The evolution of resistance to herbicides in weedy species

Agro-Ecosystems, 4 (1978) 377--385

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© Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands

THE EVOLUTION OF RESISTANCE TO HERBICIDES IN WEEDY SPECIES

PIERRE GRIGNAC

Ecole Nationale Sup$rieure Agronomique, 34060 MontpeUier Cedex (France) (Received 22 August 1977)

ABSTRACT

Grignac, P., 1978. The evolution of resistance to herbicides in weedy species. AgroEcosystems, 4: 377--385. An ecotype of Poa annua was subjected to strong selective pressure by repeated metoxuron treatments. A highly resistant biotype appeared. Under natural conditions the resistance of the biotype under selective pressure evolved more or less rapidly according to the gene flow to which it was exposed. The occurrence of herbicide resistant biotypes seems to be a general phenomenon in many weedy species. We have noticed evolution towards increased resistance to, at~azine in populations of Echinochloa crus galli, Setaria viridis, Digitaria sanguinalis and Veronica persica. No such change was observed in Chenopodium album which apparently lacked atrazine resistance genes in the population studied.

INTRODUCTION

Allogamy seems to be characteristic of many weedy species of plants. Thus, in a given agricultural environment, a weedy species is represented by a heterogeneous population of individuals which show differences in their morphology and other biological features (development rate, precocity, seed production and dormancy). The use in a cultivated field of a herbicide with a more or less prolonged residual activity will introduce a powerful selective force. A large number of the susceptible plants are removed before resistant plants allowing more rapid multiplication of their progeny. Repeated treatment of a weed population with the same herbicide can produce selection favouring the build-up of resistant biotypes. The evolution of herbicide resistant forms had already been predicted by Blackman (1950) and Harper (1956) when the use of herbicides was becoming widespread. Ryan (1970) working with Senecio vulgaris L. and Radosevich and Appleby (1973) with Amaranthus retroflexus L. comment on the evolution of some biotypes towards high resistance to triazines. Holliday and Putwain (1974) showed that ecotypes of Capsella bursa pastoris L.,

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C h e n o p o d i u m a l b u m L. and Senecio vulgaris L., weeds in English orchards,

acquire resistance to simazine when treatments are repeated for several years. Gasquez et al. (1975) found that E c h i n o c h l o a crus galli ecotypes, occurring in maize crops regularly treated with triazines, can develop normally in soil treated with atrazine whereas ecotypes from other crops are destroyed under the same conditions. Grignac (1975) presents evidence for the acquisition of a high resistance to m e t o x u r o n b y an e c o t y p e o f Poa annua and for genetic change towards triazine resistance in Setaria uiridis L. and E c h i n o c h l o a crus galli L. We followed the evolution within a Poa annua e c o t y p e repeatedly treated with metoxuron. We also estimated the resistance of different populations of weedy species treated and untreated with triazines over several years. MATERIALS AND METHODS An e c o t y p e A of Poa annua was systematically treated with m e t o x u r o n (0.3 g of A.M. per square metre). It was spontaneously present on a bare ground site which was easy to spray and relatively well isolated from other populations of the same species. Treatments were carried o u t when most plants started tillering, and with an interval between applications of three to five months according to the period o f the year. The experiment was continued for eight years during which 25 successive herbicide applications were made. Before and after every treatment plant counts were made to determine the percentage of survivors. Under laboratory conditions we compared the evolution of resistance to m e t o x u r o n of plants grown from seeds of different ecotypes. E c o t y p e A is the treated e c o t y p e and ecotypes B and C were growing spontaneously on sites close to A and were not treated with herbicide. The experimental procedure was as follows. Seeds were germinated in Petri dishes at 20°C and 18 hour day length. The seedlings were transplanted at the firstAeaf stage and further grown in a greenhouse at 20 ° C. When most plants had produced one or t w o tillers, m e t o x u r o n was applied as a spray in t w o or three doses (0.3 g, 0.4 g and 0.6 g per square metre). Twenty days after treatment the surviving plants were counted and their average vigour scored (0 = plant killed, 10 = normal plant). There were four replicates of 100 plants for each herbicide concentration and each seed source. A change in the behaviour o f e c o t y p e A was noted after the 18th treatment. Seeds were then harvested and sown on three plots whose soils did n o t contain any spontaneous Poa annua seeds. On plots I and II, only ecotype A seeds were sown. Plot I was well isolated and plot II was located near e c o t y p e B. On plot III (also isolated) a mixture of 50% seeds of A (metoxuron selected) and 50% seeds of B (unselected) was sown. These plots were n o t treated b u t every t w o or three generations (four samplings in all) seeds were harvested from all three plots to test the resistance o f the plants to m e t o x u r o n under laboratory conditions.

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*'The evolution of resistance was also followed in other weedy species. On fallow or cultivated fields seeds of the following species were harvested: Echinochloa crus galli L., Setaria viridis L., Digitaria sanguinalis L., Chenopodium album L. and Veronica persica Pois. Thirty samplings were carried out over two consecutive years. The number of triazine treatments received was known for each sampling site. The various seeds were germinated in clay pots containing either the equivalent of 5 kg/ha atrazine or no herbicide. The number of seeds per pot should have yielded 200 seedlings. Twenty to thirty days after the seeds had been sown, living plants were counted. The percentage mortality due to atrazine is expressed as the ratio between the number of living plants in the treatment and the control pots. RESULTS Table I shows the results obtained during the metoxuron resistance assays made on plants of Poa annua of ecotypes A, B and C, before and after repeated herbicide treatment of ecotype A. At the beginning of the experiment, metoxuron destroyed nearly all plants from every ecotype and it was not possible to detect differences in the susceptibility of the three ecotypes. However, a few plants survived and were sufficiently vigorous to develop and produce seeds. TABLE I

The phytotoxicity o f m e t o x u r o n to three ecotypes of P o a

Doses

annua

At the start of the study

After 18 herbicide applications

0.3 g/m =

0.4 g/m =

0.3 g/m =

% o f living vigour plants

% o f living plants

vigour

% of living plants

vigour

% of living plants

vigour

1.25 0.75 1.50

5 4.5 3.5

46.5a* 3.4b 3.2b

7.2 5.6 6.3

24.3 0 0

4.5 ---

~o~A2.5 B 3 C 3.5

6.5 6 6.5 N.S.

0.6 g/m =

N.S.

* Values followed by a different letter are significantly different (P = 0.05). **N.S., n o t significant.

After many metoxuron treatments on ecotype A there was a significant change in behaviour with the appearance of a resistant biotype. A high percentage of plants were now resistant to what would be regarded as very high concentrations of metoxuron (0.6 g/m 2). No detectable change occurred in resistance of the untreated ecotypes B and C. Table II shows the results of plant counts after metoxuron treatments on ecotype A under natural conditions.

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TABLE II The evolution of resistance to metoxuron of a P o a Treatment number

% of plants still a l i v e

Treatment number

annua

% of plants still a l i v e

ecotype repeatedly treated Treatment number

% of plants still alive 56.2 54.6

1

6.3

9

16.2

17

2 3 4

7.4 5.8 7.0

10 11 12

22.8 19.1

18 19

5 6

7.5 6.7

13 14

26.4 30.3 28.0

20 21 22

7

8.3

15

31.5

23

81.6

8

18.4

16

30.8

24 25

83.0 82.4

59.4 61.2

82.5 83.2

Least significant difference 0.05 = 5.45 (L. S. D.). There was no detectable evolution of resistance until a sudden change occurred at the eighth treatment. Afterwards there was no continuous evolution of resistance but sudden increases of resistance each followed by a plateau~ After the 21st treatment there was a final plateau, unchanged even after four subsequent treatments. After the 18th treatment of ecotype A, seeds were harvested and sown on plots on which t h e y were no longer subjected to the selective pressure of herbicide treatments. Plot I, isolated from natural populations, should receive nnly very little contamination from outside pollen, the source of m e t o x u r o n susceptibility genes. On plot II, not isolated, a higher proportion of flowers might be expected to be fertilized b y pollen from susceptible plants and ecotype A might be expected to be more exposed to the inflow of susceptibility genes. Plot III was isolated and contained mixed populations of m e t o x u r o n selected and unselected ecotypes. At the beginning of the experiment, pollen from either ecotype might be expected to fertilize the flowers with equal probability, provided the plants had the same vigour, similar competitive ability and identical pollen production. If those conditions are respected, susceptibility and resistance gene flows should be in equilibrium. Table III shows the evolution of the resistance of the populations from all three plots. Results were obtained after assays had been carried o u t under laboratory conditions. On plots I and IH, in the absence of selective pressure, there was almost identical slow loss resistance, which became significant only after the fourth sampling, i.e. after eight generations. On plot I, the slow deviation towards susceptibility can be explained in terms of imperfect isolation. Identical results were obtained on plot III, which indicates that

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T A B L E III E v o l u t i o n o f r e s i s t a n c e t o m e t o x u r o n in p o p u l a t i o n s o f P o a a n n u a ( b i o t y p e A): p e r c e n t a g e o f p l a n t s alive a f t e r t r e a t m e n t

Origin o f t h e seeds Used seed 1st s a m p l i n g 2nd sampling 3rd sampling 4th sampling

Plot I (isolated)

P l o t II (not isolated)

P l o t III (isolated)

100 % A 4 6 . 4 a* 45.6 a 43.4 a 44.3 a 36.5 b

100 % A 46.4 a 41.3 a 36.4 b 28.3 c 20.2 d

50 % A + 50 % B 47.0 a 41.0 a 44.8 a 48.8 a 26.4 c

*Values f o l l o w e d b y a d i f f e r e n t l e t t e r are significantly d i f f e r e n t (P = 0.05).

there was a balanced flow between susceptibility genes from the natural ecotype and resistance genes occurring in ecotype A plants. Plot II, not isolated, lost resistance much faster: after the second sampling, i.e. after three generations, there was a significant decline in herbicide resistance, Results from the second set of experiments are shown in Table IV. They involved seeds from different weedy species. Results are expressed in terms of percentage mortality after treating the soil with atrazine (5 kg A.M./hectaxe). To avoid overcrowding the table, only representative means from a few populations are given which illustrate the whole of the collected data. For Echinochloa crus galli, Setaria viridis and Digitaria sanguinalis there was a detectable change in herbicide resistance after the fifth or the sixth treatment and a further change after the eighth treatment. No resistance developed in Chenopodium album. Results for Veronica persica varied according to the populations tested which makes it difficult to interpret: there seemed to be a tendency towards development of resistance. DISCUSSION

The method we used in our assays to test the resistance of plants grown from seeds avoids the problems arising from the large differences between weedy species in their germination and development times. We could not recognize in Poa annua distinct morphological criteria that distinguished the plants resistant to metoxuron. The special behaviour of the resistant plants is not caused by detectable differences in phenology or morphology but presumably by some unknown physiological features. The development of resistant biotypes of Poa annua in these experiments could be considered rapid. However, conditions were chosen to maximise the chance of resistance developing. Poa annua is capable of several reproductive cycles each year; its seeds have a short life-span in the first centimeters of the

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TABLE IV P e r c e n t a g e m o r t a l i t y in p o p u l a t i o n s o f various w e e d species t r e a t e d w i t h 5 k g / h a o f atrazine. T h e p o p u l a t i o n s h a d d i f f e r e n t p r e v i o u s histories o f h e r b i c i d e a p p l i c a t i o n s Species

Rotation or c r o p

N u m b e r o f triazine t r e a t m e n t s received by the crops before seed was h a r v e s t e d

Percentage mortality

1975

1976

1975

1976

Echinochloa crus gatli

fallow grape vine grape vine grape vine maize--wheat maize--wheat maize maize

0 2 5 9 3 8 8 13

0 3 6 10 3 9 9 14

94.5 95.3 83.4 70.5 93.8 72.0 68.8 66.4

a* a b c a c c c

96.4 97.3 86.5 73.4 95.5 75.4 69.3 69.4

a a b c a c c c

Setaria viridis

fallow grape vine grape vine grape vine maize--wheat maize--wheat maize

0 2 5 9 3 8 13

0 3 6 10 3 9 14

92.8 93.4 72.5 68.8 92.2 68.5 67.8

a a b b a b b

91.5 91.8 70.4 66.5 90.0 68.0 68.5

a a b b a b b

Digitaria sanguinalis

fallow grape vine maize--wheat wheat

0 9 8 13

0 10 9 14

82.5 66.0 70.5 70.0

a b b b

78.5 67.0 68.5 71.0

a b b b

Chenopodium album

fallow grape vine grape vine maize--wheat maize

0 5 9 8 13

0 6 10 9 14

98.4 98.9 98.5 99.0 97.5

a a a a a

100 100 97.5 99.0 98.5

a a a a a

Veronica persica

fallow grape vine maize--wheat maize

0 9 8 13

0 10 9 14

87.5 70.0 65.5 59.0

a b bc c

78.5 59.5 64.0 55.0

ab c bc cd

* F o r a given species, results f o l l o w e d b y d i f f e r e n t letters differ significantly.

soil; the ecotype numerous.

was isolated; herbicide

treatments

were both frequent

and

It is difficult to interpret the plateaux followed by sudden increases in resistance; they did n o t coincide regularly with the same season of the year. The fact that resistance did n o t increase further after reaching the 81% threshold

383 may be explained if some cross-fertilization occurred inspite of our precautions. When selective pressures on b i o t y p e A, resistant to metoxuron, were stopped (cf. experiments referred to in Table III) there were variations in the decline of resistance according to the extent of isolation. Results from plot I (isolated from external pollen sources) showed that the b i o t y p e maintained its resistance level for eight generations. It colonized the site as quickly and as completely as the other spontaneous populations. The loss of resistance on plot III was similar to that taking place on plot I, indicating that pollen production and the competitive ability o f b i o t y p e A plants did n o t differ from that o f the spontaneous plants. The flow of genes from susceptible plants would appear to be balanced b y the resistance gene flow from b i o t y p e A plants. The decline in resistance occurring at the fourth sampling (i.e. after eight generations) seems to be due to the imperfect isolation that we achieved. There is no evidence that the acquisition o f resistance to m e t o x u r o n is a handicap or an advantage for the resistant b i o t y p e in comparison with the spontaneous ecotypes in a herbicide-free environment. This would explain the similarity of the populations for that characteristic (cf. Table I): initially all ecotypes had the same percentage of resistant plants. There was, however, a rapid fall of resistance on plot II which presumably gained a considerable a m o u n t of pollen from susceptible plants. After a few years the initial and usual equilibrium value of 1 to 3% resistant plants would probably be re-established. Only a minute proportion of the natural local population of Poa annua possessed resistance to m e t o x u r o n and a more or less rapid return to initial conditions is expected when herbicide treatment is stopped. There was evidence of evolution of herbicide resistance within other weed species that had been repeatedly treated with herbicides. In Echinochloa crus galli and Setaria viridis a shift towards atrazine resistance was detectable after the fifth generation i.e. faster than for Poa annua treated with metoxuron. However, atrazine has quite a long persistence in the soil and the p r o d u c t remains active t h r o u g h o u t the period of plant development. Digitaria sanguinalis showed less susceptibility to atrazine in the populations that had not been treated, b u t perceptible development o f resistance could n o t be detected until the ninth treatment. For these three species the percentage of mortality in each case reached and remained constant at ca. 66% i.e. two-thirds of the plants were susceptible to atrazine. This may be a coincidence and further studies are needed. On the other hand populations of Chenopodium album did n o t show any sign of developing resistance, even after 14 treatments. This different behaviour may be connected with the absence of a source of resistance in the populations studied and with the high level of dormancy ~ f the seeds that allows the accumulation of a seed bank conferring a certain stability on the populations. The behaviour of Veronica persica presents a special case. Germination and seedling establishment usually occur in autumn and seed production is significant

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in March--April, under meridional conditions. This species is therefore only rarely a weed in maize crops and should avoid the selective action of atrazine. Biological assays of atrazine in soft have demonstrated the presence of the herbicide when Veronica persica is germinating. In fact, there is evolution of resistance not only in populations present in the treated crops but also in populations in other crops of the rotation because of the persistence of the substance. In contrast, the maize--wheat type of rotation, where the wheat is not treated with triazines, plays no part or only a minor one in the creation of resistant biotypes for adventitious species of maize crops (three times, there is no significant effect of this rotation on the percentage of mortality, with respectively 3,3 and 9 treatments). These species are absent in the wheat crop which has a different development cycle. There is selection only during maize cultivation which includes a triazine treatment. CONCLUSION

In many species the development of biotypes resistant to herbicides seems to be quite a widespread phenomenon when treatments with the same herbicide are repeated for several years and when particular resistance genes exist in the treated population. It is difficult to estimate the importance of this problem at this moment. The evolutionary process is slow and, under natural conditions, appears to have a tendency towards an equilibrium value depending on crossfertilization and pollen exchanges. However, in many areas of Southern France triazine resistant biotypes are present in populations of Echinochloa crus galli, Setaria viridis and Digitaria sanguinalis. Herbicides of the atrazine group become less efficient and new substances must be applied. ACKNOWLEDGEMENTS

The author wishes to acknowledge the help given by Dr. P. Jacquard and Prof. J.L. Harper during the preparation of the manuscript.

REFERENCES Blackman, G.E., 1950. Selection toxicity and the development of selective weed killers. J. R. Soc. Arts, 98: 500--517. Gasquez, J., Compoint, J.P. and Barralis, G., 1975. Biologie et diff~renciations taxonomiques d'une mauvaise herbe Echinochloa crus galli L. P.B. Eur. Weed Res. Symp. 1, 1975, pp. 330--339. Grignac, P., 1974. S~lection d'un biotype de Paturin annuel (Poa annua) r~sistant au metoxuron par r~p~tition de traitements herbicides. A.C. Agriculture France, 60: 401--408. Grignac, P., 1975. D~viation g~n~tiques de biotypes de plantes adventices sous l'action r~p~t~e de traitements herbicides. Eur. Weed Res. Symp. 1, 1975, pp. 340--348. Harper, J.L., 1956. The evolution of weed in relation to resistance to herbicides. Proc. 3rd Br. Weed Control Conf., pp. 179--188.

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Holliday, R.J. and Putwain, P.D., 1974. Variation in the susceptibility to simazine in three species of annual weeds. Proc. 12th Br. Weed Control Conf., pp. 649--654. Radosevich, S.R. and Appleby, A.P., 1973. Relative susceptibility of two common groundsel (Senecio vulgaris L.) biotypes to six s-triazines. Agron. J., 65: 553--555. Ryan, G.F., 1970. Resistance of common groundsel to simazine and atrazine. Weed Sci., 18: 614--616.

RESUME Grignac, P., 1978. Evolution de la r~sistance aux herbicides chez des esp~ces adventices. Agro-Ecosystems, 4 : 3 7 7 - - 3 8 5 (en anglais). Dans u n ~cotype de Poa annua L., soumis ~ une forte pression de s~lection par des traitements r~p~t~s ~ base de metoxuron, est apparu u n biotype tr~s r~sistant ~ cette substance herbicide. En conditions naturelles, sous pression de s~lection, la r~sistance du biotype ~volue plus ou moins rapidement suivant le flux de g~nes de sensibilit~ auquel il est soumis. L'apparition de biotypes r~sistants ~ u n herbicide semble un ph~nom~ne assez g~n~ral pour de nombreuses esp~ces. Nous avons observ~ cette ~volution vers une r~sistance accrue l'atrazine dans le cas de populations d'Echinochloa crus gaUi, Setaria viridis, Digitaria sanguinalis et Veronica persica. Par contre, chez Chenopodium album aucune d~viation n'a pu ~tre raise en ~vidence, les g~nes de r~sistance ~ l'atrazine paraissent absents darts les populations ~tudi4es.