Allyl Isothiocyanate Release and the Allelopthic Potential of Brassica Napus

Allyl Isothiocyanate Release and the Allelopathic Potential of Brassica napus (Brassicaceae)Author(s): Devi N. Choesin and Ralph E. J. BoernerSource: American Journal of Botany, Vol. 78, No. 8 (Aug., 1991), pp. 1083-1090Published by: Botanical Society of AmericaStable URL: http://www.jstor.org/stable/2444897 .

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American Journal of Botany 78(8): 1083-1090. 199 1.

ALLYL ISOTHIOCYANATE RELEASE AND THE ALLELOPATHIC POTENTIAL OF BRASSICA NAPUS (BRASSICACEAE)l

DEVI N. CHOESIN AND RALPH E. J. BOERNER2 Department of Plant Biology, Ohio State University, 1735 Neil Avenue, Columbus, Ohio 43210

The allelopathic potential of Brassica species has been attributed to release of the mustard oil glycosides which they produce in large quantities. Upon hydrolysis, these glucosinolates yield isothiocyanates, compounds with strong antibiotic properties. The objective of this study was to assess whether Brassica napus, a common and widespread crop and weed crucifer, is capable of allelopathic interference, and if so, whether its glycoside derivative, allyl isothiocyanate (Al), is capable of producing this interference. Wild type and low glucosinolate-mutant B. napus were grown in low organic content soil under greenhouse conditions, and Al release into soil was monitored. Most plants released low levels of Al, though approximately 10% released much higher levels. Wild type plants released more Al than mutants. Growth of the target species, Medicago sativa, was not affected by additions of Al to soils at concentrations equal to the median and 95% quantile from the B. napus soils. In replacement series experiments, the two B. napus genotypes suppressed growth of M. sativa equally despite differences in Al release rate. In an intraspecific replacement series experiment, the two B. napus genotypes were equal competitors. Under our experimental conditions, B. napus showed no indication of being allelopathic, and Al concentrations typical of soils around B. napus plants did not inhibit target plants.

Allelopathy has been implicated as a mechanism important in determining the compo- sition and spatial pattern of plants in communities for almost half a century. The term, as originally proposed by Molisch in 1937, was intended to include both stimulatory and inhibitory interactions among plants and mi- crobes (Putnam and Tang, 1986). The more restricted, current usage considers allelopathy as the inhibitory effect of a compound being added to the environment. It is thus separated from competition, which involves the removal or reduction of some environmental resource (Rice, 1984).

Unequivocal instances of allelopathy (e.g., Weidenhamer, Hartnett, and Romeo, 1989) have proven to be difficult to document because it is both difficult to quantify the nature of the plant-allelochemical-plant interaction well and to clearly separate allelopathy from competition. For allelopathy to be proven unequivocally, one must demonstrate 1) that some compound is being released into the environ-

' Received for publication 20 December 1990; revision accepted 28 March 1991.

The authors thanks Dan Crawford, Allison Snow, Peter Curtis, and Roland Seymour for constructive comments on earlier drafts; Bruce Williamson and Jon Keeley for constructive reviews; and Amy Scherzer, Kathleen Harris, Jennifer Brinkman, Sandy Whisler, Doug Beak, and Jim Strittholt for laboratory and greenhouse assistance. This work was supported by a World Bank XVII/Indonesian Second University Development Fellowship to DNC.

2 Author for correspondence.

ment and has significant residence time there, 2) that the compound is available to the target plant for uptake, and 3) that the compound is toxic to the target plant at typical concentrations and in realistic environmental conditions.

Members of the Brassicaceae have frequent- ly been cited as being potentially allelopathic (Chew, 1988). Both Brassica nigra (Muller, 1969; Bell and Muller, 1973) and B. napus (Martin and Rademacher, 1960) have been shown to inhibit the germination and growth of other plants, though the precise inhibitory mechanism(s) is not yet clear. Brassica spp. synthesize large quantities of glucosinolates, which, upon hydrolysis, produce compounds with active antibiotic properties, most notably the isothiocyanates (mustard oils). The glucosinolates are stored in vacuoles and are converted to the active isothiocyanates by the action of the cell wall-bound enzyme myrosinase (Chew, 1988). Thus, most of the hydrolysis occurs after mechanical damage to the Brassica tissue results in the combination of the sub- strate and enzyme; however, low and variable levels of isothiocyanates are found in. intact tissues as well (Tang, 1971; Crowell, 1986).

Crucifer isothiocyanates have adverse effects on growth and survivorship of bacteria, fungi, mammats, and insects (Feeny and Rosenberry, 1982), and are, therefore, likely candidate compounds for allelopathic activity. Allyl glucosinolate (AG or sinigrin) is among the most abun-

1083



1084 AMERICAN JOURNAL OF BOTANY [Vol. 78

dant glucosinolates in many Brassica spp.; upon hydrolysis AG yields allyl isothiocyanate (AI), a compound already implicated as an allelochemical (Muller, 1969). Thus, though Bras- sica spp. are widely thought to be allelopathic, and the release of mustard oils from roots or detrital material may constitute the allelopathic mechanism (Bell and Muller, 1973), data that unequivocally demonstrate either one are still lacking.

The overall goal of this study was to inves- tigate the allelopathic potential of Brassica napus, a widespread and common crop and weed crucifer. The specific questions to be answered were: 1) How much AI is released by B. napus roots, and what is the residence time of AI in soil? 2) Are the concentrations ofAI in B. napus rhizosphere soil sufficient to produce significant inhibition of growth of neighboring plants? If so, how is this inhibition affected by the density of neighboring plants? and 3) Do wild type and low glucosinolate mutants ofB. napus exhibit the same effect on neighboring plants?

MATERIALS AND METHODS

AI release by Brassica napus -Wild type Brassica napus seeds were obtained from ag- ricultural weed populations in Idaho (F & J Seed Service, Woodstock, IL). Seeds of a low glucosinolate mutant, B. napus var. Westar, were obtained from the Agriculture Canada Research Station, Saskatoon, Saskatchewan. Glucosinolate content of the Westar mutants averaged 10.4 Amol g-1 (J. J. Capcara, personal communication), compared to a range of 186- 524 ,umolbg-1 in other genotype mixes of B. napus (Glenn, Chew, and Williams, 1988).

Seeds were planted in 1,300-cm3 pots of sterile 4:1 (v:v) sand: perlite mixture and watered with deionized water as needed. After the first true leaves emerged, a soil microbial mix was introduced into each pot by pipetting 30 ml of a forest soil slurry over the soil surface.

Forty plants of each genotype were grown for 10 wk under ambient greenhouse conditions. Nutrients were supplied by feeding plants weekly with Ruakura Solution (Smith, John- ston, and Comforth, 1983), and the pots were randomized weekly to eliminate position effects. When the plants were harvested at the end of 10 wk, > 50% of the plants were in flower and >40% bore mature fruit. Each plant was separated into root and shoot, dried at 70 C for 48 hr, and weighed. Soil from each pot was collected and stored at -5 C for later AI extraction. A complete characterization of the time course of growth and carbon allocation of the two genotypes and the effects of varying

phosphorus and sulfur supply rate are given by Choesin (1990).

Recovery of AI added to soil-A solution of 30 mg -l1 Al in hexane was mixed thoroughly into the same sand: perlite mixture as above, divided into 1,300-cm3 pots, and allowed to sit open in the greenhouse. After 10 hr, a microbial mixture was added to each pot as above. Pots were watered daily with deionized water and randomized weekly. Samples were harvested 1, 2, 6, 12, 24, and 36 hr, and 1, 2, 3, 4, 5, and 6 wk after addition of the microbial mix. Soils were stored for later extraction as above.

Analysis ofAI in soil-Two 30-g subsamples were taken from each soil sample. AI was extracted from one subsample with 20 ml of hexane. AG was extracted from the second by adding 15 ml of deionized water and then shak- ing for 24 hr at 150 rpm on a shaker table (Crowell, 1986). The AG in the water extract was then hydrolyzed to AI with myrosinase prepared from yellow mustard flour (Rodman, 1978), then transferred to hexane by repeated washings in a separatory funnel (methods fol- low Crowell, 1986). The final AI content of a soil sample was calculated as the sum of the AI from the hexane- and water-extracted subsamples. All hexane extracts were stored at 0 C.

AI concentrations were determined by gas chromatography using a Varian 3500 chromatograph equipped with a TSD NPD detector and a 60-m x 0.25-mm fused-silica column. Retention time for AI ranged from 4.42 to 5.09 min. Specific gas chromatograph operating pa- rameters are given by Choesin (1990). Stan- dard AI preparations were used to verify peak identity and construct standard curves.

Because of a lack -of both normality and ho- mogeneity of variances among sample groups, differences in AI concentrations were analyzed nonparametrically: Wilcoxon two-sample test for total AI in soil and Kruskal-Wallis H test for AI release per gram root mass (Sokal and Rohlf, 1969).

Responses of Medicago sativa to AI in soil- Medicago sativa plants were grown in a 4 x 5 factorial design, with five M. sativa densities (2, 4, 8, 16, or 32 plants per 1,300-cm3 pot) and four soil amendments (20 jg kg-1 AI added in hexane, 1 95 jg kg- I AI in hexane, hexane alone, or water). Each plant density x soil amendment combination was replicated eight times. The two Al concentrations represented the median and 95% quantile values of AI from the initial growth/AI release experiments. As



August 1991] CHOESIN AND BOERNER-IS BRASSICA NAPUS ALLELOPATHIC? 1085

AI was added to the soil using a hexane vehicle, both a hexane and a water control were nec- essary. All pots were given a forest soil slurry as above.

Plants were grown under greenhouse conditions for 12 wk and fed twice weekly with 50 ml of Ruakura Solution. Plants were harvested and weighed as above, and mass data were analyzed by analysis of variance and linear regression.

Replacement series experiments-To com- pare the interference effects of wild type and low glucosinolate mutant Brassica napus on Medicago sativa, the relative proportions of each species were varied while total plant density was held constant. Two interspecific replacement series (B. napus wild type vs. M. sativa, and B. napus mutant vs. M. sativa) and one intraspecific series (B. napus mutant vs. B. napus wild type) were established.

A large number of seeds of both participants was planted in 12,800-cm3 pots of 4:1 (v:v) sand: perlite. After the appearance of the first true leaves, the seedlings were thinned to a total of 36 evenly spaced plants per pot, of which only the 16 center plants were eventually harvested. Each series consisted of five proportional combinations (100% species A; 75% species A: 25% species B; 50% of each species; 25% species A: 75% species B; 100% species B) with five replications of each combination per series.

Pots were inoculated with 150 ml of forest soil slurry, watered daily, and fed twice weekly with 100 ml of Ruakura Solution. Plants were harvested and dried as above 10 wk after thin- ning.

Because comparing the performance of two different species in terms of absolute yield is questionable, data were analyzed both as absolute yield and as relative yield totals (RYT) (Dekker, Meggitt, and Putnam, 1983), where the relative yield of a species in a given mixture is the ratio of its yield in that proportional mixture to its yield in its pure stand.

RESULTS

AI release by Brassica napus -Total AI concentration in soil around wild type plants was significantly greater than that around mutant plants (wild type: 60.0 ? 26.5 jggkg-' vs. mutant: 23.5 ? 4.2 juggkg-', P < 0.025). There was, however, a wide range in soil AI concentration detected (2.0-558.3 jug kg-'), and the frequency distribution was strongly skewed to- ward the lower concentrations (Fig. IA).

To allow for minor differences in plant mass,

we converted AI concentrations to a per gram root mass basis. The same skew in distribution was apparent (Fig. 1 B) and suggested that the B. napus plants typically released AI at low rates but were capable of producing greater concentrations under certain conditions. AI release on a root mass basis ranged from 1.6 to 524.0 ,g/g root mass. Wild type plants released marginally larger amounts of AI on a root mass basis than did mutants (wild type: 144.4 + 34.5 ,ug/g vs. mutant: 96.2 ? 25.0 ,ug/g, P < 0.07).

Recovery of AI added to soil-Soil AI concentration decreased rapidly over the first 10- 12 hr after addition (Fig. 2). This rapid, initial loss was probably due to volatilization; the greenhouse area around the pots smelled strongly of mustard oil through that period. Only 322.1 juggkg-' AI of the original 30 mg kg-1 was present in samples extracted 11 hr after AI addition. Over the next 2 wk, extract- able AI concentrations varied between 15.3 and 177.8 jg kg-1 with no clear temporal pattern. From 2 to 6 wk, AI concentrations remained relatively constant around 30 ig kg-'. The best-fit first order decay curve for the period from 12 hr to 6 wk after AI addition was: ln Concentration = 1.942-0.00054 (time in hr), r = -0.466, r2 = 0.218, P < 0.05. Solving this equation gives an estimate of AI loss during this 6-wk period of 1.01 jg kg-1 d-1. This pattern suggested that most AI is lost by volatilization soon after release, and that microbial degradation of the small percentage of added AI remaining in the soil was relatively slow.

Responses of Medicago sativa to AI in soil- Mean M. sativa mass decreased with increasing plant density in all four soil treatments (Fig. 3). However, there were no significant differences in the slope of the log-log regression line among soil treatments. Neither regression of the density patterns nor analysis of covariance with density as the covariate demonstrated any effect of AI at either 20 or 195 jug-kg-' on M. sativa growth.

Replacement series: Brassica napus genotypes vs. Medicago sativa -There were no significant differences in the combined biomass of the two species (i.e., RYT) between the Brassica napus mutant: Medicago sativa and the B. napus wild type: M. sativa replacement series (Fig. 4). Re- placement series diagrams for these two series indicated that both B. napus genotypes were capable of suppressing M. sativa (Fig. 4). There were no significant differences between the two B. napus genotypes in biomass or relative yield




20 (

20

IL 4

0 2 20 40 60 80 100"" 100 150 200 250 300 +

Soil Al Concentration

Mutant

nr0 1=E Wild Type

8

0 6 0

b. 2 U..

2 20 40 60 80 100 100 150 200 250 300 +

Al Release Fig. 1. Frequency distributions for (A) total soil Al concentration (,g gkg-') and (B) Al concentrations corrected for

plant root mass (g g-1). N = 39 for each.

at any density combination. Similarly, there were no significant differences in M. sativa yield between the replacement series with the two different B. napus genotypes in any B. napus: M. sativa proportional combination (Table 1).

Replacement series: Brassica napus wild type vs. mutant-The replacement series diagram for the two genotypes indicated a positive com- plementation in which the performance of mixtures exceeded the average of the constit- uent genotypes in pure stands (Fig. 5). The RYT of the mixtures involving the two genotypes averaged 1.186, again suggesting a yield advantage was obtained in mixtures.

Varying the mixture proportions between the

two genotypes did not affect shoot or root pro- duction in either genotype (Table 2). There were no indications from either relative or absolute yields that there were differences in competitive ability between genotypes.

DISCUSSION Brassicas have long been known to be poor

companion plants in cultivated settings (Chew, 1988) and have been implicated as being allelopathic under natural field conditions (Mul- ler, 1969; Bell and Muller, 1973). In annual grasslands of coastal California, for example, Brassica nigra can establish and form pure stands into which other species cannot suc-



August 1991] CHOESIN AND BOERNER-IS BRASSICA NAPUS ALLELOPATHIC? 1087

100 000

c o 10000

- 1000 U

0

- 100

1 &+ . , . ,

0 1 2 3 4 5 6

Time (wk)

Fig. 2. Recovery of Al (tggkg-1) from soil over 6 wk. The Al initially added to the soil is indicated by the open diamond, and the Al remaining in subsequent extractions is indicated by closed diamonds.

cessfully invade (Muller, 1969). Bell and Mul- ler (1973) established that the failure of grasses to colonize these B. nigra stands was not due to edaphic factors, grazing, or resource competition. They erected allelopathy as the most likely mechanism of exclusion, and suggested that release of isothiocyanates from B. nigra litter might constitute the allelochemical mechanism.

For allelopathy to be an important mechanism in determining community structure, the putative allelochemical must be 1) released into the environment in measurable amounts, 2) have significant residence time in the medium into which it is released, and 3) be transported to the target plant. Bell and Muller (1973) detected Al and similar compounds in B. nigra litter leachate and dew, and suggested that it might be transported to target plants through the soil solutions. We have demonstrated an- other pathway by which a Brassica species can introduce AI into the soil environment: direct release from actively growing roots.

We are uncertain about the residence time of AI released into the soil by roots of B. napus. When we mixed AI in hexane into sterile soil, approximately 99% of the AI was lost to volatilization in 10-12 hr. We used hexane as a mixture vehicle because AI is poorly soluble in water. Much of the AI volatilization we observed may, therefore, have been caused by the presence ofthe rapidly volatilizing hexane. The residual AI that remained after the hexane had dissipated was relatively stable in soil inoculated with a natural soil microbial mix: the loss rate from residual volatilization and microbial degradation was approximately 3% per day over

3.3

3.2 - 0

3.1-

3.0-

< 2.9-

Z 2.8 * -l

2.7 -

Lu 2.6-

O 2.5- SLOPE r2

2.4 0 WATER -0.645 0.87 * HEXANE -0.502 0.45 i

2 20ug-kg1Al -0.638 0.73 195 ug-klAl -0.720 0.86

2.2 . I 0.3 0.6 0.9 1.2 1.5

LOG PLANT DENSITY Fig. 3. Relationship of log mean plant mass (mg) and log plant density of Medicago sativa after 12 wk of growth

under four soil treatments. Slopes and coefficients of determination are given for the best fit regression lines.




B.napus WILD TYPE vs M.sativa B. napus MUTANT vs M. sativa 1.2 1.00 0.91 0.96 0.93 1.00 RYT 1.2 1.00 1.19 1.12 1.03 1.00

1.0 1.0

LUA 0.8 LUA 0.8

LUA LUA > 0.6 > 0.6-

-J 0.4 I-I 0.4- LU ILUA

0.2 0.2

o . *0 | * * o,--C * . 0 25 50 75 100 0 25 50 75 100 100 75 50 25 0 100 75 50 25 0

% M. sativa * % M. sativa* O

*4 % B. napus **% B. napus

Fig. 4. Relative yield diagrams for the replacement series involving Medicago sativa vs. Brassica napus (A) Wild Type and (B) Mutant. Relative yield is expressed in relation to the yield of the pure stand of each species. On the horizontal axis, the proportion of B. napus in the mixture decreases to the right and the proportion of M. sativa increases to the right. Relative yield totals (RYT) for the mixtures are given at the top of the figures.

6 wk. As Al is released from growing Brassica slowly and not in a nonpolar solvent, its natural residence time is likely longer than our recovery experiment suggests.

In addition to demonstrating release, persistence, and transport of a putative allelochemical, to unequivocally demonstrate allelopathy one must also demonstrate the ability of the allelochemical to inhibit growth of target

TABLE 1. Mean plant mass (g) ofMedicago sativa grown in replacement series with Brassica napus mutant or wild type plants

Proportion of M. satival

100% 75% 50% 25%

Shoot mass Wild type 0.051 0.014 0.014 0.014

series (0.010) (0.002) (0.001) (0.002) Mutant series 0.051 0.016 0.013 0.014

(0.010) (0.001) (0.002) (0.002) Root mass

Wild type 0.020 0.008 0.009 0.008 series (0.005) (0.001) (0.001) (0.001)

Mutant series 0.020 0.011 0.008 0.009 (0.005) (0.001) (0.001) (0.002)

Total mass Wild type 0.070 0.022 0.022 0.022

series (0.015) (0.002) (0.001) (0.002) Mutant series 0.070 0.026 0.020 0.024

(0.015) (0.002) (0.002) (0.004) a Standard errors of the means of five replicate pots per

series/proportion combination are given in parentheses.

plants under realistic concentrations, growth conditions, and plant densities. We observed no inhibition of Medicago sativa growth when exposed to concentrations of Al similar to those present in B. napus soils at any Medicago den-

B.napus WILD TYPE vs MUTANT 1.2

1.0 *

0.8 _ u/

u1 0.6 -

-0.4 0.4 uJ Ir / ~~~*

02-

0 25 50 75 100 100 75 50 25 0

% Mutant+*

*4 % Wild Type Fig. 5. Relative yield diagram for the replacement se-

ries involving the wild type and mutant genotypes of Bras- sica napus.- Relative yield is expressed in relation to the yield of the pure stand of each genotype. On the horizontal axis, the proportion of B. napus wild type in the mixture decreases to the right and the proportion of B. napus mutant increases to the right.



August 1991] CHOESIN AND BOERNER- IS BRASSICA NAPUS ALLELOPATHIC? 1089

TABLE 2. Mean plant mass (g) of Brassica napus wild type and mutant genotypes grown together in a replacement series

Proportion of each genotypea

100% 75% 50% 25%

Shoot mass Wild type 0.152 0.192 0.199 0.158

(0.020) (0.029) (0.014) (0.014) Mutant 0.138 0.173 0.173 0.194

(0.021) (0.010) (0.018) (0.026) Root mass

Wild type 0.072 0.072 0.069 0.077 (0.017) (0.011) (0.010) (0.009)

Mutant 0.085 0.092 0.090 0.100 (0.016) (0.013) (0.014) (0.029)

Total mass Wild type 0.224 0.264 0.268 0.234

(0.026) (0.037) (0.014) (0.012) Mutant 0.223 0.265 0.263 0.294

(0.035) (0.005) (0.018) (0.046) a Standard errors of five pots per proportion combina-

tion are given in parentheses.

sity we tested. Furthermore, the pattern of density-dependent reduction in mean Medicago mass was unaffected by AI concentration. In contrast, in a study of gallic acid and hydroquinone, putative allelochemicals produced by Polygonella myriophylla, Weidenhamer, Hart- nett, and Romeo (1989) found that phytotoxicity of these compounds decreased with increasing plant density. They attributed this to "dilution" of allelochemicals, i.e., sharing of the available phytotoxin among plants at high plant density reduced the effective dose each plant received. Thus, they found that gallic acid and hydroquinone produced characteristic deviations from the expected density-biomass relationships: the maximum plant mass occurred at intermediate plant density. Plants grown at low density received higher doses of the allelochemicals, while those grown at high density received a higher intensity of resource competition. We observed no such deviations from the expected linear patterns of log biomass:log density relationships, and no differences in the slope of that relationship attributable to AI concentration.

Allelopathy may result more often from the synergistic activity of a number of compounds than from a single allelochemical (Williamson, 1990); certainly Brassica spp. typically contain more than one glucosinolate (Glenn, Chew, and Williams, 1988), and we found the pentyl- and butyl-substituted isothiocyanates to be common in our extracts as well. Thus, the failure of Al in soil to affect target plant growth, in and of itself, might be attributable to our

using Al out of the context of other root ex- udates. If that were the case, our replacement series experiments involving wild type and low glucosinolate mutants of B. napus should have demonstrated a greater ability of the wild type plants to suppress growth of the Medicago tar- gets than the low glucosinolate mutants, or even an ability of the wild type B. napus plants to inhibit growth of the mutants. However, we observed no differences in the ability of the two Brassica genotypes to inhibit Medicago, and no differences in the interference capability of the two B. napus genotypes when grown together.

Taken together, these results suggest that release of Al by B. napus does not appear to be responsible for any allelopathic effect. Al- though Bell and Muller (1973) also failed to demonstrate any appreciable effect of Al under field conditions, they still concluded that Bras- sica nigra was likely allelopathic; we find no data from our experiments to conclude the same for Brassica napus.

An alternative explanation for the dominance of Brassica spp. in the field might be that they are simply strong competitors. Bras- sica spp. often have high growth rates and are capable of preempting space before other plants become established (Bell and Muller, 1973). Brassicas also possess rrmechanisms to increase nutrient availability in their rhizosphere (Grinsted et al., 1982). Boerner and Harris (in press) found that when B. nigra plants were grown in competition with Panicum virgatum plants of equivalent total biomass, the greater belowground biomass allocation of B. nigra enabled them to take up the majority of a pulse of soluble nitrogen generated by collembolan grazing. Crowell and Boerner (1988) found that the belowground competitive effect of B. nigra on Ambrosia artemisiifolia was greater on a per unit biomass basis than was competition from conspecifics. Thus, there is ample information to suggest that Brassica dominance is, at least partially, a function of high competitive ability.

The objective of this study was to assess whether Brassica napus is capable of allelopathic interference, and if so, whether AI is the compound responsible for this interference. Our results indicate that although B. napus releases detectable amounts of Al under greenhouse conditions, and that some of that Al has long residence time in soil, Al concentrations found around mature B. napus plants did not exert any significant effect on target plant growth. Furthermore, genotypes of B. napus that dif- fered in Al release rates did not appear to have quantifiably different effects on growth of target plants, or each other, in replacement series ex-




periments. Thus, we suggest that future studies of the aggressive colonization and persistence of Brassica species in plant communities should focus on life history attributes and mechanisms other than allelopathy.

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Allyl Isothiocyanate Release and the Allelopthic Potential of Brassica Napus