Compatibility of Azoxystrobin 23 SC with Biocontrol Agents and Insecticides

An experiment was conducted to study the compatibility of azoxystrobin 23 SC with bacterial and fungal biocontrol agents and insecticides under in vitro and glass house conditions, respectively. Bacterial biocontrol agents viz., Pseudomonas fluorescens and Bacillus subtilis were compatible with azoxystrobin 23 SC even at a high concentration of 300 ppm whereas fungal biocontrol agent Trichoderma viride was inhibited by azoxystrobin 23 SC at a concentration above 15 ppm. Among the four insecticides tested for compatibility, all insecticides were physically compatible with azoxystrobin 23 SC at 125, 250 and 500 g ai ha -1 whereas dichlorvos was biologically incompatible even at the lowest concentration tested.

Since fungicides may have deleterious effects on the pathogen as well as the antagonist, an understanding of the effect of fungicides on the pathogen and the antagonist, would provide an information on the selection of selective fungicides and fungicide resistant antagonists. The idea of combining biocontrol agents (BCA) with fungicides is for the development or establishment of desired microbes in the rhizosphere (Papavizas and Lewis, 1981). Further, the antagonism of BCA was influenced by the addition of fungicides (Kay and *Corresponding author email: archanas_agri@yahoo.co.in Stewart, 1994;Naar and Kecskes, 1999). Many authors reported the compatibility of fungicides with biocontrol agents in various crops (Utkhede and Koch, 2002;Senthilvel et al., 2004;Anand et al., 2007).
In intensive agriculture, crop production is challenged by an array of insects and pathogen leading to wide variety of damage. In order to combat these maladies, often it becomes necessary to mix two or three compatible pesticides (insecticide with fungicide) in a single preparation to save time and also expenses. Under such circumstances, it is desirable to generate information regarding the compatibility of insecticide and fungicide based on their efficacy against insect pest and diseases. The compatibility of insecticides with fungicides has been reported earlier in many cases (Varalakshmi et al., 2000;Sendhilvel, 2003). Considering all these points the present study was undertaken to test the compatibility of azoxystrobin 23 SC with biocontrol agents and also insecticides.

Materials and Methods
The bacterial biocontrol agent, Pseudomonas fluorescens Migula (Pf 1) and Bacillus subtilis (SVPR4) and fungal biocontrol agents Trichoderma viride (TV1) were obtained from Department of Plant Pathology, Tamil Nadu Agricultural University, Coimbatore. Insecticides and azoxystrobin 23 SC were purchased from pesticides market at Coimbatore.

Turbidometric method
One ml of the each bacterial culture viz., P. fluorescens and B. subtilis was transferred to a 250 ml sidearm flask containing 50 ml of King's B and Nutrient Agar broth, respectively and amended with azoxystrobin 23 SC technical standard at five different concentrations viz., 100,150,200,250 and 300 ppm. The control was maintained without inoculation of bacterial culture and azoxystrobin technical standard in both. The flasks were incubated at 28 ± 1 °C in a psychotherm shaker. The optical density values of the culture broth were determined in Spectrophoto colorimeter at 610 nm at regular intervals of 6 h.

Poisoned food technique
The compatibility of Trichoderma viride with azoxystrobin was tested using lower doses unlike in bacterial biocontrol agents as the T. viride is highly sensitive to fungicide and in addition to this it is mainly delivered as soil application. Potato dextrose agar (PDA) was used as the basal medium to which calculated quantities of azoxystrobin 23 SC was separately mixed aseptically after sterilizing the medium to give required concentrations viz., 5, 10, 15, 20 and 25 ppm. For each concentration, azoxystrobin 23 SC was taken into a 100 ml Erlenmeyer flask containing 100 ml of the sterilized and molten medium, mixed thoroughly by gently swirling the flask, poured 15 ml in each sterile Petri dish and allowed to solidify. A nine mm actively growing PDA culture disc of test fungus was placed at the centre of the plate and the plates were incubated in inverted position at room temperature (28 ± 2°C). The PDA medium without azoxystrobin 23 SC and inoculated with T. viride served as control. Three replications were maintained for each concentration. The radial growth of mycelium was measured periodically at 1, 2, 3, 4 and 5 days after inoculation.

Compatibility of azoxystrobin 23 SC with insecticides
In vitro condition

Preparation of standard hard water
Standard hard water is defined as water, which provides a hardness of 342 ppm calculated as calcium carbonate. For getting this hardness, 304 mg of anhydrous calcium chloride and 139 mg of magnesium chloride were dissolved in distilled water.
This solution was used to prepare insecticide solutions for all tests.

Emulsion stability test for physical compatiblity
The test was carried out for dichlorvos, monocrotophos, carbaryl and dimethoate as prescribed by Indian Standard Specification for emulsion stability test (ISI, 1964). To 75 to 80 ml of standard hard water kept in a beaker at 30 °C, the insecticide and fungicide was added by means of Mohr's type pipette. The insecticide and fungicide mixture was added to the standard hard water @ 25 to 30 ml pouring directly into the beaker and not along the sides of the beaker. The contents of the 375 beaker were stirred with a glass rod at four revolutions per second during the addition.
The diluted emulsion was made up to 100 ml mark with water which was transferred immediately to a clean dry graduated cylinder. Then the cylinder with its contents was kept at 30°C in a thermostat for 30 min. The creaming matter at the top and sedimentation at the bottom were observed. The creamy/sedimentation above two ml was considered as unstable.

Biological compatibility under glass house condition
Grapevines cv. Muscat was raised in pots under glass house conditions. Three month old vines were sprayed with mixture containing insecticides viz., monocrotophos (1.4 ml l -1 ), dichlorvos (1 ml l -1 ), carbaryl (2 ml l -1 ) and dimethoate (2 ml l -1 ) with the three concentrations of azoxystrobin 23 SC viz., 125, 250 and 500 g ai ha -1 to test the compatibility. The trial was conducted with three replications and three pots constituted one replication. Observations on leaf injury were taken after first, second and third days after spray.

Statistical analysis
The data generated from various experiments of this study were statistically analyzed by DMRT with IRRISTAT software. The data with per cent values were subjected to arc sine transformation.

Fungal biocontrol agent
The compatibility of T. viride isolate TV1 with five different concentrations of azoxystrobin 23 SC was tested and results are presented in Table 3. From the data it was inferred that among the five concentrations tested, three concentrations namely 5, 10 and 15 was found to be compatible whereas the other two concentrations inhibited the growth of T. viride. The least mycelial growth of 52 mm was observed in case of 25 ppm as against 90 mm in case of control at five days after inoculation. Over all, from the results it was concluded that up to 15 ppm concentration, the test fungicide was compatible with T. viride. The compatibility of fungicide with T. viride has been reported by earlier workers (Anand et al., 2007;Sendhilvel et al., 2004).

Physical compatibility of azoxystrobin 23 SC with insecticides
The results on the investigations carried out to study the physical compatibility in terms of emulsion stability revealed that recommended dose of insecticides viz., monocrotophos (1.4 ml l -1 ), dichlorvos (1 ml l -1 ), carbaryl (2 ml l -1 ) and dimethoate (2 ml l -1 ) with recommended dose of azoxystrobin 23 SC at three different concentrations viz., 125, 250 and 500 g ai ha -1 did not produce any creamy matter (or) sediment more than 2.0 ml at the top or bottom of the 100 ml measuring cylinder. The results confirmed the physical compatibility of the above insecticides (dichlorvos, cabaryl, monocrotophos and dimethoate) with azoxystrobin 23 SC (Table 4). The results of present study are in agreement with findings of Anand et al. (2007) and Sendhilvel (2003) who reported physical compatibility insecticides with azoxystrobin.

Biological compatibility of azoxystrobin 23 SC with commonly used insecticides under glass house conditions
The biological compatibility of azoxystrobin 23 SC at three concentrations viz., 125, 250 and 500 g ai ha -1 with the commonly used insecticides viz., dichlorvos, cabaryl, monocrotophos and dimethoate was studied under glass house conditions. The results of the experiment are presented in Table 5. Among the four insecticides used monocrotophos exhibited good compatibility with azoxystrobin 23 SC with the concentration of 125, Azoxystrobin 125 g ai ha -1 + Monocrotophos @ 1.4 ml l -1 0.00 a 0.00 a 0.00 a -Azoxystrobin 250 g ai ha -1 + Monocrotophos @ 1.4 ml l -1 0.00 a 0.00 a 2.31 b 0.77 Azoxystrobin 500 g ai ha -1 + Monocrotophos @ 1.4 ml l -1 3.12 b 5.31 b 8.71 c 11.33 III. Carbaryl (50 WDP) Azoxystrobin 125 g ai ha -1 + Carbaryl @ 2 ml l -1 0.00 a 0.00 a 0.00 a -Azoxystrobin 250 g ai ha -1 + Carbaryl @ 2 ml l -1 7. In a column, means followed by common letter are not significantly different at the 5% level by DMRT 250 and 500 g ai ha -1 . Dichlorvos produced severe phytotoxic injury with all the concentrations of azoxystrobin 23 SC (125, 250 and 500 g ai ha -1 ) under glass house conditions revealing its incompatibility. Carbaryl and dimethoate produced no phytotoxic symptoms with azoxystrobin 23 SC at the concentration of 125 g ai ha -1 , but both these insecticides produced mild phytotoxic symptoms viz., leaf injury with azoxystrobin 23 SC at 250 and 500 g ai ha -1 concentrations under glass house conditions indicating their incompatibility with azoxystrobin above 125 g ai ha -1 . The possible reason for incompatibility of dichlorovos could be because of formation more toxic compound through chemical reaction with test fungicide. The results of the experiment are in line with findings of Bagwan (2010) who reported the compatibility of insecticides with fungicides. Hence, monocrotophos can be combined with azoxystrobin 23 SC during application.