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Exploitation of the Nematicidal Potential of Bio-and Synthetic Chemicals Against Meloidogyne incognita and Their Impact on Phytotoxicity and Nematode Reproduction.

Byline: Huma Abbas, Nazir Javed, Sajid Aleem Khan and Saeed Ahmad

Abstract

Present investigation was conducted to exploit the nematicidal potential of bio and synthetic chemicals against Meloidogyne incognita (Kofoid and White) on tomato. Effect of twenty chemicals currently available in market was evaluated against M. incognita. Hatching inhibition and juvenile's mortality of M. incognita was assessed under in vitro conditions. Four concentrations of each chemical were prepared viz., 2S, S, S/2, S/4 according to recommended dose of each chemical. Data on hatching inhibition was recorded after 2, 4 and 6 days and on mortality after 12, 24, 48 and 72 h. Maximum hatching inhibition and mortality percentage was recorded in synthetic [Cartap (Thiocarbamate), Virtako (Thiamethoxam + chlorantraniliprole)] and bio [Cure (Abamectin), Azadirachtin (Aza)] chemicals. These four chemicals were selected and evaluated further against mobility of juveniles and for their phytotoxic effect on tomato.

Minimum number of J2's were recovered in Cartap (95.67) followed by other chemicals while maximum were recovered in control (238.10). Tomato plants were examined for following symptoms yellowing or browing, wilting, necrosis and plant mortality after two months, none of the chemical was found to be phytotoxic. Efficiency of selected chemicals was evaluated at different time intervals (7, 14 and 28 days) against M. incognita on nematode reproduction parameters. A gradual decline was noted in the effectiveness of chemicals with the increase in time interval. Galling index was increased in all the chemicals after 28 days interval as compared to 7 and 14 days. The results of present investigation suggest suitable chemicals for grower having nematode problem in field to incorporate it in management strategies.

Key words: Nematicidal, hatching, mortality, phytotoxic, management.

INTRODUCTION

Management of Meloidogyne incognita is difficult due to its wide host range including more than 3,000 plant species (Abad et al., 2003). Root-knot nematodes cause severe losses in vegetables throughout the world. Yield losses upto 24% due to M. incognita and M. javanica (Treub) were reported (Kathy, 2000). Disease infestation and prevalence was 32% and 60%, respectively, due to M. incognita in Pakistan (Javed et al., 2010; Kamran et al., 2010). In Pakistan yield losses due to M. incognita and M. javanica were 40% (Anwar and Mckenry, 2012). Population density of M. incognita was reported at higher level on tomato (Kamran et al., 2013). A range of strategies employed for the management of root-knot nematodes including cultural practices, biological control, sanitation, soil amendments and host plant resistance. But unfortunately all these practices are unable to protect the crops under field conditions because these are not cost-effective and require extra labour (Kerry, 1990).

So, the most practical alternative like chemical control should be used to protect the plants under field conditions. Chemical control through nematicides is the quickest way to reduce the root-knot nematode population under field conditions in a short period of time. Though, the use of some nematicides and fumigants has been restricted due to concerns about the health hazards to humans and environment safety (Rich et al., 2004). However, chemical control still endures to be the main approach for the management of nematodes. The chemicals preferably used should possess a high rate of nematode suppression in a short time and have no phytotoxic. Information about the level of nematode infestation in the soil is a prerequisite to avoid the needless use of nematicides (Dubey and Trivedi, 2011).

Lamberti et al. (2000) reported that non-fumigant nematicides can be easily and safely applied as compared to fumigants, which are most widely used such as carbofuran, aldicarb, fenamiphos, fosthiazate cadusafos, oxamyl, ethoprop and organophosphate based nematicides.

The effectiveness of various chemicals for the management of root-knot nematodes was evaluated on sunflower cultivars. The results revealed that out of five nematicides, Rugby-10 G (cadusaphos) was most effective followed by Unihypo-3.6 G and Furadan-3 G (carbofuran) (Rehman et al., 2006). Bhosle et al. (2012) revealed that application of carbofuran and phorates in granular form were relatively effective in minimizing the root-knot nematode population and in increasing the yield of okra. Nematicides based on micro-organisms are referred as biopesticides; they have the potential to reduce the nematode population in the soil (Arora et al., 2000) and can be successfully used in integrated disease management. The role of abamectin for the management of root-knot nematode on cotton as a seed treatment was studied. The findings revealed that final nematode population density was reduced due to the treatment of abamectin (Monfort et al. 2006).

Rehman et al. (2009) incorporated various bio-products into the soil to lessen the population of M. incognita. Abamactin proved the best for reducing the invasion and development of M. incognita followed by emamectin whereas azadirachtin reduced the number of eggs per egg mass and proved to have nematostatic properties. In Pakistan, true nematicides are not available in public domain; therefore present study was conducted to exploit nematicidal potential of available bio and synthetic chemicals against M. incognita.

MATERIALS AND METHODS

Collection of diseased plants

Tomato roots and soil samples infested with root-knot nematodes were collected from the vegetable production areas of University of Agriculture Faisalabad. Root and soil samples were processed separately to assess root-knot nematode population. The roots were separated from the soil, washed and weighed. The entire root system was chopped and incubated in a mist chamber for 5 days to hatch the eggs. Soil samples were thoroughly mixed and processed by Baermann funnel techniques for 3 days to collect nematodes.

Identification

Perineal patterns of mature females were prepared for different root-knot nematode species (Jepson, 1987). At least 10 perineal patterns were examined to make the identification.

Mass culturing of root knot nematodes

The sterilization of sandy loam soil was done in oven at 120C for 20 min (Talavera and Mizukubo, 2003) and then it was stored for two weeks at 25C before using them for experimental purpose. Seeds of tomato (Lycopersicon esculentum) 'Moneymaker' were collected from Ayub Agriculture Research Institute, Faisalabad. Seeds were planted in seedling trays containing sterilized soil. Three weeks old seedlings were transplanted in earthen pots (20-cm diam.). In order to make pure culture of field population, single egg mass inoculation of M. incognita was done. Single mature egg mass was inoculated in pots around the root of young tomato seedlings. Mass culturing was done by inoculating new tomato seedlings with at least 15 egg masses, each obtained from pure culture in order to maintain sufficient inoculum for further studies.

Evaluation of inhibitory effects of bio and synthetic chemicals on hatching of M. incognita

Four concentrations (2S=Double dose, S=Recommended dose, S/2= Half dose, S/4= Quarter dose) of each chemical were prepared according to recommended dose by adding requisite amount of distilled water. For hatching test, population of M. incognita maintained on the roots of egg plant from single egg mass culture was used, eggs of M. incognita were isolated by the method of Hussey and Barker (1973). Single egg mass of uniform size containing about 250 eggs was placed in each Petri dish. Four concentrations of each chemical were added in Petri dishes. Five replications were done for each chemical and incubated at 282C in a completely randomized design. Data was recorded after 2, 4 and 6 days of incubation.

Percent egg hatching was calculated and corrected by Abbott's formula (Abbott, 1925):

t - c / 100 - c x 100 = Hatching Inhibiton (%)

Where t, percent hatching inhibition in the chemical (bio/synthetic); c, percent hatching inhibition in the control.

After each count the egg masses were washed with 1 mL of distilled water in their respective plates and transferred to fresh concentrates of chemicals.

Evaluation of nemastatic/nematicidal effects of bio-and synthetic chemicals on mortality of M. incognita

For mortality test, all the experimental protocol and conditions were similar as in experiment No. 1 except freshly hatched second stage juveniles of M. incognita were used. Juveniles of M. incognita were extracted from the eggs and 1 ml of the suspension containing 80 juveniles was placed in each Petri dish.

Juveniles mortality was calculated and corrected by Abbott's formula (Abbott, 1925):

t - c / 100 - c x 100 = Mortality (%)

Where t, percent mortality in the chemical (bio/synthetic); c, percent mortality in the control.

Juveniles were considered dead if they did not move when probed with a fine needle (Abbasi et al., 2008) and were considered alive if they moved or appeared as a winding shape (El-Rokiek and El-Nagdi, 2011).

Effect of bio and synthetic chemicals on mobility of juveniles of M. incognita and their impact on phytotoxicity

Among the bio-and synthetic chemicals which showed significant results in hatching and mortality experiments were selected and their efficacy was evaluated against nematode mobility in soil. Five hundred J2's of M. incognita were inoculated in each plastic pot (8.5 cm top diam.; 7.5 cm bottom diam.; 4.5 cm depth) of sterilized soil. Each treatment was replicated fifteen times and placed under completely randomized design (CRD). Data was recorded after three days on the basis of number of juveniles recovered, recovery percentage and % reduction over control.

For phytotoxic effect three week old seedlings of tomato cv. Moneymaker were dipped in selected chemicals for 20-30 min while the seedlings dipped in water served as control. Then the seedlings were transplanted in earthen pots (10 cm diam.). Data on phytotoxicity was recorded on the basis of yellowing or browning, wilting, necrosis, burning and plant mortality after two months.

Efficiency of bio and synthetic chemicals against M. incognita at different time intervals

Efficiency of selected bio and synthetic chemicals was evaluated at different time intervals against M. incognita. Three weeks old seedlings of tomato cv. Moneymaker were transplanted in earthen pots of (10 cm dia.) containing amended soil with bio and synthetic chemicals. At different time intervals of 7, 14 and 28 days 750 J2's of M. incognita were inoculated in each pot. Three sets of treatments with fifteen replications were placed under CRD. Data was recorded after 35 days on visual estimation of root knot nematode galling index on root system of tomato by using galling index 0-10 scale (Bridge and Page, 1980), number of egg masses were counted by staining them with phloxine B (Holbrook et al., 1983), number of females/root system were recorded by staining in boiling 0.1% acid fuchsin solution (McBeth et al., 1941) for 1 min.

RESULTS

Inhibitory effects of bio-and synthetic chemicals on hatching

Inhibitory effect of twenty bio and synthetic chemicals was evaluated against percent hatching inhibition of M. incognita. The results revealed that all the treatments varied significantly in their potential towards M. incognita. Hatching of M. incognita was significantly varied in synthetic chemicals (Table I). Among the twelve chemicals Rugby caused maximum percent hatching inhibition followed by Cartap and Virtako as compared to other chemicals after 2 days. Percent (%) hatching inhibition in each chemical was affected by concentrations. Maximum inhibition was observed in 2S and S concentrations of all the chemicals while minimum was recorded in S/4 concentration. After 4 days% hatching inhibition was higher in Rugby, Cartap and Virtako while lower in Silk and Vimax (Table I). Rugby at its all concentration caused (98.85) % hatching inhibition while Cartap and Virtako caused (95.10, 79.65) respectively after 6 days of incubation (Table I).

Regression analysis showed linear relationship between % hatching inhibition and concentrations of synthetic chemicals. The relationship showed as the concentration lowered from 2S to S/4, a significant decreased in % hatching inhibition was observed. Time duration also affected hatching inhibition percentage, as the time duration increases, a significant increase in % hatching inhibition was recorded. The relationship between Cartap, Virtako and % hatching inhibition and was observed through regression analysis (Fig.1) respectively. Effect of biochemicals on % hatching inhibition of M. incognita was also evaluated. Results revealed that % hatching inhibition was significantly varied in all the treatments (Table II). Cure and Azadirachtin caused higher % hatching inhibition as compared to other bio chemicals after 2 days. After 4 days of incubation Cure and Azadirachtin caused (67.55%, 58.30%) hatching inhibition respectively at their all concentration (Table II).

Maximum inhibition percentage was observed in Cure and Azadirachtin at their all concentration from other chemicals after 6 days of incubation (Table II). Regression analysis showed linear relationship between concentrations of bio chemicals and % hatching inhibition. Time duration also affected hatching inhibition percentage, as its higher value was reached after 6 days of incubation. Regression curves were also drawn between Cure, Azadirachtin and % hatching inhibition (Fig. 1).

Table I.-Evaluation of synthetic chemicals on % hatching inhibition of Meloidogyne incognita after 2, 4 and 6 days.

###% Hatching inhibition at different concentrations a

Treatments###Mean b

###2S###S###S/2###S/4

After 2 days

Rugby###100.00 0.00ad###100.00 0.00a###96.20 0.58b###85.00 0.55d###95.30 1.42A

Regent###30.40 0.51opq###27.40 0.40rs###20.80 0.37tu###17.20 0.37wx###23.95 1.21G

Movento###31.00 0.32op###28.00 0.32qr###18.80 0.37uvw###16.00 0.32xy###23.45 1.44G

Alance###37.00 0.71m###34.20 0.37n###25.20 0.49s###20.40 0.51tuv###29.20 1.56F

Coredor###46.60 0.51k###41.00 0.45l###32.80 0.37no###28.40 0.68qr###37.20 1.64E

Cartap###100.00 0.00a###93.00 0.32c###80.20 0.58e###74.20 0.37f###86.85 2.34B

Arrivo###58.00 0.45i###51.40 0.51j###43.40 0.51l###38.20 0.37m###47.75 1.75D

Virtako###70.60 0.60g###60.80 0.37h###48.20 0.37k###42.00 0.45l###55.40 2.55C

Steward###30.00 0.32pq###27.20 0.37rs###20.40 0.51tuv###17.20 0.37wx###23.70 1.19G

Silk###14.40 0.51y###10.60 0.40zz1###5.00 0.32z2###3.00 0.32z2###8.25 1.05I

Vimax###22.20 0.37t###18.00 0.32vwx###11.20 0.37z###8.20 0.37z1###14.90 1.28H

Actara###59.00 0.32hi###52.40 0.51j###41.60 0.40l###37.40 0.51m###47.60 1.97D

Mean c###49.93 3.55A###45.33 3.49B###36.98 3.42C###32.27 3.15D

After 4 days

Rugby###100.00 0.00ad###100.00 0.00a###100.00 0.00a###90.60 0.68b###97.65 0.95A

Regent###44.20 0.37n###39.20 0.37opq###35.00 0.32rs###27.80 0.58wx###36.55 1.39I

Movento###46.40 0.51mn###41.20 0.37o###34.20 0.37st###29.00 0.32vw###37.70 1.53H

Alance###54.80 0.66hi###49.40 0.51kl###40.40 0.24op###37.00 0.32qr###45.40 1.64G

Coredor###62.00 0.32fg###57.40 0.51h###46.20 0.58mn###38.40 0.51pq###51.00 2.14F

Cartap###100.00 0.00a###97.80 0.37a###89.00 0.71b###81.40 0.51c###92.05 1.71B

Arrivo###70.40 0.51e###64.00 0.45f###56.00 0.55h###49.20 0.37l###59.90 1.85D

Virtako###81.00 0.45c###74.80 0.58d###60.40 0.51g###52.40 0.51ij###67.15 2.61C

Steward###44.80 0.58mn###37.20 0.37qr###33.00 0.45stu###25.40 0.51xy###35.10 1.63J

Silk###30.60 0.40uv###24.80 0.37y###19.00 0.32zz1###14.20 0.37z2###22.15 1.42L

Vimax###37.00 0.45qr###32.20 0.37tu###21.40 0.51z###18.20 0.37z1###27.20 1.77K

Actara###71.20 0.58e###63.00 0.71fg###52.00 0.71jk###47.40 0.51lm###58.40 2.16E

Mean c###61.87 2.89A###56.75 3.05B###48.88 3.10C###42.58 2.94D

After 6 days

Rugby###100.00 0.00ad###100.00 0.00a###100.00 0.00a###95.40 0.51b###98.85 0.47A

Regent###48.60 0.40lm###42.40 0.51pqr###38.00 0.32tu###30.20 0.58w###39.80 1.55I

Movento###50.60 0.51kl###44.40 0.51op###39.00 0.45st###33.20 0.37v###41.80 1.49H

Alance###60.40 0.51h###56.20 0.37i###43.40 0.51opq###39.20 0.37st###49.80 2.02G

Coredor###68.40 0.51f###61.20 0.37h###52.40 0.51jk###45.20 0.37no###56.80 2.02F

Cartap###100.00 0.00a###100.00 0.00a###93.40 0.51b###87.00 0.55c###95.10 1.25B

Arrivo###76.40 0.51d###67.20 0.66f###59.40 0.68h###52.60 0.81jk###63.90 2.06E

Virtako###95.20 0.37b###88.00 0.71c###71.40 0.51e###64.00 0.55g###79.65 2.88C

Steward###47.40 0.51mn###41.00 0.55qrs###35.80 0.37uv###29.00 0.32w###38.30 1.56J

Silk###35.40 0.51uv###28.00 0.32w###25.00 0.32x###20.40 0.51y###27.20 1.27L

Vimax###40.40 0.51rst###36.00 0.55u###29.40 0.51w###23.20 0.58x###32.25 1.52K

Actara###78.80 0.37d###69.20 0.37ef###60.40 0.51h###54.60 0.51ij###65.75 2.11D

Mean c###66.80 2.90A###61.13 3.06B###53.97 3.01C###47.83 3.02D

Table II.-Evaluation of biochemicals on % hatching inhibition of Meloidogyne incognita after 2, 4 and 6 days.

###% Hatching inhibition at different concentrations a

Treatments###Mean b

###2S###S###S/2###S/4

After 2 days

Azadirachtin###53.20 0.66bd###45.80 0.66c###35.40 0.51fg###31.00 0.71hi###41.35 2.02B

Neemix###26.20 0.49k###22.40 0.51mn###18.40 0.51p###15.20 0.49q###20.55 0.98E

Neemakill###42.00 0.32d###37.80 0.49ef###30.60 0.40hi###27.00 0.71jk###34.35 1.37C

Astra###14.20 0.37q###10.80 0.37r###5.20 0.37s###3.40 0.51s###8.40 1.01F

Cure###62.40 0.93a###55.00 0.55b###42.20 0.37d###36.60 0.51ef###49.05 2.36A

Spintor###25.00 0.32klm###23.20 0.37lmn###19.40 0.51op###14.60 0.68q###20.55 0.94E

Radiant###33.20 0.49gh###29.20 0.37ij###21.60 0.40no###18.20 0.66p###25.55 1.38D

Timer###43.00 0.55cd###39.00 0.55e###30.40 0.51hi###25.80 0.66kl###34.55 1.58C

Mean c###37.40 2.38A###32.90 2.14B###25.40 1.74C###21.48 1.61D

After 4 days

Azadirachtin###70.60 0.51bd###64.40 0.51c###52.60 0.68ef###45.60 0.40g###58.30 2.26B

Neemix###43.40 0.51gh###36.00 0.55i###27.20 0.49jkl###21.80 0.58m###32.10 1.91E

Neemakill###60.40 0.51d###53.00 0.71ef###43.20 0.58gh###36.40 0.51i###48.25 2.12C

Astra###29.20 0.58jk###24.40 0.51lm###15.40 0.51n###11.80 0.37o###20.20 1.61F

Cure###80.20 0.86a###73.40 0.51b###61.20 0.58d###55.40 0.51e###67.55 2.26A

Spintor###42.20 0.58h###36.80 0.80i###26.40 0.51kl###23.00 0.55m###32.10 1.80E

Radiant###52.20 0.66f###45.40 0.51g###34.00 0.71i###29.60 0.51j###40.30 2.08D

Timer###61.40 0.93cd###55.20 0.37ef###43.40 0.51gh###37.00 0.71i###49.25 2.22C

Mean c###54.95 2.49A###48.58 2.42B###37.93 2.26C###32.58 2.10D

After 6 days

Azadirachtin###84.40 0.75bd###77.80 0.86c###66.00 0.71e###58.00 0.55gh###71.55 2.37B

Neemix###60.40 0.68fg###54.60 0.51ij###44.80 0.66l###39.40 0.51m###49.80 1.90F

Neemakill###74.20 0.66d###68.20 0.49e###55.80 0.37hi###50.40 0.51k###62.15 2.19D

Astra###33.00 0.71n###28.60 0.60o###21.40 0.51p###19.40 0.51p###25.60 1.28G

Cure###90.00 0.63a###83.40 0.51b###74.20 0.66d###68.40 0.51e###79.00 1.92A

Spintor###59.20 0.58fg###54.20 0.66ij###45.40 0.51l###38.00 0.71m###49.20 1.89F

Radiant###69.00 0.71e###62.20 0.66f###50.80 0.37k###46.00 0.55l###57.00 2.10E

Timer###75.20 0.66cd###68.60 0.51e###59.20 0.66fg###52.20 0.66jk###63.80 2.04C

Mean c###68.18 2.67A###62.20 2.55B###52.20 2.40C###46.48 2.21D

Table III.-Evaluation of synthetic chemicals on % mortality of Meloidogyne incognita juveniles after 24, 48 and 72 h.

###% Juvenile mortality at different concentrations a

Treatments###Mean b

###2S###S###S/2###S/4

After 24 h

Rugby###100.00###0.00ad###100.00 0.00a###72.26 0.55e###57.58 0.78i###82.46 4.20A

Regent###30.85###0.37pqr###30.65 0.58qr###26.83 0.37st###20.80 0.74vwx###27.28 0.97G

Movento###33.07###0.45nopq###30.04 0.69qr###23.41 0.67uv###17.78 0.50xyz###26.08 1.39G

Alance###44.12###0.53k###37.69 0.49lm###32.46 0.51opqr###23.82 0.49tuv###34.52 1.72F

Coredor###52.76###0.43j###46.53 0.58k###33.87 0.36nop###26.83 0.48st###40.00 2.35E

Cartap###90.15###0.68b###81.50 0.54c###63.41 0.53h###50.15 0.58j###71.30 3.58B

Arrivo###65.23###0.51gh###56.58 0.44i###43.92 0.63k###32.06 0.70opqr###49.45 2.90D

Virtako###75.87###0.78d###70.46 0.32ef###45.53 0.74k###34.47 0.26no###56.58 3.94C

Steward###29.65###0.75rs###22.62 0.25uvw###16.38 0.61z###11.76 0.75z1###20.10 1.57H

Silk###22.21###0.49uvw###19.79 0.50wxy###6.12 0.70z2z3###3.71 0.53z3###12.96 1.88J

Vimax###24.82###0.21tu###16.78 0.50yz###11.36 0.51z1###7.74 0.39z2###15.18 1.49I

Actara###67.84###0.53fg###56.78 0.61i###40.70 0.40l###35.88 0.58mn###50.30 2.93D

Mean c###53.05###3.31A###47.45 3.28B###34.69 2.49C###26.88 2.03D

After 48 h

Rugby###100.00###0.00ad###100.00###0.00a###77.72 0.38e###65.36 0.76gh###85.77 3.42A

Regent###35.79###0.71q###32.34###0.63r###27.28 0.65st###24.05 0.44uv###29.87 1.07G

Movento###36.40###0.54q###31.54###0.47r###25.87 0.20tu###17.56 0.41w###27.84 1.62H

Alance###56.87###0.36k###46.53###0.51mn###40.66 0.67p###24.86 0.37tuv###42.23 2.67F

Coredor###65.97###0.43gh###54.83###0.44k###40.45 0.35p###29.92 0.55rs###47.79 3.16E

Cartap###95.75###0.37b###87.04###0.36c###75.49 0.39e###66.78 0.45g###81.26 2.54B

Arrivo###77.31###0.60e###61.52###0.66ij###48.96 0.55lm###40.85 0.69p###57.16 3.17D

Virtako###83.18###0.56d###75.89###0.46e###60.70 0.47j###42.48 0.35op###65.57 3.59C

Steward###40.86###0.34p###25.26###0.58tuv###16.14 1.00w###10.88 0.48xy###23.28 2.63I

Silk###30.13###0.32rs###22.43###0.51v###10.47 0.63xy###8.05 0.52y###17.77 2.07K

Vimax###30.33###0.21r###26.47###0.86tu###16.76 0.20w###13.11 0.59x###21.67 1.62J

Actara###71.65###0.28f###63.74###0.68hi###49.56 0.50l###45.31 0.63no###57.56 2.45D

Mean c###60.35###3.19A###52.30###3.24B###40.84 2.82C###32.43 2.49D

After 72 h

Rugby###100.00###0.00ad###100.00###0.00a###92.17 0.76b###79.59 0.37c###92.94 1.92A

Regent###38.14###0.58op###33.20###0.37q###28.66 0.51s###22.68 0.52t###30.67 1.33I

Movento###44.12###0.47n###37.10###0.65p###29.49 0.40rs###18.56 0.41uv###32.32 2.19H

Alance###67.42###0.51fg###49.48###0.49kl###45.15 0.47mn###30.72 0.68qrs###48.19 3.01G

Coredor###69.49###0.49ef###61.24###0.35i###47.62 0.45lm###30.72 0.40qrs###52.27 3.38F

Cartap###100.00###0.00a###92.58###0.38b###80.62 0.33c###75.87 0.46d###87.27 2.20B

Arrivo###80.62###0.37c###70.52###0.36e###63.10 0.29hi###46.19 0.28mn###65.11 2.89D

Virtako###90.11###0.51b###80.62###0.59c###69.48 0.26ef###56.90 0.50j###74.28 2.85C

Steward###45.36###0.49mn###30.72###0.40qrs###22.68 0.41t###16.08 0.21vw###28.71 2.51J

Silk###31.74###0.74qr###23.09###0.72t###15.26 0.25w###7.63 0.04y###19.43 2.07L

Vimax###40.20###0.50o###32.36###0.84q###19.18 0.38u###11.95 0.78x###25.92 2.55K

Actara###76.49###0.37d###65.57###0.64gh###56.90 0.44j###51.96 0.70k###62.73 2.15E

Mean c###65.31###3.09A###56.37###3.24B###47.53 3.16C###37.40 3.05D

Nemastatic/nematicidal effects of bio-and synthetic chemicals on mortality

Effect of all the chemicals varied significantly on % juveniles mortality of M. incognita (Table III). After 12 h of incubation

Rugby caused maximum % juveniles mortality (71.00) at its 2S concentration followed by S, S/2 and S/4 concentrations. Juveniles mortality percentage was maximum in Rugby, Cartap and Virtako at their all concentrations after 24 h (Table III). In Rugby, Cartap and Virtako % mortality was (100.00, 95.75, 83.18) in 2S concentration while at S/4 concentration % mortality was (65.36, 66.78, 42.48) respectively (Table III) after 48 h. Mean mortality percentage was significantly higher in Rubgy (92.94) followed by Cartap (87.27) and Virtako (74.28) while lower in Silk (19.43) at their all concentrations after 72 h of incubation (Table III). Regression analysis between % juveniles mortality and concentrations of synthetic chemicals was showed in Figure 2. A linear relationship was observed in mortality and concentrations of chemicals after all time duration. Regression equations were also drawn for the chemicals which caused maximum mortality at their all concentrations after all time intervals.

Mortality of M. incognita was significantly affected by bio chemicals (Table IV). Among the bio chemicals Cure caused maximum % juveniles mortality after 12 h of incubation followed by Azadirachtin. After 24 h of incubation concentration effect remained significant, as Cure caused 76.62% mortality at 2S followed by S (70.57), S/2 (57.89) and S/4 (48.41) concentration, respectively (Table IV). In Cure and Azadirachtin % mortality was increased after 48 h of incubation at their all concentrations (Table IV). After 72 h Cure and Azadirachtin caused maximum mean mortality (75.44, 68.91), respectively, from all other chemicals at their all concentration (Table IV). The relationship between % mortality and biochemicals was observed through regression analysis. Regression equations between Cure, Azadirachtin and % mortality showed as the concentration of chemicals increased, % mortality increased significantly with the increase in time duration.

In regression analysis a progressive increase was noted in mortality with the increase in concentration (Fig. 2).

Effect of bio-and synthetic chemicals on mobility of juveniles and their impact on Phytotoxicity

Bio-and synthetic chemicals which showed significant results in mortality and hatching experiments were evaluated against mobility of M. incognita and for phytotoxic effect on tomato. Effect of bio-and synthetic chemicals on mobility of juveniles (J2s) of M. incognita was observed after three days on number of J2s recovered, recovery percentage and % reduction over control. Reaction of all the treatments varied significantly on recovery of J2s after three days (Table V). Minimum number of J2s were recovered in Cartap (95.67) followed by other chemicals while maximum were recovered in control (238.1). Maximum percentage of reduction over control was observed in Cartap (60) followed by Virtako (55), Cure (39) and Azadirachtin (34). To check phytotoxic effect, plants were examined for following symptoms yellowing or browing, wilting, necrosis, burning and plant mortality after two months. None of the chemical was found to be phytotoxic.

Efficiency of bio-and synthetic chemicals at different time intervals

Efficiency of selected bio-and synthetic chemicals was evaluated at different time intervals; 7, 14 and 28 days against M. incognita. Data on galling index, number of egg masses and number of females/root system was recorded after 35 days of inoculation. Among synthetic chemicals (Cartap, Virtako) galling index varied significantly while in bio chemicals (Cure and Azadirachtin) results were statistically non significant (Table VI). All the chemicals varied significantly (P=0.05) in their response towards number of egg masses on root system of tomato. Maximum number of egg masses were observed in control (222.5) treatment while minimum were recorded in Cartap (65.8) followed by other chemicals. A declining trend was observed in the efficiency of bio and synthetic chemicals in terms of galling index, number of egg masses and number of females/root system after 14 days.

In case of Cartap and Virtako minimum number of females (105.4, 139.1) were recorded as compared to Azadirachtin and Cure (176.8, 160.7) respectively. Galling index was increased in all the chemicals after 28 days interval as compared to 7 and 14 days. In Cartap number of egg masses was observed (132.5) after 28 days while these were (65.8, 87.4) after 7 and 14 days respectively, similar behavior was observed in all other treatments containing chemicals (Table VI).

Table IV.-Evaluation of Bio chemicals on % mortality of Meloidogyne incognita juveniles after 12, 24, 48 and 72 h.

###% Juvenile mortality at different concentrations a

Treatments###Mean b

###2S###S###S/2###S/4

After 12 h

Azadirachtin###41.20 0.37cd###38.60 0.24d###30.80 0.37f###27.20 0.37gh###34.45 1.31B

Neemix###22.40 0.24ijk###20.80 0.58kl###15.80 0.37m###12.20 0.37n###17.80 0.95E

Neemakill###33.80 0.37e###31.40 0.24f###26.40 0.24h###23.20 0.37ij###28.70 0.96C

Astra###9.20 0.37o###6.00 0.45p###1.00 0.32q###0.60 0.24q###4.20 0.84F

Cure###49.00 0.32a###44.80 0.37b###38.00 0.32d###33.80 0.37e###41.40 1.36A

Spintor###21.60 0.40jkl###20.40 0.24l###15.60 0.40m###10.80 0.37no###17.10 0.99E

Radiant###28.80 0.37g###26.00 0.32h###20.40 0.51l###15.80 0.37m###22.75 1.17D

Timer###35.00 0.32e###31.80 0.37f###26.20 0.37h###23.80 0.37i###29.20 1.03C

Mean c###30.13 1.87A###27.48 1.81B###21.78 1.70C###18.43 1.59D

After 24 h

Azadirachtin###62.93 0.59cd###57.28 0.57d###44.79 0.38h###39.54 0.37ij###51.13 2.16B

Neemix###36.92 0.57kl###32.49 0.58mn###20.60 0.40pq###17.17 0.50rs###26.80 1.89F

Neemakill###51.44 0.36ef###48.41 0.44g###37.73 0.48jkl###30.88 0.19mn###42.11 1.90D

Astra###19.39 0.51pqr###16.57 0.41s###9.72 0.61t###7.30 0.43t###13.25 1.15G

Cure###76.62 0.40a###70.57 0.45b###57.89 0.29d###48.41 0.36g###63.37 2.52A

Spintor###35.92 0.57l###33.30 0.47m###21.82 0.38p###18.58 0.52qrs###27.40 1.70F

Radiant###45.79 0.46h###40.76 0.27i###28.26 0.56o###21.82 0.38p###34.16 2.20E

Timer###53.65 0.25e###51.03 0.55f###38.74 0.47ijk###30.27 0.48no###43.42 2.18C

Mean c###47.83 2.65A###43.80 2.51B###32.44 2.31C###26.75 1.98D

After 48 h

Azadirachtin###73.46 0.62bd###68.16 0.36c###50.20 0.38h###45.10 0.38ijk###59.23 2.73B

Neemix###46.94 0.25i###42.85 0.62k###30.81 0.58o###25.30 0.49p###36.48 2.02F

Neemakill###65.10 0.36de###60.40 0.67f###43.68 0.25jk###35.10 0.57mn###51.07 2.80D

Astra###24.69 0.40p###20.81 0.59q###16.52 0.62r###13.06 0.51s###18.77 1.04G

Cure###82.04 0.66a###74.49 0.33b###62.66 0.44ef###54.07 0.67g###68.32 2.48A

Spintor###46.12 0.53ij###44.08 0.38jk###30.41 0.48o###23.67 0.60p###36.07 2.16F

Radiant###60.41 0.59f###54.89 0.59g###38.98 0.39l###34.49 0.23n###47.19 2.48E

Timer###66.32 0.64cd###61.42 0.49f###47.14 0.46i###37.55 0.37lm###53.11 2.63C

Mean c###58.13 2.73A###53.39 2.56B###40.05 2.13C###33.54 1.93D

After 72 h

Azadirachtin###82.60 0.33bd###75.96 0.37c###62.90 0.35f###54.19 0.39i###68.91 2.54B

Neemix###57.51 0.39gh###51.09 0.30j###42.17 0.54l###35.53 0.60m###46.57 1.94F

Neemakill###68.08 0.46d###63.73 0.37ef###56.26 0.51hi###50.67 0.50j###59.69 1.55D

Astra###28.70 0.52n###24.55 0.60o###17.92 0.33p###14.40 0.42q###21.39 1.30G

Cure###87.56 0.47a###80.51 0.42b###69.53 0.36d###64.14 0.62ef###75.44 2.11A

Spintor###56.26 0.51hi###50.68 0.42j###41.76 0.41l###34.50 0.42m###45.80 1.92F

Radiant###64.14 0.62ef###58.75 0.35g###45.07 0.42k###40.30 0.44l###52.07 2.24E

Timer###69.53 0.41d###65.60 0.38e###56.06 0.54hi###51.50 0.44j###60.67 1.67C

Mean c###64.30 2.72A###58.86 2.62B###48.96 2.40C###43.16 2.31D

Table V.-Mobility of Meloidogyne incognita in soil amended with bio-and synthetic chemicals and their impact on phytotoxicity.

Treatments###No. of J2 recovered###Recovery (%)###% reduction over###Phytotoxicity3

###(after 3 days)###control2

Cartap###95.671 e###19.13 e###60###Nil

Virtako###112.3 d###22.47 d###55###Nil

Abamectin###145.3 c###29.05 c###39###Nil

Azadirachtin###158.3 b###31.65 b###34###Nil

Control###238.1 a###47.63 a###-###Nil

Table VI.-Efficiency of bio-and synthetic chemicals in amended soil at different time intervals.

Treatments###After 7 days###After 14 days###After 28 days

###Galling###No. of###No. of###Galling###No. of###No. of###Galling###No. of###No. of

###index###egg###females/root###index###egg###females/root###index###egg###females/root

###masses###system###masses###system###masses###system

Cartap###2.21 d###65.8 e###77.4 e###2.80 d###87.4 e###105.4 e###4.00 d###132.5 e###163.5 e

Virtako###2.7 c###80.6 d###98.6 d###3.46 c###110.5 d###139.1 d###4.20 cd###158.5 d###184.5 d

Abamectin###3.2 b###92.5 c###107.5 c###3.73 c###132.2 c###160.7 c###4.40 bc###175.3 c###196.6 c

Azadirachtin###3.5 b###105.6 b###128.3 b###4.13 b###155.4 b###176.8 b###4.60 b###192.4 b###220.7 b

Control###5.4 a###222.5 a###237.6 a###5.20 a###216.5 a###235.5 a###5.53 a###225.6 a###243.8 a

LSD###0.36###0.42###0.36###0.31###0.35###0.44###0.31###0.36###0.39

DISCUSSION

In present investigation, nematicidal potential of various bio-and synthetic chemicals was evaluated against M. incognita. All the chemicals under in vitro studies showed different levels of hatching inhibition percentage and J2s mortality after different time duration. Several researchers reported about nematicidal potential of chemicals (Cayrol et al., 1993; Safdar et al., 2012) on hatching inhibition and J2 mortality. Nematicidal activity of different chemicals was attributed due to different mechanisms. Rugby and Cartap as belongs to organophosphate and carbamate group respectively both caused maximum reduction in nematode population under in vitro studies. Their nematicidal activity was due to the inactivation of acetylcholinesterase which is critical enzyme in nervous system of nematodes as nematode locomotion depends upon motor neurons and interneurons that use a neurotransmitter acetylcholine whose activity is stopped acetylcholinesterase (Johnson and Stretton, 1980, 1987).

Another synthetic chemical Virtako caused significant inhibition in hatching of M. incognita, its nematicidal activity attributed to thiamethoxam and chlorantraniliprole. Thiamethoxam caused reduction due to contact and by binding to acetylcholine receptor site, damaging the nervous system, ultimately paralysis and death (Yamamoto, 1999). Chlorantraniliprole belongs to anthranilic diamides having a different mode of action by liberating and exhaustion of calcium from muscle cells which results in impaired muscle cells, paralysis and death (Cordova et al., 2006). Due to its diversified activity reduction of nematode was higher in Virtako. Previously its toxicity was evaluated against insects (Cordova et al., 2006; Dinter et al., 2008). Bio chemicals also reduced nematode population by increasing mortality and inhibition percentages. Cure was the most successful chemical in reducing nematode population.

Its nematicidal potential was due to the blockage of electrical activity in nerve and muscle cells. As it belongs to avermectins that also have a role in human health and crop protection (Dybas et al., 1989). It also binds with gamma-aminobutyric acid that leads to the condition of hyperpolarisation and paralysis (Bloomquist, 1996). Another bio chemical Azadirachtin also caused maximum increase in mortality and decrease in hatching of M. incognita as compared to other chemicals. As a chemical compound, azadirachtin belongs to limonoid group that is secondary metabolite present in neem (Kosma et al., 2011). Its nematicidal activity was also due to presence of alkaloids, quercetin, kaemferol and limnoids (Khan et al., 1974; Alam, 1993).

Bio-and synthetic chemicals which caused significant mortality and hatching inhibition were evaluated against mobility of M. incognita and for phytotoxic effect on tomato. Cartap, Virtako, Cure and Azadirachtin were selected from bio and synthetic chemicals and tested further. Rugby was not selected due to its phytotoxic effects recorded on Poa annua (McClure and Schmitt, 2012). This was also included in the list of those chemicals whose utilization was banned from February 2008 by French agriculture ministry. Due to its promising nematicidal activity it was evaluated as a standard to check the potential of other chemicals. Phytotoxicity is actually the assessment of temporary or long lasting damage to the plant caused by a chemical compound or pesticide (Short, 1981). In the present study none of the chemical was found to be phytotoxic on their recommended doses after two months.

Garcia-Hernandez et al. (2001) reported the phytotoxic effects of chemicals above recommended doses but not at recommended. Phytotoxic effects of different chemicals were evaluated on different crops in different studies (Raymond et al., 2002; Fanigliulo and Sacchetti, 2008). Recovery of juveniles was assessed after three days from soil in Cartap, Virtako, Cure and Azadirachtin. As first two days of contact with the host are crucial for the penetration of nematodes (Nwauzor and Fawole, 1992), so minimum recovery of juveniles from the chemicals indicates a population reduction at a critical period. In our results recovery percentage was decreased in all the chemicals tested as similar with the findings of others (Lei et al., 2010; Saad et al., 2011; Moosavi, 2012).

Cartap, Virtako Cure and Azadirachtin showed a decreasing trend in efficacy with the increase in time interval. Maximum population of nematodes was observed after 28 days interval in all chemicals. Chemicals were applied in soil as a single dose before transplantation of tomato plants. A direct relation was observed between efficacy of chemicals and time by (Deliopoulos et al., 2010), as with the passage of time, the efficacy of chemicals decreased with the increase in nematode population. Degradation of chemicals from the soil was attributed in three ways viz., leaching, chemical and biological degradation (Dunn and Noling, 2003). Susceptibility of chemicals varied towards degradation, organophosphate and carbamates were found to be more susceptible to biological degradation (Laveglia and Dahm, 1977). Biological include microbial degradation caused by bacteria (Cain and Head, 1991), fungi (Jones, 1976) and algae (Zuckerman et al., 1970).

Galling index, number of females and number of egg masses were higher at third time interval due to decrease in efficacy of chemicals. So efficiency and time are negatively correlated. It may be concluded from these findings that bio and synthetic chemicals have nematiticidal potential against M. incognita through diversified mechanisms.

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