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`E-ISSN: 2278-4136
`P-ISSN: 2349-8234
`JPP 2018; 7(5): 2974-2977
`Received: 24-07-2018
`Accepted: 26-08-2018
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`Gunda VNS Madhu Kiran
`Department of Plant Pathology,
`College of Agriculture, Vellayani,
`Kerala Agricultural University,
`Kerala, India
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`Thara SS
`Assistant Professor, College of
`Agriculture, Vellayani, Kerala
`Agricultural University, Kerala,
`India
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`Jyothi KR
`Department of Plant Pathology,
`College of Agriculture, Vellayani,
`Kerala Agricultural University,
`Kerala, India
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`Correspondence
`Gunda VNS Madhu Kiran
`Department of Plant Pathology,
`College of Agriculture, Vellayani,
`Kerala Agricultural University,
`Kerala, India
`
`Journal of Pharmacognosy and Phytochemistry 2018; 7(5): 2974-2977
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`Studies on compatibility of biocontrol agents with
`chemical fungicides for integrated management of
`Alternaria leaf spot of cabbage
`
`Gunda VNS Madhu Kiran, Thara SS and Jyothi KR
`
`Abstract
`The compatibility of fungal biocontrol agents (Trichoderma viride and T. harzianum) and bacterial
`biocontrol agents (Bacillus subtilis) was assessed with chemical fungicides viz., propiconazole,
`hexaconazole, tebuconazole and copper oxychloride. Studies on the compatibility of these effective
`fungicides and bioagents revealed that the triazole fungicides viz., propiconazole, hexaconazole,
`tebuconazole completely inhibited the growth of two bioagents viz., T. viride and T. harzianum at all the
`three concentrations but compatible with B. subtilis at all the three concentrations. The percentage of
`inhibition of T. viride with copper oxychloride was 61.4, 74.4 and 80 % and with T. harzianum was 65.5,
`80.0 and 85.5% at 0.1%, 0.2% and 0.4% concentrations respectively. Copperoxy chloride found to be
`incompatible with B. subtilis as inhibition zone increased with increase in the concentration of fungicide.
`
`Keywords: Fungal antagonists, bacterial antagonists, poisoned food technique, disc diffusion method.
`
`Introduction
`Alternaria leaf spot of cabbage is the most destructive disease and cause tremendous yield
`losses worldwide. Most of the plant diseases were controlled by utilization of either by
`utilization of chemical fungicides or by fungal and bacterial antagonists. In several disease
`management strategies, the addition of fungicide at reduced rate in combination with
`biocontrol agents has significantly enhanced disease control compared to treatments with
`biocontrol agents alone (Buck, 2004) [4]. Hence the present study was undertaken to test the
`compatibility of biocontrol agents viz., Trichoderma viride, T. harzianum and Bacillus subtilis
`with commonly used fungicides at different concentrations under in vitro conditions for the
`control of Alternaria brassicicola causing Alternaria leaf spot of cabbage.
`Mclean et al. (2001) stated that sporulation of T. harzianum (C52) was completely inhibited by
`the tebuconazole (0.05%) and mancozeb (0.1%). Pandey et al. (2006) [12] reported that both
`hexaconazole and tebuconazole fungicides showed 100% inhibition of mycelial growth of both
`T. viride and T. harzianum under in vitro conditions at 500 ppm concentration. Bagwan (2010)
`[1] conducted an in vitro experiment and observed the compatibility of fungicides with T. viride
`and T. harzianum and reported that propiconazole, tebuconazole, hexaconazole and
`chlorothalonil were incompatible with both bioagents as they shown 100% inhibition at 0.2%
`concentration. Percentage inhibition with copper oxychloride was 34.8% and 32.6% with T.
`viride and T. harzianum respectively at 0.2% concentration.
`Bhai and Thomas (2010) [3] stated that T. harzianum was not inhibited by copper oxychloride
`at 0.25% concentration. Gaur and Sharma (2010) [6] studied the compatibility of copper
`oxychloride with T. viride and T. harzianum and reported that the percentage of inhibition was
`26.27% and 36.37% with T. viride and 38.07% and 48.03% with T. harzianum at 500 ppm and
`1000 ppm respectively. Madhusudhan et al. (2010) [9] reported that two Trichoderma isolates
`T2 and T4 were completely incompatible with propiconazole and hexaconazole as they
`showed 100% inhibition at 500 and 1000 ppm concentrations. Sarkar et al. (2010) [14] tested
`the compatibility of propiconazole, hexaconazole and tebuconazole with T. harzianum at 5, 10,
`25, 50, 100, 200, 300 ppm concentrations and reported that all the three fungicides completely
`inhibited the growth of bioagent at 200 and 300 ppm concentration and at the remaining
`concentrations hexaconazole found to be more toxic compared to propiconazole and
`tebuconazole. In addition to that, compatibility was also tested with copper oxychloride and
`found to be moderately sensitive as the percentage of inhibition was 60% and 50% at 300 ppm
`and 200 ppm concentrations.
`Madhavi et al. (2011) [8] tested the compatibility of different fungicides with T. viride and the
`percentage of inhibition reported with highly sensitive fungicides propiconazole (0.1%),
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`Journal of Pharmacognosy and Phytochemistry
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`
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`Hexaconazole (0.2%), tebuconazole (0.15%) and moderately
`sensitive copper oxychloride (0.3%) was 93%, 94.4%, 94.4%
`and 62.9% respectively. Pandya et al. (2012) [13] studied the
`compatibility of propiconazole (0.05%) with T. harzianum
`and reported that as an incompatible combination as mycelial
`growth was completely inhibited. Zalte et al. (2013) [18]
`reported that B. subtilis was compatible with propiconazole at
`0.2% concentration. Singh et al. (2015) [16] reported that T.
`viride was compatible with copper oxychloride at 500 ppm
`but mycelial growth was least inhibited at 1000 and 1500
`ppm. At 2000 ppm concentration of copper oxychloride the
`growth of the bioagent was completely inhibited.
`Dhanya et al. (2016) [5] observed the compatibility of T. viride
`with hexaconazole under in vitro conditions and reported that
`mycelial growth was completely
`inhibited at 0.1%
`[15] conducted an
`concentration. Sharma et al. (2016)
`experiment under in vitro conditions and determined the
`compatibility of T. harzianum with propiconazole and
`observed that the interaction was incompatible as the growth
`of Trichoderma spp. was completely prevented at 31.2, 62.5
`and 125 ppm concentrations of the fungicide.
`Gayatri et al. (2016) [7] reported that T. viride and T.
`harzianum showed 21.01% and 13.37% inhibition with
`copper oxychloride and found to be least sensitive but the
`sporulation was prevented. In contrast to this B. subtilis was
`reported to be most sensitive to copper oxychloride as it
`showed 89.36% inhibition. Mareeswaran and Asir (2016) [10]
`reported that T. viride was incompatible with propiconazole
`and hexaconazole as it shown 100% and 89.06% inhibition
`over control at 10 ppm concentration but compatible with
`copper oxychloride at 10 ppm concentration as both bioagents
`grown completely without any inhibition and also reported
`that B. subtilis was compatible with hexaconazole and
`propiconazole compared to copper oxychloride. Barooah
`(2016) [2] reported that propiconazole and hexaconazole were
`fully incompatible with T. viride and T. harzianum at 0.1%
`concentration but compatible with B. subtilis at 0.1%
`concentration.
`
`Materials and methods
`Four fungicides propiconazole, hexaconazole, tebuconazole at
`0.05%, 0.1% and 0.2% concentrations and copper oxychloride
`at 0.1%, 0.2% and 0.4% concentrations were selected to test
`the compatibility with biocontrol agents.
`
`a) In vitro evaluation of compatibility of fungicides with
`fungal antagonists
`The compatibility of fungicides with fungal biocontrol agents
`was tested using poisoned food technique. In order to study this,
`50 ml of double strength PDA medium and 50 ml of distilled
`water were taken in two separate conical flasks and sterilized in
`an autoclave. Under aseptic conditions in laminar airflow
`chamber required concentration of chemical is added to the
`sterile distilled water and stir well. Thereafter the fungicide
`suspension added to the 50 ml melted and cooled double strength
`PDA medium and stirred well. Then 15 ml of the poisoned
`medium was poured into the petriplate and biocontrol agent was
`inoculated at the centre of the petriplate. For each treatment three
`replications were maintained and incubated at room temperature.
`Biocontrol agent placed at the centre of unamended media plate
`served as control.
`Per cent inhibition of the bioagent over control was determined
`as described by Vincent (1927) [17].
`
`
`
`
`Where
`I = Per cent growth inhibition
`C = Growth of bioagent in control plate
`T = Growth of bioagent in treatment plate
`
`b) In vitro evaluation of compatibility of fungicides with
`Bacterial antagonists
`The compatibility of fungicides with bacterial biocontrol
`agents was tested using Disc diffusion method. In order to
`study this, 15 ml of the nutrient agar was poured in the
`petriplate and allowed to solidify it under aseptic conditions
`in laminar airflow chamber. Overnight culture of B. subtilis
`(0.1 ml) was spread over the nutrient agar with the help of
`spreader. Fungicide solutions of required concentrations
`(propiconazole, hexaconazole, tebuconazole at 0.05%, 0.1%
`and 0.2% concentrations and copper oxychloride at 0.1%,
`0.2% and 0.4% concentrations) were prepared in separate test
`tube and 5mm diameter filter paper discs were prepared.
`There after these discs were dipped in fungicide solutions at
`required concentrations and placed on the nutrient agar and
`properly labelled it. Discs dipped in normal water and placed
`on the nutrient agar which served as control. For each
`treatment three replications were maintained and incubated at
`room temperature. Diameter of the inhibition zone was
`recorded and compared it with control treatment.
`
`Results
`a) In vitro evaluation of compatibility of fungicides with
`fungal antagonists
`The compatibility of T. viride and T. harzianum were tested
`with
`the
`fungicides propiconazole, hexaconazole, and
`tebuconazole at 0.05%, 0.1% and 0.2% concentrations and
`copper oxychloride at 0.1%, 0.2% and 0.4% concentrations
`using poisoned food technique. The results showed that the
`triazole
`fungicides viz., propiconazole, hexaconazole,
`tebuconazole completely
`inhibited
`the growth of
`two
`bioagents viz., T. viride and T. harzianum at all the three
`concentrations. The percentage of inhibition of T. viride with
`copper oxychloride was 61.4, 74.4 and 80 % and with T.
`harzianum was 65.5, 80.0 and 85.5% at 0.1%, 0.2% and 0.4%
`concentrations respectively (Table 1, Plate 1-2).
`the
`test
`to
`An
`in vitro experiment was conducted
`compatibility of
`four
`fungicides viz., propiconazole,
`hexaconazole, and tebuconazole, copper oxychloride with
`three bioagents viz., T. viride, T. harzianum and B. subtilis
`which were effective against A. brassicicola. The results
`showed
`that
`triazole
`fungicides viz., propiconazole,
`hexaconazole and tebuconazole at 0.05%, 0.1% and 0.2%
`concentrations were incompatible with T. viride and T.
`harzianum as it showed cent per cent inhibition (Table 24).
`Compatibility of both T. viride and T. harzianum was tested
`with hexaconazole (0.05%) and
`tebuconazole (0.05%)
`fungicides and reported
`that both combinations were
`incompatible by Pandey et al. (2006) [12] as they showed cent
`percentage inhibition. The results of the present study were
`also in accordance with Bagwan (2010) [1]. Sarkar et al.
`(2010) [14] revealed that T. harzianum was incompatible with
`propiconazole, hexaconazole and tebuconazole at 300 ppm
`concentration.
`In the present study, the fungicide copper oxychloride were
`incomapatible with two fungal bioagents viz., T. viride and T.
`harzianum as they recorded more than 50% inhibition at three
`concentrations 0.1%, 0.2% and 0.4%. Madhavi et al. (2011)
`[8] reported that growth of T. harzianum was highly sensitive
`with copper oxychloride as it showed 62.9% inhibition at 0.3
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`Journal of Pharmacognosy and Phytochemistry
`
`per cent concentration. Singh et al. (2015) [16] stated that
`copper oxychloride was incompatible with T. viride at 0.1,
`0.15 and 0.2% concentrations. In in vitro study, copper
`oxychloride was incompatible with B. subtilis as the zone of
`
`inhibition increased with increase in the concentration of
`fungicide which is similar with the results of Mareeswaran
`and Asir (2016) [10].
`
`Table 1: Effect of fungicides on the growth of T. viride and T. harzianum
`
`Fungicides
`
`Propiconazole
`Tebuconazole
`Hexaconazole
`
`T. viride
`100
`100
`100
`
`0.05%
`T. harzianum
`100
`100
`100
`
`Copper oxychloride
`*Mean of four replications
`
`0.1%
`
`61.4
`
`65.5
`
`Percentage inhibition *
`0.2%
`0.1%
`T. viride T. harzianum T. viride T. harzianum
`100
`100
`100
`100
`100
`100
`100
`100
`100
`100
`100
`100
`0.4%
`85.5
`
`0.2%
`
`74.4
`
`80
`
`80
`
`Plate 1: Compatibility of T. viride with effective fungicides
`
`Plate 2: Compatibility of T. harzianum with effective fungicides
`
`b) In vitro evaluation of compatibility of fungicides with
`Bacterial antagonists
`The compatibility of B. subtilis was tested with the fungicides
`propiconazole, hexaconazole, Tebuconazole at 0.05%, 0.1%
`and 0.2% concentrations and copper oxychloride at 0.1%,
`0.2% and 0.4% concentrations by disc diffusion method. The
`results were expressed in inhibition zone in diameter and 5
`mm indicates 0% inhibition as it was the diameter of the disc
`
`placed in the petriplate. The inhibition zone of triazole
`fungicides i.e., propiconazole, hexaconazole and tebuconazole
`was 5 mm at all the three concentrations. The diameter of
`inhibition zone observed in copper oxychloride treatment was
`7, 8 and 11 mm at 0.1, 0.2 and 0.4% concentration (Table 2,
`Plate 3).
`The compatibility of B. subtilis was also tested under in vitro
`and found to be compatible with three triazole fungicides viz.,
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`Journal of Pharmacognosy and Phytochemistry
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`propiconazole, hexaconazole and tebuconazole at 0.05, 0.1
`And 0.2% concentrations and these results were in accordance
`with Mareeswaran and Asir (2016) [10].
`
`
`Table 2: Compatibility of effective fungicides with B. subtilis
`
`
`Fungicides
`
`Propiconazole
`Tebuconazole
`Hexaconazole
`
`
`Copper oxychloride
`*Mean of four replications
`
`
`Inhibition Zone (mm) *
`0.05%
`0.1%
`0.2%
`5
`5
`5
`5
`5
`5
`5
`5
`5
`0.1%
`0.2%
`0.4%
`7
`8
`11
`
`8. Madhavi GB, Bhattiprolu SL, Reddy VB. Compatibility
`of biocontrol agent Trichoderma viride with various
`pesticides. J Hortic. Sci. 2011; 6(1):71-73.
`9. Madhusudhan P, Gopal K, Haritha V, Sangale UR, Rao
`SVRK. Compatibility of Trichoderma viride with
`fungicides and efficiency against Fusarium solani. J Pl.
`Dis. Sci. 2010; 5(1):23-26.
`10. Mareeswaran J, Asir RPS. Compatibility of biocontrol
`agents with selected agrochemicals commonly used in tea
`plantation. Current biotica. 2016; 10(2):104-111.
`11. Mclean KL, Hunt J, Stewart A. Compatibility of
`biocontrol agent Trichoderma harzianum C52 with
`selected fungicides. N. Z. Plant Prot. 2001; 54:84-88.
`12. Pandey KK, Pandey PK, Mishra KK. Bioefficacy of
`fungicides against different fungal agents for tolerance
`level and fungistatic behaviour. Indian phytopath. 2006;
`59(1):68-71.
`13. Pandya JR, Sabalpara AN, Chawda SK, Waghunde RR.
`Compatibility
`of
`Trichoderma
`harzianum with
`fungicides. Bioinfolet. 2012; 9(4):695-696.
`14. Sarkar S, Narayanan P, Divakaran A, Balamurugan A,
`Premkumar R. The in vitro effect of certain fungicides,
`insecticides and biopesticides on mycelial growth in the
`biocontrol fungus Trichoderma harzianum. Turkish J
`Biol. 2010; 34:399-403.
`15. Sharma D, Sharma R, Puri S. Compatibility of biocontrol
`agents with fungicides. Bioscan. 2016; 11(4):2863-2866.
`16. Singh C, Sharma A, Sharma N. Compatibility of
`Trichoderma viride and its interaction with different
`fungicides. Int. J Tech. Res. Appl. 2015; 3(6):253-257.
`17. Vincent JM. Distortion of fungal hyphae in the presence
`of certain inhibitors. Nature. 1927; 159:800.
`18. Zalte A, Gade RM, Shitole AV, Belkar YK. Management
`of tomato damping off by using plant growth promoting
`microorganisms. J Pl. Dis. Sci. 2013; 8(2):200-203.
`
`
`Plate 3: Compatibility of B. subtilis with selective fungicides
`
`
`
`
`References
`1. Bagwan NB. Evaluation of Trichoderma compatibility
`with fungicides, pesticides, organic cakes and botanicals
`for integrated management of soil borne diseases of
`soyabean (Glycine max (L.) Merril). Int. J Plant Prot.
`2010; 3(2):206-209.
`2. Barooah. Mycology and Microbiology. Ann. Sci. Rep.
`2016; 4:38-46.
`3. Bhai RS, Thomas J. Compatibility of Trichoderma
`harzianum with fungicides, insecticides and fertilizers.
`Indian Phytopath. 2010; 63(2):145-148.
`4. Buck JW. Combination of fungicides with Phylloplane
`yeasts for improved control of Botrytis cinerea on
`geranium seedlings. Phytopath. 2004; 94:196-202.
`5. Dhanya MK, Anjumol KB, Murugan M, Deepthy KB.
`Compatibility of Trichoderma viride with Pseudomonas
`fluorescens with plant protection chemicals and fertilizers
`in cardamom. J Trop. Agric. 2016; 54(2):129-135.
`6. Gaur RB, Sharma RN. Biocontrol of root rot in cotton
`and compatibility of potential bioagents with fungicides.
`Indian J Plant Prot. 2010; 38(2):176-182.
`7. Gayatri B, Umamaheswari R, Rao MS, Prabu P, Priti K,
`Grace GN, et al.
`Impact of commonly used
`agrochemicals on different
`fungal and bacterial
`bioagents. J oilseeds res. 2016; 33(1):62-67.
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