Research Journal of Agricultural Sciences 2012, 3(2): 500-505

A New Carboxynilide Group Fungicide against Paddy Sheath Blight

M K Prasanna Kumar, D K Sidde Gowda, K T Pandurange Gowda and K Vishwanath
Zonal Agricultural Research Station, VC Farm, (UAS, GKVK, Bangalore) Mandya 571 504, Karnataka, India
e-mail: babu_prasanna@rediffmail.com

ABSTRACT
Thifluzamide, a new fungicide of carboxynilide group was tested for the efficacy against rice sheath blight during 2005, 2006 and 2009. Among, different test concentrations, Thifluzamide 24% SC @ 90 and 110g ai/ha was found effective in reducing the disease severity and increasing the yield. Carboxinililde group fungicide was found effective both as preventive and curative activity without any symptoms of phytotoxicity such as, vein clearing, necrosis, epinasty, hyponasty etc on rice plants. The cost of managing the disease by two applications is covered by the extra yield achieved due to suppression of the disease, where it was found significantly superior to the standards. Residues of Thifluzamide and its metabolite in grain, straw and soil were found below the quantifiable limit at the test doses. Key words: Thifluzamide, Carboxynilide, Sheath blight, Rhizoctonia solani Sheath blight, Rhizoctonia solani Kuhn (Teleomorph: Thanatophorus cucumeris (Fran) Donk. has assumed the status of economically important disease in paddy, and has become a major constraint to rice production during the last two decades (Kobayashi et al. 1997). The disease is prevalent in all rice growing countries especially in South and Southeast Asia and causes significant yield losses (Premalatha 1990, Roy 1993, Rajan and Naidu 1986, Ramakrishnan 1971). Yield loss up to 50% has been recorded in susceptible rice cultivars when all the leaf sheaths and leaf blades were infected (Lee and Rush 1983). The emergence of R. solani as economically important rice pathogen has been attributed to the intensification of ricecropping systems with the development of new short-stature, high-tillering, high-yielding varieties, high plant densities and increased level of fertilizers and other inputs (Chahal et al. 2003, Siddiq 1999) and these factors promote disease spread by providing favourable micro climatic condition due to dense leaf canopy with an increase leaf-to-leaf and leafto-sheath contact (Savary et al. 1995). Both seedlings and adult plants are equally affected but loss is much more when the disease appears in seedlings. The older plants are attacked in flooded conditions and swampy rice fields (Dodman and Flentje 1970, Kannaiyan 1987, Shimamoto 1995). The infection and spread of disease before the flag leaf stage revealed 20% grain loss. Further, a strong relationship between the severity of symptom and yield reduction was reported among cultivars (Marchetti and Bollchi 1991). S Sheath blight can be effectively controlled with the application of systemic fungicides. However, bio-fungicides and resistant varieties are the other options of control management but, are not at par with chemical control. These fungicides are very popular and are at the peak of its usage which may lead to reduced residual period and efficacy due to increased virulence of R. solani. Thifluzamide is a member of the carboxamide class of fungicides which is single-site inhibitors of the succinate ubiquinone reductase or succinate dehydrigenase (Sdh) complex in the respiratory chain (FRAC 2007) interfere with fungal respiration via their inhibitory effect on succinate dehydrogenase within the tricarboxylic acid cycle. (OReilly 1995). This compound was reported effective against Basidiomycete fungi in particular along with efficacy on some Ascomycetes and Rhizoctonia solani. This molecule is registered for use in rice, turf, potatoes, coffee and strawberries in Brazil, Mexico, Colombia, Venezuela, Japan, Korea, China and Vietnam. Since, no fungicide has been registered with this unique modes of action for the control of sheath blight of paddy, Thifluzamide can fit into resistance management system by integrating in spray schedules in potential rice growing tracts. Hence, the present study was conducted to characterize Thifluzamide 24 % SC under field conditions against Rhizoctonia solani.

MATERIALS AND METHODS

The experiment was laid out in red sandy loam soils with unique soil properties (pH 5.9 to 6.2, 0.30% organic matter, 25.0kg ha-1 of available N, 24.23kg ha-1 of P2O5 and 215.55kg ha-1 of available K2O). The plots were flooded with water and ploughed until any soil aggregates were broken up. Excess water was drained out and the field was partitioned into 7 blocks. The efficacy of the fungicide against sheath blight was investigated under field conditions at Zonal Agricultural Research Station, Mandya, Karnataka during Kharif 2005, 2006 and 2009. The experiment was laid out in a randomized block design with seven treatments replicated four times. Uniform plant population was maintained for each plot, with spacing of 20 10cm between rows and plants, respectively. The experimental site was selected based on intensive cultivation of rice where, sheath blight disease a major yield constraints to the

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Kumar et al. farmers. Treatments consisted of a rice plant (cv. IR-64) sprayed with different doses of Thifluzamide 24% SC (75. 90, 110g ai/ha) along with recommended national standard fungicides. Two sprays of chemical with desired concentration were given on 50th, 65th and 80th days after planting (DAP). The first spray was applied as prophylactic spray at jointing stage and second spray was applied as curative sprays when the third to fourth leaf from the top shows symptoms. For prophylactic trails fungicides were sprayed before disease appearance and curative trials were taken at 37.5% disease incidence. The causal organism was artificially built up at tillering stage using the fresh sheath blight infected bits on the rice seedlings by placing between the tillers just above the water line at 38 DAP. The sclertotial bodies produced by the isolated cultures of R. solani were also used for inoculation at the sheath region near water level. A popular sheath blight susceptible variety, IR-64 was used as check in all three years. The disease assessment was carried out at 14 days after fungicide application. In each trial two observations were recorded. The first observation was made soon after prophylactic spray and second after curative spray. The disease severities were subjected for 0-9 scale using standard evaluation system for rice developed by International Rice Research Institute (SES 2002). Further, the disease severity was calculated using the formula. Subsequently, the data on disease severity and yield parameters were collected and were subjected to appropriate statistical analysis: 0 - No infection 1 - Vertical spread of the lesions up to 20% of plant height 3 - Vertical spread of the lesions 21 - 30% of plant height 5 - Vertical spread of the lesions 31 - 45% of plant height 7 - Vertical spread of the lesions 46 - 65% of plant height 9 - Vertical spread of the lesions > 65%of plant height
Disease severity % = Sum of disease grades No. of infected tillers/ hill Total No. of Tillers Max. disease grades No. 100 of tillers assessed

In each treatment, ten plants selected at random. The total number of leaves and those showing phytotoxicity parameters viz leaf injury, wilting, vein clearing, necrosis, epinasty hyponasty were recorded before spray, and at 3, 7, and 15 days after spraying. Further, the percentage data were subjected for 0-10 score (Fiebig 2002). The studies on residues were carried out during 2005 at two doses (90 and 180g ai/ha) with an untreated control. The residue analysis was done at International Institute of Biotechnology and Toxicology (IIBAT), Chennai which was sponsored by Nagarjuna Agrichemicals Limited. Five hundred grams of test samples (straw, grain and soil) were collected and sent to the IIBAT laboratory for residue analysis. The extraction and column clean-up were done as per the protocol developed by IIBAT. Shimadzu High Performance Liquid Chromatograph system equipped with LC-10 ATvp pump and SPD-10Vvp UV-VIS detector connected to CBM-101 module using CLASS-LC-10 software was used for Thifluzamide analysis. The reference compound Thifluzamide, Metabolite-1 (MON 24000-CH2OH) and Metabolite-2 (MON 24000-COOH) supplied by Nagarjuna Chemicals with a purity of 99.95%, 94.59 % and 100%, respectively.

Table 1 Efficacy of new fungicide Thifluzamide 24% SC as prophylactic spray against R. solani under field conditions 2005 2006 2009 Dose g Treatments Disease % control Disease % control Disease % control over a. i/ha severity over untreated severity over untreated severity Untreated Thifluzamide 24% SC 60 18.67 (25.57) 60.28 22.33(28.19) 64.17 25.67 (30.41) 56.98 Thifluzamide 24% SC 75 14.67 (21.55) 68.79 23.00 (28.64) 63.10 24.67 (29.76) 58.66 Thifluzamide 24% SC 90 14.00 (21.97) 70.21 20.67 (26.97) 66.84 22.33 (28.20) 62.57 Thifluzamide 24% SC 110 12.00 (20.22) 74.47 19.33 (26.03) 68.98 21.33 (27.50) 64.25 Validamycin 3% L (St.I) 37.5 25.33 (30.16) 46.10 40.00 (39.22) 35.83 28.00 (31.93) 53.07 Hexaconazole 5 EC (St.II) 50 22.67 (28.38) 51.77 32.67 (34.84) 47.59 25.33 (30.21) 57.54 Untreated control 47.00 (43.26) 0.00 62.33 (52.17) 0.00 59.67 (50.58) 0.00 SE m 1.40 1.33 0.74 CD @ p=0.05 4.27 3.45 2.26 CV % 8.76 5.76 3.9
Figures in parenthesis are Arcsine transformed; St.I: Standard-I, St.II: Standard-II

For calibrating the limit of detection, different concentrations of Thifluzamide and its metabolites (5.0 -0.5 ppm) were prepared in residue grade methanol by diluting the stock solution which, was used for injecting to HPLC along with standards. A calibration curve was plotted for concentration of the standard injected against area observed. The lowest limit of detection (LOD) was established as 0.05ppm for Thifluzamide and its metabolites. Residual content was calculated with the formula: Residue content (g/g) = ABC DE

Where, A-Peak area of samples (V-sec) B-Volume of sample (ml) C-Concentration of the standard (g/ml) D- Peak area of standard (V-sec) E- Weight of the sample (g)

RESULTS AND DISCUSSION

Sheath blight (R. solani) infection dominated during all the years. The infection was severe in 2006 and 2009 when it was promoted by high relative humidity and high

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Carboxynilide Group Fungicide against Paddy Sheath Blight temperature. During the study, disease severity was 47.0, 62.33 and 59.67% which helped in characterization of the new fungicide. The increased disease pressure in the present study was attributed to natural incidence and artificial inoculation of the pathogen. In all the three field trials Thifluzamide 24% SC provided excellent control of sheath blight with exceptional long lasting activity against sheath blight.

Table 2 Impact of new fungicide Thifluzamide 24% SC as curative spray rice sheath blight disease and yield 2005 2006 2009 Dose Treatments g a. Disease % control Yield Disease % control over Yield Disease % control Yield i/ha severity over untreated kg/ha Severity untreated kg/ha severity over untreated kg/ha 14.50 18.75 17.44 Thifluzamide 24% SC 60 53.23 3967 53.13 4451 58.54 4642 (21.94) (25.62) (24.67) 14.50 11.75 10.88 Thifluzamide 24% SC 75 53.23 4094 70.63 5002 74.15 4870 (21.77) (19.98) (19.24) 11.00 10.00 9.13 Thifluzamide 24% SC 90 64.52 4105 75.00 4843 78.31 5043 (19.27) (18.39) (17.57) 8.50 9.00 8.31 Thifluzamide 24% SC 110 72.58 4138 77.50 5379 80.24 5212 (16.87) (17.43) (16.75) 10.50 10.00 11.63 Validamycin 3% L (St.I) 37.5 66.13 3797 75.00 5010 72.36 4997 (18.71) (18.43) (19.91) Hexacomazole 5 EC 10.50 10.00 11.94 50 66.13 3627 75.00 4740 71.62 4970 (ST.II) (18.71) (18.43) (20.16) 31.00 40.00 42.06 Untreated control 0.00 3145 0.00 4179 0.00 4342 (28.68) (39.21) (40.42) SE m 1.5 124.43 0.67 123.9 0.61 100.1 CD @ p=0.05 4.41 369.55 1.98 367.8 1.80 308.6 CV % 13.78 6.48 5.93 5.2 5.37 3.6
Figures in parenthesis are Arcsine transformed; St.I: Standard-I, St.II: Standard-II

Table 3 Cost Benefit ratio of Thifluzamide 24% SC application against sheath blight of paddy Treatments Dose g Grain yield Total returns B:C Marginal Total cost Total returns Marginal a i/ha Kg/ha (Rs.) ratio cost (Rs.) (Rs.) (Rs.) returns (Rs.) Thifluzamide 24% SC 60 3967 43637 1.897 1000 23000 29679.000 9042 Thifluzamide 24% SC 75 4094 45034 1.937 1248 23248 32225.000 10439 Thifluzamide 24% SC 90 4105 45155 1.921 1500 23500 32215.000 10560 Thifluzamide 24% SC 110 4138 45518 1.910 1832 23832 32609.000 10923 Validamycin 3% L (St.I) 37.5 3797 41767 1.816 1000 23000 25939.000 7172 Hexaconazole 5 EC (ST.II) 50 3627 39897 1.750 800 22800 22399.000 5302 Untreated control 3145 34595 1.573 22000 12595.000 0.00
B:C ratio- benefit cost ratio; Total returns = Cost of grain @ Rs. 10/kg and straw Rs. 1/kg

Thifluzamide 24% SC @ 90 and 110g ai/ha was found highly effective as prophylactic spray (14.0 and 12.0%) with per cent disease control over untreated of 70.21 and 74.47, respectively during kharif 2005. The untreated plots recorded 47.0% disease severity, which confirms the disease pressure during the experimentation. The standard fungicides Validamycin 3% L @ 37.5g ai/ha and Hexaconazole 5 EC @ 50g ai/ha were found inferior even with Thifluzamide 24% SC @ 60g ai/ha. Similarly, during kharif 2006 and 2009, the test fungicide @ 90 and 110g ai/ha recorded a disease control 66.84 and 68.98, 62.57 and 64.25%, respectively over untreated (Table 1). The disease in untreated plots was higher compared to kharif 2005 which recorded 62.33 and 59.67%, respectively in 2006 and 2009. The mean results proved the fact that Thifluzamide can give good control of the disease as protective fungicide. As expected, the fungicide sprayed after appearance of the disease symptoms exhibited good control of the sheath

blight. Consistent results were observed in all the three trials with application of thiflzamide @ 90 and 110g a.i./ha recording 11.0 and 8.5, 10.0 and 9.0, 9.13 and 8.31 during 2005, 2006 and 2009, respectively. However, the standard fungicides were also performed better as curative sprays. The intensity of fungicide caused significant differentiation in yields of tested varieties by treatment variants (Table 2). Two applications of Thifluzamide 24% SC @ 90 and 110g ai/ha revealed lower disease intensity with higher yields in all the test years (Table 2). Comparing the economic benefits of the extra yield produced by application fungicide, in spite of recording lower disease incidence it proved its effectiveness in maximizing and stabilizing the net yield (4105 and 4138 kg/ha at 90 and 110g ai/ha). Taking into account, the cost of cultivation of transplanted paddy ~Rs. 22,000/ acre and grain cost Rs. 1000/quintal and straw cost Rs. 1000/tonnes. The total cost of controlling sheath blight using

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Kumar et al. Thifluzamide @ 90 and 110g a i/ha is ~1500 and ~1832 which has given the marginal return of Rs. 10560 and 10923, respectively (Table 3). Thifluzamide was tested for its phytotoxic effect on rice at four different doses (60, 75, 90 and 110g a i/ha). All the test doses did not cause any phytotoxic effect on rice viz leaf injury, wilting, vein clearing, necrosis, epinasty and hyponasty when applied as foliar spray after 3, 7 and 15 days. Analysis of paddy grains, straw and soil collected from Mandya showed residues of Thifluzamide and its metabolite below the quantifiable limit at the test doses. The residue of the test chemical in paddy grains while similar results were obtained in paddy straw and soil (Table 4). Calibration of Thifluzamide and its metabolites were done by injecting the sample concentration of 5.0 -0.05ppm and response in area was plotted V-sec and the data is presented in table-5. Thifluzamide @ 90g a i/ha in paddy grains were not found as per the standard MRL values of WHO/FAO. While, under rule 65 of the prevention of Food Adulteration Act 1965 of Government of India is 0.5 ppm. The test chemical did not show any traces in paddy grains and the proposed PHI was found to be 28 days.

Table 4 Residues of Thifluzamide and its metabolites in paddy grain Peak area (V-sec) Results (g/g) Volume Weight of Treatment Thifluzamide of sample Thifluzamide MB-1 MB-2 sample (g) MB-1 (ml) residue residue T0 R1 ND ND ND 50 3 ND ND T0 R2 ND ND ND 50 3 ND ND T0 R3 ND ND ND 50 3 ND ND T1 R1 ND ND ND 50 3 ND ND T1 R2 ND ND ND 50 3 ND ND T1 R3 ND ND ND 50 3 ND ND T2 R1 ND ND ND 50 3 ND ND T2 R2 ND ND ND 50 3 ND ND T2 R3 ND ND ND 50 3 ND ND

Average residue (g/g) Thifluzamide MB-2 MB-1 MB-2 residue ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND

Standard concentration of Thifluzamide : 0.05ppm-1126 (V-sec); Standard concentration of metabolite-1 (MB-1): 0.05 ppm-794 (V-sec) Standard concentration of metabolilte-2 (MB-2) : 0.05 ppm-1283 (V-sec); ND- Not detectable Table 5 Calibration details of Thifluzamide and its metabolites

Injected Response in area (V-sec) concentration(ppm) (Thifluzamide and Thifluzamide Metabolite-1 Metabolite-2 Metabolites) 5.0 104853 57182 120887 2.5 51813 28776 59072 1.0 20847 11409 23757 0.1 2286 1383 2538 0.05 1204 732 1316
Correlation coefficient: 0.999

Thifluzamide and its metabolites peak time report obtained through chromatography indicated that there is an increase in peak time with the decrease in area of response which is directly related with metabolite/chemical concentration. The peak time ranged between 3.624-3.782 for Metabolite I, 9.543-10.121 for Metabolite II and 11.83512.294 for Thifluzamide @ 5.0-0.05 g/ml concentration, respectively. Intensive rice cultivation with no crop rotation and apt seasonal weather factors has increased the occurrence of sheath blight. Yield reduction due to disease infections can avoid by chemical control measures. The chemical control should be need based on by looking into the severity of the disease, variety, and other abiotic factors. In future, the inclination will most likely be towards management of disease under integrated plant protection methods whereby the fungicide dose and time of application are calculated based on the resistance level of the variety, the prevailing abiotic factors of the rice ecosystem (Soovli and Koppel

2009). In UK, it has been found that fungicide treatment is effective when the infection level is visually more than 5% of leaf area (Cook et al. 1999) for instance, lowers intensity infection and yield loss is found to be smaller than the cost of fungicidal application. In a rice ecosystem, in each season, more than one disease is observed and hence new fungicidal groups like oryzastrobin Qol are gaining importance as they are broadspectrum fungicides providing effective control against rice sheath blight and blast (Stammler et al. 2007). However, the broad spectrum fungicides may not give sufficient protection when the disease severity is very high. At present the ruling chemicals viz Hexaconazole, Propiconazole, Validamycin, Carbendazim which are extensively used for the management of sheath blight disease (Chien and Chu 1973, Wakae and Matsura 1975, Viswanathan and Mariappan 1980a, b, Das and Mishra 1990, Van Eechout et al. 1991). Further, laboratory studies on two isolates of R. solani from rice and potato showed significant variation in response to different concentrations of fungicides (carbendazim, carboxin, pencycuron, Propiconazole and Validamycin) (Thind and Aggarwal 2005). Lore et al. (2005), Biswas (2002) evaluated and reported effectiveness of new fungicide Pencycuron (Moncern 250 EC) against rice sheath blight in Punjab and West Bengal. Our investigations also revealed the effectiveness of Thifluzamide 24% SC against R. solani causing sheath blight in paddy. To support our results the data of 2005 trials were obtained from 16 locations which are part of All India Coordinated Research Project, Directorate of Rice Research, Hyderabad including

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Carboxynilide Group Fungicide against Paddy Sheath Blight Mandya centre and locations across India were pooled (Fig 1). Thifluzamide @ 90 and 110g a i/ha was found highly effective at all locations tested and strongly supports our data conducted for three years. Profenophos treated plots. While comparing with standards Thifluzamide was superior as prophylactic spray, but with the same chemical as curative performed slightly better over prophylactic spray (Fig 2). The difference in the efficacy as prophylactic and curative is very less; Thifluzamide can be applied as curative treatment against sheath blight when the third to fourth leaf from the top shows symptoms and extent of damage caused on a particular variety. In addition to its efficacy and increasing yield at higher dosage, Thifluzamide was found to have phytotonic effect with appreciable green with luxuriant growth of rice plants. The best time to apply chemicals is at the jointing stage, during which time the percentage tiller infected was highly correlated with sheath blight at wax ripeness stage: percentage yield loss depended on disease index at wax ripeness (CPC 2005). Prophylactic application targeted at this stage proved as the right stage of application against initial infection by Rhizoctonia solani. Singh and Singh (2009) observed that the application of Validamycin at tillering stage of rice crop seems to have positive effect in enhancing the efficacy. Fungicides having least residues in food, feed and soil are being encouraged in present day agriculture. Thifluzamide at higher doses (90 and 180g a i/ha) did not show any residues in grains, straw and soil and proposed PHI being 28 days which is found to be less than acceptable limit. Yi and Lu (2006) found that probenazole residues in soil, brown rice, and water were undetectable at levels of recommended and doubled dosage with an interval of 63 days. This study signifies that fungicide Thifluzamide at 90 and 180g a i/ha recommended for management of paddy sheath blight, which could be considered as safe to paddy plants, human beings and soil environment. These would contribute to provide the scientific basis of using this fungicide. Thifluzamide 24 % SC a new group fungicide was found effective both as preventive and curative activity without any symptoms of phytotoxicity, reducing the disease severity and increasing the yield. The cost of managing the disease by two applications is covered by the extra yield achieved due to suppression of the disease, where it was found significantly superior to the standards. Residues of Thifluzamide and its metabolite in grain, straw and soil were found below the quantifiable limit at the test doses.

Fig 1 Multi-location traits across the country on efficacy of thifluzamide against sheath blight

Fig 2 Comparison of prophylactic and curative sprays with thifluzamide

Thus the present studies reveal that Thifluzamide 24% SC @ 90 and 110g a i/ha were found highly effective in controlling paddy sheath blight. The fungicide applied both as preventative and curative measure performed significantly superior over standards (Hexaconazole and Validamycin). Devi et al. (1987), found the best curative and protective effect of Validamycin against sheath blight of rice whereas, maximum grain yield was obtained from

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