CS256197B1 - Method of indication and quantitative evaluation of herhicidal efficacy of photosynthesis inhibiting agents - Google Patents

Abstract

The solution concerns a method of indicating and quantitatively evaluating the herbicidal efficacy of photosynthesis-inhibiting substances by comparing the radioactivity of leaf segments treated with the test substances with the radioactivity of untreated control segments after light exposure under the influence of 14C-labeled carbon dioxide. The solution can be used in the research of new herbicides, in the quantitative evaluation of the degree of their selectivity, photosynthesis inhibition, resistance of some biotypes as well as the influence of additives on the potentiation of herbicidal effects.

Description

256197

The invention relates to a method for the indication and quantitative evaluation of a herbicidal activity of photosynthesis inhibiting substances. The photosynthesis inhibitors form the predominant part of herbicidal active substances. Before the introduction of the new herbicide into the soil, the tens of thousands of compounds are biologically tested for herbicidal activity. The majority of commercially available herbicides belong to photosynthesis inhibitors by their mechanism of action. With their inhibitory effect, they gradually eliminate the vital biomass assimilation processes, which ultimately manifests itself in plant death [Buchel, K. H .: Fist. Sci. 3, 89-110 (1972)].

The most widely used screening methods for assessing herbicidal efficacy are mainly based on the phytotoxicity monitoring of the bonitory scale, or when refining the results, with a time-related loss of biomass, depending on the concentration of the investigators in comparison with the untreated controls [Bradbury, D. T .: Proc. 10th Bri-tish Weed Control Conf. 1970, 986-996 (1970); Saggers, D. T.: Herbicidal Acadeus Press London, ed. AUDUS, 447-473 (1976)]. These methods are time-consuming, energy-intensive, require large glass spaces, thereby limiting the limited range of test compounds. Herbicidal symptoms often manifest themselves in a delayed manner, blocking the test areas for a longer period of time. The method of application and the preparation of spray solutions require at least some coagulation amounts of the test components, which imposes higher demands on the preparation and availability of these compounds. Considering capacity constraints, these methods are used in the systematic research of known herbicidally active moieties and the target of research into the synthesis of novel herbicidal compounds where the occurrence of herbicidal active agent is more likely. These methods are indispensable for refining the evaluation of herbicidal activity under practical application to treated cultures in the respective weed community.

Looking for new herbicidally effective groups, tens of thousands of compounds need to be tested with strong, efficient structures. For these purposes, there is a need for very fast, working, energy and spatially non-test methods that are as close as possible to the practical application conditions by the minimum amount of test substance required. These requirements only partially satisfy some of the so-called "in vitro" methods, based, for example, on the inhibition of Hill's reaction-isolated chloroplasts, possibly photo-synthesis on leaf segments by monitoring the mass loss of biomass dry matter, as diving of floating treated segments [Moreland, DE, Hill, KL: 1B, 229-236 (1962); Šesták, Z., Čadský, J.: Methods of Photosynthetic Production Studies, Academia, 1966, p. 396; Truelove, B.al. Weed Sci. 22, 15-17 (1974); Saltzman, S., Heuer, B., Pestic. Sci. 16, 457-462 (1985)]. Said methods are labor-intensive, since in terms of weight of evidence they require large statistical files and do not allow automation of results evaluation. Other methods utilize intact microorganisms, lower plant organisms as well as in combination with fluorescent methods to determine herbicidal efficacy [Addison, D.A., Bardsley, C.E., Weedci. 16, 427-429 (1968) to Bohme, H. et al., Feed Sci. 29, 371-375 (1981)], or by measuring CO 2 by IR spectrophotometry, optionally by radiometric method (DD212 985-A). The quantitative evaluation of photosynthesis inhibition is drawn by a large error, as the amount of carbon dioxide CO2 consumed is very low compared to the total amount of carbon dioxide CO2. A method based on the inhibition of the Hill reaction using a suitable redox indicator for the detection of nanogram quantities of inhibitors [Kováč, J., Henselova, M .: Pho-tosynthetica 10 (3), 343-374 (1976)]. the routine test method, unless the mechanism of action is based on inhibition of other photosynthetic processes. The method does not include changes in efficacy caused by the inhibitor trans-port to the site of action.

Similar method [Okut, T., Kawahara, H., Tomita, G .: Plant cell physiol. 12 (4), 559-566 (1971)], based on the evaluation of the inhibition of the Hill reaction by measuring the oxygen content of an isolated chloroplast by an oxygen electrode, does not indicate active substances interfering with other photosynthetic processes.

The aforementioned drawbacks of the test procedures for indicating and evaluating the herbicidal activity of a large number of aspects of photosynthetic process inhibition removes the method of indication and quantitative evaluation of the herbicidal activity of the photosynthesis inhibiting agents of the invention. The present invention is based on the fact that the solution of the test substance is topically applied to the cut out plant leaf segments, which, together with symmetrical but untreated control segments, are placed in the photosynthetic chamber where they are exposed to light exposure under the action of radioactive 14 CO 2. After exclusion, the radioactivity of the treated untreated segments is measured and the presence of photosynthesis inhibiting substances is indicated from the decrease in the radioactivity of the treated segments compared to the control segments and the photosynthesis inhibition in% is quantitatively evaluated. The advantage of the method of indicating and quantifying the herbicidal efficacy of photosynthesis-inhibiting substances is that, in view of the high sensitivity of the radiometric method, the decrease in radioactivity in treated leaf segments can already be detected with a short light exposure. This makes it possible, with the use of automatic radiometric technique, not to test a large number of specimens in several milligrams in a short time, but also to quantitatively evaluate the selectivity coefficient quickly using the seedling of the grapevine plant materials as well as the impact and final biological treatment. herbicides.

The accompanying figure shows an apparatus for photosynthesis in an atmosphere containing 14 CO 2 formed by a photosynthetic chamber 2 made of plexiglass with a washer 1 for receiving segments, wing nuts 3 and valves 15, 9 for sealing, with an opening 7 inserting the syringe with the needle 5 through the rubber stopper 4, the needle 5 of the filling paper being attached to the needles of the syringe 5. In addition to the photosynthesis device, the membrane pump 10 for recirculating the atmosphere of the photosynthetic chamber 2 and the sorbent 11 is also non-assimilated or 14CO 2 adsorbed.

These examples illustrate, but do not limit the invention. Example 1 Radiometric indication of photo synthesis inhibitors based on urea triazine, diazine and carbamate herbicides.

Apparatus: automatic liquid scintillation counter radioactivity Rack-Beta LKB; device for photosynthesis in an atmosphere containing 14CO2, Fig. 1; Hamilton syringe (10 mm3).

Solutions and chemicals: aqueous solution of radioactive sodium carbonate (Na 2 O 4 CO 3), conc. 11.1 mg. cm -1 with total activity of 37 MBq, NaOH solution conc. 2 mol .. 11, solution H 2 SO 4 conc. Ethanol alcohol solutions of herbicide-photosynthesis inhibitor compounds - conc. 2 mg. cm "3. Active substances: 2-ethylamino-4-chloro-6-tert-butylamino-1,3,5-triazine (terbutylazine), 2-ethylamino-4-methylthio-6-tert-butyl-amino; 1,3,5-triazine (terbutryn), N, N-dimethyl-N- (3-chloro-4-tolyl) urea (chlorotoluron), N-4- (4-chlorophenoxy) phenyl-N , N-dimethylurea (chloroxuron), 4-amino-3-methylthio-6-tert-butyl-1,2,4-triazin-5-one (metribuzin),

B 2-Chloro-4-ethylamino-1 H -isopropylaimino-1,3,5-triazine (atrazine), N - (4) -methoxyphenoxy) -phenyl) -N, N -dimethyl-difluoro (difenoxuron), N - (3,4-dichlorophenyl) -N-methoxy-N-methylurea (linuron), N- (4-bromophenyl) -N-methylurea (metobromuron), N, N-dimethyl-N- (4-isopropylphenyl) ) -urea (isoproturon), O- (3-methoxycarbonylanilino) -N- (3-methyl-phenyl-carbamate (feumedifam), 2-methylthio-4,6-bis-isopropylamino-1,3,5- triazine (prometryn), 3-isopropyl-1-hydro-2,1,3-benzthiadiazine-4-yl-2,2-dioxide (bentazone), 4-chloro-2-oxo-3-benzothiazolinoc acid ( benazolin), N- (4-chloro-phenyl-N-methoxy-N-methyl-urea (monolinuron), 3-cyclohexyl-5, & -trimethylenuracil (lenacil), 4-amino-1-phenyl-S-chloro-B -pyridazone (chloridazone), 1,1-dimethyl-3-trifluoromethylphenyl-urea (fluometuron) From the leaf area of the bean (Phaseolus vulg.) at the development stage of the first two leaves, a pair of circular segments of the leaf nerve were formed so that preserve the symbiosis of the At least four bices were cut out of each bishop. The segments were deposited on filter paper discs (0 cm) uniformly wetted with distilled water, placed on a glass backing, and the segments were adhered to the wet backing by gentle pressing, thereby reducing the evaporation of water from the segment surface and achieving the desired fixation of the segments on the backing surface. The surface of the segment of the respective sample-control pair was applied with a capillary micro-pipette (5 mm 3) of the ethanol solution of the substance to be examined, and the segmentamisa washer 1 was inserted into the photosynthetic chamber 2 after the occlusion of the photosynthetic chamber 2 with the cover 12 with the rubber seal. 3 and valves 8, 9, a rubber stopper 4 pierced by a syringe needle 5 at the end of which a filter wick 6 has been attached has been removed from the opening 7 of the photosynthetic chamber 2. The syringeS is partially filled with dilute sulfuric acid solution, the needle is exchanged for 25B197 needle with load 5, and 5 mm 3 of a solution of radioactive sodium carbonate were deposited at the end of the filter paper wick 6. Then a rubber stopper 4 with a syringe 5 and a needle 8 was inserted into the opening 7 of the photo-synthetic chamber 2. After closing the valve 8, 9 the photo-synthetic chamber 2 was covered with a black paper and a drop of sulfuric acid solution was expelled ] from the syringe 5 the knQ6 was wetted and the sulfuric acid released from the sodium carbonate from the radioactive 14CO2 into the chamber of the photosynthetic chamber 2. After 15 minutes of equilibration of the photosynthetic chamber 2, the segments were exposed to 30-minute exposure to light fluorescent lamp (3 x 40 W), placed 20 cm of pad 1 with segments. Upon termination of the exposure by opening the valves 8, 9 and switching on the air pump membrane 10 of the photo-synthetic chamber 2, it was recirculated through the absorber 11 with the sodium hydroxide solution. The absorption of the non-assimilated 14CO2 took 30 minutes. Then, the rear end of the photosynthetic chamber 2 was opened and the washer 1 with the segments was extended. The segments were blotted into scintillation counting vials with 10 ml of liquid scintillation counter TST-31. Radioactivity measurements were made on an automatic liquid scintillation counter of the Rack-Beta LKB radioactivity (another equivalent device may be used). The decrease in radioactivity, indicating the onset of photosynthesis inhibition and hence naherbicidal activity, is shown in Table 1.

Table 1 Radiometric indication of biological activity of herbicides on bean leaf segments expressed as% photosynthesis inhibition

Radioactivity tested Radioactivity

Inhibition of photosynthesis effective controls, seg. ošetr. seg. (cpm) (i - ^ -) 100 (%) substance (cpm) Terbutryn 31 445 75 99,8 Prometryn 30 060 85 99,7 Ghloridazon 31 665 90 99,7 Atrazine 30 055 95 99,7 Difenoxuron 29 905 105 99.6 Lenacil 29 470 170 99.4 Fluometuron 29 435 175 99.4 Phenmedipham 30 565 210 99.3 Isoproturon 31115 235 99.2 Bentazone 31 765 245 99.2 Monolinuron 30 230 260 99.1 Metobromuron 29 705 315 98 , 9 Metribuzin 29 970 335 98,9 Terbutylazine 30 795 425 98,6 Linuron 31 305 550 98,2 Chlortoluron 30 385 605 98,0 Chlorroxuron 31 205 815 97,4 Benazolin 31400 995 96,8 Example 2 Concentration limits 0,001 to 2 , 00 mg. The micro-quantitative radiometric evaluation of the individual solutions was applied by pipette to the cut out segments of the herbicide activity leaves, expressed as a bean-like procedure in the photosynthesis example, from the active compound concentration 1 and evaluated in the application solution after light exposure. from the measured radioactivity of the treated and untreated segments photosynthesis inhibition Procedure: Aqueous solutions in% were prepared as shown in Tables 2 and 3. The final herbicide formulations based on the result are an average of six parallel active agents of atrazine and bentazone in determination. 256197 9 10

Radiometric evaluation of photosynthesis inhibition (%) versus atrazine concentration in application solution

Table 2

Atrazine concentration (mg. Cm-3j radioactivity control segment (c. M.)

And the radioactive treated segment (c.p.m.)

B photosynthesis inhibition ° / o (1 -? -) · 100 0.001 25 030 20150 19.5 0.002 24 750 16 530 33.2 0.004 24 710 11 970 51.6 0.020 26 045 8 190 68.6 0.040 25 150 5 147 79.5 0.400 26 570 305 98.9 2.000 24 775 125 99.5 Table 3 Radiometric evaluation of photosynthesis inhibition (% j depending on bentazone concentration in delivery solution Radioactivity radioactivity inhibition of bentazone photosynthesis control segment treated segment (%) (mg. cm'3) (cpm) (cpm) p 1 1 1ΠΠ AB [ia * 1UU 0.001 25 970 21 960 15.4 0.002 26 160 19 310 26.2 0.004 25 035 13 745 45.1 0.020 24 030 8 980 62 6 0.040 24 220 5 980 75.3 0.400 25 005 1 990 92.0 2.000 26 970 270 99.0 Example 3

Radiometric evaluation of the potency of the herbicidal effect by the additive.

Procedure: The bean leaf segments were treated with aqueous solutions of the final product-tested Zeazine herbicides (bentazone active) without addition or addition of additive, at graded concentrations of atrazine and bentazone. It prepared a 0.2% sodium additive solution (Sloviol-R containing 16% polyvinyl alcohol). The inhibition of photosynthesis of graded concentrations of the respective herbicides obtained by dilution with water and an aqueous solution of the additive was compared. The next procedure was the same as in the previous examples. The potentiating effect of the additive is shown in Tables 6 and 7.

Table 4

Potentiating the herbicidal effect of atrazine-photosynthesis in graded additives of Sloviol-R expressed by herbicide concentrations by inhibition

Conc. atrazine (mg. cm-3) (c. p. m) radioactivity control segment (c.p.m.)

A radioactivity treated segment (c. P. M)

B photosynthesis inhibition (%) (1- ^ (1) .100 0.001 27,050 18,905 0.002 25,110 14,095 0.004 25,750 9,640 0.020 26,330 62,295 0.040 25,175 1,995 0.400 26,970,190 2,000 26,145 75 30,143, 962.6 76.1 92.199.399.7 25B197

Potentiating the herbicidal effect of bentazone with Sloviol-R additive, expressed by photosynthesis inhibition at graded herbicide concentrations 11 12

Table 5

The concentration of bentazone radioactivity and control segment of radioactivity treated segment inhibition of photosynthesis (mg. Cm-3) (cpm) (cpm) (1 D -1. 100 AJ 0.001 24 070 18 150 24.6 0.002 25 300 16 002 36.8 0.004 25 120 11 300 55,0 0,020 25 025 7 150 71,4 0,040 26 130 4 700 82,0 0,400 25 055 155 99,4 2,000 26 035 80 99,7 T abu 1 'ka 6 Comparison of photosynthesis inhibition by atrazine and additive addition Concentration 0,001 0.002 0.004 0.020 0.040 0.400 2.000 atrazine (mg. Cm'3) inhibition (%} 20 34 52 69 80 99 99 without additive inhibition (%) 31 44 63 77 93 99 99 with additive T abu 1 'ka 7 Comparison of photosynthesis inhibition bentazone without addition and additive addition Concentration 0,001 0,002 0,004 0,020 0,040 0,400 2,000 bentazone (mg. cm'3) inhibition (%) 16 27 46 63 76 93 99 no additive inhibition (%) 25 37 56 72 83 99 99 additive example 4 solutions of the final product bentazo-radiometric \ t the activity of α-trazine and bentazone on the segments of finazule, maize and barley.

Procedure: Gradient concentrations of aqueous solutions of the final atrazine product and segments of barley leaves, graded water concentrations, were applied to the acacia bean leaf segments. Inhibition of photosynthesis was evaluated radially from the radioactivity values of treated and control samples as in the previous examples. The selectivity of the tested herbicides is evident from the results of Tables2. 3, 8 and 9 from a comparison of photosynthesis inhibition (%) as a function of the concentration of these herbicides after treatment of the bean, maize and barley segments.

Claims (1)

256197 Radiometric evaluation of photosynthesis inhibition (%) versus atrazine concentration on maize leaf segments. 13 14 Table 8 Concentration of aatrazine (mg. Cm-3) Radioactivity control segment (cpm) Radioactivity treated segment (cpm) Photosynthesis inhibition (%) 2.0 18 665 15 330 17.9 2.5 17 545 11 220 36.1 5.0 18 025 495 97.5 10.0 17 985 195 98.9 Table 9 Radiometric evaluation of photosynthesis inhibition (%) versus concentration of bentazone (mg. Cm 3) concentration of bentazone on barley segments Inhibition of photosynthesis (%) Radioactivity control segment (cpm) Radioactivity Treatment Segment (cpm) 2.0 18 110 13 705 24.3 2.5 17 995 9 445 47.5 5.0 17 025 305 98.2 10.0 18 445 70 99.6 SUBJECT Type of indication a a quantitative evaluation of photosynthesis inhibiting substances, wherein a test substance solution is applied to the leaf segment of the plant, which, together with the symmetrical untreated control segments, is subjected to a radioactive 14CO2 light exertion radiometric The determination of the activity of treated and untreated segments, whereby the presence of photosynthesis inhibiting agents indicates a decrease in the activity of the treated segments compared to the untreated segment, and the photosynthesis inhibition in% is quantitatively evaluated from the decrease in radioactivity. 1 sheet of drawings