Structural and Biochemical Characterization of Dinitroaniline-Resistant

and little or no metabolism of the herbicide occurs in the plant (10). For example .... greatly affected even at 10"8. M trifluralin. .... (arrows) al...
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Chapter 25

Structural and Biochemical Characterization of Dinitroaniline-Resistant Eleusine

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Kevin C. Vaughn and Martin A. Vaughan Southern Weed Science Laboratory, Agricultural Research Service, U.S. Department of Agriculture, Stoneville, MS 38776

Dinitroaniline-resistant goosegrass (Eleusine indica) biotypes were first discovered in South Carolina and have now been found in several southern states where trifluralin is used for weed control in cotton. Two kinds of resistant biotypes have been noted: one is highly-resistant (R) and is unaffected even by saturated solutions of dinitroaniline herbicide, whereas an intermediate-resistant (I) biotype is only 50X resistant to trifluralin and less than 10X resistant to oryzalin compared with the susceptible (S) biotype. Both R and I biotypes are cross-resistant to phosphoric amide herbicides. Tubulin from the R is able to polymerize into microtubules even in the presence of oryzalin, whereas that of the S biotypes cannot. Western blots of tubulin from the R biotype reveal twoβ-tubulinisotypes whereas only one form is noted in the S biotype. Because the R biotype is hypersensitive to the microtubule-stabilizing agent taxol, it is likely that the R biotype is resistant by having hyperstabilized microtubules. The I biotype has no gross alteration in tubulin nor extreme sensitivity to taxol, indicating that this biotype has a different resistance mechanism than the R. The dinitroaniline herbicides, trifluralin and pendimethalin, have been utilized in greater than 80% of the cotton acreage in the Southern United States because of their very effective weed control in this crop (1). Many of these fields are essentially in cotton monoculture and hence the continued use of these herbicides has constantly selected out those weeds most tolerant of these herbicides. Under such a selection pressure, the appearance of weed biotypes resistant to dinitroaniline herbicides is expected (2). The first report of a resistant biotype did not appear until 1984, Mudgeetal. (3) described the occurrence of dinitroaniline-resistance in Eleusine indica in counties in South Carolina where cotton is extensively cultivated. Since that initial report, dinitroaniline-resistant Eleusine has been detected throughout the midsouth (H. LeBaron, personal communication). The occurrence of dinitroaniline-resistance in Eleusine is quite alarming, because it is one of the world's ten worst weeds (4) and dinitroaniline herbicides are a cost-effective way of controlling this weed in cotton. Even more alarming is This chapter not subject to U.S. copyright Published 1990 American Chemical Society

In Managing Resistance to Agrochemicals; Green, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1990.

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the apparent fitness of this biotype: Murphy el al (5) found virtually no significant difference between the two biotypes when grown under non-competitive conditions. This makes the potential of this resistance problem much greater than triazine resistance, in which the resistant mutant is much less competitively fit in the absence of herbicide.

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Mechanisms of Tolerance/Resistance to Dinitroaniline Herbicides Crop plants in which dinitroaniline herbicides are used are tolerant of the herbicide by a number of mechanisms. Some researchers have turned their attention to these in the hope of finding a mechanism for dinitroaniline resistance in Eleusine. Lipids. Dinitroaniline herbicides are effective on small-seeded, lipid-poor species. Hilton and Christiansen (7) examined the level of seed lipid and the susceptibility of a plant to trifluralin and found a good correlation between the two. These authors concluded that the herbicides would be compartmentalized into the lipid bodies of the seed and away from the growing tip of the plant. Upadhyaya and Nooden (8) even found that there is a differential between susceptible and tolerant species in the uptake of oryzalin into the membrane system, indicating that more than the seed lipids may be involved in determining dinitroaniline sensitivity. Chernicky (9) investigated the possibilities that alterations in the amount of lipid is involved in the resistance of Eleusine to dinitroaniline herbicides. Both susceptible (S) and resistant (R) biotypes had less total lipid than tolerant crop species and even most sensitive weed species. The S biotype had actually 36% more total lipid in the roots than the R biotype (a result opposite to what one would expect if higher lipid content correlates dinitroaniline resistance). Translocation and Metabolism. All of the dinitroaniline herbicides are poorly translocated from the roots to the shoots in both susceptible and tolerant species and little or no metabolism of the herbicide occurs in the plant (10). For example, carrot, a species highly tolerant of dinitroaniline herbicides, 89% of the C -trifluralin applied to the soil was still in the form of the parent compound after one month. All of the metabolites were found in the soil as well as the plant, indicating that all of the metabolites were either chemically or microbially produced. Chernicky (9) found that there were some slight differences between the R and S biotypes in translocation of C -trifluralin to shoots of root-fed Eleusine seedlings, whereas C -oryzalin was equally translocated in both R and S biotypes. Because the R biotype is resistant to both herbicides, it is likely that the differences in trifluralin translocation have relatively little to do with dinitroaniline resistance. However, these data might suggest that a small tolerance to trifluralin may have allowed selection of progenitors of the R biotype as tolerant survivors from initial herbicide treatment. A similar case scenario was reported by Gressel e_t al. (11) in a triazine-resistant Brachvstvlon: the biotype had developed resistance both by metabolism of the herbicide and by an alteration of the site of action (the 32 kD protein). Because field selection of herbicide-resistance generally is noted after many years of use, the probability of a biotype acquiring several tolerance/resistance mechanisms is high. To our knowledge, no one has investigated herbicide metabolism in the dinitroaniline-resistant Eleusine and due to the low metabolism of these herbicides in general, even in tolerant plants, it is unlikely that much metabolism is occurring. Powles e_t al (12) have found that a diclofop-methyl resistant biotype of Lolium rigidum shows an amazing range of cross-tolerance to sulfonylurea, other propionic acid herbicides, and trifluralin. From studies with cytochrome P450 inhibitors, these workers concluded that the mechanism of this cross resistance is

In Managing Resistance to Agrochemicals; Green, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1990.

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due to metabolism of these herbicides by the plant mono-oxygenase system. Because the dinitroaniline resistant Eleusine does not have a similar pattern of cross-resistances to the Lolium [see the study of Mudge gt aj. (3) for example], it is doubtful that, if metabolism is involved, it is the same type as the Lolium. Calcium. A number of mitotic disrupter herbicides cause an efflux of calcium from the mitochondria into the cytoplasm (13). Calcium is known to destabilize microtubules and is thought to be involved in the catostrophic loss of microtubules during mitosis so that existing arrays are destroyed and new conformations of microtubules may be made. Although these herbicides may disrupt the calcium efflux into mitochrondria, the calcium effects occur at 100-1000 X more herbicide than is required to cause mitotic disruption (14), indicating that this may be a secondary consequence of the herbicide. Calcium localization may be easily made using the electron microscope, using pyroantimonate to precipitate calcium into an electron opaque precipitate (15). When the pyroantimonate procedure (15) was utilized to detect calcium distributions in control and trifluralin-treated Eleusine. loss of calcium from the mitochondria was noted in both R and S biotypes at 10 M (Fig. 1) but not at lower trifluralin concentrations (not shown). Because both biotypes were affected and then only at this high concentration, we conclude that the efflux of calcium from the mitochondria is not related to herbicide resistance. Moreover, this efflux does not even seriously disrupt mitosis in the R biotype, as no mitotic disruption is noted at this level of herbicide (16). Microtubules. Tubulin, the major protein constituent of the microtubule, is the primary site of action of dinitroaniline herbicides. Strachan and Hess (17) and Morejohn et al. (18) have isolated tubulin from Chlamvdomonas and higher plants respectively and have established a binding of herbicide to the tubulin dimer and an inhibition of tubulin polymerization into microtubules in vitro. These data are the strongest evidence that tubulin is the principle site of action of the dinitroaniline herbicides. Microtubules have a number of functions in the higher plant cell, the most important of these being the movement of chromosomes during cell division, the formation of the cell plate, and the determination of cell shape (19). As a consequence of dinitroaniline herbicide treatment, mitosis is arrested at prometaphase, cells become isodiametric, and cell plates are either misshapened or non-existant (13). Microtubule loss after treatment also has a dramatic effect on the gross morphology of the seedling root (13, 15). The loss of cortical microtubules in the zone of elongation makes these cells isodiametric rather than elongate, whereas the inhibition of cytokinesis makes these cells larger. As a consequence, a characteristic club-shaped root is formed, similar to the classic disrupter colchicine. Each of the tubulin genes is found in multiple copies in most organisms (e.g. 19), which makes the probability of all (or most) of the tubulin genes mutating to a new resistant form rather low. Because naturally-selected triazine resistant weeds and lab-selected animal cell line resistant to microtubule disrupters are unfit, one would expect mutants resistant to herbicides that act similarly also to be selectively unfit. In the case of the Eleusine. however, the resistant biotype is comparatively as fit in a number of growth parameters as the susceptible biotype (5). Despite this, our research shows that there apparently is an alteration in tubulin present in the R biotype that may account for dinitroaniline resistance in Eleusine. Structural and Cross-Resistance Studies In an effort to determine the magnitude of the resistance to dinitroaniline herbicides, we (15) utilized root tip squashes to determine changes in the mitotic indices of the R and S biotypes. Dinitroaniline herbicides disrupt mitosis at prometaphase, due to a loss of microtubules. Chromosomes fail to arrange at the

In Managing Resistance to Agrochemicals; Green, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1990.

In Managing Resistance to Agrochemicals; Green, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1990. 5

Figure 1. Electron micrographs of pyroantimonate precipitation of calcium (arrow) in Eleusine root tip mitochrondria in the presence and absence of trifluralin. A. S biotype. B. R biotype. C. S biotype grown 24 h in 10 M trifluralin. D. R biotype grown 24 h in 10" M trifluralin. Bar = 0.5 um. m = mitochrondrion.

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metaphase plate and do not undergo anaphase movement. As a consequence, cells spend a longer period in mitosis so that the chances of observing a given cell in mitosis increases. When the R biotype was treated with up to 10 M trifluralin for 24 h, no effect on the mitotic indices of the treated seedlings was noted, whereas there was a significant increase in the mitotic indices of the S biotype even at 10 M trifluralin (15). No disruption of mitosis was noted in electron microscopic observations of the R biotype, although the S biotype appeared to be greatly affected even at 10" M trifluralin. These differences in sensitivity as measured by mitotic indices indicate a 1000-10,000-fold increase in resistance to these herbicides. Root tips squashes were also used to examine the cross-resistance of the R biotype to other mitotic disrupters. Mudge el al. (3) had previously shown that field applications of all of the dinitroaniline herbicides were ineffective in controlling the R biotype, and this was confirmed using root tip squashes as well (20). The phosphoric amide herbicide amiprophosmethyl also inhibits polymerization of tubulin into microtubules (21) and causes the same kinds of mitotic disruption as the dinitroaniline herbicides. The R biotype is also cross-resistant to this herbicide (20). Because the structure of amiprophosmethyl and trifluralin are quite different, it is likely that mechanisms of resistance based upon translocation and/or metabolism of herbicides is unlikely (but see 16 for an exception). The R biotype is sensitive to many other mitotic disrupters including the classic disrupters, colchicine, vinbastine, and podophyllotoxin (20). Actually, the R biotype appears more sensitive (~ 10-fold) to compounds that cause multipolar mitosis (propham, chloropropham) or disturb cell plate formation (caffeine). This hyper-sensitivity may be due to the already misarranged cell plate formation in the R biotype that is more easily exacerbated than those of the S biotype. As noted below, there may be another reason for this difference that is related to the stability of the microtubules in the R biotype. Because dinitroaniline herbicides are one of the most effective methods of weed control in cotton, the development of resistance to dinitroaniline herbicides in Eleusine was especially serious. Moreover, many of the herbicides that are effective on the R Eleusine [see Mudge §1 al. (3)] are much more costly or more detrimental to the growth of the cotton than the dinitroaniline herbicides. In an attempt to find a solution to this problem, Figliola ei al. (22) isolated two pathogenic fungi, Bipolaris setariae Saw. and Piricularia grisea (Cke.) Sacc. from infected Eleusine seedlings and found that those pathogens could be used as biocontrol agents of both the S and R biotypes of Eleusine. Neither of these pathogens infected any of the major crops of the Carolinas (soybean, tobacco, peanut, and cotton). So, when released, biocontrol agents should be an important part of the weed control program.

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Comparisons of Tubulin and Microtubules in R and S Biotvoes With the elimination of many of the other possible mechanisms for resistance, we began an investigation of the site-of-action, tubulin, for differences that might be responsible for dinitroaniline resistance. Extracts of the R and S biotypes of Eleusine were fractionated by stepwise increases in polyethylene glycol concentration and the various fractions were monitored for the presence of tubulin and other proteins by electrophoresis and Western blotting. By making small increases in PEG concentration, one fraction contained virtually all the recognizable tubulin and was > 85% pure as determined by Coomassie blue staining of denaturing gels (23, 24). This is comparable in purity to the protocols of More John gt al. (25), but is a much faster method and allows

In Managing Resistance to Agrochemicals; Green, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1990.

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concentration of tubulin even from a relatively poor source like the 1-month-old Eleusine plants. Tubulin may be polymerized into microtubules in vitro by incubation of a relatively concentrated protein extract in the presence of GTP and high concentrations of dimethylsulfoxide (25). When extracts of the R and S biotypes were subjected to this protocol in the presence of the dinitroaniline herbicide oryzalin, only the R biotype formed recognizable microtubules whereas the S biotype formed only small fragments or ring-like structures that may be small aggregates of tubulin (Fig. 2). Both biotypes were able to form microtubules in the absence of oryzalin (23, 24). These data indicate that the tubulin of the R biotype is different than the tubulin of the S biotype in its sensitivity to dinitroaniline herbicides. To further examine differences between the R and S biotypes, we ground root tips from 5-day-old seedlings under liquid nitrogen and then homogenized in Laemmli (26) solubilization buffer in the presence of the three protease inhibitors recommended by Morejohn el al. (27). These precautions were taken to assure that the tubulin was not degraded by proteases during the isolation. The extracts were centrifuged in microfuge tubes and the supernatant was applied to either Laemmli (26) or Blose (28) formulation gels. The extracts were blotted to nitrocellulose and probed with commercially available monoclonal antisera to a-tubulin and p- tubulin, as well as to a polyclonal antiserum that recognizes both tubulin forms (29, 30). On one-dimensional gels, only one a-tubulin form is found in both biotypes and only one tubulin form in the S biotype. In the R biotype, however, two ^-tubulin bands are noted (Fig. 3). These two ^-tubulin bands vary in mobility, depending on the buffer and gel system utilized, but in virtually every system two are resolved. This does not appear to be a simple geographical variant, as R biotypes obtained from North Carolina, Arkansas, and South Carolina have the tubulin doublet even though they are geographically widely separated. The only exception to this point is the so-called intermediate-resistant (I) biotype found in a few counties of South Carolina (see below), which has similar tubulin profiles to the S biotype. Although equal numbers of seedlings were ground and these were of nearly equal fresh weight and protein content, the extracts of the R biotype always give a more intense staining in Western blots, indicating that there may be more tubulin, as well as the new form. Southern blot analysis indicates a novel set of ^-tubulin genes in the R biotype, not found in the S biotype (M. D. Marks and D. P. Weeks, unpublished). These data indicate that there is a the novel form of 0-tubulin present in the R biotype and it may be responsible for the insensitivity of the tubulin of the R biotype to dinitroaniline herbicides. Mechanism for the Resistance Although our original thought was that the alteration in 0-tubulin would prevent binding of the dinitroaniline herbicide, it appears that a more complex mechanism is responsible for the resistance. In animal cell lines resistant to microtubule disrupters such as colchicine, the cell lines have hyperstabilized microtubules (31) so that the turnover of subunits at both plus and minus ends of the microtubule does not occur as frequently and the destabilizing effects of the disrupter are also minimized. Along with this increase in hyperstability, the cell lines are more sensitive to agents that hyperstabilize microtubules, such as taxol, than the disrupter-sensitive lines. This has been explained using a model of stability and instability (31). When the R and S biotypes were treated with taxol, the R biotype was 100 X

In Managing Resistance to Agrochemicals; Green, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1990.

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Figure 2. Electron micrographs of in vitro polymerized microtubules from R (A) and S (B) biotypes of Eleusine in the presence of 10" M oryzalin. Although good microtubules are formed in the extracts of the resistant biotype, only fragments or structures that represent limited polymers of tubulin were found in the S. Inset shows a higher magnification of an individual microtubule (polymerized without oryzalin) revealing the "beaded" structure typical of microtubules. Bar = 0.5 5

In Managing Resistance to Agrochemicals; Green, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1990.

Dinitroaniline-Resistant Eleusine

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Figure 3. Example of Western blots of R and S Eleusine extracts probed with anti-sea urchin tubulin that recognizes both a-and ^-tubulins. The R biotype has two ^-tubulin bands, but only one a-tubulin in one dimensional gel extracts.

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more sensitive to this compound than the S biotype (32). This was determined by examining the response of microtubules (by immunofluorescence) and other cellular structures (by electron microscopy) to taxol over 10 to 10 M taxol treatments. In the taxol-treated R biotype, the microtubule arrays included star-like structures, where the microtubules radiated from a central region, to multipolar spindles, and extensive cortical microtubule arrays (Fig. 4). The S biotype was affected but generally only wall abnormalities were noted, similar to that noted previously in ultrastructural studies of the R biotype grown on water (15). Because of the similarity between the ultrastructure of the taxol-treated S biotype and the untreated R biotype, it is conceivable that the taxol-treated S-biotype was also dinitroaniline resistant. Treatment of the S biotype with taxol and subsequently with trifluralin or oryzalin revealed little or no mitotic disruption. Similar results have been obtained by others who used taxol as a protectant for other mitotic disrupter herbicides, such as the phosphoric amide herbicide cremart (33). These studies established that (a) the microtubules of the R biotype are hyperstabilized and (b) phenocopies of the R biotype in structure and in insensitivity to microtubule disrupters could be induced by treating the S biotype with taxol. From this, one could conclude that a major factor in dinitroaniline-resistance in Eleusine is the hyperstability, presumably caused by the novel 0-tubulin form in the R biotype. This does not eliminate the possibility that the binding of dinitroaniline herbicides is altered in the R biotype and we hope to investigate this possibility as well. The dinitroaniline herbicides are relatively insoluble in water (and consequently probably in the cytoplasm) so that a mechanisnrof resistance based upon hyperstability would probably allow for a rather complete resistance to these compounds. Other compounds, like colchicine, are much more water-soluble and, at the high concentrations of these compounds required to elicit effects in all plants (34) , little difference in selectivity between the biotypes is noted (20). When small differences were noted, the R biotype was always slightly more resistant (unpublished). In the case of the carbamate herbicides, the initial effect is to depolymerize microtubules and then new mini-spindles are formed directing the chromosomes to many poles (S. Wick, unpublished observations). The hyperstability in the tubulin of the R biotype appears to promote the assembly at many sites, as the frequency of multipolar mitosis is greater in the R biotype than the S (20). A similar explanation may be offered for the greater effect of caffeine on the R biotype than the S biotype (20). The phragmoplast microtubules arrays require a rapid polymerization and depolymerization of the microtubules to orient and allow for fusion of the golgi-derived vesicles in the forming cell plate. Hyperstabilized microtubules may prevent proper movement of the microtubules and hence orientation of the cell plate. This may be the reason for the abnormal extensions of the cell wall, similar to partial cell plates, sometimes found in the R biotype grown on water (15). Caffeine affects the fusion of vesicles in the cell plate so that the combined effects of hyperstabilized microtubules and lack of vesicle fusion result in binucleate cells in the R biotype after caffeine treatment (20). Thus, the hyperstability hypothesis can explain the patterns of cross-resistance and sensitivity as well as the resistance to dinitroaniline herbicides. Tissue cultures of both biotypes of Eleusine can be initiated by a simple combination of plant hormones. Surprisingly, when the cultures were tested for resistance, there was no difference in the susceptibility (as measured by growth) even though the regenerated plants retained the characteristics of the parental line (35) . Because hormone concentrations greatly affect the stability of microtubules, it is possible that the hormones used in these cultures have altered microbubule

In Managing Resistance to Agrochemicals; Green, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1990.

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Figure 4. Electron micrograph of the effects of taxol on the R biotype of Eleusine. The walls in this figure have extensive groups of microtubules (arrows) along the wavy wall (w). Bar = 0.5 /im.

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stability so the herbicides have no additional effect, i.e. 2,4-D reduces the stability of the microtubules. We are currently investigating the tissue cultures using electron microscopy to determine what the effect of tissue culture has upon the microtubule display as well as what effects dinitroaniline herbicides have on the tissue culture.

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Intermediate-Resistant Biotvoe In the initial description of dinitroaniline-resistance in Eleusine. Mudge el al. (3) reported that biotypes from some counties could be controlled by high rates of dinitroaniline herbicides although normal field rates were relatively ineffective at control. Recently, we have investigated this intermediate-resistant (I) biotype in more detail (36). The level of resistance to trifluralin based both upon seedling growth and changes in mitotic indices is much less than the R biotype, around 50 X . The pattern of cross-resistance is also much different from the R biotype. While the I biotype is highly resistant (1000 X) to isopropalin, it is only slightly (10X) resistant to oryzalin. There is no consistent structural or solubility characteristic of the dinitroaniline herbicide that correlates with the resistance of the I biotype (36). The I biotype does not make abnormal cell walls nor is it more sensitive to taxol than the S bioype, indicating that the microtubules of the I biotype are not hyperstabilized. Similarly, there is no obvious ^-tubulin doublet in this biotype. At present, we are not sure why the I biotype is resistant to these compounds. This biotype is very similar in level of resistance and cross-resistance to the Chlamvdomonas mutants of James el aj. (37) that appear not to be tubulin mutants but rather map to loci involved with flagellar function, possibly microtubule-associated proteins. Thus, resistance to dinitroaniline herbicides does not necessarily involve alterations in tubulin, although this is one potential mechanism. When the I biotype was self-pollinated and the progeny were only of the I phenotype (unpublished), so these individuals are not a hybrid of S and R biotypes. So far, the resistant biotypes obtained from other areas have all been of the highly-resistant (R) rather than I type (unpublished). Although all of the biotypes arose under the same situations (cotton monoculture and dinitroaniline herbicide as a pre-plant herbicide treatment) only the populations from two counties in South Carolina apparently have this lower level of resistance. As our case scenarios on other occurrences of dinitroaniline resistance increase, we may be able to relate this to certain sets of unique environmental conditions that may have allowed selection of one type of resistance phenotype over another. Acknowledgments Thanks are extended to B. J. Gossett, N. D. Camper, D. Marks and D. Weeks who supplied published and unpublished reports for preparation of this manuscript. Ms. Ruth Jones provided excellent technical assistance during these experiments. This work was supported by a USDA Competitive Grant No. 86-CRCR-1933 to K. C. Vaughn.

Literature Cited 1. 2. 3.

Parka, S.J.; Soper, Q.F. WeedSci.1977, 25, 79-87. Gressel, J.; Segel, L.A. J. Theor. Biol. 1978, 75, 349-371. Mudge, L.C.; Gossett, B.J., Murphy, T.R. Weed Sci. 1984,32,591-594.

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