Bioefficacy of fungicides , biocontrol agents and botanicals against leaf spot of turmeric incited by Colletortricum capsici

Efficacy of ten fungicides with three different concentrations, that is, 500, 1000 and 1500 ppm were studied in vitro against Colletortricum capsici by poisoned food technique. Results revealed that Propiconazole was significantly superior to all other fungicides by inhibiting (100%) growth followed by Hexaconazole (96.55%) and Chlorothalonil (86.78%) over control after 10 days of incubation. Biocontrol agent namely, Trichoderma viride, Gliocladium spp., Trichoderma harzianum, Trichoderma koningii and Pseudomonas fluorescens were evaluated for their efficacy against C. capsici in vitro. Percent inhibition of the test pathogen ranged from 41.18 to 53.33%. T. harzianum, was found to be the most effective antagonist which caused growth inhibition of 53.33%. It showed the possibility of the most ecofriendly and inexpensive method of control of the leaf spot disease. A total of eleven plant extracts which were locally available were evaluated in vitro against C. capsici and were tested applying poisoned food technique. Overall percent inhibition of growth of the fungus ranged from 7.30 to 76.15%. Polyalthia longifolia recorded maximum inhibition of 76.15% at 15% concentration followed by rhizome extract Curcuma longa with 66.88% and Allium cepa 63.96%. Whereas, Aegale marmelos and Parthenium hysterophorus showed minimum mean inhibition of 20.86 and 28.66%, respectively. The results from pot culture experiment revealed that Propiconazole (0.05%) was found to be significantly superior among all the treatments. The next best fungicide was Hexaconazole (0.10%) and Chlorothalonil (0.15%). Among plant extracts, P. longifolia (15%) was superior over all other plant extracts. In the case of biocontrol agents, T. viride was superior over T. harzianum in pot culture experiment.


INTRODUCTION
Turmeric (Curcuma longa L.) is one of the most important spice crop cultivated in India.It is a rhizomates herbaceous perennial plant of the family Zingiberaceae.It has versatile uses in flavoring, dye making, drug preparation, cosmetics and medicine (Dixit et al., 2002).Turmeric is officially entered in the Ayurvedic Pharmacopoeia of India, Pharmacopoeia of the People's Republic of China and in Japanese standards of herbal medicines.India's annual production of turmeric is about 59.86 lakh tonnes on area of 892,213 ha.In Maharashtra, the area under turmeric cultivation is 6146 ha with production of 8,503 tonnes (Anonymous, 2011).This herbal plant is highly prone to several fungal diseases (Naidu, 1988;Pruthi, 2000).Among them, leaf spot caused by Colletotrichum capsici (Syd.)Butler and Bisby which is the most important disease of turmeric resulting *Corresponding author.E-mail: drgpjagtap@gmail.com.
to huge losses of 25.83 to 62.12% fresh weight and 42.10 to 62.10% dry weight of mother and finger rhizomes, respectively (Hudge and Ghogul, 2010).By considering economic importance of the crop and the disease, the present investigation was undertaken to evaluate fungicide, botanicals and bio-agents to assess their efficacy against C. capsici in vitro.

In vitro evaluation of fungicide
A total of ten fungicides viz.Carbendazim, Copper oxy chloride, Propiconazole, Mancozeb (Dithane M-45), Copper hydroxide, Chlorothalonil, Strobilurine, Hexaconazole, Triadimefon and Propineb which had been reported earlier as effective against the test pathogen (C.capsici) by several research workers on different crops including turmeric was included in the present study.Each fungicide at different concentrations was evaluated in vitro by applying poisoned food technique (Nene and Thapliyal, 1993) and using Potato Dextrose Agar as basal culture medium.
The required quantity of each fungicide was taken and thoroughly mixed with the medium and cooled (45°C) PDA in conical flasks to obtain desired concentrations.The fungicides amended PDA was then poured (15 to 20 ml plates) in sterilized Petri plates (90 mm diameter) under its different concentrations, replicated for three times, on solidification of PDA, all plates were inoculated by placing in the centre a 5 mm uniform mycelial disc obtained from the 7 days old culture of C. capsici grown on PDA.Plates containing PDA without any fungicide served as control.All these plates were then incubated at room temperature (28 ± 2°C) in BOD for a week or till the control plates were fully covered with mycelial growth of the test pathogen.
Observations on radial growth and sporulation of the test pathogen were recorded and percent growth inhibition of the test pathogen over control was calculated by the formula of Vincent (1927).The bioefficacy of these fungicides was evaluated at different concentration: 500, 1000 and 1500 ppm.

Evaluation of bioagents against C. capsici in vitro
The antagonistic potential of bioagents was assessed against C. capsici by dual culture technique on PDA medium as per procedure described by Stack et al. (1986).Bioagents were collected from Department of Plant Pathology, MKV, Parbhani, Maharashtra, India.For this, 20 ml of sterilized PDA was poured in each Petri plate and allowed to solidify.A 5 mm disc of C. capsici was cut and placed at one end of plate with the help of sterilized cork borer and inoculating needle.Contrarily, a 5 mm disc of the respective bioagent was used.Control, without inoculation of the bioagent was maintained, simultaneously.Observations regarding colony diameter of C. capsici and bioagent was recorded 10 days after inoculation by incubating at 28 ± 2°C temperature.

In vitro evaluation of plant extract
The antifungal properties of various plant species, botanicals were evaluated against C. capsici in vitro, applying poisoned food technique (Nene and Thapliyal, 1993).A total of 11 different plant species (Ashoka, Babhul, Bael, Parthenium, Neem, Beshram, Mehandi, Tulsi, Turmeric, Onion and Lantana) free from any disease were selected on the basis of their antifungal properties reported earlier and easy availability round the year in the field.
Fresh and healthy leaves of selected plant species were collected from the field and campus of Marathwada Krishi Vidyapeeth, Parbhani and brought to the laboratory for further studies.The leaves were first washed under running tap water to remove dirt material adhering to the surface.One hundred gram leaves from each sample were then grounded with sterile water (100 ml) in a mixture grinder.After thorough grinding, the extract was first filtered through muslin cloth and then through Whatman filter paper number 1. Later, the extract was passed through zeitz filter to free them from bacterial contamination.The extract was then used as standard plant extract solution of 100% concentration or 1:1 ratio.
The inhibitory effects of aqueous extract of the botanicals were assessed against the pathogens by poisoned food technique.5, 10, 15 ml of plant extract was mixed in 95, 90, 85 ml of PDA medium, to prepare 5, 10 and 15% of plant extract containing medium.
A mycelial disc of fungus, 5 mm in diameter was cut from periphery of 7 to 10 days old culture of fungus and aseptically inoculated unto the medium; each set was replicated three times.The control was run side by side by using only sterilized PDA medium.The Petri plates were inoculated at 28 ± 2°C temperature in BOD incubator and observations on mycelia growth were recorded and percent growth inhibition over control was calculated as per formula of Vincent (1927).

Integrated evaluation of fungicides, plant extracts and bioagents (pot culture)
The effective fungicides, plant extracts and antagonistic organism were tested against leaf spot disease in a pot culture experiment.Four fungicides, three plant extracts and two bioagents were thus selected on the basis of in vitro experiment with three replications and CRD design and were used for comparison.
One month old turmeric plants were raised in earthen plant pots in the glasshouse in sterilized soil + compost + sand mixture (2:1:1) in 22.5 cm clay pots.The plants were inoculated first to create disease symptoms.The effective fungicides, plant extracts and antagonistic organisms were sprayed on plant after the inoculation period of one week.The plants, uninoculated with the pathogens were used as control.Three replications were performed.Three sprays of each treatment were taken at an interval of 15 days.The plants were maintained in the glasshouse conditions.The intensity, severity and percent disease control of leaf spot was recorded after 15 days of last spray of the treatment, that is, fungicides, plant extracts and antagonistic microorganisms.
The disease incidence was calculated by using the formula as given by Mayee and Datar (1986).The percent disease control was calculated as per formula of Vincent (1927).

RESULTS AND DISCUSSION
Results (Table 1) revealed that all the fungicides tested were significantly effective in inhibiting the mycelial growth of the test pathogen as compared to the control (untreated).Among the fungicides, Propiconazole, Hexaconazole, Chlorothalonil and Azoxystrobin were found to be most effective at all concentrations, showing 100 (89.98), 96.55 (87.73), 86.78 (72.43) and 86.35% (72.10) mean inhibition respectively (Table 2).
The effect of Propiconazole was higher than all others; it completely inhibited the fungal growth at all concentrations.The Hexaconazole was second with 89.66 (71.31), 100 (89.98) and 100% (89.98) inhibition at 500, 1000 and 1500 ppm, respectively, followed by Propiconazole was observed to be effective in inhibition of growth of C. capsici in the present study which was also reported as effective by earlier workers (Chidanandaswamy, 2001;Singh et al., 2003;Swamy and Kulkarni, 2003;Ekbote, 2005;Gorawar et al., 2005;Kothikar, 2006).

Evaluation of bioagents against C. capsici in vitro
The present study was undertaken to evaluate the bioefficacy of fungal and bacterial biocontrol agents.Trichoderma viride, Gliocladium spp., Trichoderma harzianum, Trichoderma koningii and Pseudomonas fluorescens were tested in vitro applying dual culture technique given by Stack et al. (1986).The percent inhibition of mean colony diameter were studied after 10 days of inoculation.The result obtained are given is Table 3.The T. harzianum was observed as the most effective than other bioagents with percent inhibition of 53.33 (46.90) followed by T. viride with 52.32 (46.32).Gliocladium spp. was the least effective for control with 42.18% (39.91) inhibition (Plate 2).Kothikar (2006), Shrinivas (2006), Tasiwal (2008), Tiwari et al. (2008) and Deshmukh (2010)   Plate 1.In vitro evaluation of fungicides at 500 (A), 1000 (B) and 1500 ppm (C) concentration on radial growth of C. capsici.Plate 2. In vitro evaluation of bioagents on radial growth of C. capsici.
be used for management of leaf spot disease.

Evaluation of plant extracts against mycelial growth of C. capsici
The antifungal properties of eleven species of botanicals were evaluated in vitro against C. capsici using PDA as basal medium as par Poisoned Food Technique (Nene and Thapliyal, 1993).Locally available plant species on the basis of their antifungal properties reported earlier by research workers were included in the present study and results are presented in Table 4 and mean of three concentrations is presented in Table 5.
Result revealed that all eleven leaf extract tested at 5, 10 and 15% concentrations were found to be significantly effective in checking the mycelial growth of the C. capsici as compared to the control.Percent inhibition of the test pathogen with all plant extracts ranged from 7.30 to 49.63 at 5%, 20.85 to 56.82 at 10% and 34.44 to 76.15 at 15% concentration, respectively.Among the leaf extract, Ashoka (Polyanthia longifolia), rhizome extract of turmeric (Curcuma longa) and bulb extract of onion (Allium cepa) was found most effective over other botanicals with mean percent inhibition of 76.15, 66.88 and 63.96% at 15% concentration, respectively.Whereas beal (Aegale marmelos) was found with minimum effect with a percent inhibition of about 43.11, 48.63 and 58.71% at 5, 10 and 15% concentration, respectively.The mean of all three concentrations was also calibrated and presented in Table 5.It was observed that the mean colony diameter in plate treated with beal (A.marmelos) was found to be maximum (71.22 mm).The Ashoka, Turmeric and Babhul showed least mean colony diameter with 35.21, 40.95 and 44.86 mm, respectively.It was observed that Ashoka followed by turmeric and Babhul were most effective with mean percentage inhibition of 60.86, 54.49 and 50.15%, respectively (Plate 3).Whereas, Beal followed by Parthenium were found to be least effective with a percent inhibition of 20.86 and 28.66, respectively.The results obtained on efficacy of P. longifolia against C. capsici in the present study are in consonance with the earlier findings reported by several investigators viz., Shivapuri et al. (1997) and Nduagu et al. (2008).

Integrated evaluation of fungicides, plant extracts and bioagetns (pot culture)
The results of efficacy of fungicidal, botanicals and bioagents spray against leaf spot of turmeric C. capsici under pot culture experiment followed under glass house condition.It is evident from Table 6 that, fungitoxins differed significantly in managing leaf spot of turmeric.All the fungitoxins, significantly reduced disease incidence as compared to control.Amongst fungicides, minimum disease incidence was recorded by Propiconazole after third spray.After third spray, the percent disease incidence of Propiconazole was observed to be 25.64%, among fungicides, Azoxystrobin was least effective having the percent disease incidence of 34.45%.Regarding the percent disease incidence of disease after the third spray of botanicals, Ashoka (P.longifolia) was most effective with percent disease incidence of 39.59%.Among bioagents, T. harzianum followed by T. viride showed 44.43 and 48.32% disease incidence, respectively.All the fungicides, botanicals and bioagents significantly reduce the disease severity as compared to control.The data is presented in Table 7.  Disease severity at the end of the third spray was seen in Propiconazole (24.42%) followed by Hexaconazole (27.80%).Among botanicals, Ashoka (P.longifolia) showed least disease severity (38.49%).The disease severity recorded by the spray of bioagent, that is, T. harzianum and T. viride was 43.27 and 46.75, respectively.The percent disease control of the leaf spot disease is shown in Table 8.
It was observed that Propiconazole (60.09%) was superior over other fungicides, followed by Hexaconazole (55.57%) in controlling the disease.Ashoka showed highest percent disease control (43.52%), among other plant extracts used.T. harzianum with 34.28% disease control was found to be superior over the T. viride (28.59%).After the disease symptoms were prominently observed, three sprays were given for each fungitoxins, at an interval of 15 days.The observations were recorded after every spray treatment.Three types of observation were recorded viz., percent disease incidence, percent disease severity and percent disease control.Among fungitoxins tested, Propiconazole 0.05% was significantly superior among all other treatment given followed by Hexaconazole (0.10%), Chlorothalonil (0.15%) and Azoxystrobin (0.15%).Among plant extracts used, P.  longifolia was found superior over other plants extracts.Among bio agents, T. viride was found superior in pot culture over T. harzianum.The present findings regarding the superiority of Propiconazole over other fungitoxins to control the disease in plant are in conformity with the results of Chidanandaswamy (2001), Singh et al. (2003), Ekbote (2005) and Theerthugiri and Ramanujam (2009).

Table 1 .
Evaluation of fungicides against C. capsici in vitro (Poisoned Food Technique).
*Mean of three replications; figures in parenthesis are angular transformed values.
screened biocontrol agents against C. capsici causing leaf spot in turmeric in vitro and reported that T. koningii, T. harzianum and T. viride were best in inhibiting the growth of C. capsici.The present results confirms that T. harzianum and T. viride inhibit the growth of C. capsici causing leaf spot in turmeric and thus can

Table 2 .
Mean colony diameter and inhibition percentage of C. capsici as influenced by fungicides.
*Mean of three concentration of each fungicide; figures in parenthesis are angular transformed value.
*Mean of three replications of each treatment; figures in parenthesis are angular transformed value.

Table 4 .
Efficacy of plant extract on the mycelial growth of C. capsici in vitro (Poisoned Food Technique).

Table 5 .
Mean colony diameter and inhibition percentage of C. capsici as influenced by plant extracts.Mean of three concentrations of each plant extract; figures in parenthesis are angular transformed values. *

Table 6 .
Efficacy of various fungitoxins on disease incidence of leaf spot disease of turmeric caused by C. capsici in pot culture experiment.
*Mean of three replications of each fungitoxins.

Table 7 .
Efficacy of various fungitoxins on disease severity of leaf spot disease of turmeric caus ed by C. capsici in pot culture experiment.
*Mean of three replications of each fungitoxins.

Table 8 .
Efficacy of various fungitoxins on percent disease control of leaf spot disease of turmeric caused by C. capsici in pot culture experiment.
*Mean of three replications of each fungitoxins.