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Crop Protection 28 (2009) 923–927

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Crop Protection

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Effect of isoxaflutole on the growth, nodulation and nitrogenfixation of chickpea (Cicer arietinum L.)

Avishek Datta a,c,*, Brian M. Sindel a,c, Paul Kristiansen a, Robin S. Jessop a,c, Warwick L. Felton b

a School of Environmental and Rural Science, University of New England, Armidale, New South Wales 2351, Australiab Nyrang Avenue, Tamworth, New South Wales 2340, Australiac Cooperative Research Centre for Australian Weed Management, Australia

a r t i c l e i n f o

Article history:Received 5 September 2008Received in revised form14 July 2009Accepted 19 July 2009

Keywords:IsoxaflutoleChickpeaPhytotoxicityGrowthNodulationNitrogen fixation

* Corresponding author at: Haskell AgriculturaNebraska-Lincoln, Concord, NE 68728-2828, USA. Tel402 584 3859.

E-mail address: adatta2@unl.edu (A. Datta).

0261-2194/$ – see front matter � 2009 Elsevier Ltd.doi:10.1016/j.cropro.2009.07.009

a b s t r a c t

Isoxaflutole at 75 g/ha is registered in Australia for the control of several broadleaf weeds in chickpea.Although isoxaflutole provides satisfactory control of problematic weeds, under certain conditions cropinjury can occur. A controlled-environment experiment was conducted to investigate the extent ofisoxaflutole damage on overall growth, nodulation and nitrogen (N) fixation capacity of one isoxaflutoletolerant and one isoxaflutole sensitive chickpea cultivar. Chickpea cultivars were grown under differentsoil nitrate (NO3

�) levels (0 and 1.5 mM), rhizobia treatments (inoculated and uninoculated control) andherbicide rates [0 and 75 g/ha (recommended rate)]. Crop growth, nodulation and N fixation in responseto isoxaflutole were assessed 42 days after herbicide treatment. Isoxaflutole at the recommended ratestunted plant growth (29%) and reduced shoot (22%) and root (50%) dry weights of the sensitive cultivarwhereas isoxaflutole had no effect on the growth parameters of the tolerant cultivar. Isoxaflutole causedmore root damage than shoot damage in the sensitive cultivar. Nodulation (number and dry weight) wasalso less in the sensitive cultivar when isoxaflutole was applied. Isoxaflutole reduced total N content ofthe sensitive cultivar by 21%. At the recommended rate of isoxaflutole, the herbicide reduced the Nfixation capacity of the sensitive cultivar by 51% compared with the tolerant cultivar (33%) even thoughboth cultivars produced identical amounts of fixed N in the presence of the herbicide. The results suggestthat there is a substantial loss in the N fixation capacity of both the tolerant and sensitive chickpeacultivars when isoxaflutole is applied at the recommended field rate. Further work is needed to examinewhether the N benefits of chickpea rotations are depleted with the use of isoxaflutole under fieldconditions.

� 2009 Elsevier Ltd. All rights reserved.

1. Introduction

Chickpea (Cicer arietinum L.) is one of several pulses suited to thefine-textured, neutral-to-alkaline clay soils of the eastern croppingzone of both Western Australia and Eastern Australia (Siddiqueet al., 1993). Chickpea is a crop that provides a cash income fromgrain, requires minimal nitrogen (N) fertiliser through its ability tofix atmospheric N and in a crop rotation can improve the N nutri-tion and yield of subsequent cereals (Doughton et al., 1993). Theinclusion of legumes in rotations with cereals or other crops canimprove soil structure, provide a break in disease and cereal pestcycles and increase the options available for weed control (Peopleset al., 1992). The presence of weeds within crops can reduce crop

l Laboratory, University of.: þ1 402 584 3810; fax: þ1

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yields, hinder harvest operations and contaminate produce (Powleset al., 1996), and herbicides are commonly used for weed control inpastures and crops (Lemerle et al., 1996). One of the major obstaclesin growing chickpea successfully is its poor ability to compete withweeds. Severe crop losses are possible in weedy situations and thelack of registered post-emergence herbicides for broadleaf weedsreduces the options for weed management (Knights, 1991).

In Australia, isoxaflutole at 75 g/ha is registered for use in chick-peas to control several broadleaf weeds [e.g. Indian hedge mustard(Sisymbrium orientale Torn.), sowthistle (Sonchus oleraceus L.),capeweed (Arctotheca calendula L. Levnys.), prickly lettuce (Lactucaserriola L.), wild radish (Raphanus raphanistrum L.) and turnip weed(Rapistrum rugosum L. All.)]. The herbicidal activity of isoxaflutole insusceptible species is associated with the development of a charac-teristic bleaching of foliar tissue following treatment (Pallett et al.,2001). The biochemical target of isoxaflutole is the enzyme4-hydroxyphenylpyruvate dioxygenase (HPPD), inhibition of whichresults in a depletion of plastoquinone, which is a co-factor of

A. Datta et al. / Crop Protection 28 (2009) 923–927924

phytoene desaturase (Pallett et al., 1998; Viviani et al., 1998). Thiseffect results in a depletion of carotenoids and an absence of chlo-roplast development in emerging foliar tissues which appearbleached and stunted (Luscombe and Pallett, 1996).

One of the principal benefits of growing a legume crop is thesymbiotic fixation of N. Thus it is important to consider any possibleeffects herbicides could have, either directly on rhizobium species,or indirectly by affecting the rhizobium–plant symbiosis (Bertholetand Clark, 1985). Residual levels of some herbicides have beenfound to inhibit nodulation (Eberbach and Douglas, 1989) and Nfixation (Koopman et al., 1995) by legumes and may have negativeeffects on the N balance of legume–cereal rotations. Mallik andTesfai (1985) found that trifluralin, 2,4-DB, alachlor, glyphosate andmetribuzin adversely affected nodulation and N fixation in soybean(Glycine max L.) when applied at 5 and 10 times normal rates. Singhand Wright (1999) examined the impact of three pre-emergenceherbicides (terbutryn/terbuthylazine, trietazine/simazine andprometryn) and a post-emergence herbicide (bentazone) onnodulation, symbiotic N fixation, growth and yield of pea (Pisumsativum L.) and reported that all pre-emergence herbicidesdecreased nodulation, total nitrogenase activity, net photosyn-thesis, root and shoot dry weight, N content and seed yield. Kumaret al. (1981) found that root nodule initiation of chickpea was notaffected by either simazine or prometryn herbicide but the laterproduction and growth of new nodules was reduced to differentdegrees by various treatments and concluded that the reductions innodulation with the herbicides appeared to be primarily a case ofgeneral root growth reduction. When examining the effects ofchlorsulfuron on chickpea, Anderson et al. (2004) reported that thepresence of the herbicide in the soil reduced the nodulation andN fixation.

Previous field and greenhouse trials showed that the chickpeacultivar Kyabra was amongst the most tolerant and Yorker was oneof the most sensitive cultivars to isoxaflutole (Datta et al., 2007).The objectives of this research were to (1) assess the growth,nodulation and N fixation of chickpea with and without isoxa-flutole, and (2) determine if these effects vary between isoxaflutolesensitive and tolerant chickpea genotypes.

2. Materials and methods

The experiment was conducted in growth chambers at theUniversity of New England, Armidale, New South Wales, Australia.The experiment was set up as a completely randomised design with2� 2� 2� 2 factorial combinations: 2 chickpea cultivars (the iso-xaflutole tolerant cultivar, Kyabra and the sensitive cultivar, Yorker),2 isoxaflutole rates [0 and 75 g/ha (recommended rate)], 2 NO3

�

levels (0 and 1.5 mM) and 2 rhizobia treatments (seeds were eitherinoculated with rhizobia or left as an uninoculated control). Therewere four replications.

The pots were placed in a controlled-environment room with25/15 �C temperatures (photoperiod approximately 12 h day/12 hnight), and light intensity of approximately 800 mmol/m2/s. Seedswere surface sterilised to kill any existing bacteria by immersionin 0.2% mercuric chloride (HgCl2) for 3 min followed by six rinses insterile water. Five seeds of each chickpea cultivar were grown ina free-draining sand system without any N content in a 14 cmdiameter plastic pot. The sand medium was sterilised by heating for8 h at 80 �C. Pots were sterilised by soaking overnight with sodiumhypochlorite. The seeds with the inoculation treatment wereinoculated with the recommended Rhizobium culture (containingMesorhizobium ciceri strain CC1192, obtained from New SouthWales Department of Primary Industries, Tamworth, Australia)before sowing and seeds were planted at a depth of 2.5–3 cm.Isoxaflutole was applied at 1 day after sowing (DAS) using a gas

operated boom-sprayer through TeeJet 11003 flat-fan nozzles(Spraying Systems Co., Wheaton, IL, USA) at an operating pressureof 300 kPa delivering a volume of 84 L/ha. At 7 DAS, plants werethinned to three per pot. Pots were flushed with 150 mL of distilledwater or 150 mL of nutrient solution on alternate days after sowing.The nutrient solution contained 0 and 1.5 mM NO3

� as KNO3. Othermacro and micro nutrients used as a basal treatment were:0.25 mM CaCl2, 0.125 mM K2SO4, 0.5 mM MgSO4$7H2O, 0.13 mMKH2PO4, 0.13 mM K2HPO4, 22.4 mM Fe-EDDHA, 1.2 mM MnSO4$H2O,0.08 mM ZnSO4$7H2O, 0.05 mM CuSO4$5H2O, 0.002 mM CoSO4$7H2Oand 0.02 mM Na2MoO4$2H2O.

Plants were harvested at 42 days after herbicide treatment. Atharvest, growth parameters (plant height, shoot and root dryweights), nodulation (nodule number, nodule dry weight andaverage nodule weight) and amount of N (shoot N, root N and totalN) were measured. Before harvest, plant heights were recorded bymeasuring from the ground level to the highest growing point.Plant shoots were harvested at ground level and roots were thenhand washed. The numbers of nodules per plant were counted aftercareful hand-washing of the roots. Dry weights of nodules, shootsand roots were determined after drying for 48 h at 80 �C. Averagenodule weight was calculated from nodule weights and nodulenumbers. Dried chickpea seeds, roots and shoots were ground tofine powder of <0.05 mm size using a mechanical grinder andanalysed for shoot, root and total N with a Carlo Erba NA1500 solidsample analyser. The amount of N taken up by the plant was alsocalculated. An estimate of the amount of N fixed by the chickpeaplants was made for plants grown without the addition of fertiliserN according to the following equation (Anderson et al., 2004).

N fixed ¼ ðTotal N of inoculated plants� Seed NÞ� ðTotal N of uninoculated plants� Seed NÞ

Data were analysed using the analysis of variance (ANOVA)function of R 2.3.0 (R Development Core Team, 2006) and P values�0.05 were considered significant. Variances were checked byplotting residual versus fitted values to confirm the homogeneity ofthe data. No transformations were necessary. Means for significanttreatment effects were compared using the standard error of thedifference (SED) at 5% level of probability (Webster, 2007). Thetreatment combination means presented for a variable are based onthe highest order of factorial combination that is significant in theANOVA. Where this is less than the maximum factorial combina-tion, the tables have been generated by pooling the data across thenon significant factors. The uninoculated treatments were includedas controls to check for contamination. The data for nodule number,nodule dry weight and average nodule weight were not normallydistributed because of the high frequency of zero values in theuninoculated treatment. As a result, the uninoculated treatmentsfor these parameters were not included to generate the ANOVA dueto their nil contribution to variation.

3. Results

Pre-emergence application of isoxaflutole caused injury andreduced plant height, shoot and root dry weights of the sensitivecultivar. Visible crop injury symptoms included yellowing of leaveson the lower and middle branches, shedding of leaves, stunting andfor the sensitive cultivar, necrosis. Chickpea plant height was influ-enced by interaction between isoxaflutole� cultivar (P< 0.001)(Table 1). Plant height of the tolerant cultivar was unaffected withthe recommended herbicide rate but plant height of the sensitivecultivar was reduced by 29% with the same herbicide rate. Shoot dryweight of chickpea was influenced by interactions between isoxa-flutole� cultivar (P< 0.05) and cultivar� nitrate (P< 0.05) (Table 1).

Table 1Effect of isoxaflutole on the growth parameters (plant height, shoot dry weight androot dry weight) of a tolerant and sensitive chickpea cultivar 42 days after herbicidetreatment.

Cultivar Plant height(cm)

Shoot dry weight (g/pot) Root dry weight(g/pot)

Isoxaflutole(g/ha)

Isoxaflutole(g/ha)

Nitrate concentration(mM)

Isoxaflutole (g/ha)

0 75 0 75 0 1.5 0 75

Tolerant 21.4 20.6 0.53 0.51 0.50 0.54 0.30 0.27Sensitive 21.8 15.5 0.50 0.39 0.38 0.51 0.26 0.13SEDa 0.52 0.03 0.03 0.02

a Standard error of the difference between two treatment means.

A. Datta et al. / Crop Protection 28 (2009) 923–927 925

Shoot dry weight of the tolerant cultivar was unaffected with therecommended rate of herbicide but shoot dry weight of the sensitivecultivar was reduced by 22% with the same herbicide rate. Shoot dryweight of the tolerant cultivar was unchanged with the addition of1.5 mM nitrate whereas shoot dry weight of the sensitive cultivarwas increased by 34% with the same amount of nitrate. Root dryweight of chickpea was also influenced by a two-way interactionbetween isoxaflutole� cultivar (P< 0.05) (Table 1). Root dry weightof the tolerant cultivar was similar with the zero and recommendedrates of herbicide whereas root dry weight of the sensitive cultivarwas reduced by 50% with the recommended herbicide rate (Table 1).

Nodule number of chickpea was significantly affected by a cul-tivar� isoxaflutole� nitrate interaction (P< 0.05) (Fig. 1). Nodulenumber of the tolerant cultivar was decreased by 27% with the

Isoxafl

Nod

ule

num

ber (

per p

lant

/g ro

ot d

ry w

eigh

t)

100

150

200

250

0 75

0 mMTolerant cultivar

100

150

200

250

0 mMSensitive cultivar

Fig. 1. Effects of isoxaflutole and nitrate on the nodule number of a tolerant and sensitive cherrors of the means.

addition of 1.5 mM nitrate in the zero herbicide treatment and 47%with 1.5 mM nitrate at the recommended herbicide rate but it wasunaffected with the recommended herbicide rate at both 0 and1.5 mM nitrate levels compared with the nil herbicide treatment.Nodule number of the sensitive cultivar was reduced by 41% with1.5 mM nitrate without any herbicide and 33% in the presence ofherbicide when N was applied. Nodule number of the sensitivecultivar was similar under the zero and recommended herbicidetreatments at 1.5 mM nitrate level but was decreased (27%) at 0 mMnitrate level when isoxaflutole was applied at the recommendedrate. Nodule dry weight of chickpea was influenced by a two-wayinteraction between isoxaflutole� cultivar (P< 0.01) (Table 2).Nodule dry weight of the tolerant cultivar was unaffected with therecommended rate of herbicide whereas it was significantlyreduced (52%) in the sensitive cultivar with the same herbicide rate.Average nodule weight of chickpea was also affected by a two-wayinteraction between isoxaflutole� cultivar (P< 0.01) (Table 2).Average nodule weight of the tolerant cultivar was similar with orwithout the recommended rate of herbicide but it was significantlyreduced (42%) in the sensitive cultivar with the addition of herbi-cide (Table 2).

Shoot N of both chickpea cultivars was unaffected by any of thetreatment combinations (data not presented). Root N of chickpeawas affected by a two-way interaction between isoxaflutole�cultivar (P< 0.001) (Table 3). Root N of the tolerant cultivar wasunaffected with the herbicide whereas it was significantly reduced(40%) in the sensitive cultivar with the same herbicide rate (Table 3).At the recommended herbicide rate, root N was far higher inthe tolerant cultivar than the sensitive cultivar. Total N content

utole (g/ha)

0 75

1.5 mMTolerant cultivar

1.5 mMSensitive cultivar

ickpea cultivar 42 days after herbicide treatment. Vertical bars represent the standard

Table 2Effect of isoxaflutole on the nodulation (nodule dry weight and average noduleweight) of a tolerant and sensitive chickpea cultivar 42 days after herbicidetreatment.

Cultivar Nodule dry weight(mg/g root dry weight)

Average nodule weight (mg)

Isoxaflutole (g/ha) Isoxaflutole (g/ha)

0 75 0 75

Tolerant 209.7 174.9 1.21 1.16Sensitive 258.0 123.7 1.31 0.76SEDa 20.5 0.10

a Standard error of the difference between two treatment means.

Table 4Effect of isoxaflutole and nitrate on the amount of fixed N of a tolerant and sensitivechickpea cultivar 42 days after herbicide treatment.

Cultivar Amount of fixed N (mg/g plant dry weight)

Isoxaflutole (g/ha) Nitrate concentration (mM)

0 75 0 1.5

Tolerant 5.5 3.7 5.9 3.1Sensitive 7.1 3.5 6.0 4.6SEDa 0.46 0.46

a Standard error of the difference between two treatment means.

A. Datta et al. / Crop Protection 28 (2009) 923–927926

of chickpea was influenced by a two-way interaction betweenisoxaflutole� cultivar (P< 0.05) (Table 3). Total N content of thetolerant cultivar was unchanged between the nil and the rec-ommended herbicide rates. Total N content of the sensitivecultivar was significantly reduced by 21% with the recom-mended herbicide rate compared with the zero herbicide rate(Table 3).

The amount of N fixed by chickpea was influenced by the two-way interactions between isoxaflutole� cultivar (P< 0.01) andcultivar� nitrate (P< 0.05) (Table 4). The recommended rate ofisoxaflutole significantly reduced the amount of fixed N by both thetolerant and sensitive cultivars but the reduction was higher in thesensitive cultivar (51%) compared with the tolerant cultivar (33%)(Table 4). The sensitive cultivar fixed more N compared with thetolerant cultivar in the absence of herbicide. The sensitive cultivarwas more affected by the herbicide. The tolerant cultivar fixed 47%less N at the 1.5 mM nitrate compared with the zero nitrate treat-ment whereas the amount of N fixed by the sensitive cultivar wasreduced by 23% with 1.5 mM nitrate (Table 4).

4. Discussion

When isoxaflutole was applied at the recommended rate, thesensitive chickpea cultivar showed more evidence of phytotoxicitythan the tolerant cultivar. Inhibition of shoot growth of the sensi-tive cultivar was observed whereas shoot growth of the tolerantcultivar was unaffected with the recommended rate of isoxaflutole.Cultivar differences in isoxaflutole tolerance have previously beenreported for chickpea (Felton et al., 2004; Datta et al., 2007, 2008,2009a) and maize (Zea mays L.) (Sprague et al., 1999). Luscombeand Pallett (1996) reported stunting of shoots and bleaching of leaftissue of maize when treated with isoxaflutole. Sprague et al. (1999)reported that maize hybrids more tolerant to isoxaflutole were ableto metabolise it more rapidly than the more sensitive hybrids. Inplants, soil, or water, a diketonitrile (DKN) derivative is produced bythe opening of the isoxazole ring of isoxaflutole (Pallett et al., 1997,1998) and this DKN is the herbicidally active form known to inhibitHPPD in plants. This DKN undergoes degradation to an inactive

Table 3Effect of isoxaflutole on the plant N (root N and total N) of a tolerant and sensitivechickpea cultivar 42 days after herbicide treatment.

Cultivar Root N (mg/g root dry weight) Total N (mg/g plant dry weight)

Isoxaflutole (g/ha) Isoxaflutole (g/ha)

0 75 0 75

Tolerant 12.1 11.1 31.1 30.1Sensitive 13.4 8.1 34.5 27.4SEDa 0.80 1.7

a Standard error of the difference between two treatment means.

benzoic acid derivative in treated plants and the extent of thisdegradation is correlated to the degree of susceptibility, being mostrapid in tolerant maize and slowest in susceptible Abutilon theo-phrasti Medik. (velvetleaf) (Pallett et al., 1998).

The growth, nodulation and N fixation parameters of thetolerant cultivar with respect to isoxaflutole application weregenerally better compared with the sensitive cultivar. The sensitivecultivar was short with low shoot and root dry weights where rootdry weight was reduced more than shoot dry weight under theherbicide treatment. The most striking effect of the herbicide wason the root dry weight of the sensitive cultivar (50% reduction).Other factors associated with root development such as nodulenumber, nodule dry weight, root N, total N and fixed N were alsolower in the presence of isoxaflutole due to a smaller root systemof the sensitive cultivar. When examining the effects of isoxaflutoleand nitrate on the growth and nodulation response of a tolerantand sensitive chickpea cultivar, Datta et al. (2009b) also observedmore root dry weight reduction than shoot dry weight underincreased nitrate levels and herbicide rates. In this experiment, theroots came into direct contact with the soil-applied isoxaflutoleand therefore probably exhibited effects earlier than the shoots.The shoot and root dry weight reductions in the sensitive cultivarcaused by isoxaflutole may have further reduced the ability of theplant to absorb nutrients and contributed to the lack of responseto N.

The lower number of nodules in the presence of isoxaflutolesuggests an impedance of nodule formation, whereas low noduledry weight and average nodule weight likely indicate an effect onnodule development or maintenance. Similarly, Datta et al. (2009b)reported that isoxaflutole reduced the number of nodules ofa tolerant and sensitive chickpea cultivar. They also concluded thathigher levels of nitrate decreased overall nodule numbers inchickpea. In their studies, nodule dry weight of the sensitivecultivar was decreased with increasing nitrate levels and herbiciderates, which is similar to what we have concluded from this study.Herbicide-induced reductions in nodulation may be the result ofinjury to the root system, or to Rhizobium before or during infection.The decline in nitrogenase activity may also be due to a herbicide-induced reduction in supplies of photosynthates to the nodules,physiological damage to the plant root or nodules or, physiologicaldamage to Rhizobium either before or after inoculation (Eberbachand Douglas, 1989).

The plant N relationships may have been affected by isoxaflutolein the sensitive cultivar and addition of N did not compensate forthe problem. The recommended rate of isoxaflutole reduced theamount of N fixed by both the tolerant and sensitive cultivars withthe reduction being greater in the sensitive cultivar. The amount offixed N was also reduced in the sensitive cultivar when nitrate wasadded. The decreased amount of fixed N with isoxaflutole is mostprobably associated with lower root and shoot growth, nodulation,nodule dry weight and total N content. Anderson et al. (2004)reported a 70% reduction in the amount of N fixed by chickpea

A. Datta et al. / Crop Protection 28 (2009) 923–927 927

plants when chlorsulfuron was present in the soil compared withplants grown without chlorsulfuron. Herbicides can reducephotosynthetic productivity, thereby potentially restricting theamount of photosynthate available for nodulation and N fixation(Singh and Wright, 1999). However, it is possible that herbicidescan also decrease the nodulation capacity of rhizobia (Madhaviet al., 1993). According to Eberbach (1993), herbicides may affectthe legume–rhizobia symbiosis in a number of ways including: (1)direct effects on the host plant (e.g. reduction in root biomass,leading to fewer infection sites, or in carbohydrate supply toexisting nodules); (2) direct effects on rhizobial survival or growth,leading to a decreased potential for rhizobial infection of root hairs;(3) an inhibition or inactivation of the biochemical signalling byeither rhizobia or plants required to initiate nodule development;and/or (4) an inhibition of nodule development by reducing thecapacity for cell division. All of these mechanisms have thepotential to reduce the efficiency of the legume–rhizobia symbioticrelationship and therefore the amount of N fixed.

A reduction in the amount of fixed N in chickpea crops due tothe application of isoxaflutole can negatively affect the potential Nbenefits associated with including chickpeas in rotation withcereals. Grain legume crops are often associated with an N ‘sparing’effect in which soil mineral N not taken up by legumes activelyfixing N, remains available to subsequent cereal crops (Ahmad et al.,2001). The performance of the tolerant chickpea cultivar wascomparatively better for some growth, nodulation and N fixationparameters. Further experiments are required to determinewhether the adverse effects of the herbicide observed in this earlygrowth study occur to the same degree under field conditions andwhether these effects continue through to harvest and negativelyimpact on grain yield.

Acknowledgements

We thank the Cooperative Research Centre for Australian WeedManagement and the University of New England for financialsupport of this work, and Ted Knights, Tamworth AgriculturalInstitute, Department of Primary Industries, New South Wales,Australia for supplying seeds of the genotypes.

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