Biotechnology for Crop Protection - American Chemical Society

Chapter 1

Downloaded by SETON HALL UNIV on April 3, 2015 | http://pubs.acs.org Publication Date: November 22, 1988 | doi: 10.1021/bk-1988-0379.ch001

Biorational Herbicide Synergists J. Gressel and Y. Shaaltiel Department of Plant Genetics, The Weizmann Institute of Science, Rehovot, IL-76100, Israel Compounds added to herbicides in order to rationally suppress the plants' tolerance mechanisms can synergize the herbicides. Tridiphane suppresses glutathione-S-transferase and thus prevents detoxification of chloro-s-triazine herbicides as well as some other pesticides, that are degraded by glutathione conjugation. Monoxygenase inhibitors can prevent many oxidative herbicide detoxifications. Our own work on biochemical, physiological and genetic studies of paraquat resistance led to a rationale for synergists. Paraquat rapidly, but transiently, inhibited photosynthesis of a resistant weed while permanently inhibiting the wild type. Chloroplasts of the resistant biotype dominantly and pleiotropically inherited constitutively elevated levels of superoxide dismutase, ascorbate peroxidase and glutathione reductase, enzymes engaged in detoxifying the active oxygen species generated with paraquat. Inhibition of these enzymes could lower the required threshold for phytotoxicity, synergizing the herbicides. The first two enzymes contain copper and the first also zinc. Copper and zinc chelators inhibited the enzymes, synergizing paraquat and other active oxygen generating herbicides, in all weed species tested.

We are blessed, in the past number of years, with a better and better understanding of the modes of action and the modes of resistance to herbicides. This is especially true of the photosystem II inhibiting herbicides (1} but also of the dinitroanilines such as trifluralin (2) and particularly with the herbicides affecting amino acid 0097-6156/88/0379-0004$06.25/0 • 1988 American Chemical Society

In Biotechnology for Crop Protection; Hedin, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.


1. GRESSEL AND SHAALTIEL

Biorational Herbicide Synergists

5

biosyntheses. The latter includes the EPSP-synthase (enolpyruvylshikimate phosphate synthase) inhibiting herbicides such as glyphosate (3), the sulfonylureas and imidazolinones inhibiting acetolactate synthase (4,5) and bialophos and glufosinate inhibiting glutamine synthase (6,7). The understandings of the biochemical underpinnings of modes of action and modes of resistance have allowed the rapid advances in biotechnology of engineering herbicide resistant crops (8, 9), the subject of the following three chapters. The greatest advance in this area, the engineering of a gene for an enzyme degrading bialophos and glufosinate occurred too late to be included in this meeting. This is unfortunate as it is a prime example of a simple enzyme, with a readily available herbicide conjugating substrate (acetyl co-enzyme A) that confers full resistance to agricultural rates of herbicide with a large margin of error at very low levels of gene expression (7). The advantages of such low expression levels of this type of herbicide degradases in having the least possible deleterious effects on crop yields has been discussed at length (8, 9). Much of our new understanding of modes of herbicide action and resistance emanate from industry and industry-academia collaboration. The gaining of this information is not always altruistic; most of the information on modes of crop resistance to herbicides comes from the metabolic studies required for registration. There is a huge variation in company policies on how much of this information is available to the scientific community. One area where our information is far too limited is in the mode of resistance to herbicides in weeds that are normally tolerant to the herbicide in question. These are the weeds that are normally not controlled by a given selective herbicide, at the time it was first tested; not weeds that evolved resistance. As there is no need to have metabolism and toxicology studies on such weeds, this information is to a large extent awaiting elucidation. As we shall see below, such information is of critical importance for the biorational choice or design of herbicide synergists. We know much more about the modes of evolved resistance to herbicides, and much of our first intuitions on synergies derived from these studies. Synergists are any combination which has a greater effect together than the sum of the components. Various methods of proving synergies between compounds have been proposed (10-13) and are needed when both compounds are active by themselves. From an industrial registration point of view synergists can be divided (sometimes arbitrarily, as we shall see) into two groups: combinations of active herbicides which are synergistic with each other; herbicide -adjuvant combinations where one of the components is not phytotoxic. One can obtain pesticide synergists by random screening of additives using sublethal rates of the pesticide. This is not the subject of this chapter. We term "biorational" as any choice of possible synergists that is based on our understanding of the mode of resistance of the pest. Adding a compound to the herbicide which we can presume on the basis of biochemical knowledge, will suppress resistance, is "biorational". The other side of the coin is the biorational protection of crops against herbicides (Table I): e.g. the use of an auxin conjugation inhibitor 2,6-dihydroxyacetophenone (14)


6

BIOTECHNOLOGY FOR CROP PROTECTION


to protect against glyphosate based on the researchers' understanding of the multitude of glyphosate actions in plants (15), is a case of such biorational protection. Biochemical Limitations of Synergy. The duty of the biorationally chosen synergist is to prevent the degradation of a herbicide, or the phytotoxic products produced when herbicide is present. When a weed performs no such degradation there is nothing to synergize. The classic selective herbicide of the previous generation (e.g. atrazine) is not overly phytotoxic to corn at 5 kg/ha; it kills some weed species at 30 g/ha, some at 100 g/ha, or 300 g/ha, but the full 1-2 kg/ha are used in the field because there are some pernicious weeds (mainly grasses in this case), that degrade atrazine (far more slowly than maize, but still degrade it) and the high rates are needed to control these species. A synergist blocking atrazine degradation probably would not lower the 30 g/ha required to kill the species usually controlled by that rate. The fully active synergists should be expected to bring all species down from the 1-2 kg/ha rate towards the much lower rates. This lowering of rates should work equally well with the newer generation of herbicides (e.g. sulfonylureas). There is far too much toxic carryover from a wheat field treated with 30 g/ha chlorsulfuron to allow cultivation of many crops, and many weeds remain suppressed. If all the weeds in wheat could be controlled by the carryover level remaining, then there would be no carryover the following year. This requires ascertaining why some weeds are more tolerant to the herbicide than others; information not fully available. It is known that wheat, a resistant species degrades chlorsulfuron (16, 17) so it can be expected that some weeds do the same, but at a lower rate. The main biorational problems will be in finding selective synergists. If a weed and crop detoxify the herbicide or its toxic products by the same means, finding a synergist will indeed be daunting. For this reason it is envisaged that many synergists will be used when the crop is not present (no-till, certain pre-emergence or directed sprays) because of this biochemical limitation. Advantages of synergists. The main advantage of synergists is that they should allow the farmer to substantially lower the herbicide rates used. The foremost interest of the farmer is cost-effective weed-control. If the synergist is less or equally expensive in a combination using far less herbicide, giving the same quality of weed control, then that criterion is met. In many cases it is expected that the spectrum of weeds controlled will be increased by addition of a synergist. There are many other short and long range advantages. With lower herbicide rates there should be less carryover, allowing a larger choice of crops for the following year. The lower the herbicide rate, the less the chance of the herbicide reaching ground water before being metabolized by soil micro-organisms. If one were to use a fifth of the present herbicide rates, the chance of a herbicide appearing in ground waters is probably less than a fifth, as the capacity to degrade would not be oversaturated. The prospects of synergists hurting industry are not as gloomy as some have assumed. Synergist sales will replace some of the decreased





herbicide sales; synergists will make less market-competitive herbicides more competitive because of lower total price; the lessened carryover will interest the farmers; industry will have less ecological combats with agencies over residue and toxicology problems. From this it is clear that there need not be a massive tonnage reduction in production.... and some reduction is better than being forced to remove a herbicide from the market-place. Synergists and the Evolution of Herbicide Resistance. Selection pressure is the most important factor controlling the rate at which herbicide resistant populations will evolve in a susceptible species (18). Selection pressure is a more or less direct function of herbicide rate. The first and most wide-spread weeds to evolve atrazine resistance were Senecio sp. Amaranthus spp. and Chenopodium spp.^ These are controlled by exceedingly low atrazine doses, 30-100 g/ha, i.e. the selection pressure exerted by 1-2 kg/ha is very great. The last species to evolve resistance were the grasses (19), that are controlled by only the highest levels, i.e. the selection pressure for grasses was lower than that for broadleaf species. Had synergists such as tridiphane (see below) been used earlier to lower atrazine rates (and thus lower the selection pressure), it would be expected that the evolution of the resistant weeds would be considerably delayed. One could make the same predictions for synergized sulfonylurea herbicides. Herbicide-Herbicide

Synergistic

Combinations.

A computer survey of the patent literature made a year ago came up with 515 recent patents claiming herbicidal synergies. A perusal of the abstracts of 45 of them, chosen as a sample, showed that the patent community uses a broader definition of synergy than used here; they believe that there is a synergism when two herbicides control more weed species than each separately. This would better be termed "complementarity". The overlap of control range allows a lowering of herbicidal rates, which may or may not be due to a metabolic synergy. Some synergies appeared by rational discovery. From metabolic class inhibitor and herbicides that are detoxified

fortuitously (atrazine-tridiphane) and not what we now know, tridiphane is a may have other rational future uses with by the same mechanism.

Tridiphane-atrazine. Tridiphane was initially developed as a grass controlling herbicide, and it was later found that atrazine and tridiphane synergistically control grasses in combination (20). From indirect evidence showing the stoppage of atrazine catabolism it was initially thought that tridiphane acts by inhibiting the specific glutathione-S^transferase for atrazine (21). It was later found that a glutathione-S-transferase conjugates tridiphane with glutathione (22): 0

tridiphane

reduced glutathione

OH

S-(tridiphane)GSH


7

8


This is similar to the first step in atrazine degradation: reduced CCH^-CH-I^

g l u f Q t h i o n e

\' ι}" 3

2


atrazine

GlutathioneS-transferase

HO Ν Ha ^C-CH -CH -ÇH

CH3) -CH-NH 2

2

2

Ν V-S-CH -CH

*

0

CH -CH -NH

(

COOH

2

ν—Ν CH -CH -fÎH 3

2

I C-N-CH -COOH 0 H 2

(NON-TOXIC)

The tridiphane-glutathione conjugate is a far more potent inhibitor of the glutathione-S-transferase measured than tridiphane alone Fig.lA). Tridiphane synergizes atrazine, killing weeds in maize in the ield, yet the maize is unaffected. This is surprising as maize utilizes a glutathione-S-transferase to degrade the atrazine and this glutathione-S-transferase is inhibited by the tridiphane-glutathione conjugate. This inhibition of glutathione-S-transferase activity is less strong than with weeds (Fig.lA). A kinetic analysis showed that the tridiphane-glutathione conjugate is then a competitive inhibitor of glutathione binding to glutathione-S-transferase with a 4 times higher affinity for the enzyme from weeds vs. maize (22). These data are probably not enough to explain why tridiphane is inactive as a synergist in corn: i.e. why is corn not killed. Young corn leaves have 6 times more glutathione than Set&ria, which could partially explain the difference.... but older maize and Setaria leaves have the same concentration (22). Tridiphane does not kill older maize plants. The answer probably lies in a rapid catabolism of the tridiphaneglutathione complex in maize which does not occur in Setaria (Fig. IB) (22). Thus, we see in balance that maize is able, by virtue of having more glutathione, less tridiphane-glutathione conjugate, with less activity and faster degradation of the conjugate, maize can "save itself" from tridiphane synergism of atrazine.

Î

As stated before, the choice of tridiphane to synergize atrazine was not "biorational". If we use the information to synergize other pesticides, this will be "biorational" by our broad definition. Many pesticides have glutathione conjugation as the first step in their downfall from toxicity. From our understanding above, tridiphane must first be conjugated by the target organism. The glutathione-Stransferase must then be inhibited, and the tridiphane-glutathione conjugate must be stable for tridiphane to synergize other pesticides in other pests. Indeed the tridiphane conjugate prevented degradation of a number of pesticides (Table II). Tridiphane was also able to synergize E P T C and alachlor in maize (20). The mode of synergy has not been checked, but both of these herbicides are degraded by glutathione conjugation. Tridiphane was also excellent at synergizing the insecticide diazinon against houseflies. Tridiphane had a twenty fold greater rate of conjugate formation with glutathione than diazinon has, and diazinon does not seem to compete with this step (23). The LD50 with normal houseflies was reduced threefold from near 0.09 /ig diazinon per fly to about 0.03 μ% diazinon per fly - but 20 μ% tridiphane per fly was required for this (23). Houseflies have other mechanisms of diazinon degradation and that may be the reason lor the small increment of synergy with much tridiphane. Tridiphane may be more useful with roaches, which only degrade diazinon by glutathione conjugation (24). Tridiphane would also be very useful


GRESSEL AND SHAALTIEL


Table I. Biorationally Protecting Against Glyphosate Action by 2, 6-Dihydroxyacetophenone (DHP) on Teucrium canadense


Free IAA content

Plant height

Treatment

100 56 92 88

Water (control) 0.2mM glyphosate 0.5mM 2,6-DHP glyphosate + DHP

(% of control) 10 20 101 74

Source: Calculated from data of Lee and Starratt (14). A. Inhibition of GSH conjugation B. Catabolism of conjugate Ε I 1 I 1 I —1 1 1 1 jool r Tridiphane alone ! 90
^ , υ ι \ 1 0 0.001 0.01 0.1 /iM Tetcyclacis

10 OO

Figure 3. Tetcyclasis inhibition of chlortoluron catabolism in cotton. Note the much greater level of activity than that of aminobenzotriazole (Figure 2). Source: Redrawn from data in Cole and Owen (36).


14



myosuroides evolved in England with cross resistances to isoproturon, methabenzthiazuron, pendimethalin, terbutryne, diclofop-methyl and chlorsulfuron (45). The researchers "rationally" decided to test monoxygenase inhibitors to suppress resistance, as chlortoluron (Fig. 3,4) is among the herbicides. Indeed, aminobenzotriazole quite effectively reduced the resistance, i.e. synergized the herbicides (46). Aryl-acylamidase inhibitors. Rice is resistant to the herbicide propanil due to a rice specific aryl-acylamidase which degrades the herbicide. Various carbamate and organophosphorothioate insecticides were found to be incompatible with propanil because they prevented propanil degradation. This was found to be due to a direct inhibition of this enzyme (47, 48). Such insecticides or their derivatives can be "rational" synergists when this enzyme is found to be active in degrading herbicides in weeds, and must be suppressed. Inhibitors of the oxygen detoxiûcation pathway. A few major groups of herbicides kill plants by photogeneration of active oxygen species. The triazines, uracils, phenylureas, and some of the pyridazinones block electron transport at the reducing side of photosystem II of photosynthesis just before plastoquinone reduction il). Some phenolic herbicides act at a nearby site on photosystem II. The bipyridilliums drain electrons from photosystem I, probably from ferredoxin (49). A large group of nitro-diphenylethers require light to generate active oxygen species although it is clear from the diverse findings in the literature that either; no bulk pigment acts as the photoacceptor in all systems; or that different pigments act in different systems (50). It is also not clear which active oxygen species is generated first. As soon as there is more active oxygen generated than the endogenous detoxification system can cope with (Fig. 4), there is membrane lipoxidation. This results in water loss, a general breakdown in the electron transport systems, and the release of the chemically transformed solar energy as a variety of active oxygen species which can be confused with the first one generated. Singlet oxygen can be produced, and so can chlorophyll radical. This self-amplifying chain reaction of generation of active oxygen, membrane lipoxidation and water leakage leads to the rapid desiccation caused by these herbicides. The stronger the light, the faster this chain reaction. Certain environmental xenobiotics: SC>2, O 3 , Ν Ο and some fungal toxins such as cercosporin can have the same effects. ' χ

Organisms have endogenous oxygen radical detoxification systems, that probably co-evolved with photosynthesis and aerobic respiration. They include superoxide dismutase and the ability to produce and recycle oxy-radical quenching agents such as glutathione and ascorbate. Plants also produce carotenes and α-tocopherol which quench these radicals. This detoxification system is especially necessary to cope with energy "leakages" from the photosynthetic electron chain. For a herbicide to be toxic, it must produce more radicals than can be quenched by this native system before the herbicide is dissipated. Chloro-s-triazine herbicides inhibit photosystem II in isolated thylakoids at the same concentration in all species. Despite, this, high rates are required in the field to kill grasses, due to the glutathione-S-transferase degradation described in an earlier section. This leads to a balance between the amount of herbicide remaining with the ability of the plant to detoxify radicals until the herbicide is degraded. As much of the "action" of producing oxygen radicals is in the chloroplast both





15

with and without herbicide, and most active species such as singlet oxygen and superoxide have very short diffusion distances, it is natural to assume that the chloroplast has its own concerted anti-oxidant defence mechanisms. This includes the slowly recyclable carotenes, as well as a pathway for oxygen detoxification which recycles its components. This pathway has been elucidated by many, and put in context especially by Halliwell (51) and Asada (52). The first enzyme is superoxide dismutase (Fig.4 ) which dismutates the superoxide to peroxide. The peroxide is less energetic and can diffuse, but is still dangerous; it can react with ferrous iron and form hydroxyl radicals (the Fenton reaction) which are extremely energetic. If the peroxide is slowly formed from superoxide, the excess superoxide can react with the ferric ions formed by the Fenton reaction, re-reducing them and making them available to form more hydroxy! ions. The Fenton reaction coupled with the iron recycling reaction is termed the HaberWeiss reactions (51). It is thus imperative for the chloroplast to rapidly detoxify both the superoxide and the peroxide but alas, chloroplasts contain no catalase. They do contain ascorbate peroxidase and ascorbate, which can adequately compete with the Fenton reaction to remove the potentially dangerous peroxide. The dehydroascorbate formed is reduced back to ascorbate by glutathione, either spontaneously or by a dehydroascorbate reductase. The oxidized glutathione is recycled back to glutathione by glutathione reductase, utilizing NADPH (51, 52). Some electrons must pass through the photosynthetic electron chain to produce NADPH for this reaction, or the system will break down. This system will recycle both glutathione and ascorbate used quench singlet oxygen and other radical reactions. Our interest in the system began when it was intimated that superoxide dismutase levels might be responsible for different levels of paraquat tolerance (53-55). As it is, some weeds such as Amaranth us spp. are killed by 30 g/ha paraquat and others at a variety of rates up to the 1-2 kg/ha used in weed control. The paraquat resistant Conyza spp. that evolved were only controlled by >15 kg/ha paraquat (56, 57). In the meantime, a second theory was promulgated to explain this paraquat resistance; that paraquat was sequestered before it could reach green tissue. This was mainly based on radioautographic evidence, obtained 4 h after paraquat was treated through cut petioles (57, 58). By this time sensitive plants have usually whithered under strong light. We believe that the radioautographic evidence shows that resistant plants can actively sequester paraquat or its metabolites, but that paraquat does get to the chloroplast, and that elevated levels of the enzymes of the Halliwell-Asada pathway keep the plant alive until paraquat is actively sequestered. The evidence that we obtained to support this view allowed us to rationally choose compounds that synergize paraquat and other active oxygen generating herbicides. The lines of research that led us to believe that elevated levels of the Halliwell-Asada pathway have primary responsibility for increased tolerance to active oxygen are as follows: (a) Kinetic evidence showed that paraquat sprayed on leaves of the paraquat resistant biotype rapidly but transiently inhibited photosynthesis (Fig. 5A). This shows that paraquat did reach the chloroplasts, affected them, but the plant remained alive while the paraquat was dissipated. In contrast, photosynthesis was inhibited just as rapidly in the sensitive wild type, but irreversibly so (Fig. OA).


16


••OH" * 0 H

ozone+hÔ

#

hydroxyl radicals

hv*e"

4^PS-lw f I k

.Paraquat · / 0 " * s ^ H 2 0 2 2

>T I A PS-i / #

Y I /V

Waquat *

v

x

Peroxidev Superoxide A dismutase ρ K

/K

0 ^ H O^ 2

2

^Dehydroascorbate^Glutathione (redL

J,NADP*

Spontaneous Spontaneous λ' or Glutathione reductase Dehydroascorbate reductase J\ Ascorbate' Glutathione(oxr NADPH x

S0

Plastoquinone


°2 PSI

#

PS ÎE

hv+e"

Âcifluorfen+unknown •---- +0 / photo-receptor \ s

2 >

Ozone?

2

ÎÂcifluorfen+unknown Λ photo-receptor*^'' S hv*e"

Figure 4. Scheme of interactions between active oxygen generating xenobiotics and the oxygen detoxification system. Light intensity, the rate constants of photogeneration of active oxygen, dissipation of the xenobiotics and the levels and rate constants of the enzymes interact to determine whether a plant will be spared or killed.

Figure 5 (A) Evidence that paraquat transiently affects chloroplasts of resistant Conyza and (B) that there are constitutively elevated levels of the Halliwell-Asada active oxygen detoxification pathway in the chloroplasts. A. Resistant and susceptible plants of Conyza bonariensis were sprayed to runoff with 0.1 mM paraquat and whole leaves were removed for measurement of photosynthesis at times thereafter as an estimation of paraquat arriving at, and affecting chloroplasts. Simultaneous measurements of stomatal aperture were made to ascertain that the stomates remained open. Source: Data redrawn from (60). B. Normal enzyme levels without paraquat treatment) in resistant and susceptible Conyza )onariensis. Source: Collated and drawn from (57, 61).

Î




17


(b) Isolated intact (Class A) chloroplasts of the paraquat sensitive Conyza bonariensis biotype evolved 3 times more ethane than the resistant type under high light (59). This is a sign of resistance to membrane damage due to oxidant "leakage" from photosynthesis. When paraquat was added, ethane evolution from the sensitive biotype tripled; it also increased in the resistant biotype, but to less than the normal level of the sensitive biotype. These data indicate that the primary mechanism for tolerating paraquat is in the chloroplast. (c) We measured the levels of the three major enzymes in the pathway in isolated intact chloroplasts and found tham to be constitutively elevated in the resistant Conyza biotype (Fig. 5B). Resistance was dominantly inherited under the control of a singe gene (60). The levels of the three enzymes were elevated in the F j generation. The critical correlation was in the Fo generation where we immunologically determined the levels of two of the enzymes in single plants that were tested for resistance / susceptibility to paraquat. All the resistant plants had elevated enzyme levels and all the sensitive plants had normal enzyme levels (Fig. 5B). This lack of F 2 segreation is a very strong correlative phenomenon. These data are supported by others' findings that induced paraquat tolerance can be correlated with increased chloroplast superoxide dismutase and glutathione reductase (62). Interspecific comparisons have correlated acifluorfen tolerance to increased levels of ascorbate and α-tocopherol (63), and to increased superoxide dismutase (64). (d) If high levels of the Halliwell-Asada pathway enzymes confer resistance to paraquat, they should confer increased tolerance to other herbicides and oxidant stresses. Indeed we and others have shown such correlations (Table V) in a variety of species. The level of resistance to paraquat is usually higher than to other herbicides, probably because the other herbicides remain active in the plant for longer durations. e

The above data supply considerable evidence for the involvement of the Halliwell-Asada pathway and its products in tolerance to oxidant generating herbicides. It only remains to be shown that suppression of the pathway suppresses tolerance. This would be further evidence for the involvement of the pathway. Any compound suppressing the pathway would have considerable possibilities as a synergist. The plastid superoxide dismutase and ascorbate peroxidase are both copper containing enzymes (52). The former contains zinc and the latter tightly bound iron, and thiol groups. The later enzymes of the pathway contain thiol groups as well (52). Thiol binding reagents and compounds which complex copper and/or zinc should inhibit the pathway. A suppression by thiol biding reagents at the plant level would hardly be good evidence as the plant has many thiol containing enzymes. Such compounds usually have high mammalian toxicity and would thus have little potential as synergists. There are few copper containing enzymes and fewer yet zinc containing enzymes in plants. Thus compounds which bind them should serve as somewhat specific synergists. We tested a variety of relatively specific copper and zinc chelators in in vitro, cellular, tissue and whole plant systems (Table VI). It is clear that they all severely affected the plants when used with active oxygen generating herbicides. None of them had any obvious effects on plants at the concentrations used (68). Their effects were probably due to the chelation of copper


18


Table V. Herbicide Cross Tolerances to Oxidative Stresses and Relations with .the Halliwell-Asada pathway


species

cross tolerances

paraquat

high SOD,GR,AP atrazine acifluorfen photoinhibitioη elevated SOD/GR so paraquat elevated SOD/GR paraquat elevated SOD/GR paraquat elevated GR

Conyza, bonariensis

paraquat

Lolium perenne Lolium perenne SOn Nicotiana tabacum

cv Florida cotton

enzymatic correlations

primary tolerance

drought

ref.

3.

2

(65)

0») m (65) (65) (63)

ÔD, superoxide dismutase; GR, glutathione reductase; AP, ascorbate peroxidase, k Jansen, Canaani, Shaaltiel, Gressel (unpub. results).

Figure 6. Synergism of paraquat by diethydithiocarbamate, a copper chelator. Paraquat sensitive Conyza bonariensis plants of slightly different sizes were sprayed to run-off 24h before photographing. Source: From (66).



£s J CO

M


^ O O O M O M T f i O O O O f M M H C O 00 Η Ο τ-Η Η Η Η Μ

W C 1

Biotechnology for Crop Protection - American Chemical Society

Recommend Documents