H'm/ Research. 1986, Volume 26, 167 172

Appearance of a biotype of the weed, Hordeumglaucum Steud., resistant to the herbicide paraquat

S. B. POWLES Department of Agronomy.Waile Agrieutlurat Research Institute.University of Adelaide. South Australia

Accepted 22 June 1985

Summary: Resume: Zusammenfassung

A biotype of the grass weed Hordeum gtaucumSteud. infesting a site at Willaura, Victoria,Atlstralia has resistance to paraquat. Applicationof the recommended rate of paraquat does notcausedeathoftheresistant biotype at any stage ofgrowth. The LD i, for the resistant biotype is 6-4kg active ingredient ha ' which is 250 timesgreater than for the normal susceptible biotype(25 g active ingredient ha '). Growth of theresistant biotype is checked by paraquat with aclear dosage response evident. The paraquatresistant biotype is also resistant to diquat but isnormally affected by herbicides with differentmodes of action. In addition to continued foliagegrowth of the resistant plants after paraquatapplication, seeds of these plants can germinateand seedlings elongate in the dark whereas seedsof susceptible plants germinate but there is nofurther growth. This suggests that sttidies of themechanism(s) conferring resistance will have toconsider both the efFcct of paraquat on thechloroplast and a non-photosynthetic eflect oncell growth.

Vn biotype de ta mauvaise herbe Hordeum glau-eum Sleud. resistant a t'tiertiicide paraquat

Un biotype de la graminee Hordeum glaucumSteud, a Willaura. Victoria. Australie. s'estmontre resistant au paraquat. L'application de ladose preconisee de paraquat nc provoque pas la

mort de ce biotype, quel qu"en soit le stadevegetal. La LD.so pour le biotype resistant est 6,4kg matiere active ha"', c'est-a-dire 250 fois plusgrande que pour le biotype normal sensible (25 gmatiere active ha '). Le paraquat provoque chezle biotype resistant une inhibition de eroissancequi se rapporte a la dose, Le biotype resistant auparaquat Test egalement au diquat mais reagitnormalement envers les herbicides a mode d*ae-tion ditferente. Non seulement la croissancefoiiaire continue normaiement apres une appli-cation dc paraquat chez les plantes resistantes,mais les graines sont eapables de germer et lesjeunes plants de s'allonger a l'obscurite, tandisque les graines de plantes sensibles germent aTobscuritc mais ne croissent pas. II semble doneque les etudes des mecanismes qui produisent laresistance devront examiner I'influencc du para-quat sur le chloroplaste ainsi qu'un effet non-photosynthetique sur la croissance cellulaire.

L'elier das Auftreten einesgegen Paraquat resislen-tcn Bioivps von Hordeum glaucum Steud.

Bei Willaura. Victoria (Australicn) iritt ein gegenParaquat resistcnler Biotyp von Hordeum gtuu-I'lim Steud. auf. Die Application der normaler-weise empfohlenen Dosicrung Paraquat totet denresistenten Biotyp in keinem Wachstumsstadiumab. Die Ldsofurden resistenten Typ betragt 6.4 kgai ha '; dies ist 250 mai mehr als beim normalsensiblen Typ (25 g ai ha '). Das Wachstum desresistenten Biotyps wird durch steigende Dosenvon Paraquat beeintrachtigt. Der gegen Paraquatresistente Typ ist auch gegen Diquat unempfin-dlich. weist aber gegcnlibcr Herbiziden mitanderen Wirkungsmeehanismen die normaleEmpiindlichkeit auf. Resistente Pfianzen zeigennach Paraquatbehandlung ein weitergehendesBlattwachstum. Ihre Samen keimen und dieSamlinge entwickein sich im Dunkehi weiter.wahrend die Samen sensibler Pflanzen zwar kei-

168 S. B. Pon-les

men, sich aber nicht weiterentwickeln. DieseBeobachtungcn weisen darauf hin. dass bei For-schungen zur Aufkliirung der Resistenzmechanis-men, sowohl die Wirkung von Paraquat auf dieChloroplasten als auch einen nicht photosynthe-tisch wirksamen Effekt auf das Zellwachstumberiicksichtigen miissen.

Introduction

Weed populations with resistance to a particularherbicide are being increasingly reported in thenorthern hemisphere. There is now widespreadresistance among several weed species to thetriazine type herbicides (Bandeen, Stephenson &Cowett, 1982; Gressel c/tj/., 1982), However onlyone studied weed species, Conyza bonariensis (L.)Cronq,. has been confirmed to have developedresistance to a bipyridyl herbicide (Youngman &Dodge, 1981; Harvey & Harper, 1982).

Bipyridyl herbicides were used successfully ona mixed cereal cropping and sheep grazing enter-prise at Willaura (stale of Victoria. Australia)over a 15-year period to control winter-growingweeds in fields of lucerne {Medicago .saliva L.).Throughout this period good contro! wasachieved of the widespread and important grassweeds, known locally as barley grasses {Honteumleporinum Link, Hordeum gtaueum Steud. andHordeum marimtm Huds. var. (^s^t.) gussoneanum(Part.) Thel. ( = //. hystcrix Roth.}). However in1981, following application of the recommendedrate of 200 g ai ha ' paraquat, poor control ofbarley grass was evident in one of four fields sownto lucerne. Again in 1982 the same rate ofherbicide was ineffectual in controlling barleygrass. Applications in 1982 of 230 g ai ha '.followed 2 weeks later by 280 g ai ha ' wereineffectual. Samples of the barley grass popula-tion were identified as H. gtaueum and wereconfirmed to survive applications up to 1 6 kg aiha"' paraquat (Warner & Mackie, 1983). Barleygrass populations infesting three other lucernefields on this farm showed no evidence of resis-tance and were controlled by the recommendedrate of paraquat. Noother instances of resistanceto paraquat were reported in the area.

Tbe appearance of herbieide resistance is a newphenomenon in Australia, A weed showing herbi-cide resistance is of economic and scientificsignificance, and studies were commenced on thisresistant population to establish a dose mortality

curve and the effect of applications at differentgrowth stages.

Materials and methods

PtanI nialerial

Seeds of H. gtaucum were collected at Willaurafrom the population showing survival under highrates of application of paraquat. Seedlings weregrown at a density of four plants per pot, in potscontaining 2 / of loamy soil. Seeds of H. gtaucumcollected at Palmer in South Australia (500 kmfrom Willaura). with normal susceptibility toparaquat, were grown as control plants. Follow-ing germination and emergence in a glasshouse,the plants were maintained outdoors for theremainder of the experiment. Plants were grownthroughout the winter months, corresponding tothe normal growth period for this species. (Ade-laide has a mediterranean climate with a cool, wetwinter growing season.)

Herbieide apptication

The plants were sprayed using a hydraulic pres-sure nozzle (Hardi Tee-Jet 8001 I-E) mountedover a variable speed continuous belt circulatingat a speed of l'43 m s"'. Tbe spray mixture(herbicide plus 0 2% surfactant) was pumped viaa regulator and filter to the nozzle at a pressure of200 kPa and directed at the target plants 26 cmbelow the nozzle. Tbe output of tbe nozzle at theheight of the foliage was evenly dispersed over a38 cm fan width, as was verified using a patterna-tor. Total spray output with this nozzle, pressureand belt speed was 100 / ha '.

The plants were treated while in deep shade andwithin 30 min of sunset and were then placed indarkness until the following day. Tbis procedurewas adopted to ensure a maximum effect from theparaqual (Brian & Headford, 1968). Following a12-b dark period the treated and control plantswere moved outdoors and kept under the naturalprevailing conditions.

Measuremenls

Dry matter was measured 21 days after herbicidetreatment by harvesting the above-ground greentiller and ieaftissue from eacb pot, drying for 24 hat 80 C and measuring tbe total dry weight of the

four plants. In experiments determining the efTectof herbicide treatment on survival the plants weremaintained through anthesis. Determination ofleaf area (10 days after treatment with paraquat)was made by harvesting the above-ground greenleaf tissue (leaf tissue bleached by paraquat wasexcluded) of four plants in each pot and measur-ing tbe total leaf area (Paton electronic leaf areameter).

Results

Plant appearanee following paraquat apptteation

Application to the foliage of 200 g ai ba ' (i.e., tberecommended rate of a commercial preparationof paraquat) caused death of susceptible //.gtaucum but not of the resistant biotype fromWillaura. The typical paraquat toxicity symp-toms of rapid (within 24 h) and massive bleachingof green tissue, followed by plant death, occurredwith the susceptible H. glaucum. However, withtbe resistant biotype only localized damage andlimited chlorophyll bleaching occurred. Theoldest leaves and leaf tips of individual leaveswere partially bleached whereas the young leavesand the younger sections of mature leavesremained green. The most striking differencebetween the susceptible and resistant biotypeswas that the young emerging leaves of theresistant biotype remained green, and continuedto grow after herbicide treatment. Although thegrowth rate was checked because of some bleach-ing and death of older tissue, the emerging leaftissue led rapidly to new growth.

Survival o/ H. g\nucurv\ following paraquat uppli-cation

Effect of plant age. It was of interest to know ifthe difTerence in resistance between resistant andsusceptible biotypes changed with leafage. Bothresistant and susceptible plants were treated atthe rate of 200 g ai ha"' . The effeets of tbeherbicide were assessed by the survival of treatedplants and the production ofviable seed. Treat-ment of the susceptible biotype. at different stagesof development (from single leaf through to post-anthesis), always resulted in death. By contrast.the resistant biotype was not killed and set viableseed (data not shown).

Hordeum glaucum resismni to paraqual 169

Table I EITecl of paraquat on survival of H. gkiucunt

Appliciilion ratekg ai ha '

Susceptible biotype0025OOS010-2

Rcsisliinl biotvpe020-40-81-63-24-86-4

No. of planissprayed

2930

200200

212275243250241233177

No.survived

11000

21227524325022519072

survival

38000

100100100100938241

PUmts were sprayed ai Ihe three tiller stage.

Effect oftterbicide rale. The effects of sprayingyoung susceptible and resistant plants at differentrates of paraquat is shown in Table 1. Tbe plantswere sprayed 36 days after seeding when at thethree tiller stage. An application of 50 g ai ha 'paraquat (I /4 the recommended applicationrate), or higher, resulted in death of the suscep-tible population. The LDsn for this populationwas approx. 0 025 kg ai ha '. However theWillaura biotype showed no mortality until tbeapplication rate reached 3 2 kg ai ha ', witb theLDs,i being around 6 4kgai ha '.Thus the lethaldose of paraquat required for H. gtaueum infest-ing the field at Willaura is some 250 times higherthan for //. gtaucum collected in South Australia.

Effect of paraquat application on dry matterproduction. Tbe results presented above indieatethe resistant biotype is only killed by paraquatapplied al extremely high rates; and it is alsoimportant to know if the recommended rate willreduce the vigour of the weed. Plants sprayed atthe one, two and three tiller stage showed reducedgrowth dependent upon the dose of paraquatapplied. Figure I shows the efTect of differentrates of paraquat on the dry matter production ofthe resistant biotype. Tbe plants were sprayed atthe three different stages of development andharvested 21 days later, along with unsprayedplants as a control. Increased doses of paraquatcaused further reduetion in plant weight witb asignificant response to herbicide dosage evident(Fig. I).

170 S. B. Powtes

Table 2 Efiect of par;tqual (200 g ai ha ') :ipplicii at anthesis on subsequent spike development iiiid seed germinaiion of the resistant H.glaucum

Mature spikes coliecled from 25 plants

Bleached, sierife spikes

Control(LInsprayed)

1144_

"germination

78

Sprayed

993

200

germination

780

Effect of paraquat apptication on teaf area. Aswas found above for the dry matter (Fig. I) theapplication of the recommended rate (200 g aiha"') resulted in approximately 43"o loss of greenleaf tissue as a result of bleaching. Increasingdoses of paraquat caused little further loss of leafarea. In contrast, treatment of the susceptible //.

(a)

6-4

V'\%. I EffeLt of increasing dosageof paraquat on the dry matterproduction (21 days alter trcalmcnt) of the resistant biotype ofHordi'uni gkuicuni sprayed at the single (a), two (b) or three (c)tiller stage. Bars are least significant difTerence at 5",, level.

glaucum at 200 g ai ha ' caused total leafdestruction (results not shown).

Effect of application at anttwsis on subsequentinflorescence devetopmcnt. The effeet of sprayingresistant plants at anthesis on the subsequentproduction of spikes is shown in Table 2. In theseexperiments, uniformly-sized plants were treatedat anthesis with 200 g ai ha ' paraquat andcompared with unsprayed plants for productionof spikes. Spraying did not reduce the number ofspikes that developed but did cause approxima-tely 10",, of the spikes to bleach and these spikesdid not develop seed. The remainder of the spikesdeveloped normally. Except for the bleachedspikes the seed from spikes of both unsprayed andtreated plants had the same germination rate ofaround 78",,. Application of the same rate ofparaquat to susceptible H. gtaucum eaused plantdeath without the production of any viable seed(results not shown).

Effect of atternalive tierbicides lo paraquat.Table 3 shows that the resistance to paraquatevident in the Willaura biotype of H. gtaueum isspecific to the bipyridyl herbicides. Application ofthe recommended rate of three herbicides withdifferent modes of aetion caused death of both theWillaura biotype and H. glaueum collected in

Tablt 3 Effect of application iif uliernalivc herbicides toparaquat on survival of H. glaucum

Herbicide

Diquat (bipyridyl)DiuronFluazifop-butylGlyphosate

Ratekg ai ha '

022-50-20-36

% stjrvival

Susceptible

60000

Resistant

100000

Plants (minimum of 50 plants treated) were sprayed at theihree liller stage.

H o r d e u m g l a u e u m resistant to paraqual 171

South Australia. The bipyridyl herbieide diquat(recommended for dicotyledonous species) wasineffective in controlling the resistant biotype andonly partially successful in controlling the suscep-tible biotype.

Effect of paraquat on seed germination. Seeds ofthe resistant and the suseeptible biotypes wereincubated in Ihe dark at 19 C on filter paper prc-soaked with water containing low concenirationsof herbicide. In these experiments only the activeingredient (methylviologen) was used to eliminatethe possibility that effeets may be due to additio-nal compounds present in commercial prep-arations of paraquat herbicide. Percentage germi-nation, shoot and root length were assessed 10days after incubation. Seed from the resistantbiotype and the susceptible biotype showed simi-lar rates of germination (assessed as emergence ofthe shoot and root tip) of appro.ximately 78'\, atall levels of methylviologen. Inhibition of theelongation of the seminal root was evident bymethylviologen with equal inhibition of rootgrowth in seed from resistant or susceptibleplants.

There was a difference between seeds of theresistant and the susceptible biotypes in the

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EE

c0)

o-

60.50^

40

30

20

10

n.

°c60

50

40

30

20

10

^

SI

--

IS-1 1

20 40

* — * —20 40

UN

1 1 1 1

60 60 100 120

1 M-. 1 4

60 80 100 120

Methylvioloqen

(o)

1 1

140 160

(b)

t m J

140 IGO

Fig. 2 t.cngih |mm} of tbe primary shoot of seeds of resislant (a)and susL'(jpiible(b)biotypesofW.t;/i;u( mil incubated for 10daysin ihe durk at 19 C in liifferenL motariiies of meihvKiulogen.Values are means ± stand^ird deviations of 10 repliL-aLcs. Eauhrepliciile »ns 10 seeds.

ability of the primary shoot to elongate in thepresenee of herbicide. With the seed from plantsresistant to paraquat the emerging shoot was ableto elongate at levels of methylviologen from 0 lo155 fiM. There was no significant differenee in thelength of the primary shoot of seeds incubated atlow concentrations of methylviologen, althoughat 155 /IM inhibition of shoot length was evident(Fig. 2a). By contrast, with seeds from thesusceptible biotype, incubation even at 15 /mmethylviologen resulted in significant inhibitionof shoot elongation and at high concentrationvery little shoot elongation occurred (Fig. 2b).

In other experiments seeds of resistant andsusceptible H. gtaucum were placed on the soilsurface and the pots sprayed with paraquat at 200g ai ha "'. There was no effect of paraquat on %germination or emergence (data not shown)confirming that paraquat is inactivated by con-tact with soil.

Discussion

It Is clear that the biotype of H. gtaueum atWillaura is resistant to the bipridyl herbicides.When plants grown from seed collected at this sitewere spayed at the recommended rates of para-quat there was 100",, survival regardless of the ageof the plants at spraying. The same treatmentsapplied to H. glaucum collected from a separatelocation resulted in death of all plants.

While paraquat does not cause death of theresistant biotype it does cause some damage toIhe foliage of the plants. Leaf tips and oldestleaves dessieate and bleach. However, theyounger leaves are unaffected and lead to newgrowth with the result that within approximately4 weeks the effects of paraquat are difficult todetect. The amount of leaf damage to the resistantbiotype is greater at higher rates of paraquat witha dose response for leaf damage and growthevident (Fig. I). Treatment of normal barley grassresults in total leaf bleaching and rapid death ofthe plant.

It is clear that the accepted practice of employ-ing paraquat at the recommended rate of 2()0gaiha ' (applied to young, actively growing weeds) isineffective in controlling the resistant biotypeinfesting the farm at Willaura, Similarly, the useof paraquat at anthesis (spray-topping to preventseed set) is ineffective with the resistant biotype as

172 S. B. Powtes

it can still produce fertile seed after treatment attwice the recommended rate (Table 2).

The resistance evident in the Willaura biotypeof H. glaucum is specific to the bipyridyl herbi-cides. Table 3 shows that herbicides with differentmodes of action are effective in controlling thisweed. Therefore, from a practical viewpoint.control of the paraquat resistant biotype can beobtained by the use of a non-bipyridyl herbicide.

The herbicidal effect of paraquat is muchgreater in the presence of light (Brian & Head-ford, 1968) consistent with its mode of action asan acceptor of electrons at photosystem onewithin the chloroplast (Youngman & Dodge,1981). The observation (Fig. 2a) that the shoot ofgerminating seeds of the resistant biotype couldgrow in the dark in the presence of paraquat,whereas the susceptible type could not (Fig. 2b) isof eonsiderable importance. This indicates thatparaquat has effects in addition to disruptingphotosynthesis and shows that the resistant bio-type displays resistance also to this (unknown)site of action.

The appearance of the resistant biotype withinthe cereal growing region of southern Australiamay cause problems to farmers. Studies are inprogress to determine the current areas of infes-tation by this resistant biotype and its possibledispersion into areas where bipyridyl herbicide iswidely used. Other studies are underway todetermine the genetical basis of the resistance andthe physiological mechanism(s) involved.

Acknowledgment

The encouragement of Dr R. Knight and Profes-sor C.J. Driseoll is gratefully acknowledged. Thiswork was initiated while the author was theholder of a Research Fellowship (Agriculture)from the Reserve Bank of Australia and conti-nued by a grant from the Wheat and the BarleyIndustry Researeh Councils.

(Received 14 Febrttary 1985)

References

BANIHI:N J.D.. STEPHKNSON G.R. & O)WL:TT E .R . (1982)Discovery ynd distribution of herbicide-resistani weeds inNurih America. In: Herbicide Ri'si.sianre in Plums, (eds H.M.Le Baron and J. Gressel), pp. y- 30. John Wiley & Sons, NewYork.

BRIAN R . C . & HKADFORD D.W.R. (1968) The effect of environ-meni on the activily of hipyridylium herbicides. Proceedings9lh Brili.sh Weed Conlrol Conference. 108 114.

GRI:SSI[L J,, AMMON H.U., FOGELFORS H. , GASQUKZ J.. KAYQ.O.N. & KHB H . (1982) Discovery and distribution ofherbicide-resistanl weeds outside North America. HerbicideResistance in Plants, (eds H.M. Le Baron and J. Grcssel). pp.31 56. John Wiley & Sons. New York.

H. RViv B . M . R . & H A R P L R D . B . (1982) Tolerance to bipyridi-lium herbicides. In: Herbicide Resistance in Planis. (eds H.M.Le Baron & J. Gressell, pp. 275 356. John Wiley & Sons.New York.

WARM.R R.B. & MACKLL W . B . C . (1983) A barley grassHorileuD! leporinum sppl. glaucum Steud. population lolerantto paraqual (Griimoxone). Australian Weed Research News-letter. 3t. 16.

YouNCiMAN R.J. & Dontiv A.D. {\9^\] On the mechanism ofparaqual resistance in Cony:a spp. Provccdings Sth Intcrna-lional Congress of Photosynthesis, (ed. G. Akoiinoglou), Vol.VI. pp. 537 544. Balbar International Science Services.