Short Communication
Azoxystrobin Activity on Rhizoctonia solani and its Efficacy Against Rice Sheath Blight Subramanian Sundravadana and Devadason Alice, Department of Plant Pathology, Sasthan Kuttalam, Department of Agricultural Entomology, and Ramasami Samiyappan, Center for Plant Protection Studies; Tamil Nadu Agriculture University, Coimbatore - 641 003, India _____________________________________________________________________________ ABSTRACT Sundravadana, S., Alice, D., Kuttalam, S., and Samiyappan, R. 2007. Azoxystrobin activity on Rhizoctonia solani and its efficacy against rice sheath blight. Tunisian Journal of Plant Protection 2: 79-84. Sheath blight, caused by Rhizoctonia solani is one of the major diseases affecting rice crop. We investigated the effect of azoxystrobin on R. solani growth (in vitro) and its efficacy against rice sheath blight, under field conditions. The results revealed that azoxystrobin at 1, 2, and 4 ppm, completely inhibited mycelial growth of R. solani. Field study showed that foliar spray of azoxystrobin at 125, 250, and 500 g/ha significantly suppressed (> 64%) the development of sheath blight and enhanced yield level (> 60%). For controlling sheath blight disease, the optimum rate of azoxystrobin was 125 g/ha. Our results showed also that there was no residue in rice matrices at harvest time and no phytotoxicity at the highest concentration of azoxystrobin (500 g/ha). Key words: Azoxystrobin, chemical control, mycelial growth, residue, Rhizoctonia solani, rice, sheath blight
_____________________________________________________________________________ Sheath blight is the most damaging disease of rice. It has the potential to cause severe economic loss. Heavily infected plants produced poorly filled grains (9). The disease is caused by Rhizoctonia solani. Under favorable conditions, infected plants build up a huge population of fungal sclerotia in the soil and rice cropping becomes uneconomic. Fungicides are commonly used in fields where sheath blight is a limiting
production factor. The strobilurin fungicides were introduced recently against a broad range of pathogens (Phytophthora capsici, Alternaria alternata, Ascochyta rabiei, Puccinia hemerocallidis and Phomopsis sp.) (11, 1, 3, 6, 4). These fungicides reduced disease development and subsequent inoculum production and increased yield. The objectives of this study were to evaluate in vitro activity of azoxystrobin against mycelial growth of R. solani, its efficacy on sheath blight incidence under field conditions, and its harvest time residues. R. solani was isolated from infected rice sheath. Then, the isolate was incubated on potato dextrose agar (PDA) medium at 28
Corresponding author: S. sundravadana
[email protected]
Accepted for publication 29 November 2007
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± 2°C. The fungicide used in the experiments is a concentrate suspension of Azoxystrobin (Amistar). A 6 mm diameter agar disk from the margin of an actively growing culture of R. solani was incubated in Petri dishes containing PDA amended with azoxystrobin at concentrations of 0, 0.25, 0.5, 1, 2, and 4 ppm at 28 ± 2°C, 70% RH for 3 days. Inhibition of the radial growth was expressed in percentage, as indicated in the following fomula: Mi = (Mc-Mt) / Mc X 100, with Mi = Percent of mycelial growth inhibition, Mc = Control colony diameter, and Mt = Colony diameter in medium amended with azoxystrobin. Sclerotia of 7 day-culture of R. solani were dipped in 100 ml beaker, containing different concentrations (0, 0.25, 0.5, 1, 2, and 4 ppm) of azoxystrobin in an aqueous solution, then seeded separately at the center of each Petri plate containing 2% RLH (%)
=
agar and incubated at 28 ± 2°C, 70% RH. Radial growth of mycelia was measured 5 days later. Field experiment was laid out with rice (ADT43) under randomized block design, with three replications in 5m x 4m plots. The efficacy of azoxystrobin at six concentrations (0, 31.25, 62.50, 125, 250 and 500 g/ha) was evaluated and compared to carbendazim (500 g/ha). Three sprays were applied, since the initial appearance of sheath blight and following a 15 day interval. Sheath blight incidence was evaluated with the Relative Lesion Height (RLH) (8). Therefore, Percent Disease Index (PDI) was calculated as indicated below (12). The phytotoxic effect of azoxystrobin was recorded on plants after each spray in the field trials. The grain yield was recorded from individual treatment and expressed in per hectare, as indicated below:
Lesion height (cm) Plant height (cm)
x 100
Sum of numerical rating PDI
=
Maximum disease grade x
Total number of plants observed
At harvest time, azoxystrobin residues in rice matrices (grain, straw, husk and bran) were determined using high performance liquid chromatography (HPLC). The rice matrices were extracted with acetonitrile: water (90:10 v/v) and partitioned with an equivalent volume (100 ml) of dichloromethane plus ½ equivalent volume of 5% NaCl solution in a separating funnel for about 1 min. The sample extract was cleaned up with a cartridge (Florisil column clean up), the cartridge was conditioned with 5 ml of hexane: dichloromethane (50:50 v/v) before sample loading (250 ml) and washing with 5 ml of dichloromethane and ethyl acetate (90:10 v/v). Azoxystrobin Tunisian Journal of Plant Protection
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was eluted from the cartridge using 5 ml of dichloromethane and ethyl acetate (70:30 v/v). The elute was evaporated (on a rotary evaporator at 40°C) to near dryness and redissolved the residue in a 10 ml of acetonitrile and an aliquot (20 µl) was injected into the HPLC. Azoxystrobin was separated on a Lichro sphere ® 60-RP column using a mobile phase of acetonitrile: water (80:20 v/v) at a flow rate of 1 ml/min and at a wavelength of 255 ηm. A recovery assay was carried out using rice matrices plus 0.05, 0.1 or 0.2 ppm of azoxystrobin. Azoxystrobin at three concentrations (1, 2 and 4 ppm) effectively inhibited in vitro mycelial growth of R. solani. 80
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Whereas, at 0.25 and 0.5 ppm mycelial growth inhibition over control ranges from 33 to 39% from mycelia and 66 to 81% from sclerotia (Table 1). Similarly, azoxystrobin inhibited the spore germination and mycelial growth of
Alternaria alternata and Cladosporium macrocarpum on wheat and Ascochyta rabiei on chickpea (1, 3). Strobilurins have been shown to inhibit the germination and pre-penetration growth of several plant pathogenic fungi (10).
Table 1. Effect of azoxystrobin on the in vitro mycelial growth of R. solani Mycelial growth Azoxystrobin concentration (ppm)
From Inhibition sclerotia over control (mm) (%) 0.25 60.23c 33.08 25.21c 66.00 0.5 54.67b 39.26 13.90b 81.26 1.0 0.00a 100.00 0.00a 100.00 2.0 0.00a 100.00 0.00a 100.00 4.0 0.00a 100.00 0.00a 100.00 Control 90.00d 84.20d * In a column, means followed by a common letter are not significantly different at the 5% level by DMRT From mycelia (mm)
Inhibition over control (%)
Table 2. Efficacy of azoxystrobin on sheath blight incidence and grain yield of rice
Fungicide dose (g/ha) Azoxystrobin 31.25 Azoxystrobin 62.5 Azoxystrobin 125 Azoxystrobin 250 Azoxystrobin 500 Carbendazim 500
Percent Diseases Index (%) Time after transplanting (days) 30 45 60 75 2.20a 14.23d 32.81d 42.21d (7.95) (22.06) (34.85) (40.44) 2.20a 12.36c 29.40c 38.45c (7.95) (20.46) (32.71) (38.19) 2.20a 7.82a 15.63a 23.53a (7.95) (15.98) (22.88) (28.51) 2.20a 7.35a 15.36a 23.12a (7.95) (15.44) (22.66) (28.21) 2.21a 7.35a 15.00a 23.10a (7.75) (15.44) (22.35) (28.19) 2.22a (8.55)
9.23b (17.48)
25.35b (30.36)
33.25b (34.98)
Disease reduction over control (%)
Yield (q/ha)
8.84
40.42
19.30
45.92
64.98
62.49
69.23
62.52
69.23
62.58
36.20
55.32
2.20a 21.44e 47.71e 67.40e 33.21 (8.37) (27.53) (43.68) (55.54) ∗ In a column, means followed by a common letter are not significantly different at the 5% level by DMRT * Values in parentheses are arcsine–transformed values Control
rice yield (Table 2). Since there were no significant differences among 125, 250 and 500 g/ha rates in term of decline sheath blight incidence and yield response, we consider 125 g /ha as the optimum rate for sheath blight control. Besides, all azoxystrobin concentrations did not produce any phytotoxic effect. Therefore,
In control plots, sheath blight incidence increased progressively from PDI 2.20 (30 days) to PDI 67.40 (75 days). Azoxystrobin at 31.25 and 62.5 g/ha slightly suppressed the sheath blight development, whereas at 125, 250 and 500 g/ha, it reduced more than 64% disease incidence and increased more than 60% Tunisian Journal of Plant Protection
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foliar application of azoxystrobin effectively control the diseases incidence like the agents of southern stem rot (Sclerotium rolfsii), head blight (Fusarium graminearum), and leather rot (Phytophthora cactorum) (14, 5, 13). Azoxystrobin means recovery from three fortified (0.05, 0.1 and 0.2 ppm) rice matrices (grain, straw, husk and bran) were 86.96%, 84.71% and 82.36%, respectively (Table 3). Therefore, under the specified conditions no interfering peaks were observed on the chromatogram of the samples. The instrument did not detect residues of azoxystrobin even at the highest applied concentration. Then, we
can suppose that the residues of azoxystrobin in rice matrices were below the limit of detection of the instrument (0.004 µg/g of sample). Considering the U.S. Environmental Protection Agency (EPA), the maximum residue limits (MRLs) for azoxystrobin in rice grain, straw and hulls are, respectively 5, 12 and 20 ppm (7). Therefore, it seems that azoxystrobin in our trials was rapidly metabolized in rice plants. Chen et al. (2) obtained a similar result in Chinese cabbage. Hence, foliar application of azoxystrobin to control sheath blight is very unlikely to cause acute and chronic health risk.
Table 3. Recovery percentage of azoxystrobin in fortified rice samples Azoxystrobin recovery (%) Rice matrice Grains Straw Bran & Husk
0.05 ppm
0.1 ppm
0.2 ppm
80.12 85.12 80.00
82.86 81.32 84.90
97.91 87.69 82.18
Mean recovery (%) 86.96 84.71 82.36
Recovery factor (RF) 1.15 1.18 1.21
* Determinability of the instrument = 0.004 µg/g of sample.
____________________________________________________________________________ RESUME Sundravadana S., Alice D., Kuttalam S., et Samiyappan R. 2007. Activité de l’azoxystrobine sur Rhizoctonia solani et son efficacité contre la brûlure de la gaine foliaire du riz. Tunisian Journal of Plant Protection 2: 79-84. La brûlure de la gaine foliaire, causée par Rhizoctonia solani, est l’une des principales maladies du riz. Nous avons étudié l’effet de l’azoxystrobine sur la croissance in vitro de R. solani et son efficacité contre cette maladie en plein champ. Les résultats ont révélé que l’azoxystrobine à 1, 2 et 4 ppm, inhibe complètement la croissance mycélienne de R. solani. L’étude au champ a montré que la pulvérisation foliaire de l’azoxystrobine à 125, 250 et 500 g/ha supprime significativement (> 64%) le développement de la brûlure de la gaine foliaire et augmente le rendement (> 60%). Pour contrôler cette maladie, la dose optimale d’azoxystrobine était de 125 g/ha. Nos résultats ont indiqué aussi qu’il n’y avait pas de résidus dans les plantes du riz au moment de la récolte et pas de phytotoxicité avec la plus haute concentration d’azoxystrobine (500 g/ha). Mots clés : Azoxystrobine, brûlure de la gaine foliaire, croissance mycélienne, lutte chimique, résidus, Rhizoctonia solani, riz
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ﻤﻠﺨﺹ ﺘﺄﺜﻴﺭ ﺍﻷﺯﻭﻜﺴﻴﺴﺘﺭﻭﺒﻴﻥ.2007 . ﺴﻭﺒﺭﺍﻤﺎﻨﻴﺎﻥ ﻭﺩﺍﻓﺎﺩﺍﺼﻭﻥ ﺃﻝﻴﺱ ﻭﺴﺎﺴﺜﺎﻥ ﻜﻭﺘﺎﻻﻡ ﻭﺭﺍﻤﺎﺴﺎﻤﻲ ﺴﺎﻤﻴﻴﺎﺒﺎﻥ،ﺼﻨﺩﺭﺍﻓﺎﺩﺍﻨﺎ Tunisian Journal of Plant Protection . ﻭﻨﺠﺎﻋﺘﻪ ﻀﺩ ﻝﻔﺤﺔ ﺍﻝﻐﻤﺩ ﺍﻝﻭﺭﻗﻲ ﻝﻸﺭﺯRhizoctonia solani ﻋﻠﻰ ﺍﻝﻔﻁﺭ 2: 79-84.
ﺘﻤﺕ ﺩﺭﺍﺴﺔ ﺘﺄﺜﻴﺭ. ﻤﻥ ﺃﻫﻡ ﺍﻷﻤﺭﺍﺽ ﺍﻝﺘﻲ ﺘﺼﻴﺏ ﺍﻷﺭﺯRhizoctonia solani ﺩ ﻝﻔﺤﺔ ﺍﻝﻐﻤﺩ ﺍﻝﻭﺭﻗﻲ ﺍﻝﺘﻲ ﻴﺤﺩﺜﻬﺎ ﺍﻝﻔﻁﺭ ﺘﻌ ﺃﻅﻬﺭﺕ ﺍﻝﻨﺘﺎﺌﺞ ﺃﻥ. ﻓﻲ ﺍﻝﺒﻠﻭﺭ ﻭﻨﺠﺎﻋﺘﻪ ﻀﺩ ﻫﺫﺍ ﺍﻝﻤﺭﺽ ﻓﻲ ﺍﻝﺤﻘلR. solani ﺍﻷﺯﻭﻜﺴﻴﺴﺘﺭﻭﺒﻴﻥ ﻓﻲ ﻨﻤﻭ ﺍﻝﻔﻁﺭ ﻭﺒﻴﻨﺕ ﺍﻝﺩﺭﺍﺴﺔ ﻓﻲ ﺍﻝﺤﻘل ﺃﻥ ﺍﻝﺭﺵ. ﺠﺯﺀ ﺒﺎﻝﻤﻠﻴﻭﻥ ﻴﺜﺒﻁ ﻜﻠﻴﺎ ﺍﻝﻨﻤﻭ ﺍﻝﻐﺯﻝﻲ ﻝﻠﻔﻁﺭ4 ﻭ2 ﻭ1 ﺍﻷﺯﻭﻜﺴﻴﺴﺘﺭﻭﺒﻴﻥ ﺒﺘﺭﺍﻜﻴﺯ ( ﻤﻥ ﺘﻁﻭﺭ ﻝﻔﺤﺔ ﺍﻝﻐﻤﺩ ﺍﻝﻭﺭﻗﻲ64%
