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Crop Protection 63 (2014) 15e25

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Crop Protection

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Management of the root-knot nematode Meloidogyne graminicolainfesting rice in the nursery and crop field by integrating seedpriming and soil application treatments of pesticides

Mujeebur Rahman Khan*, Ziaul Haque, Nida KausarDepartment of Plant Protection, Faculty of Agricultural Sciences, Aligarh Muslim University, Aligarh 202002, U.P., India

a r t i c l e i n f o

Article history:Received 27 August 2013Received in revised form17 April 2014Accepted 29 April 2014

Keywords:CarbosulfanChemical controlChlorpyriphosPhorateRoot-knot nematode

* Corresponding author. Tel.: þ91 571 2901524; faxE-mail addresses: mrkhan777in@yahoo.co.in

(M.R. Khan).

http://dx.doi.org/10.1016/j.cropro.2014.04.0240261-2194/� 2014 Elsevier Ltd. All rights reserved.

a b s t r a c t

The study evaluates the effects of seed priming (5 g or ml kg�1 seed) and soil application (2 kg or l ha�1)of eight organophosphate pesticides on rice root-knot disease caused by Meloidogyne graminicola. Seedpriming (SP) or soil application (SA) of phorate, carbofuran and chlorpyriphos (1000 J2 of M. graminicolakg�1 soil) suppressed galling in the rice nursery by 92 and 99%, 80 and 88% and 76 and 80%, respectively,over control. Relatively similar decreases in the galling were recorded when this nursery was grown forfour months in the sterilized soils in earthen pots. Rice cv. PS-5 grown in naturally infested soil in earthenpots (1000 J2 kg�1 soil) became stunted, showing chlorotic foliage, and terminal galls developed on theroots. The treatment of SP þ SA 15 þ 30 days after planting (DAP) with phorate, carbosulfan, andchlorpyriphos significantly suppressed the root-knot development and improved the plant growth of riceover the controls (P � 0.05). The overall effect of the SP þ SA 15 DAP treatments was marginally weakerthan the SP þ SA 15 þ 30 DAP treatments but statistically on par. Under field conditions, the greatestdecrease in the galling occurred due to SP þ SA 15 þ 30 DAP of phorate (69�71%) and SP þ SA 15 DAP (65�67%) followed by carbosulfan and chlorpyriphos. The yield of rice plants was also highest with phorate(32�36% and 29�34%) over the control during the two years of the study. The soil population ofM. graminicola decreased by 58e84% over four months due to the phorate treatments. The study dem-onstrates that seed priming with phorate effectively controls nematode infections in the nursery and thatsoil application at 15 DAP (2 kg ai ha�1) prevents root-knot development in an infested field underirrigated conditions. Use of SP þ SA 15 DAP may enable to avoid one soil application of phorate in thefield.

� 2014 Elsevier Ltd. All rights reserved.

1. Introduction

Root-knot nematode, Meloidogyne spp. is one of the mostdevastating and widespread nematode pests of agricultural crops(Sasser, 1989). The nematode has exceedingly wide host range andattacks almost all cereal, vegetable, pulse, fiber, fruit and beveragecrops (Bridge et al., 2005). Rice is the world’s most important staplefood and is cultivated in around 162 mha annually with an annualglobal production of 464 mmt (FAOSTAT, 2013). Rice is cultivated infive major ecosystems viz., irrigated, deep water, upland, lowlandand rainfed rice. About 53% of the world’s rice is grown underirrigated conditions (having good water control) that provides 75%

: þ91 571 2703516., mrkhan.amu@gmail.com

of total global production (Bridge et al., 2005). Rainfed lowland rice(31% of the world rice area) is entirely dependent on rainfall,whereas the deep water areas (35%) occurs in the river deltas.Upland rice area (13%) is also rainfed but without surface wateraccumulation (Bridge et al., 2005).

Rice is quite susceptible to root-knot nematode and is attackedby Meloidogyne incognita, Meloidogyne graminicola, Meloidogynetriticoryzae, Meloidogyne javanica, Meloidogyne oryzae and Meloi-dogyne arenaria (Gaur and Pankaj, 2010). Amongst these species,M. graminicola is a primary pest of rice and poses a substantialthreat to rice cultivation in particular Southeast Asia where around90% of the world rice is grown and consumed (Dutta et al., 2012).Rice root-knot nematode, M. graminicola causes terminal, hookshaped or spiral galls which are characteristic symptoms of theinfection of this nematode species (Khan et al., 2012). In India, riceis grown in all five major ecosystems (Jairajpuri and Baqri, 1991).M. graminicola is highly damaging to upland, rain-fed lowland

M.R. Khan et al. / Crop Protection 63 (2014) 15e2516

(Prot et al., 1994) and irrigated rice cultivation (Netscher and Erlan,1993). In India, this nematode has been found to cause yield lossesof 16e32% in rainfed and upland rice (Prasad et al., 2010).

There are various methods of nematode management that mayprove effective against rice root-knot nematodes (Gaur and Pankaj,2010). Despite the known deleterious effects of chemicals, pesti-cides are still the most effective means of nematode managementin rice ecosystems (Khan et al., 2012). In view of limited availabilityof rice cultivars resistant to M. graminicola (Dutta et al., 2012),pesticides are preferred by farmers, as they provide instant results,whereas other disease management practices only begin to have arecognizable impact over considerable time. Pesticides can functionthrough contact and/or via systemic action, and are appliedthrough different modes, such as seed (Jain and Gupta, 1990), bareroot-dipping (Jain and Bhatti, 1991) and nursery bed treatments(Jain and Gupta, 1990), but soil application is a universal method(Jain and Bhatti, 1988).

Pesticides from the organophosphate group have been found tobe effective against M. graminicola, though the degree of controlachieved may vary greatly with the mode of application (Khan andJairajpuri, 2010). The application of carbosulfan and chlorpyriphosat concentrations of 0.1 and 0.2%, respectively, to rice seedlingsthrough root-dipping treatments for 6 h was found to significantlysuppress galls and increase plant growth (Khan et al., 2012).Seedlings grown from carbosulfan-treated seeds show fewer gallsand egg masses, and this treatment reduces the nematode popu-lation by up to 82% (Rahman and Das, 1994). Soil application ofphorate and carbosulfan at 1 kg a.i. ha�1 or more also significantlyreduces galling on rice plants (Prasad and Rao, 1976, 1977a,b).

A critical analysis of the available information reveals that mostof the studies in this area have been carried out using phorate andcarbosulfan with multiple treatments, whereas other organo-phosphate pesticides have seldom been tested againstM. graminicola in rice. In addition, the effects of differentmodes andtimes of application have not been adequately studied. In the pre-sent study it has been attempted to target the chemicals preciselyand specifically againstM. graminicola by applying the pesticides onseeds. Farmers in India who realize the significance of rice root-knot nematode generally apply pesticides at 2e3 kg a.i. ha�1

atleast twice, one or two days before the irrigation (Prasad et al.,2010). Despite of giving two high doses of pesticides, the root-knot disease persists in the field because of rapid percolation ofthe chemicals beyond the root zone of plants under saturatedconditions resulting from irrigation. In the present study, weattempted to avoid the loss of pesticides in soil by giving the soiltreatments 2e3 days after irrigation when the soil is only wet notflooded. However, a major objective of the study was to reduce thecost of management and to prevent environmental contaminationby reducing the dose and the number of treatments of the pesti-cides. In the present study, we first evaluated the effectiveness ofseed priming (SP) and soil application (SA) of the pesticides tocontrolM. graminicola in the rice nursery. Thereafter, persistence ofSP and SA treatments was tested by allowing the nursery to grow inthe nematode infested and non-infested soil for four months.Management of root-knot in a nursery shall prove a much effectivetactic in preventing the spread of M. graminicola as well as con-trolling the nematode attack in rice cultivation. To improve effec-tiveness, soil application treatments were integrated with SP.Hence, in the present study, the effectiveness of different combi-nations of seed priming and soil application (after 15 and 30 days ofplanting of seedlings) using eight organophosphate pesticides, viz.,phorate, carbosulfan, malathion, dimethoate, monocrotophos,dichlorvos, methyl parathion and chlorpyriphos was examinedagainst M. graminicola Golden and Birchfield. The effect of the SPand soil application (SA) treatments was first evaluated in relation

to the control of root-knot in a rice nursery. Thereafter, the evalu-ationwas done under pot conditions, followed by a field trial in twoconsecutive years to ascertain the effectiveness of the three pesti-cides found to be most effective pesticides tested in the pot trial.

2. Materials and methods

2.1. Rice nursery

Certified seeds of rice Oryza sativa L. cv. PS-5 (Pusa Sugandha-5)were procured from an authorized dealer in Aligarh, India. Thiscultivar is widely cultivated in the northern India and has beenfound to be highly susceptible to M. graminicola (Khan and Anwer,2011). The seeds were soaked inwater for 12 h and then transferredto a clean muslin bag. The bag was hung in the shade for 24 h tofacilitate seed germination. Thereafter, the seeds were sown in25 cm-diameter earthen pots containing 2 kg of autoclaved soil(four parts sandy loam plus one part farm yard manure). The potswere watered daily. The seedlings reached transplantation stage(four leaves and 12�15 cm height) in 4 weeks.

2.2. Pesticides and their doses

The chemical pesticides phorate (Ambuja Agrochem, India),carbosulfan (FMC, India), malathion (Insecticides India Ltd),dimethoate (Vallabh Pesticides Ltd., India), monocrotophos(Unique Farm Aid, India), dichlorvos (Crop Life Science Ltd., India),methyl parathion (Crop Chemicals India Ltd.) and chlorpyriphos(Century, India) were procured from an authorized pesticide dealerin Aligarh. The pesticides were applied to the pot soil (1 kg) at therecommended dose: 25 mg phorate 10G (equivalent to 2 kg a.i.ha�1); 10 ml carbosulfan 20 EC (2 l a.i. ha�1); 720 ml malathion 50 EC(0.06%, 3 l a.i. ha�1); 100 ml methyl parathion 50 EC (0.08%, 4 l a.i.ha�1); 100 ml dimethoate 30 EC (0.05%, 1.5 l a.i. ha�1); 400 mldichlorvos 76 EC (0.05%, 1.6 l a.i. ha�1); 1000 ml monocrotophos 36SL (0.06%, 1.7 l a.i. ha�1) and 25 ml chlorpyriphos 20 EC (0.1%, 2 l a.i.ha�1) per pot. In the field trial, phorate, carbosulfan and chlorpyr-iphos were applied at 2 kg or l a.i. ha�1. For SP, the pesticides wereapplied at 5 g or ml kg�1 dry seeds.

2.3. Pot trial

Bulk soil was collected in JulyeAugust in two consecutive yearsfrom the same rice fields, which showed symptoms of root-knotcaused by M. graminicola. Infected rice roots with galls were alsocollected. Ten females were excised from the galled tissue, and theirperineal patterns were prepared to identify the Meloidogyne spe-cies associated with the galls (Yik and Birchfield, 1979). The soil wasthoroughly mixed, and the population of M. graminicola and othernematodes was estimated by processing five soil samples of 500 geach soil using Cobb’s decanting and sieving method (modified),followed by the Baermann funnel technique (Southey, 1986).Nematode larvae were picked from each soil isolate, and temporarymounts were prepared to identify M. graminicola and other nem-atode genera on the basis of the morphological characters of ju-veniles and to count their relative populations (Siddiqui, 2000). Theaverage populations of M. graminicola estimated in the bulk soilwere 1267 � 79 and 1306 � 54 J2 kg�1 soil in the two study years,respectively. The population of other nematodes present in soilranged from 131�26 and 107� 38 larvae kg�1 soil, which includedRadopholus, Helicotylenchus, Hoplolaimus, Tylenchorhynchus, Roty-lenchus, Rotylenchulus, Hirschmanniella, Pratylenchus, Ditylenchus,Trichodorus and some free-living nematodes (fast moving), amongothers. A mixture of 1 kg of nematode-infested soil (1267 � 79 and1306 � 54 J2 kg�1 soil) and 250 g of autoclaved farm yard manure

Table 1List of treatments used in the nematode infested and non-infestedsoil.

S. No. Treatments

1. Control2. Pesticide (Seed Priming, SP)3. Pesticide (15 DAP)4. Pesticide (30 DAP)5. Pesticide (15 þ 30 DAP)6. Pesticide (SP þ 15 DAP)7. Pesticide (SP þ 30 DAP)8. Pesticide (SP þ 15 þ 30 DAP)

M.R. Khan et al. / Crop Protection 63 (2014) 15e25 17

was added to clay pots (15 � 15 cm diameter and height). Hence,the resultant nematode population was approximately 1000M. graminicola J2 kg�1 soil. A population of 800e1000M. graminicola J2 kg�1 soil has been found to cause severe gallingand significant damage in susceptible rice cultivars (Prasad et al.,2010). For the non-infested treatment (control), the soil and farmyard manure mixture (4:1) was autoclaved at 15 kg cm�3 pressureat 121 �C for 15�20 min. The pots filled with nematode-infestedand sterilized soil were used to conduct the following twoexperiments.

2.4. Effect of pesticide treatments on root-knot in the nursery andmature plants of rice

Seeds of rice cv. PS-5wereweighed and soaked inwater for 12 h.The soaked seeds were transferred to a muslin cloth bag and hungfor 2 h to drain off excess water. Arabica gum was then applied tothe seeds for adhesiveness on seed surface, followed by seedpriming with pesticides at 5 g or ml kg�1 dry seed. The seeds weregently mixed to achieve a uniform distribution of the pesticide.Next, the seeds were transferred to a wet muslin bag and hung inthe shade to facilitate germination. After 22 h, the seeds were sownin earthen pots filled with either nematode-infested soil or steril-ized soil (autoclaved, non-infested). The seedlings, 4 weeks aftersowing, were collected from the nursery pots and examined for gallformation and egg mass production. Seedlings from each nurserytreatment were then planted in pots filled with sterilized soil andgrown for 4 months.

To evaluate the effectiveness of pesticides on the root-knot inmature plants, seedlings of rice cv. PS-5 with or without seedpriming were transplanted into pots filled with nematode-infestedsoil (1000 J2 kg�1) or sterilized soil (1 seedling/pot). The pesticideswere applied to the pots at the doses indicated above eitherthrough SP or SA at 15 DAP, 30 DAP, 15 þ 30 DAP, SP þ 15 DAP,SP þ 30 DAP or SP þ 15 þ 30 DAP. Five pots (replicates) for eachtreatment were maintained and arranged in a completely ran-domized block design on the roof-top, where they received uniformsunlight. The seedlings were watered immediately after trans-plantation, which continued until harvesting. The plants weregrown for 4 months. During the experimental period, the plantswere regularly observed for any visible symptom attributable toinfection byM. graminicola and/or the nematicides. The plants wereharvested 4 months after transplantation to determine the lengthof their roots and shoots, the dry weight of their roots and shoots,the number of galls and egg masses/root system, and the soilpopulation of M. graminicola.

2.5. Field trials

Field trials to ascertain the effectiveness of the soil application ofthree pesticides, viz., phorate, carbosulfan and chlorpyriphos,which had proved the most effective against the root-knot nema-todes in rice in the pot trail, were conducted in naturally infestedfarmer’s fields during two consecutive years. Two fields, one ofapproximately 0.6 ha (75 � 83 m) infested with M. graminicola(1091 � 87 J2 kg�1 soil) and a non-infested field (94 � 27 J2 kg�1

soil) of 0.6 ha (71 � 85 m), were included in this study. The soil inboth fields was a sandy loam containing 75.8% sand, 13.6% silt and10.6% clay, with a pH of 7.9, a water holding capacity of 45%, anorganic carbon content of 0.017% and an organic matter content of1.8%. In each of the fields, 72 plots (5 � 10 m) were laid down. Theadjoining plots were separated by a bund 50 cm wide and 25 cmhigh to check over-flooding of water among the plots. Each plot hadan independent irrigation channel. In the second year, two otherfields with similar dimensions and soil types were included in the

experiment, subjected to identical treatments. In these fields, thenematode population consisted of 1176 � 95 M. graminicola J2 kg�1

soil (high-infested) and 89 � 41 J2 kg�1 soil (low-infested andconsidered as non-infested). All of these fields had a history of ricecultivation for the last 8�10 years.

The fields were prepared following standard agronomic prac-tices for rice cultivation. The four week old seedlings of rice cv. PS-5raised in sterilized soil were planted in plots under saturated(flooded) conditions. Phorate, carbosulfan and chlorpyriphos wereapplied at the doses and treatments described above. The SAtreatments were applied when the field soil was wet (2�3 daysafter irrigation, without any standing water). For each of the threepesticides, 16 treatments were maintained which are enlistedin Table 1. Of these, half treatments (8 in nos.) were maintained inthe field having around 1100 J2 of M. graminicola kg�1 (infested orhigh infested soil), and remaining 8 treatments in an adjoining fieldwith around 100 nematode larvae (non-infested or low infestedsoil). Each treatment was represented by 3 replicate plots, whichwere randomly distributed within an infested or non-infested field.Weeds were removed manually, and irrigation (drain) was appliedat 2 and 3 months of transplanting. All of the plants in a plot, 4months after sowing, were harvested, dried for 2 weeks andthrashed separately with a mechanized thrasher to determine thegrain yield (with seed husks, without grain milling).

2.6. Root galls and egg masses

In the pot experiment, the roots were examined at harvest tocount the numbers of galls and egg masses/root system, whereas inthe field experiment, 3-month-old plants (10 plants/plot) wererandomly and carefully uprooted from each replicate plot todetermine galling and egg mass production. In addition, maturefemales were excised from the galled tissue, and the perinealpattern of the females was prepared to verify the association ofM. graminicola with the galls.

2.7. Soil population of M. graminicola

The soil population of juveniles of M. graminicola was deter-mined using Cobb’s decanting and sieving method (modified),followed by Baermann’s funnel technique (Southey, 1986). In thepot experiment, soil (500 g/pot) was collected from 5 pots in eachtreatment at harvest (4 months after transplantation), and wasprocessed separately using the method described above to deter-mine the population of M. graminicola kg�1 soil. The nematodepopulation in the field was determined at planting and 1, 2, 3 and 4months (harvest) after planting. On each occasion, 500 g of soil wasrandomly collected from 10 points within a replicate plot andprocessed separately to determine the nematode population, asdescribed above.

Table 2Effect of seed priming and soil application of organophosphate pesticides on thegalling and egg mass production on nursery of rice cv. PS-5 raised in the soil infestedwith Meloidogyne graminicola (1000 J2 kg�1 soil).

Pesticides Galls per root system Egg masses per rootsystem

Seedpriming

Soilapplication

Seedpriming

Soilapplication

#Non-infested soil (control) 00 (0.7) 00 (0.7) 00 (0.7) 00 (0.7)Infested soil (infested control) 30.2 (5.5) 21.8 (4.7) 30.2 (5.5) 21.8 (4.7)Infested soil þ chlorpyriphos 7.2 (2.7)* 4 (2.1)* 4.0* (2.1) 00* (0.7)Infested soil þ carbosulfan 6.0 (2.5)* 3.2 (1.9)* 3.6* (3.0) 00* (0.7)Infested soil þ dichlorvos 14.2 (3.8)* 7.2 (2.7)* 13.4* (3.7) 8.3* (2.9)Infested soil þ dimethoate 19.4 (4.4)* 10.2 (3.2)* 16.2* (4.0) 8.2* (2.9)Infested soil þ malathion 24.3 (4.9) 14.4 (3.8)* 19.4* (4.4) 13.6* (3.7)Infested soil þ methyl

parathion26.5 (5.1) 17.2 (4.2)* 23.6* (4.9) 16.2* (4.0)

Infested soil þ monocrotphos 29.8 (5.5) 20.8 (4.6) 28.8 (4.5) (5.4)/20.4Infested soil þ phorate 2.3* (1.6) 00 (0.7)* 0.4* (0.9) 00* (0.7)LSD P � 0.05 1.7 1.3 1.6 1.2F-values Pesticides (df ¼ 8) 23 29 36x 31x

Each value is the mean of five replicates. Values in parentheses are transformedvalues [sqrt (x þ 0.5)]. Values followed by the superscripts * are significantlydifferent from the infested control at P � 0.05. F-values followed by x are significantotherwise not significant (NS) at P � 0.05. # The data on the effect of pesticidetreatments in non-infested soil (low infested) were pooled after analysis to form acommon control (non-infested control) because the treatment differences were notsignificant at P � 0.05.

M.R. Khan et al. / Crop Protection 63 (2014) 15e2518

2.8. Statistical analysis

The observations made from 10 plants in a plot were averagedand considered as one replicate. The observations made in 5replicate pots (e.g., for plant growth, galls, egg masses, soil pop-ulations) or 30 root systems or points (for galls, egg masses andsoil populations; 10/plot) and 3 replicate plots (grain yield) wereaveraged to calculate means. The two years of data from the potand field experiments were analyzed separately. The data (5 or 3replicates/treatment) on the plant growth and yield were analyzedthrough a two-factor analysis of variance (ANOVA), and least sig-nificant differences (LSDs) were calculated to identify significanteffects of different treatments at a probability level of P � 0.05using MINITAB 11.0 for Windows XP. The data on plant growthvariables from the pot experiment were additionally analyzed bysingle factor ANOVA to identify significant differences between thetreatment factors viz., SP, SA 15 DAP, SA 15 þ 30 DAP etc. Whereasin the field experiment, significant treatment factors were

Table 3Effect of seed priming and soil application (in nursery) of organophosphate pesticides on tgrown in sterilized soil in pots without any further pesticide treatment.

Pesticides Galls per root system Egg mas

Seed priming Soil application Soil app

#Non-infested soil (control) 00 (0.7) 00 (0.7) 00 (0.7)Infested soil (infested control) 47.8 (6.94) 38.2 (6.2) 47.8 (6.9Infested soil þ chlorpyriphos 12.4* (3.5) 7.2* (2.7) 6.2* (2.5Infested soil þ carbosulfan 9.3* (3.1) 4.0* (2.1) 3.0* (1.8Infested soil þ dichlorvos 25.2* (5.0) 19.0*(4.4) 17.3* (4Infested soil þ dimethoate 23.6* (4.9) 18.0* (4.3) 15.2* (3Infested soil þ malathion 31.5* (5.6) 24.3* (4.9) 22.4* (4Infested soil þ methyl parathion 34.0* (5.8) 22.8* (4.8) 29.5* (5Infested soil þ monocrotphos 45.5 (6.7) 35.2 (5.9) 46.5 (6.8Infested soil þ phorate 4.3* (2.1) 1.7* (1.4) 1.6*(1.4LSD P � 0.05 2.4 1.9 1.5F-values Pesticides (df ¼ 8) 15x 21x 19x

Each value is the mean of five replicates. Values in parentheses are transformed values [sqinfested control at P� 0.05. F-values followed by x are significant otherwise not significan(low infested) were pooled after analysis to form a common control (non-infested contr

identified with the help of two factor ANOVA followed by Tukey’sTest. To simplify the tables and figures, the data on the effect ofpesticide treatments on plant growth, yield, galls, soil populationetc. in non-infested soil (low infested) were pooled after analysisas a common control (non-infested control) because the treatmentdifferences were not significant at P � 0.05. The data on thenumbers of galls and egg masses and the soil population ofM. graminicola (5 replicates for the pot experiment and 30 repli-cates for the field experiment, 10/plot) were analyzed via single-factor ANOVA, and LSDs were calculated at P � 0.05. Thepercent variation over the control was also calculated. The data onthe soil populations and other parameters are presented ingraphical form. The bars or points in the figures are marked forsignificance at P � 0.05 according to Tukey’s Test. Since, the potexperiments in two years were done under the identical condi-tions viz., rice cultivar, soil type, nematode infestation level,manuring, irrigation, location of pots, months etc., the year dif-ferences in the data were non-significant at P � 0.05, hence thedata from two years were pooled.

3. Results

3.1. Effect of pesticide treatments on root-knot in the rice nurseryunder pot conditions

The nursery of rice cv. PS-5 raised in the nematode-infested soil(1000 J2 kg�1 soil) developed approximately 30 terminal galls and22 egg masses (Table 2). Pesticide treatments, however suppressedthe galling and egg mass production in the 4-week old ricenursery. The greatest decrease in the galls (92 and 99%) and eggmasses per root system (100%) was recorded due to soil applica-tion of phorate over control. Next in the effectiveness were car-bosulfan, chlorpyriphos and dichlorvos (Table 3). The plants rearedin the nursery in nematode-infested soil that were later grown innon-infested soil for 4 months showed a greater number of galls(58%) and egg masses (72%) in comparison to the values recordedin the nursery (Table 3). However, the effect of the pesticidetreatments (administered to the nursery bed) on galling, egg massproduction and soil population of M. graminicola in mature plantswas quite similar to that observed in the nursery stage (Tables 2and 3). The lowest number of galls per root system was recor-ded following the SA or SP of phorate (97 and 91% decrease) overcontrol (Table 3). Overall, SA in nursery soil was 3e9% (notinvariably significant at P � 0.05) and 6�16% (P � 0.05 or 0.01)

he galling and eggmass production onmature plants of rice cv. PS-5 transplanted and

ses per root system Soil population of M. graminicola J2 kg�1 soil

lication Seed priming Seed priming Soil application

00 (0.7) 00 (0.7) 00 (0.7)) 38.2 (6.2) 2424 (49.2) 2724 (49.2)) 2.2* (1.6) 107* (10.3) 00* (0.7)) 0.6* (1.0) 00* (0.7) 00* (0.7).2) 7.2* (2.7) 32* (17.9) 136* (11.6).9) 11.5* (3.4) 194* (13.9) 141* (11.8).7) 14.4* (3.8) 562* (23.7) 302* (17.3).4) 21.2* (4.6) 577* (24.0) 596* (24.4)) 36.4 (6.0) 2681 (51.7) 2623 (51.2)) 0.2* (0.8) 00* (0.7) 00* (0.7)

1.1 10.6 11.325x 62x 71x

rt(xþ 0.5)]. Values followed by the superscripts * are significantly different from thet (NS) at P� 0.05. # The data on the effect of pesticide treatments in non-infested soilol) because the treatment differences were not significant at P � 0.05.

Fig. 1. Effect of seed priming and soil application of some organophosphate pesticides on the galling caused by Meloidogyne graminicola in rice cv. PS-5 under pot conditions. Errorbars indicate the standard error. Bars with different alphabet are significantly different at P � 0.05 according to Tukey’s Test. DAP ¼ Days after planting.

M.R. Khan et al. / Crop Protection 63 (2014) 15e25 19

more effective than SP in suppressing the development of galls innursery (4-week-old seedlings) and mature plants (4-month-oldplants), respectively.

3.2. Effect of pesticide treatments on root-knot and plant growth inrice under pot conditions

3.2.1. SymptomsPlants grown in pots filled with nematode-infested soil (1000

M. graminicola J2 kg�1 soil) were stunted and showed chloroticfoliage, and their roots displayed terminal galls. The pesticide

Fig. 2. Effect of seed priming and soil application of some organophosphate pesticides on eError bars indicate the standard error. Bars with different alphabet are significantly differe

treatments suppressed the nematode infection and greatestreduction in the numbers of galls and egg masses following SA at15 DAP was recorded for phorate (37 and 20%, P � 0.05). The orderof effectiveness of the pesticides against the galling was:phorate > carbosulfan > chlorpyriphos > methyl parathion >

malathion > dimethoate > dichlorvos > monocrotophos (Figs. 1and 2). The SA 30 DAP of above eight pesticides decreased thegalls and egg masses 2�5% less than the 15 DAP treatment (Figs. 1and 2). Treatments consisting of two soil applications of the pes-ticides at 15 and 30 DAP were 4e7% more effective than the 15DAP treatment in reducing the number of galls and egg masses per

gg mass production by Meloidogyne graminicola in rice cv. PS-5 under pot conditions.nt at P � 0.05 according to Tukey’s Test. DAP ¼ Days after planting.

Table 4Effect of soil application of pesticides at different combinations of seed priming, 15 and 30 days of planting (DAP) on shoot and root length (cm) of rice cv. PS-5 grown in soil infested with Meloidogyne graminicola (1000 J2 Kg�1

soil) in pots.

Treatments Seed priming (SP) 15 DAP 30 DAP 15 þ 30 DAP SP þ 15 DAP SP þ 30 DAP SP þ 15 þ 30 DAP LSD P � 0.05

Shootlength

Rootlength

Shootlength

Rootlength

Shootlength

Root length Shoot length Root length Shootlength

Root length Shootlength

Root length Shootlength

Rootlength

Shootlength

Rootlength

#Non-infestedsoil (control)

55.0 21.9 55.0 21.9 55.0 21.9 55.0 21.9 55.0 21.9 55.0 21.9 55.0 21.9 2.9 1.3

Infested soil(infestedcontrol)

40.3 (�27) 16.5*(�23)

40.3 (�27) 16.5*(�23)

40.3 (�27) 16.5* (�23) 40.3 (�27) 16.5* (�23) 40.3 (�27) 16.5* (�23) 40.3 (�27) 16.5* (�23) 40.3 (�27) 16.5* (�23) 2.4 1.1

Infested soil þchlorpyriphos

c42.1 (4.5) b17.3 (4.2) b43.9** (9.0) b 17.4(4.8)

b44.5**(10.9)

c17.2 (3.6) a46.1** (15.2) a17.7** (6.6) a46.5**(16.1)

a17.9** (7.8) b44.8**(11.7)

a17.7** (6.0) a47.7**(19.2)

a18.1** (9.0) 3.0 0.9

Infested soil þcarbosulfan

42.0 (4.5) 17.1 (3.0) 45.3** (12.4) 17.6**(5.4)

44.7**(11.4)

17.3 (4.8) 46.3** (15.7) 17.9** (7.8) 47.3** (18.0)

18.3** (10.8) 45.2**(11.9)

17.9** (6.6) 48.2**(19.4)

19.0** (14.4) 2.8 1.0

Infested soil þdichlorvos

b41.4 (3.2) b17.1 (1.8) a41.7 (4.0) a17.2 (3.6) a41.8 (4.2) a17.5 (4.2) a42.1 (5.7) a17.5 (5.4) a42.2 (5.5) a17.4 (5.4) a42.0 (4.7) a17.2 (4.8) a43.8 (8.5) a17.8 (7.2) 2.7 0.8

Infested soil þdimethoate

a41.9 (4.0) a16.6 (1.8) a40.6 (1.5) a17.0 (1.6) a40.9 (1.8) a16.9 (1.8) a41.6 (4.0) a17.3 (3.0) a42.0 (4.2) a17.1 (1.8) a41.4 (2.5) a17.0 (2.4) a42.7 (6.7) a17.4 (4.8) 2.9 1.2

Infested soil þmalathion

a42.2 (4.5) a17.0 (3.0) a42.3 (5.5) a17.3 (4.2) a43.0 (7.0) a17.3 (4.2) a44.1** (10.4) a17.5 (6.0) a45.3 (13.2) a17.6 (6.6) a44.2 (9.5) a17.3 (5.4) a46.0**(12.9)

a17.9** (7.8) 2.6 1.0

Infested soil þmethylparathion

a41.2 (2.7) a17.1 (1.2) a41.8 (3.7) a16.9 (1.2) a41.2 (3.0) a17.0 (1.8) a42.1 (4.8) a17.3 (4.2) a42.7 (6.0) a17.4 (4.8) a41.9 (4.2) a17.2 (4.8) a43.0 (7.2) a17.8 (7.8) 2.4 1.2

Infested soil þmonocrotphos

a41.0 (2.5) a17.3 (3.0) a42.0 (4.2) a17.1 (3.0) a41.3 (3.2) a17.2 (2.4) a42.2 (5.0) a17.4 (4.8) a42.2 (4.5) a17.3 (4.2) a41.8 (4.2) a17.1 (3.6) a42.9 (6.5) a17.7 (6.0) 2.3 1.1

Infested soil þphorate

d42.3 (5.0) c17.5 (4.2) c46.5**(16.2)

c17.9**(6.8)

c46.1 (15.0) c17.6 (6.0) b49.1** (22.4) a19.3** (17.4) a50.1**(24.1)

a19.6** (18.0) b47.9**(18.7)

b18.8**(13.8)

a52.0**(29.1)

a20.0**(21.0)

2.6 1.3

LSD P � 0.05 2.8 1.5 3.0 0.9 3.1 1.0 3.5 1.1 3.2 1.3 3.4 1.0 3.3 1.3 e e

F-valuesNematode 43 38 49x 37x 67x 56x 87x 73x 54x 47x 35x 31x 207x 98.3x e e

Pesticides NS NS NS NS NS NS 16x 14x 20 17 NS NS 22.4x 18x e e

Interaction NS NS NS NS NS NS 7.3x 5.1x 7.8 6.5 NS NS 10.5x 9.4x e e

Each value is the mean of five replicates. Values in parenthesis are percent variation with respective control. Values within column followed by the * are significantly different from the non-infested control at P� 0.05. The valuewithin a column followed by ** are significantly different from the respective infested control at P � 0.05. F-values followed by x are significant otherwise not significant (NS) at P � 0.05. Values within row followed by differentalphabet are significantly different at P� 0.05 according to Tukey’s Test. SP¼ Seed priming. # The data on the effect of pesticide treatments in non-infested soil (low infested) were pooled after analysis to form a common control(non-infested control) because the treatment differences were not significant at P � 0.05.

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Table 5Effect of soil application of pesticides at different combinations of seed priming, 15 and 30 days of planting (DAP) on dry weight of shoot and root (DAP) of rice cv. PS-5 grown in soil infested withMeloidogyne graminicola (1000 J2Kg�1 soil) in pots.

Treatments Seed priming (SP) 15 DAP 30 DAP 15 þ 30 DAP SP þ 15 DAP SP þ 30 DAP SP þ 15 þ 30 DAP LSD P � 0.05

Shoot dryweight

Rootweight

Shoot dryweight

Rootweight

Shoot dryweight

Rootweight

Shoot dryweight

Rootweight

Shoot dryweight

Root weight Shoot dryweight

Rootweight

Shoot dryweight

Rootweight

Shootdryweight

Rootweight

#Non-infestedsoil (control)

3.60 1.78 3.60 1.78 3.60 1.78 3.60 1.78 3.60 1.78 3.60 1.78 3.60 1.78 0.15 0.08

Infested soil(infestedcontrol)

2.46* (�31) 1.48*(�16)

2.46* (�31) 1.48*(�16)

2.46*(�31)

1.48*(�16)

2.46*(�31)

1.48* (�16) 2.46* (�31) 1.48* (�16) 2.46*(�31)

1.48*(�16)

2.46* (�31) 1.48* (�16) 0.16 0.10

Infested soil þchlorpyriphos

d2.60 (5.2) d1.50 (2.7) c2.70** (9.3) c1.60**(8.2)

c2.68**(8.9)

c1.58**(8.8)

c2.76**(12.1)

b1.61**(10.2)

a3.12** (26.6) a1.69** (12.6) b3.01**(22.2)

b1.62**(9.5)

a3.15** (28.6) a1.70**(13.3)

0.13 0.09

Infested soil þcarbosulfan

f2.61 (5.6) c1.53 (4.8) d2.86**(15.7)

b1.64**(12.2)

d2.78**(12.9)

b1.62**(11.6)

c2.93**(19.4)

b1.63**(12.9)

a3.25** (27.7) a1.70** (16.3) b3.15**(28.2)

b1.65**(10.9)

a3.12** (25.4) a1.67**(12.2)

0.17 0.11

Infested soil þdichlorvos

b2.60 (4.8) a1.50 (2.7) b2.60 (5.6) a1.53 (3.4) b2.59 (4.8) a1.51 (6.1) b2.65 (7.7) a1.56 (8.8) a2.99** (20.2) a1.60 (8.8) a2.91**(16.9)

a1.57 (7.5) a3.01** (20.4) a1.61 (10.2) 0.14 0.08

Infested soil þdimethoate

b2.51 (2.00 a1.50 (2.7) b2.52 (2.2) a1.51 (3.4) b2.50 (0.8) a1.50 (2.7) b2.52 (1.6) a1.53 (4.1) a2.73** (9.7) a1.54 (4.1) a2.67 (7.3) a1.51 (4.1) a2.78** (12.9) a1.52 (4.8) 0.15 0.09

Infested soil þmalathion

c2.54 (2.8) a1.51(4.10)

c2.63 (6.9) a1.55 (6.1) c2.60 (5.6) a1.54 (4.8) c2.66**(8.10)

a1.60**(10.2)

a3.05** (21.8) a1.63 (12.2) b2.84**(13.3)

a1.59 (8.2) a3.11** (24.4) a1.64**(13.6)

0.18 0.12

Infested soil þmethylparathion

c2.50 (1.20 a1.48 (1.4) b2.54 (2.8) a1.50 (3.4) a2.52 (2.0) a1.49 (2.8) a2.60 (4.9) a1.55 (7.5) a2.90** (16.9) a1.56 (7.5) a2.74 (8.9) a1.55 (5.4) a2.96** (18.2) a1.60 (8.8) 0.13 0.10

Infested soil þmonocrotphos

b2.52 (2.4) a1.53 (4.8) b2.55 (3.4) a1.55 (4.1) a2.56 (4.0) a1.54 (4.8) a2.61 (5.2) a1.60 (9.5) a2.66 (6.0) a1.59 (6.1) a2.7 (8.9) a1.58 (6.8) a2.71 (8.50 a1.62(10.2)

0.15 0.12

Infested soil þphorate

f2.60 (5.2) c1.55 (6.1) d2.96**(18.8)

a1.68**(14.0)

c2.83**(12.9)

b1.60**(8.8)

b3.05**(23.0)

a1.75**(15.0)

a3.42** (27.5) a1.76** (15.3) b3.26**(25.5)

a1.69**(14.2)

a3.45** (29.1) a1.77**(15.5)

0.11 0.09

LSD P � 0.05 0.17 0.11 0.15 0.08 0.21 0.09 0.24 0.10 0.23 0.09 0.22 0.10 0.24 0.11 e e

F-valuesNematode 39.5x 38.6x 40.5x 39.5x 27.8x 25.5x 55x 42x 44x 39x 11.7x 7.5x 15.3x 10.2x e e

Pesticides NS NS NS NS NS NS NS NS 18.5x 16.9x 6.9x 4.5x 18.5x 14.2x e e

Interaction NS NS NS NS NS NS NS NS 5.9x 3.2x NS NS 9.2x 7.1x e e

Each value is the mean of five replicates. Values in parenthesis are percent variation with respective control. Values within column followed by the * are significantly different from the non-infested control at P � .05. The valuewithin a column followed by ** are significantly different from the respective infested control at P� 0.05. F-values followed by x are significant otherwise not significant (NS) at P� 0.05. Values within row followed by differentalphabet are significantly different at P� 0.05 according to Tukey’s Test. SP¼ Seed priming. # The data on the effect of pesticide treatments in non-infested soil (low infested) were pooled after analysis to form a common control(non-infested control) because the treatment differences were not significant at P � 0.05.

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M.R. Khan et al. / Crop Protection 63 (2014) 15e2522

root system. The SP alone did not affect nematode galling or eggmass production, but it significantly improved the effectiveness ofthe soil treatments. The SP þ SA 15 DAP of phorate reduced thegalling (65%) and egg mass production (36%) in rice plants overthe control (P � 0.05). The decline in galling and egg mass pro-duction with SP þ SA 30 DAP was 1e7% less than the SP þ 15 DAPtreatment, but with SP þ 15 þ 30 DAP treatment, the decrease was2e11% greater than the former (occasionally significant atP � 0.05).

3.2.2. Effects on plant growth and biomass production in riceA marked reduction (P � 0.05) of all growth parameters of rice

cv. PS-5 was recorded in the plants grown in nematode-infestedsoil (Tables 4 and 5). However, treatment with the pesticideschecked the suppressive effect of the nematodes, leading to im-provements of plant growth that varied with the treatment regimeand pesticide. The SA of phorate, carbofuran or chlorpyriphosresulted in significant increases in the length and dry weight of riceplants grown in nematode-infested soil over the control (P � 0.05);these effects were greatest under the 15 þ 30 DAP regime (8�25%),followed by the 15 DAP treatment (5�14%) in comparison toinfested control. The SP alone did not prove significantly effective inenhancing the plant growth of nematode infested plants comparedto the control. The greatest improvement in the plant length anddry weight of the shoots and roots was recorded due toSP þ 15 þ 30 DAP with phorate (20�40%), followed by the triotreatment with chlorpyriphos (14�28%), carbosulfan (12�25%) andmalathion (7�23%), compared to the control (P � 0.05). The effectof the SP þ 15 DAP treatment on the length and dry weight of theshoots and roots of the rice plants was statistically equal to that ofthe SP þ 15 þ 30 DAP regime, resulting in a greatest increase(18�37%) in the plant growth parameters due to phorate applica-tion (P � 0.05, Tables 4 and 5). The SP þ 30 DAP treatment was 3e7% less effective than the SP þ 15 DAP regime in promoting thegrowth of rice plants, and the differences of the two treatmentswere variably significant (P � 0.05).

Fig. 3. Effects of seed priming and soil application of some organophosphate pesticides on thbars indicate the standard error. Bars with different alphabet are significantly different at P

3.2.3. Soil population of M. graminicolaAt the harvest stage (4 months), the soil population density of

juveniles of M. graminicola in the control pots (untreated) hadincreased to more than four times the initial inoculum density(1000 J2, Fig. 3). The population density of the nematodes in potstreated with different pesticides was considerably lower than un-treated pots (Fig. 3). The M. graminicola population was 39% lowerin the pots that received 15 DAP SA with phorate over control(P � 0.05). Effects of 30 DAP treatments on the nematode pop-ulations was relatively equivalent to the 15 DAP regime. Dual soilapplication of the pesticides (15 þ 30 DAP) caused 15e76% reduc-tion in the nematode population and lowest population wasrecorded in phorate treatment (Fig. 3). When SP treatment wascombined with the SA 15 DAP and 15 þ 30 DAP regimes, thenematode populations were 77 and 81% (carbosulfan) and 40 and43% (chlorpyriphos) lower than the control, respectively (P � 0.05).

3.3. Effect of pesticide treatments on root-knot and plant growth inrice under field conditions

3.3.1. Gall formation and egg mass productionThe plants of rice cv PS-5 grown in the nematode-infested field

showed extensive terminal galls which varied in number from 73 to81 galls per root system, while 61 to 69 egg masses were recordedin the two years. Application of pesticides suppressed galling in amanner that varied with the pesticide and treatment regime(Table 6). In a single soil application of the pesticides, galling was32e49% lower than the control with around 9�13% greater effect ofSA 15 DAP treatments. The galling was 62 and 66% lower on theplants that received SA 15 þ 30 DAP of phorate in the first andsecond year, respectively. However, lowest number of galls per rootsystem was recorded due to application of SP þ SA 15 þ 30 DAP ofphorate (49e71% and 47e69%) followed by SP þ SA 15 DAP car-bosulfan (41�67% and 37�65%) over control in the first and secondyear, respectively. The pesticide treatments were found to beequally effective in suppressing egg mass production by

e soil population of Meloidogyne graminicola in rice cv. PS-5 under pot conditions. Error� 0.05 according to Tukey’s Test. DAP ¼ Days after planting.

Table 6Effect of soil application of phorate, carbosulfan and chlorpyriphos at differentcombinations of seed priming, 15 and 30 day of transplanting on galling, egg massproduction and grain yield of rice cv. PS-5 grown in soil infested with Meloidogynegraminicola (around 1000 J2 kg�1 soil) under field condition.

Treatments (Days afterplanting, DAP)

I Year II Year

Galls Eggmasses

Yield(kg/ha)

Galls Eggmasses

Yield(kg/ha)

#Non-infested soil (control) e e a4050 e e a3945Infested soil (control) a73 a61 d2925* a81 a69 d2880*Phorate (Seed Priming, SP) a67 a58 c3120 a73 a63 c3050Phorate (15 DAP) d37** d28** b3470** d40** d31** b3350**Phorate (30 DAP) c43** c35** c3220** c51** c40** c3140**Phorate (15 þ 30 DAP) e25** f19** a3825** e31** e23** a3720**Phorate (SP þ 15 DAP) e24** 21** a3985** 27** e22** a3855**Phorate (SP þ 30 DAP) d33** d30** b3655** d41** d35** b3545**Phorate (SP þ 15 þ 30 DAP) e21** g12** a4025** f24** f13** a3925**Carbosulfan (SP) a69 a60 c3095 a76 a66 c3005Carbosulfan (15 DAP) c44** c37** b3350** c52** c41** b3280**Carbosulfan (30 DAP) b53** b44** c3165** b57** b51** c3125**Carbosulfan (15 þ 30 DAP) d33** e25** b3620** 39** d31** b3460**Carbosulfan (SP þ 15 DAP) d34** d26** b3505** d43** d30** b3685**Carbosulfan (SP þ 30 DAP) d38** c33** b3460** c48** c42** b3495**Carbosulfan

(SP þ 15 þ 30 DAP)

d29** e20** a3825** e32** e25** a3755**

Chlorpyriphos (SP) a68 a59 c3025 a78 a64 c3080Chlorpyriphos (15 DAP) c47** c35** c3140** c52** c45** b3200**Chlorpyriphos (30 DAP) b53** b46** c3080 b64** b51** c3050Chlorpyriphos (15 þ 30 DAP) d39** d31** c3275** c48** d39** b3255**Chlorpyriphos (SP þ 15 DAP) c43** c36** c3350** c50** d38** b3350**Chlorpyriphos (SP þ 30 DAP) c44** c37** c3285** c52** c41** b3200**Chlorpyriphos

(SP þ 15 þ 30 DAP)

d37** d26** b3485** d43** d34** b3395**

LSD P � 0.05 11.2 6.7 306 12.6 6.5 318F-valueNematode (df ¼ 1) 480x 166x 149x 77.1x 197x 102x

Pesticides (df ¼ 9) 82.1x 56.2x 586x 71.3x 51.2x 614x

Interaction (df ¼ 9) 8.5x 12.4x 32.4x 8.3x 5.6x 21.2x

Each value is the mean of five replicates. Values followed by the superscripts * aresignificantly different from the non-infested control at P� 0.05. The values followedby a are significantly different from the infested control P � 0.05. F-values followedby x are significant otherwise not significant (NS) at P � 0.05. SP ¼ Seed priming,DAP ¼ Days after planting. # The data on the effect of pesticide treatments in non-infested soil (low infested) were pooled after analysis to form a common control(non-infested control) because the treatment differences were not significant atP � 0.05.

M.R. Khan et al. / Crop Protection 63 (2014) 15e25 23

M. graminicola (Table 6). The suppressive effect of the differentregimes on the galling and egg mass productionwas in the order ofSP þ SA 15 þ 30 DAP > SP þ SA 15 DAP > SP þ SA 30 DAP > SA15 þ 30 DAP > SA 15 DAP > SA 30 DAP > SP (Table 6).

3.3.2. Grain yieldRice cv. PS-5 grown in nematode-infested fields exhibited grain

yield reductions of approximately 26 and 29% in the two years,respectively, over control (non-infested field, Table 6). Applicationof the pesticides checked the suppressive effect of M. graminicola,leading to increases in the yield that varied with the treatment. Soilapplication of three pesticides at 15 DAP significantly improved theyield of rice grown in nematode-infested plots with greatestenhancement occurred in phorate treatment (16 and18% in twoyears) over control (P � 0.05). A significant increase in grain yieldunder the SA 30 DAP treatment was recorded for only phorate (10and 8.6%) and carbosulfan (8.2 and 7.5%) in the first and secondyear, respectively (P� 0.05). The rice grain yield obtained followingdual soil treatments ranged from 13 to 31%with a maximal increaseoccurred due to phorate application over the control. The combi-nation of SP with SA resulted in yield enhancements of 19e38% and17�35% (SP þ SA 15 þ 30 DAP), 13�36 and 11�32% (SP þ SA 15DAP), and 12�25 and 11�23% (SP þ SA 30 DAP) over the control in

the two years, respectively, with the relatively greatest increasebeing observed for phorate and smallest for chlorpyriphos.

3.3.3. Soil population of M. graminicolaOne month after planting, the M. graminicola population in the

soil was almost double the initial population and further increasedto approximately four times the initial population at harvest (4months, P � 0.05. Fig. 4). The seed priming treatment did not affectthe nematode population. The plots that received SA of pesticidesat15 DAP had 41 and 51% lower nematode population over initialpopulation in the two study years, respectively, with relativelygreatest effect being observed for phorate and lowest for chlor-pyriphos (P � 0.05, Fig. 4). Under the 30 DAP treatment (recordedjust before the pesticide application), the one-month nematodepopulationwas equal to that in the control. However, in the secondmonth, the population was 30e48% (first year) and 32�42% (sec-ond year) less than one-month population (P � 0.05). At the thirdand fourth months, the M. graminicola populations were signifi-cantly (P� 0.05) greater than the second-month population, exceptunder phorate treatment. Following the SA 15 þ 30 DAP treatment,the nematode populations in the first month were equal to thoseobtained after the 15 DAP treatment, but in the third month, thenematode population was 23 and 19% lower than one-monthpopulation in two study years, respectively (Fig. 4). A combina-tion of SA and SP of the pesticides provedmuchmore effective thanthe treatments without SP (Fig. 4). The greatest decrease in thenematode population i.e., 27�84% and 24�79% was recorded underthe SPþ SA 15þ 30 DAP and SPþ SA 15 DAP regime, especially withphorate in both years.

4. Discussion

The first experiment, examining the development of root-knotin the nursery, demonstrated the potential risk of nursery rearingof rice cv. PS-5 in nematode-infested soil. Infested nurseries are oneof the major sources of the dissemination and subsequent estab-lishment of M. graminicola in uninfested paddy fields (Yik andBirchfield, 1979). The nursery rearing of plants in soil infestedwith approximately 1000 J2 of M. graminicola kg�1 soil resulted inthe development of 30 galls/root system, and this infestation led tosevere root-knot disease (over 50 galls) in mature plants grown for4 months in non-infested soil. The root-knot nematodeM. graminicola is one of the few nematode pests that are highlydamaging in both nurseries and the main field (Bridge et al., 2005).Application of the pesticides reduced the nematode infestation inthe nursery to a varying extent. Among the eight tested organo-phosphates, phorate, carbosulfan and chlorpyriphos proved to behighly effective against M. graminicola, and their SA or SP treat-ments suppressed the galling by 76e99% in the nursery, leading to87�100% control of the nematodes inmature plants when grown innon-infested soil. The greatest suppression in the galling, egg massproduction and the soil population of M. graminicola was observedusing phorate, followed by carbosulfan and chlorpyriphos.Although the SP was found to be 4�12% less effective than the SAtreatment, but with regards to the dose, SP has emerged econom-ically as well as ecologically a much superior treatment to controlM. graminicola in rice nurseries, because SP treatment requires avery small amount of the pesticides.

Plants of rice cv. PS-5 grown in nematode-infested soil in pots(1000 M. graminicola J2 kg�1) or fields (approximately 1100 J2 kg�1

soil) developed terminal, hook-shaped or spiral galls. The spiral orhook like galls are specifically caused by M. graminicola and areconsidered a characteristic symptom of the nematode in rice (Khanet al., 2012). Severe galling also occurred under field conditions,indicating that rice cv. PS-5 was highly susceptible to the nematode

Fig. 4. Effects of seed priming and/or soil application of phorate, carbosulfan and chlorpyriphos on the soil population ofMeloidogyne graminicola in rice cv. PS-5 in different monthsin two years under field conditions. Bars with different alphabet are significantly different at P � 0.05 according to Tukey’s Test. DAP ¼ Days after planting.

M.R. Khan et al. / Crop Protection 63 (2014) 15e2524

infestation. A population of approximately 800e1000 J2 ofM. graminicola can incite severe galling in rice in sandy loam, whichwas the type of soil examined in the present study (Prasad et al.,2010). The plants grown in M. graminicola-infested soil exhibiteda 16�32% reduction in plant growth parameters (pot conditions)and a 26�28% reduction in grain yield (field condition) over thecontrol. In sandy loam soils, such as the soil in the present study,M. graminicola causes greater damage than the clay loam, resultingin yield losses that may reach 72% (Gaur and Pankaj, 2010; Khanand Anwer, 2011).

The eight organophosphates evaluated to determine theireffectiveness against M. graminicola under pot conditions showedvarying effects. The order of pesticide effectiveness regarding thesuppression of galls and promotion of plant growth was as follows:phorate > chlorpyriphos > carbosulfan > parathion >

dimethoate > dichlorvos > monocrotophos. Prasad et al. (2010)observed greater effectiveness of soil application of phorate at4.5 kg a.i. ha�1 in suppressing M. graminicola compared to carbo-sulfan or chlorpyriphos. However, in the present study, differentmode, time and dose of application of the pesticides were used. Soilapplication of the pesticides at 15 DAP resulted in greater nematodecontrol and subsequent plant growth improvements in rice grownin pots compared to the 30 DAP treatments, but the difference wasnot significant at P � 0.05. Seed priming alone did not significantlysuppress root galling, but the combination of this treatment withsoil application greatly improved the effectiveness of pesticideapplication. The SP þ SA 15 þ 30 DAP regime using phorate pro-vided the highest degree of nematode control, although the resultsof the SP þ SA 15 DAP treatment were statistically on par with theformer treatment. Phorate is a nematicide that acts via contact,though also showing some systemic properties (Johnson, 1985),and its soil application has proved to be highly effective against a

large number of soil nematodes (Khan and Jairajpuri, 2010). Hence,the nursery-reared plants grown from SP treated seeds containedsome pesticides in their system due to the partial systemic action,which would have suppressed nematode penetration in the rootsfor 15 days when phorate was applied in the soil. By 15 days,seedlings become established in the soil, and considerable numbersof lateral roots emerge, providing a substrate for nematode feeding.Soil application of phorate at this stage would have induced mor-tality in the juvenile nematodes and suppressed their ability toinvade the roots. The second application, at 30 DAP, would haveserved as a booster dose of the nematode suppressant, causingmortality in the juvenile nematodes that escaped the first dose orbelonged to the second generation. The root galling and soil pop-ulation of nematodes observed in the pots receiving the SP þ SA 15DAP and SP þ SA 15 þ 30 DAP treatments were statisticallyequivalent. This indicates that the SA 15 DAP treatment causedmortality to a large proportion of the initial nematode populationand the remaining larvae at least partially lost infectivity (Johnson,1985). Thus, the effectiveness of the SP þ SA 15 DAP treatment wasconsiderably closer to the SP þ SA 15 þ 30 DAP treatment. Othertested nematicides, especially carbosulfan and chlorpyriphos, alsosignificantly suppressed nematode pathogenesis, but their overallperformances were significantly inferior than phorate (P � 0.05).

The field trial involving phorate, carbosulfan and chlorpyriphostreatment also showed significantly greater effectiveness of the SA15 DAP treatment, with or without seed priming, than SA 30 DAPtreatments in both years of the study (P � 0.05). This shows thatearly application of the pesticides, just at the stage when lateralroots (feeding sites) are emerged, proved more effective in pro-tecting the root system from nematode invasion than treatment ata stage when plants had already become established (30 days). Riceplants have been found to be more susceptible to nematodes at an

M.R. Khan et al. / Crop Protection 63 (2014) 15e25 25

early stage of growth than at later stages (Plowright and Bridge,1990). The approximately 50% decreases observed in the numbersof galls, eggmasses and juveniles ofM. graminicola in the soil due tothe SA 15 DAP treatment with phorate indicates that the activeingredient of this pesticide persisted in the soil or plant roots forthe rest of the crop duration (3.5 months) and conferred protectionto the rice plants throughout the season. Carbosulfan is a systemicpesticide, but it did not provide a degree of nematode control equalto that of phorate, revealing that the active ingredient in phoratewas more toxic to M. graminicola than carbosulfan, although boththe pesticides were organophosphate chemicals.

Field trials demonstrated a greater effectiveness of the SP þ SA15 DAP and SP þ SA 15 þ 30 DAP regimes using phorate, sup-pressing galling by 65e67% and 69�73 and improving grain yieldsby 32e36% and 35�38, respectively. As the differences betweenthese two treatments were marginal, the former will be moreacceptable to farmers. The farmers who realize the economicconsequences of M. graminicola infestation in paddy generallyapply 2-3 soil treatments with pesticides to control the nematode.Hence, use of SP þ15 DAP treatment would enable to save atleastone dose of soil application that would considerably reduce the costof management and prevent contamination of soil and food. Inaddition, SP treatment with phorate also greatly contributed to thecontrol of nematode infestation in the nursery. Based on the pre-sent study, a treatment consisting of seed priming (5 g kg�1 dryseed weight) and soil application of phorate (2 kg ai ha�1) 15 daysafter planting can effectively control root-knot development underirrigated conditions. Soil application of the pesticide should beperformed when fields are wet, without standing water (2�3 daysafter irrigation), so that the chemical does not quickly percolateinto deeper soil layers, but remain in the root zone for a greaterduration to allow contact with the nematode and the simultaneousabsorption by the roots.

Acknowledgments

The financial assistance from the University Grant Commission(UGC) through a research project (No. 41-1197/2012-SR) to carryout the research work of the present publication is acknowledgedwith thanks.

Appendix A. Supplementary data

Supplementary data related to this article can be found at http://dx.doi.org/10.1016/j.cropro.2014.04.024.

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