The effect of aldicarb and benomyl on root-knot nematode and rootrot in subterranean clover

S.H. PungAC, M.J. BarbettiB and K. Sivasithamparam A

ASoil Science and Plant Nutrition Group, The University of Western Australia, Nedlands,Western Australia6009BPlantPathology Branch, Department of Agriculture, Baron-Hay Court, South Perth, Western Australia 6151cPresent address: Insect Pathology, Department of Primary Industry, St John's Avenue, New Town,Tasmania 7008

Abstract

The role of Me/oidogyne arenaria and mot rot fungi in thedecline of subterranean clover in affected fields wasassessed bysoilapplication of the fungicide, benomyl, andthe nematicide, aldicarb. M. arenaria appeared to tie a sig­nificantcause of poorproductivity of subterranean clover.Aldicarb inhibited root-knot nematodes and increased plantvigour. Aldicarb increased the incidence of Rhizoctoniaspp, Neither chemicalaffected the incidence of Fusariumoxysporum. A combination of aldicarb and benomylincreased the incidence of Pythium irregulare.

Introduction

Subterranean clover (Trifolium subterraneum L.) isthe most important pasture legume in the southernand eastern Australian temperate regions where ithas been sown in more than 16 million ha of drylandpasture (Gladstones and Collins 1983). Root rot,caused by one or more of several fungi, is a seri­ous problem in subterranean clover growing in thetemperate areas of southern Australia (Barbetti etal. 1986).

Field samplings (Pung et al. 1988) have shown theroot rot and root-knot nematode (Meloidogyne eren­area (Neil) Chitwood) are prevalent in subterraneanclover pastures in the southwest of Perth. Root rotin the field may be reduced by fungicides. Benomylreduces infection of roots of red clover by root rotpathogens such as Fusarium species (Skipp 1986).Aldicarb is an effective nematicide and insecticidebut neither it nor any of its metabolites is toxic tosoil bacteria or fungi even at higher than recom­mended dosages (Spurr and Sousa 1974).

This study investigated the effect of the fungicide,benomyl, on root rot fungi and the nematicide, aldi­carb, on M. arenaria and assessed the role of M.erenerle and root rot fungi in the decline of subter­ranean clover in an affected field.

Methods

Intact cores (15 cm diam.) of sandy loam soil werecollected in June 1988 from a subterranean cloverpasture in Dardanup (160 km south of Perth) whereheavy M. arenaria infestation had been recorded in1986 (Pung et al. 1988). Subterranean clover rootrot tends to diminish when the field soil is disturbed(Barbetti and MacNish 1978), so soil cores were col-

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lected intact to preserve inoculum concentrationsdown the soil profile. The soil cores were transferredto free-draining pots and air-dried for about 4 weeksin the shade to kill weeds. Soil cores were treatedas required with benomyl (a benzimidazole systemicfungicide and/or aldicarb (a non-volatile, systemiccarbamate nematicide/insecticide).

Pots were drenched with 400 mL of a suspensioncontaining 2 g Benlate Wettable Powder (500 g/kgbenomyl) per litre of deionized water. Temik 150Ggranules (150 g a.i.Ikg aldicarb) were mixed withriver sand. The aldicarb/sand mixture was spreadand mixed into the soil surface (10 mm deep) at300 mg aldicarb/pot, and the pots wetted with 20 mLdeionized water. There were four treatments con­sisting of control (nil treatment), aldicarb, benomyland benomyl plus aldicarb. Fifteen replicates werearranged in a randomised complete block design.Subterranean clover cv. Woogenellup seeds weresurface sterilised in 95% ethanol for 5 min and20 germinated seedlings planted 1.5 cm deep ineach pot and thinned to 10 per pot after 7 days.Pots were kept at 25/20°C (day/night) in a phyto­tron (165 W/m2) and watered to free draining onalternate days.

After 6 weeks, seven replicates were harvestedand all plants were found to be free of root-knotsymptoms. Hence the shoots of plants from theremaining eight replicates were removed. Pieces oftomato (cv. Tartura) roots, 20 mm long, containingegg masses of M. arenaria, were placed 20 mmdeep at the centre of each pot and covered with soil.Forty-five days after the first chemical applicationthe cores were retreated with benomyl suspension(200 mL) and aldicarb/sand mixture (300 mg/pot),then reseeded as before. Forty days later the plantswere harvested and weighed.

In order to determine the fungi associated with theroots, two whole root systems from each replicatein each treatment were washed and cut into pieces(10 to 15 mm long), mixed thoroughly and thendivided into two portions. One portion was rinsedin sterile deionised water, then shaken vigorouslyin sodium hypochlorite solution (1.25% availablechlorine) for about 2 min, rinsed in three washesof sterile deionised water and blotted dry. Fortyroot pieces were plated onto 2% water agar plus100 units/mL of streptomycin sulphate (supplied byBoehringer) and 20 pieces onto 3 P agar which had50 ppm potyrnlxln'B sulphate, 50 ppm penicillin and

Australasian Plant Pathology Vol. 20 (4) 1991

100 ppm pimaricin (all antibiotics supplied by SigmaChemicals) (Eckert and Tsao 1962), and incubatedat 25°C for 4 days. The other portion of the rootpieces was washed vigorously three times in steriledeionised water and 20 root pieces were then floatedon sterile pond water in a Petri dish with 5 rootpieces to each dish. The dishes were then incub­ated at about 20°C and the roots were examinedat 24, 48 and 72 h intervals for the presence ofPhytophthora clandestina Taylor, Pascoe andGreenhalgh.

The remaining root systems were assessed for theextent of galling and root rot, and tap root lengthswere measured. The extent of gall formation wasassessed on individual plants using the followingscale: 0 = unaffected root; 1 = 1-10; 2 = 11-25;3 = 26-50; 4 = 51-75 and 5 = 76-100 galls per rootor % root galled (Pung 1990). The extent of root rotwas assessed according to the following scale:o = no discoloration; 1 = superficial light brown dis­coloration; 2 = deep seated brown discoloration withno obvious physical damage; 3 = dark brown dis­coloration with some stunting and 4 = severe rootstunting or root rotted off. Means of both root rot andgall ratings were calculated for each treatment, andthen converted into root rot and gall indices, respec­tively, using the formulae, root rot index = (meanof root rot score for each treatment/4) x 100 and gallindex = mean of nematode gall score for eachtreatment/5) x 100.

Results

Only the aldicarb treatment increased fresh shootweight (Table 1) but all chemical treatmentsincreased fresh root weight. Chemical treatmentsdid not affect the level of root rot. Aldicarb andaldicarb plus benomyl prevented the production ofnematode galls. Benomyl increased the number ofgalls on the lateral roots but not on the tap roots.Benomyl did not affect the percentage of tap orlateral roots galled. Aldicarb and aldicarb plusbenomyl treatments increased tap root length butonly aldicarb plus benomyl increased total rootlength.

On water agar, F. oxysporum was frequently isol­ated in all treatments (Table 2). Rhizoctonia spp.were common in soil cores treated with aldicarb. Theincidence of Rhizoctonia spp. in roots from thebenomyl plus aldicarb treatment was lower than thatin the aldicarb treatment.

Other fungi isolated from root pieces on 2% wateragar, in a decreasing frequency of occurrence, werePhoma medicaginis Malbr. & Roum., F. avenaceum(Corda:Fr.) Sacco Gliocladium roseum Bainier,Trichoderma spp., Mortierella spp. Penicillium spp.,Mucor spp. and F. equiseti (Corda) Sacco On the 3 Pagar medium, which is selective for Pythiaceousfungi, almost all Pythium isolated belonged toPythium irregulare Buisman (Table 2). This fungus

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is a pathogen commonly associated with root rot ofsubterranean clover. The incidence of P. irregularein the aldicarb treatment was approximately doublethat in the control and benomyl treatments. How­ever, the incidence of P. irregulare in the aldicarbplus benomyl treatment was about four times thatin the control and benomyl treatments.

P. clandestina, an important subterranean cloverroot rot pathogen, was not detected on root piecesthat were floated in sterilised pond water.

Discussion

Soil cores used in this study were obtained in 1988.Although high nematode populations were recordedat the same site in 1986 (Pung et al. 1988), thenematode population in 1988 was undetectable. Thedrying of soil cores may have adversely affectednematodes as well as some fungi resulting in littleor no nematode infection and the absence of P.clandestina.

The increase in fresh shoot weight from aldicarbapplication and the increase in fresh root weight andthe prevention of nematode gall formation from thealdicarb and aldicarb/benomyl treatments, togethersuggest that M. arenaria is a major cause of the poorsubterranean clover productivity observed at theDardanup site (Pung et al. 1988). However, apartfrom the inhibition of the nematode, aldicarb mayhave had a direct effect on the growth of plants byenhancing the activities of plant hormones, hencestimulating plant growth (J.M. Fisher, pers. comm.).This may account for some of the increased vigourobserved in plants grown in the aldicarb-treatedsoil.

Benomyl may have increased gall production byreducing root rot and consequent reduction of com­petition for infection sites. Benomyl is also knownto stimulate hatching of some nematodes (J.M.Fisher, pers. comm.). Although benomyl can con­trol Fusarium spp. (McGee and Kellock 1974), it hadno effect on the incidence of F. oxysporum in thisstudy. Benomyl had no effect on the incidence ofthe fungi isolated.

Benomyl inhibited, while aldicarb increased, theincidence of Rhizoctonia spp. This is consistent withthe observations of Hide and Corbett (1974) andLeach and Frank (1982). R. solani can metaboliseand degrade aldicarb (Jones 1976) and is stimulat­ed by aldicarb in nutrient rich media (Hoffman andBollen 1987; Spurr and Sousa 1974). Aldicarb hadno effect on the incidence of F. oxysporum. The com­bined aldicarb and benomyl applications resulted ina higher incidence of P. irregulare. Both P. irregulareand Rhizoctonia spp. are known to be pathogenicto subterranean clover seedlings (Wong et al.1985a;b). Spurr and Sousa (1974) showed thataldicarb and some of its metabolites, aldicarb sul­phoxide and aldicarb sulphone, were toxic to insectsand root-knot nematode, but had no adverse effect

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Table 1 Effect of aldicarb and benomyl on plant growth and root rot of subterranean clover, and gallproduction by Meloidogyne arenaria

AldicarbControl Aldicarb Benomyl + Benomyl

Plant fresh weight (g)Shoot weight/shoot 1.095aA 20410b 10430a 1.902aRoot weight/root 0.551c 0.966d 0.765d 0.767d

Root rot index (%)Tap root 84.8ab 87.3a 68.5b 72.3abLateral root 72.0c 67.0c 56.0c 60.8c

No. galls per root (% index)Tap root 12.6a Ob 14.0a ObLateral root 39.2c Oe 67.6d Oe

% root galled index (%)Tap root 20.6a Ob 24.0a ObLateral root 52.0c ad 72.6c ad

Tap root length (cm) 10.8a 16.9b 11.3a 1604bTotal root length (m) 64c 87c 70c 112d

ANumbers in the same row not followed by the same letter are significantly different (P = 0.05) using Dun­can's Multiple Range Test. There were four replicates in each treatment.

Table2 The number of roots containing the various fungi (from a total of 40 root pieces being examined)isolated from roots of subterranean clover grown in soil treated with aldicarb and/or benomyl

AldicarbFungus Control Aldicarb Benomyl + Benomyl

Water agarFusarium oxysporum 29 20 27 28Fusarium avenaceum 3 a 5 8Fusarium equiseti 1 a a aPhoma medicaginis 5 3 11 5Gliocladium roseum 1 2 2 aRhizoctonia spp. 8 28 3 13Trichoderma spp. 2 a 1 aMortierella spp. a 1 2 aPenicillium spp. a a 1 aMucor spp. a a 1 a

3 P. agarPythium irregulare 6 12 7 25Pythium spp. 1 a a 1

on F. oxysporum, R. solani, Pythium debaryanumHesse or the bacteria tested.

Acknowledgements

This work was supported by a studentship awardfrom the Wool Research Trust Fund on the recom­mendation of the Australian Wool Corporation.

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Manuscript received 16 July 19l>O, accepted 14 May 1991.

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