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`Asian Journal of
`Crop science
`
`ISSN 1994-7879
`
`science
`alert
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` OPEN ACCESS
`
`Asian Journal of Crop Science
`
`ISSN 1994-7879
`DOI: 10.3923/ajcs.2018.174.179
`
`Research Article
`Compatibility Trichoderma harzianum with Systemic and Two non
`Systemic Fungicides of in vitro
`
`1Ajay Tomer, 2Ramji Singh and 3Durga Prasad
`
`1Department of Plant Protection, School of Agriculture, Lovely Professional University, Jalandhar-Delhi, G.T. Road (NH-1), 144411, Phagwara,
`Punjab, India
`2Department of Plant Pathology, Sardar Vallabhbhi Patel University of Agriculture and Technology, 250110 Meerut, UP, India
`3Department of Plant Pathology, Bihar Agriculture University Sabor Bhagalpur, Bihar, India
`
`Abstract
`Background and Objectives: Trichoderma spp. is a soil inhabitant fungus with an ability of inhibiting plant pathogens and immunity
`and growth enhancer in plants. Based on the available literatures, the objective of this investigation was planned to study the
`compatibility of Trichoderma harzianum along with two systemic and two non-systemic fungicides in vitro. Materials and Methods: To
`isolate the biocontrol agent i.e., T. harzianum from soil, soil samples were collected from crop fields of crop research centre (CRC) of SVPUA
`and T Meerut, India. Four fungicides viz Mancozeb, Thiram, Carboxin and Propiconazole at 25, 50, 75 and 100 ppm were tested for their
`compatibility with Trichoderma harzianum by poisoned food technique and inhibition (%) by individual fungicides were noticed and
`recorded at an interval of each 24 h. Data were subjected to analysis using appropriate statistical methods, analysis of variance and
`treatment means were differentiated using Fischerʼs t-test in Completely Randomized Design (CRD) in laboratory. Results: It was evident
`that all the four concentrations of Mancozeb were highly compatible with almost negligible toxic effect against Trichoderma harzianum
`in vitro. As there was no or very little (0.00, 0.00, 5.19 and 7.03) inhibition of radial growth of Trichoderma harzianum due to Mancozeb
`at 25, 50, 75 and 100 ppm concentrations, respectively. Thiram was less compatible than Mancozeb. Carboxin and Propiconazole were
`toxic and incompatible with Trichoderma harzianum. Conclusion: Two non-systemic fungicides i.e., Mancozeb and Thiram were found
`to be compatible, as compared to systemic fungicides viz. Carboxin and Propiconazole which exhibited acute toxicity for growth of
`Trichoderma harzianum in vitro.
`
`Key words: Biocontrol agent, radial growth, plant pathogens, Trichoderma harzianum, Mancozeb, Thiram, non-systemic fungicides, carboxin and
`propiconazole
`
`Citation: Ajay Tomer, Ramji Singh and Durga Prasad, 2018. Compatibility Trichoderma harzianum with systemic and two non systemic fungicides of
`in vitro. Asian J. Crop Sci., 10: 174-179.
`
`Corresponding Author: Ajay Tomer, Department of Plant Protection, School of Agriculture, Lovely Professional University, Jalandhar-Delhi, G.T. Road (NH-1),
`144411, Phagwara, Punjab, India Tel: +91-9779312649
`
`Copyright: © 2018 Ajay Tomer et al. This is an open access article distributed under the terms of the creative commons attribution License, which permits
`unrestricted use, distribution and reproduction in any medium, provided the original author and source are credited.
`
`Competing Interest:
`
` The authors have declared that no competing interest exists.
`
`Data Availability:
`
` All relevant data are within the paper and its supporting information files.
`
`

`

`Asian J. Crop Sci., 10 (4): 174-179, 2018
`
`INTRODUCTION
`
`Agriculture in modern era depends largely on the use of
`agrochemicals, for managing plant diseases and to enhance
`crop productivity. Agrochemicals are capable of minimizing
`the threats from diseases and enhancing crop yields, however
`at the same time pose serious threats to human health along
`with environmental hazards. This has resulted in an increasing
`interest in biological control as a promising alternative or a
`supplemental way of reducing the use of agro-chemicals.
`Some naturally occurring soil bacteria and fungi have shown
`great potential to inhibit plant pathogens, hence, biological
`control involving the use of such beneficial micro-organisms
`for plant protection is being considered as a viable substitute
`to reduce the use of agrochemicals in general and pesticides
`in particular1.
`Management of plant diseases by the use of antagonist
`micro-organisms might be an effective means2. A large
`number of plant diseases have been successfully managed
`through fungal and bacterial antagonists2-5. Trichoderma sp.
`have been used in the management of plant diseases. The
`duration and degree of active disease control can be extended
`by using chemicals and biological control agents (antagonists)
`together as a mixed formulation in integrated disease
`management system. In a mixed formulation even reduced
`amount of the fungicide may weaken the pathogen and
`render its propagules more susceptible to subsequent attack
`by the antagonists6. Chemical protectants may be effective
`even under such climatic conditions where antagonists are
`less effective, while an active biological control agent can
`prophylactically colonize wounds or senescent plant tissue
`and ultimately protect them against pathogenic infection7.
`Usually fungicidal resistant or tolerant isolates of bio-agents
`are readily screened and obtained through selection on
`pesticide containing media8. Trichoderma is being used as a
`biological component in the integrated disease management
`of soil borne pathogen of cardamom (Elettaria cardamomum
`Maton.) viz. capsule rot and rhizome rot9,10. There
`is
`considerable pressure from environment active groups and
`scientists to give lesser emphasis on use of chemicals and
`more emphasis on promotion of biological methods for
`management of crop pests and diseases. Though the use of
`fungicides is necessary at present and it will continue to be in
`near future too, however their use can be minimized as a long
`term solution to the crop health problem because they are
`hazardous and also eliminate natural enemies and beneficial
`micro flora. In addition, several pesticides are quite expensive
`and several of them are inducing pesticide resistance and
`thereby posing high risk of pest resurgence. Thus, todayʼs
`
`need is to use eco-friendly compounds that can be fitted well
`into the holistic management strategy of the disease and
`compatible with commonly used antagonists viz. Trichoderma
`spp. and Pseudomonas spp. However, meager informationʼs
`are available on the compatibility of these commonly used
`plant protection chemicals with Trichoderma harzianum, the
`bio-control agent. Hence, present study was undertaken to
`test the compatibility of T. harzianum with commonly used
`and recommended dosages of such fungicides in vitro so that
`such chemicals can be used in a compatible manner as a
`mixture with bio-control agents.
`
`MATERIALS AND METHODS
`
`Determination of Trichoderma harzianum compatibility
`with different fungicides in vitro: Compatibility of four
`fungicides (two non-systemic) viz. Mancozeb and Thiram, (two
`systemic) Carboxin and Propiconazole were tested. Each
`fungicides were tested at 25, 50, 75 and 100 ppm
`concentrations against Trichoderma harzianum by poisoned
`food technique in vitro. Initial experimental work was started
`on 7 November, 2014. The PDA plates were inoculated with
`Trichoderma harzianum to prepare at least 7 day old culture/
`inoculums to be used for cutting 3 mm mycelial discs of test
`fungus. Required amount of all the test fungicides i.e., 25, 50,
`75 and 100 mg L(cid:42)1 of basal medium in case of Mancozeb,
`Thiram and Carboxin and 25, 50, 75 and 100 mL L(cid:42)1 of basal
`medium in case of Propiconazole were added in conical flasks
`(250 mL capacity), containing 100 mL pre-sterilized basal
`media (PDA) to obtain 25, 50, 75 and 100 ppm concentration
`of each fungi toxicants and mixed thoroughly by shaking the
`flask prior to pouring in sterilized Petri plates. After pouring in
`Petri plates, the medium was allowed to be cool and solidified
`over night. After solidification, 3 mm mycelial discs from 7
`days old culture of Trichoderma harzianum grown on PDA
`plates was placed in center of each Petri plate containing PDA
`with different concentration of test fungi toxicants. The PDA
`medium mixed with Sterilized distilled water only served as
`check. Inoculation of plates with mycelial bits of Trichoderma
`harzianum was done on 15 November, 2014. Three
`replications were maintained for each treatment. After
`inoculation with mycelial disc of Trichoderma harzianum, Petri
`plates were incubated at 28±2(cid:40)C in BOD incubator and
`arranged in a fashion of Completely Randomized Block Design.
`Observations were recorded on radial growth of Trichoderma
`harzianum at an interval of each 24 h upto 10 days. Final
`observations on radial growth of Trichoderma harzianum
`were recorded on the evening of 25th November, 2014.
`
`175
`
`

`

`Asian J. Crop Sci., 10 (4): 174-179, 2018
`
`91.86
`88.89
`86.67
`84.44
`98.14
`97.03
`95.92
`94.81
`93.70
`92.97
`89.63
`86.30
`7.03
`5.19
`0.00
`0.00
`(%)
`
`Inhibition
`
`90.00
`7.33
`10.00
`12.00
`14.00
`1.67
`2.67
`3.67
`4.67
`5.67
`6.33
`9.33
`12.33
`83.67
`85.33
`90.00
`90.00
`
`(mm) 240 h
`
`growth
`Radial
`
`92.22
`89.63
`87.78
`85.56
`98.52
`97.41
`96.30
`95.19
`94.08
`93.33
`90.37
`86.67
`7.78
`5.56
`0.00
`0.00
`(%)
`
`Inhibition
`
`90.00
`7.00
`9.33
`11.00
`13.00
`1.33
`2.33
`3.33
`4.33
`5.33
`6.00
`8.67
`12.00
`83.00
`85.00
`90.00
`90.00
`
`(mm) 192 h
`
`growth
`Radial
`
`94.81
`92.22
`90.00
`87.41
`99.08
`98.33
`97.41
`96.30
`94.44
`93.70
`90.74
`86.67
`15.56
`11.11
`6.67
`3.33
`(%)
`
`Inhibition
`
`90.00
`4.67
`7.00
`9.00
`11.33
`0.83
`1.50
`2.33
`3.33
`5.00
`5.67
`8.33
`12.00
`76.00
`80.00
`84.00
`87.00
`
`(mm) 168 h
`
`growth
`Radial
`
`95.92
`90.77
`90.77
`86.15
`98.97
`97.95
`97.69
`97.43
`96.92
`92.31
`89.74
`84.11
`23.08
`18.46
`10.77
`6.15
`(%)
`
`Inhibition
`
`65.00
`3.67
`6.00
`6.00
`9.00
`0.67
`1.33
`1.50
`1.67
`2.00
`5.00
`6.67
`10.33
`50.00
`53.00
`58.00
`61.00
`
`(mm) 120 h
`
`growth
`Radial
`
`97.66
`95.32
`91.23
`87.14
`98.82
`97.67
`97.07
`96.49
`95.32
`94.16
`92.40
`88.30
`38.60
`31.58
`22.81
`15.79
`(%)
`
`Inhibition
`
`57.00
`1.33
`2.67
`5.00
`7.33
`0.67
`1.33
`1.67
`2.00
`2.67
`3.33
`4.33
`6.67
`35.00
`39.00
`44.00
`48.00
`
`(mm) 96 h
`growth
`Radial
`
`100.00
`99.20
`97.98
`96.76
`100.00
`98.78
`97.56
`98.78
`97.98
`96.76
`94.32
`91.88
`56.10
`46.34
`39.02
`34.15
`(%)
`
`Inhibition
`
`41.00
`0.00
`0.33
`0.83
`1.33
`0.00
`0.50
`1.00
`0.50
`0.83
`1.33
`2.33
`3.33
`18.00
`22.00
`25.00
`27.00
`
`(mm) 72 h
`growth
`Radial
`
`100.00
`99.68
`99.40
`98.62
`100.00
`100.00
`100.00
`99.68
`99.54
`99.22
`98.94
`96.91
`67.70
`58.47
`40.01
`30.78
`(%)
`
`Inhibition
`
`21.67
`0.00
`0.07
`0.13
`0.30
`0.00
`0.00
`0.00
`0.07
`0.10
`0.17
`0.23
`0.67
`7.00
`9.00
`13.00
`15.00
`
`(mm) 48 h
`growth
`Radial
`
`100.00
`100.00
`100.00
`100.00
`100.00
`100.00
`100.00
`100.00
`100.00
`100.00
`100.00
`100.00
`96.55
`92.95
`70.01
`55.02
`(%)
`
`Inhibition
`
`CD at 5 % = Concentration ( A) = 0.2800, Incubation periods (B) = 0.4081, CXH = 1.154
`Control
`
`6.67
`0.00
`0.00
`0.00
`0.00
`0.00
`0.00
`0.00
`0.00
`0.00
`0.00
`0.00
`0.00
`0.23
`0.47
`2.00
`3.00
`
`(mm) 24 h
`growth
`Radial
`
`100
`75
`50
`25
`100
`75
`50
`25
`100
`75
`50
`25
`100
`75
`50
`25
`(ppm)
`Concentration
`
`Thiram
`
`Propiconazole (25EC)
`
`Carboxin 37.5%
`
`Mancozeb
`Fungicides
`
`Table 1: Effect of different concentrations of Mancozeb, Thiram, carboxin and propiconazole on radial growth of Trichoderma harzianum in vitro
`
`Experiment was continued only upto 10 days (25 November,
`2014) because radial growth of Trichoderma harzianum in
`the Petri plates maintained as check (Containing basal
`medium without any fungicide) occupied full growth upto the
`periphery of Petri plate. On the basis of radial growth in
`respective treatments, inhibition (%) in the radial growth of
`Trichoderma harzianum was calculated by using the following
`equation:
`
`(cid:117)
`
`100
`
`C(cid:16)
`
`C T
`
`Inhibition (% )
`
`(cid:32)
`
`Where:
`C = Growth of fungus in control ( PDA mixed with sterilized
`distilled water)
`T = Growth of fungus in respective treatments (PDA mixed
`with respective concentration of test fungicide )
`
`Statistical analysis: This experiment was conducted following
`two factorial completely randomized design. Two factors
`consisted fungicides concentrations (17) and incubation
`periods (8). Three replications were maintained for each
`treatment. Average of data was analyzed using two way
`analysis of variance using OPSTAT1.EXE software . The data
`given in percentage were first transformed into angular value
`and then analyzed for test of significance11,12.
`
`RESULTS
`
`The data presented in Table 1 indicated that all the four
`concentrations of Mancozeb i.e., 25, 50, 75 and 100 ppm
`(mL L(cid:42)1) were highly compatible with Trichoderma harzianum
`in vitro. There was absolutely no inhibition in radial growth of
`T. harzianum at 25 and 50 ppm concentration of Mancozeb
`after 192 and 240 h of incubation respectively on basal media
`containing these concentrations of Mancozeb. However a
`minimal inhibition in radial growth at 75 and 100 ppm
`concentrations of Mancozeb, respectively was noticed, after
`240 h (10 days) of incubation on basal media i.e., PDA
`containing these concentrations of Mancozeb. Thiram, was
`comparatively more toxic to Trichoderma harzianum than
`Mancozeb and next in the order of compatibility/toxicity. In
`the initial period of incubation i.e., upto 48 h, there was
`absolute inhibition (%) in the radial growth of
`Trichoderma harzianum but with increasing in the days of
`incubation on Thiram containing basal media, mycelial growth
`of antagonist (Trichoderma harzianum) get started and quite
`visible also. At 72 h of incubation on basal media containing
`25, 50, 75 and 100 ppm concentration of Thiram, there was
`
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`
`

`

`Asian J. Crop Sci., 10 (4): 174-179, 2018
`
`quite high level or absolute inhibition in radial growth of
`T. harzianum, whereas at 96 h, inhibition in radial growth was
`further reduced than those noticed at 72 h. At the end of
`experiment i.e., at 240 h (10 days) of incubation on basal
`media containing 25, 50, 75 and 100 ppm concentration of
`Thiram, the level of inhibition in radial growth of T. harzianum
`was 84.44, 86.67, 88.89 and 91.86% which was quite less than
`the inhibition noticed at 72 and 96 h.
`Two systemic fungicides i.e., Carboxin and Propiconazole
`were found to be more toxic than the two non-systemic
`fungicides
`(Mancozeb and Thiram) at all the
`four
`concentrations (25, 50, 75 and 100 ppm) tested. However,
`among two systemic fungicides, the Propiconazole was more
`toxic for Trichoderma harzianum than Carboxin. At 24 h of
`incubation on basal media, containing at 25, 50, 75 and
`100 ppm concentration of carboxin, there was absolute
`inhibition (%) in radial growth of Trichoderma harzianum.
`Level of inhibition at 48 h of incubation was comparatively less
`than those noticed at 24 h. From 72 h onward upto 192 h, the
`inhibition in radial growth ranged between 84.11-97.98% at
`25, 50, 75 and 100 ppm concentration of Carboxin. After
`240 h, the inhibition (%) in radial growth were lowest. There
`was gradual decrease in the level of inhibition with increasing
`the period of incubation. In case of Propiconazole, there was
`absolute inhibition in radial growth of Trichoderma harzianum
`at 25, 50, 75 and 100 ppm concentration, respectively. After 72
`h onward up to 192 h, the inhibition in radial growth ranged
`between 95.19-99.08% at 25, 50, 75 and 100 ppm
`concentration, respectively of Propiconazole. After 240 h, the
`inhibition (%) in radial growth of Trichoderma harzianum were
`lowest but still it was above 90% at all the concentrations (25,
`50, 75 and 100 ppm ) of Propiconazole. Overall, it was noticed
`that the two non-systemic fungicides i.e., Mancozeb and
`Thiram were found safer. Two systemic fungicides viz.
`Carboxin and Propiconazole exhibited acute toxicity for
`growth of Trichoderma harzianum in vitro. Compatibility
`couldnʼt be measured beyond 10 days as the Petri plates
`containing basal medium without any fungicides filled
`completely with radial growth of antagonist within 10 days.
`
`DISCUSSION
`
`During this study, two systemic fungicides i.e., Carboxin
`and Propiconazole were found to be comparatively more toxic
`and incompatible at all the concentrations tested i.e., 25, 50,
`75 and 100 ppm, respectively, against Trichoderma harzianum.
`Two non-systemic fungicides i.e., Mancozeb and Thiram were
`found to be compatible with the antagonist at all the
`concentrations tested. However, among the two systemic
`
`fungicides, Propiconazole was quite higher toxic than
`Carboxin at each concentration. Two systemic fungicides viz
`Carboxin and Propiconazole remained highly toxic after 10
`days also, whereas one non-systemic fungicides i.e., Mancozeb
`was found to be compatible and safer even at starting period
`also i.e., 24 h. Thiram, an universally accepted and widely used
`seed dressing fungicide was found to be less compatible with
`Trichoderma harzianum than Mancozeb. However if go on
`with the level of inhibition in radial growth, Thiram can be
`considered as more compatible than Carboxin, as itʼs toxicity
`lesser against Trichoderma harzianum than that of Carboxin.
`Thus, non-systemic fungicides i.e., Mancozeb and Thiram
`and one systemic fungicide i.e., Carboxin may be considered
`safer as compared to Propiconazole which exhibited acute
`toxicity for growth of Trichoderma harzianum. Bagwan13
`reported that thiram (0.2%), copper oxychloride (0.2%) and
`mancozeb (0.2%) were found comparatively safer against
`Trichoderma harzianum and Trichoderma viride as compared
`to other fungicides. However some other fungicides like
`captan,
`tebuconazole, vitavax, propiconazole and
`chlorothalonil were toxic to both the species of Trichoderma.
`These findings are in conformity with the findings of present
`study. Madhavi et al.14, also evaluated the compatibility of
`Trichoderma viride with 25 different pesticides in vitro where
`they tested six chemicals as seed-treatment. T. viride showed
`a high compatibility with the insecticide, imidacloprid (7.6 cm
`mycelial growth), followed by Mancozeb (6.3 cm) and
`Tebuconazole (3.7 cm). Contact fungicides, viz., Pencycuron
`and Propineb were found to be fully compatible with T. viride.
`In addition, 10 herbicides were also tested by this group and
`it was noticed that, the fungus Trichoderma viride was highly
`compatible with Imazethapyr (9.0 cm) followed by 2,4-D
`Sodium salt (8.9 cm) and Oxyfluorfen (6.5 cm) while it was
`totally
`incompatible with systemic
`fungicides
`like
`Carbendazim,
` Hexaconazole,
` Tebuconazole
` and
`Propiconazole. This report is also in accordance with the
`present study. Correa and Soria1, also observed that out of four
`systemic fungicides and two non-systemic fungicides tested
`in vitro for compatibility with potential bio-agent, Mancozeb
`was found highly compatible with Trichoderma harzianum.
`Thus the findings of present study is supported by the
`findings of Olga and Marcelo also. Ranganathswamy et al.15,
`also tested the compatibility of fungicides with biological
`control agents, i.e., Aspergillus niger, Trichoderma viride,
`T. koningii, T. harzianum and T. virens. Among the fungicides,
`Azoxystrobin was less toxic and compatible up to 400 ppm.
`Captan, Propineb and Azoxystrobin can be used for mixed
`formulation of chemical and bioagents at 200-400 ppm
`depending upon the Trichoderma species. Pencycuron can
`
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`
`

`

`Asian J. Crop Sci., 10 (4): 174-179, 2018
`
`with Trichoderma harzianum but this study suggested that
`Thiram is comparatively safer than carboxin for mixing with
`antagonist. Propiconazole should never be used for mixing
`with Trichoderma harzianum.
`
`SIGNIFICANCE STATEMENT
`
`This study discovers that Mancozeb was compatible with
`Trichoderma harzianum hence these two can be mixed
`together for seed treatment or spraying also. These findings
`can be beneficial for the agro
`industries
`involved
`in
`manufacturing of fungi toxicants and production of bio-
`control agents. This will also help the farmers by providing
`them an alternate method for minimizing chemicalʼs use in
`agriculture. This study will help the researcher to uncover the
`critical areas of developing consortia of chemicals and micro-
`organists for plant disease management. Thus a new theory
`on integrated use of fungi toxicants and antagonist may be
`arrived at.
`
`REFERENCES
`
`incorporated with Trichoderma spp. even at a
`be
`concentration of more than 400 ppm for seed treatment in the
`integrated management system. Rubayet and Bhuiyan16
`conducted an experiment to test the compatibility of three
`fungicides namely Provax-200 (Carboxin), Rovral 50 WP
`(Iprodione) and Bavistin 50 WP (Carbendazim) against T 10
`isolate of Trichoderma harzianum in vitro and reported that it
`was compatible with Provax-200 and Rovral 50 WP only at
`lower concentration which are in accordance with current
`findings with respect to compatibility of carboxin with
`Trichoderma harzianum, Tapwal et al.17 reported that among
`five fungicides viz., dithane M-45, ridomil, captaf, blue copper
`and bavistin, only captaf and blue copper were compatible to
`some extent with T. viride. Nandeesha et al.18 also observed
`that out of four systemic fungicides and two non-systemic
`fungicides tested in vitro for compatibility with potential
`bioagent, mancozeb was found highly compatible with
`Trichoderma harzianum. An integrated management strategy
`was developed for collar rot of groundnut under glass house
`conditions. Thus the findings of present study is supported by
`the findings of Nandeesha et al.18. Kumar and Singh19
`conducted an experiment to determine in vitro and in vivo
`sensitivity of T. viride to chemical fungicides (hexaconazole,
`propiconazole, crossman, carbendazim and mancozeb) which
`are usually applied in cultivation of crops to reduce the
`severity of a number of plant pathogens. They reported that
`hexaconazole, propiconazole, crossman and carbendazim
`were not compatible with the T. viride at recommended dose
`or even at lower dosages. Whereas, mancozeb was found
`moderately compatible with T. viride at recommended dose
`(2000 ppm). Present findings also suggest mancozeb as
`compatible and propiconazole as
`incompatible with
`Trichoderma harzianum. Meena et al.20 conducted an
`experiment to test the compatibility of 5 fungicides viz.
`Carbendazim Mancozeb Carboxin+Thiram Hexaconazole and
`Propiconazole I with Trichoderma spp. and found that
`mancozeb and carboxin+thiram were compatible. These
`findings also support the findings of present studies.
`
`CONCLUSION
`
`This study observed that Mancozeb was compatible with
`Trichoderma harzianum hence these two can be mixed
`together for seed treatment or spraying also. Thiram and
`Carboxin were also compatible with Trichoderma harzianum
`but Mancozeb should be preferred over these two. In the
`situation where Mancozeb is not required, then Thiram and
`Carboxin can also be mixed but Mancozeb should be
`preferred. Previously, carboxin have been in use for mixing
`
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