemicals present in the water agar can be easily extracted and recovered by organic solvents. One additional benefit of ECAM is that it allows a clear view of the allelopathic effects on the root development as the experiment progresses.


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VI. Field Screening Field screening has been extensively used to assess rice allelopathy on aquatic weeds (Dilday et al., 1994, 1998; Olofsdotter & Navarez, 1996; Hassan et al., 1998; Kim & Shin, 1998; Kim et al., 1999). In the United States alone, approximately 12,000 accessions on Hetheranthera limosa (Sw.) Willd. and 5000 accessions on Ammannia coccinea Rottb. have been evaluated (Dilday et al., 1994, 1998). Generally, five to seven seeds of each donor accession are sown in a 0.75 x 0.75 m grid with two replications. Receiver weeds are naturally and uniformly infected. Donor allelopathic activity is recorded at the panicle initiation stage by measuring: the weed-free radius from the base of the rice plants to the outermost edge of the area of activity, defined as the area around the rice plant where no weed growth appears or a reduced weed stand is present; and the percentage of weed control in the radial area compared with a control with slight or no allelopathic activity. Strong allelopathic accessions are selected and confirmed with three or four replications. It is essential to demonstrate allelopathic activity in the field, However, it would be very difficult to eliminate the influence of competition when assessing the allelopathic potential of a crop in the field (Wu et al., 2000a). Olofsdotter & Navarez (1996) claimed that weed reduction in the rice field could be due to rice/weed competition and to allelopathy. The contribution of allelopathy to the observed inhibition of weeds could not be easily assessed in the field. Laboratory bioassays can be designed to eliminate all possible alternative interferences in a controlled environment, so that complex field conditions can be varied one at a time to search for mechanism interactions (Inderjit & Dakshini, 1995). Olofsdotter & Navarez (1996) proposed that field screening be followed by laboratory bioassays in order to obtain reliable results. In addition, field screening for the allelopathic trait against weeds is time consuming, requires large amounts of space, and can be costly (Mattice et al., 1999).

VII. Chemical Screening Crop accessions containing high levels of atlelochemicals are more likely to possess strong allelopathic capability (Wu et al., 1999, 2000d, 2001). Direct chemical screening can differentiate between allelopathic and nonallelopathic accessions. Following an initial chemical screening, potential accessions are selected and screened further in the greenhouse or the field (Mattice et al., 1999), thereby reducing the amount of time and space required in other screening bioassays. Results from chemical screening also help to explain the experimental data of other bioassays and indicate the likely genetic control of the differential allelochemical production between crop varieties (Wu et al., 1999). One of the disadvantages of chemical screening is that it requires the preknowledge of responsible allelopathic agents associated with the suspected donor species so that the chemical screening of particular compound(s) can be undertaken (Fay & Duke, 1977; Wu et al., 2000c, 2000d, 2001). It also depends on the availability of analytical expertise and expensive facilities. Direct chemical screening of a large collection of crop accessions can therefore be costly. It is always important to eliminate the obvious alternate explanations before embarking on months, possibly years, of laboratory chemical work and field tests (Romeo & Weidenhamer, 1999). It is essential that chemical screening be combined with other screening bioassays under a controlled environment or in the field. V I I I . Conclusions A number of bioassays are available to evaluate crop allelopathic potential, the selection of which are dependant upon the research objective, bioassay species, the availability ofana-

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lytical instruments, and funding. In their discussion of bioassays in the study of allelopathy, Leather & Einhellig (1985: 142) concluded: "There is no perfect hioassay that will meet all the requirements for detecting bioactivity of allelochemicals and it would be prudent to use several for each case of suspected allelopathic interactions." The combination of several screening bioassays in sequence is therefore essential in order to establish conclusive proof of crop allelopathy on weeds. Evaluating thousands of crop accessions for allelopathic activity in weed suppression is extremely expensive. A specific approach from biological screening under laboratory conditions to greenhouse experiment and then to further field testing is proposed in order to reduce the requirements for time, space, and labor (Wu et al., 2000b). After the preliminary screening by laboratory bioassays, a small proportion of promising genotypes with strong allelopathic activity could be selected for greenhouse and further field testing in order to verify laboratory results, thereby substantially reducing the requirements for time, labor, and space in a large screening project. Screening bioassays that facilitate the extraction and characterization of allelochemicals are needed for understanding the chemical and later genetic basis for the observed differential allelopathic activity between crop accessions. More chemical screening research is expected in the course of finding chemical explanations for the differential allelopathic effects between accessions. Bioassays using agar as a growth medium are very helpful in the detection and identification of allelochemicals (Pederson, 1986; Wu et al., 2000a). To date, all cases of alleged allelopathy appear to involve a complex of chemicals that interact synergistically (Rasmussen et al., 1977; Einhellig, 1986; Blum, 1996). Preference must therefore be given to the screening of multiple allelopathic agents (Wu et al., 2000d, 2001). Selection of crop varieties based on their allelopathic weed-suppressing capability is perhaps one of the best strategies for taking advantage of allelopathy (Wu et al., 1999). An allelopathic variety is likely to be able to suppress weeds by natural exudation of hioactive allelochemicals into their vicinity, thereby reducing dependence upon synthetic herbicides. Modem DNA technology makes it easier to locate genetic markers associated with allelopathic activity. However, much more screening work still needs to be done before any breeding programs can be initiated to develop crop cultivars with allelopathic potential. IX. A c k n o w l e d g m e n t s

The authors are thankful for the financial support from the Grains Research & Development Corporation of Australia. X. Literature Cited

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