Effect of fungicides , plant extracts / botanicals and bioagents against damping off in brinjal

Brinjal damping off incited by Pythium ultimum Trow. is one of the most important and destructive disease of brinjal, which can cause mortality upto 90% (pre -emergence seed rot and post emergence seedling mortality). Being soil borne, P. ultimum is very difficult to manage with fungicides alone and also uneconomical. Therefore, the present in vitro studies were undertaken to test bioefficacy of the nine fungicides (each at 500, 1000 and 1500 ppm), ten plant extracts / botanicals (each at 10, 15 and 20 %) and seven bioagents against P. ultimum. The experiments were designed with CRD and all the treatments replicated thrice. Results reveal that all the fungicides, botanicals and bioagents tested were found effective and were fungistatic against the test pathogen and significantly inhibited its growth over untreated control. Of the fungicides tested, Metalaxyl was found most effective and recorded 84.22% mean growth inhibition of the test pathogen. The second and third best fungicides found were Captan + metalaxyl and carbendazim + Mancozeb with mean growth inhibition of 82.42 and 62.88%, respectively. The rest of the fungicides tested recorded mean growth inhibition in the range of 24.50 to 52.79%. Of the botanicals evaluated, garlic was found most effective and recorded significantly the highest mean mycelial growth inhibition (94.83%). The second and third best botanicals found effective were Adulsa (75.53 %) and Datura (60.65 %). The rest of the botanicals tested recorded mean growth inhibition in the range of 20.82 to 56.83%. Of the bio-agents evaluated, Trichoderma viride was found most effective and recorded significantly highest mean mycelial growth inhibition (69.44%). The second and third best bioagents found effective were Trichoderma koningii (67.32%) and Trichoderma hamatum (63.99%); the rest of the bioagents also recorded significant inhibition of the test pathogen. Results reveals that seed treatment of captan (at 1.5 g/kg) + metalaxyl (at 3g/kg seed)+ garlic extract (at 100ml/kg soil) +soil application of T. viride (at 25g/kg soil) was the most effective treatment which could be practiced on large scale for management of damping off disease in brinjal and other solanaceous vegetable crops.

known to have ayurvedic medicinal properties and good for diabetic patients.It has also been recommended as an excellent remedy for those suffering from liver complaints (Shukla and Naik, 1993).For brinjal, the following diseases have been reported this includes fungal diseases viz., damping off, Phomopsis, blight and wilt, viral diseases viz., mosaic and mottled dwarf and mycoplasma of leaf.Many fungi prevalent in soils can cause damping-off.Fursarium spp., Pythium spp., and Phytophthora spp.are most active in cool, wet soils whereas, Cylindrocladium spp.and Rhizoctonia spp.are more common in warm, wet soils.
The infected tissue become soft and water soaked, the collar portion rots and the seedlings ultimately collapse and die.The guaranteed supply of quality seedlings in required quantities is a major pre requisite for stabilized production of Brinjal.While raising seedlings in beds, the farmers face major problem of damping off incited by Pythium spp.
The damping off in brinjal is caused by Pythium spp., including P. aphanidermatum, P.irregulare and P. ultimumTrow, which can cause pre -emergence damping off and results in seed rot before the plants emerge out of the soil.The post emergence damping off phase is characterized by infection of the young tissues of the collar at the ground level.The pathogen is a soil borne with wide host range and almost worldwide distribution.The disease seems very difficult to control by conventional chemical means, due to its wide host range.Further, the use of chemicals/fungicides alone for the control of P. ultimum has been found to be impracticable and uneconomical.Therefore, an integrated disease management approach that encompass the use of chemicals, biocontrol agent, and plant extract could be the most economical and effective strategy for controlling the damping off and other soil borne plant diseases.Considering economic importance of the crop and losses caused by disease damping off in Brinjal, the present investigation was undertaken.

MATERIALS AND METHODS
The pathogen was isolated from the damping-off brinjal plants collected in nursery beds in Kharif, from the Department of Horticulture, VNMKV, Parbhani, Maharashtra, India in 2011.The Brinjal seedlings on nursery beds showing the symptoms of damping off (Plate 1) were collected in the polythene bags, labelled and brought to the laboratory.These samples were processed after surface sterilization (0.1% HgCl 2 ) for isolation of P. ultimum Trow.The isolate of the test pathogen were purified, numbered and maintained on potato dextrose agar (PDA) slants and stored at 8 to 10C in a refrigerator.

Efficacy of fungicides
The efficacy of fungicides against P. ultimumTrow.was evaluated in three concentrations (500,1000 and1500 ppm) in vitro by applying poisoned food technique (Nene and Thapliyal, 1993) and using PDA as basal medium.The experiment was conducted by Completely Randomized Design with ten treatments and three replications.
100 ml PDA medium was poured in 250 ml capacity sterile glass conical flask and sterilized at 15 lbs pressure for 15 min.Required quantity of test fungicides for 500, 1000 and 1500 ppm was calculated and added in the sterilized PDA medium separately and mixed thoroughly.
This fungicide amended PDA medium with different concentrations of the test fungicides was poured (20 ml/plate) in sterilized glass Petri dish (90 mm.dia) and allowed to solidify at room temperature.The plates were inoculated by pure culture of P. ultimum Trow.For this purpose, 5 mm disc of one week old culture was cut with a sterilized cork borer.The disc was lifted and transferred aseptically in the centre of Petri plates containing the medium with test fungicides.Three plates per treatment per replication were maintained.The PDA plates without fungicides were also inoculated with the test pathogen and maintained as a uninoculated control.All the plates were incubated at 26 + 2C.
The observations on colony diameter were recorded after a week of incubation.Per cent inhibition of the test pathogen was calculated by using the formula of Vincent (1927) and the data was statistically analysed: Where, PI= percent of inhibition, C= growth in control plates, T= growth in plates treated with fungicides.

Efficacy of plant extracts
Ten botanicals were evaluated in vitro at 10, 15 and 20% each for their fungistatic against P. ultimum Trow.by poisoned food technique (Nene and Thapliyal, 1993).The experiment was conducted by completely randomized design with 11 treatments and three replications.
Leaves/ rhizomes of the test botanicals were washed first in tap water, then in distilled water.Then 100 g of plant tissues + 100 ml distilled water were crushed (1:1 w/v) in mortar and pestle.The extract was filtered through double layered muslin cloth.The filtrate thus obtained was centrifuged at 5000 rpm for 15 min.The supernatant was collected and pellet was discarded.The supernatant obtained was strained through whatman No.1 filter paper and filtrate thus obtained was used as stock solution (100% conc.).
Aqueous plant extract (100%) were poured at 10, 15 and 20 ml each and separated into 100 ml autoclaved and cooled PDA in conical flask.The plant extract amended PDA was poured (each 20 Plate 2. Mass multiplication of P. ultimum (B) on sand : maize medium.ml/plate) in sterile glass Petri plates (90 mm dia.) and allowed to cool.Five mm disc of P. ultimum Trow.was placed on the center of the solidified PDA plate under aseptic conditions.The PDA plates without plant extract and inoculated with the test pathogen served as untreated control.
These Petri plates were incubated at 26±2C till the growth of the test pathogen in control plate was fully covered.The radial mycelial growth in all the plates was recorded and percent inhibition of mycelial growth over control was calculated by applying the formula (Vicent, 1927): Where, C= growth of test fungus in control plates and T= growth of test fungus in treatment plates.

Efficacy of bioagents
The antagonistic potential of seven bioagents viz, T. viride, T. harzianum, T. koningii, T. hamatum, Gliocladium virens, Bacilius subtilis and Pseudomonas fluorescence against P. ultimum Trow.was evaluated in vitro by Dual culture technique (Stack et al., 1986) on PDA medium.The experiment was conducted by completely randomized design with eight treatments and three replications.Autoclaved and cooled PDA medium was poured at 20 ml/plate in Petri plates (90 mm) and allowed to solidify.The plates were inoculated with 5 mm disc of 7 days old culture of biocontrol agents as well as 5 mm disc of 7 days old culture of P. ultimum Trow.at equidistance and exactly opposite with each other on PDA in plates.For bacteria, antagonist were streaked with the help of sterilized inoculating needle at one end of the PDA Petri plate.After 24 h of incubation, just opposite to the bacterial streak, a 5 mm disc of the test pathogen was placed.The PDA plates inoculated in center with the disc of the culture of the test pathogen only served as control.A triplicate set of inoculated PDA plates per treatment per replication was maintained and all the treatments were replicated thrice.
All these plates were incubated at 26±2 C in incubator.Observations on radial mycelial growth of the fungal pathogen and biocontrol agents was measured and per cent inhibition of the test fungus (Pythium ultimum Trow.) was calculated by applying formula given by Arora and Upaddhyay (1978) as follows:

Integrated evaluation of fungicides, botanicals, and bioagents in pot culture
Pot culture experiment was conducted to integrate the effective fungicides, bioagents and botanicals for integrated management of damping off (Pythium ultimum) disease in brinjal Cv.Hadgaon local.The experiment was conducted by Completely Randomized Design with eleven treatments and three replications.
Pathogen multiplied on sand: maize medium (Plate 2) was mixed with autoclaved (30 lbs for 30 min) potting mixture soil: sand: FYM (2:1:1) (at 25 g / kg potting mixture) and filled into pots, (disinfected with 5 % solution of copper sulphate) watered lightly and incubated at room temperature for two weeks in screen house.Within this period test pathogen multiplied in the pots.Those fungicides, plant extract /botanicals and bioagents found effective in in vitro studies were used for integrated disease management (IDM) experiment.The effective test fungicides viz., Metalaxyl (alone) and Captan (in combinations) were applied as seed treatment as detailed above.The 20% aqueous crude extract of garlic bulk was applied (alone and in combination) as soil drenching (100 ml/kg soil).The carrier based preparation of bioagent T. viride was applied (alone and in combination) in the soil at 25 g/kg soil, as detailed above.The fungicide treated seed of brinjal Cv.Hadgaon local were sown (6 seeds /pot) as per the treatment details.For T3, T4 and control treatments, the surface sterilized (0.1%HgCl 2 ) seed of brinjal Cv.Hadgaon local were sown (6 seeds/pot).The suitable untreated control with soil and surface sterilized seed sown of brinjal Cv.Hadgaon local was maintained.Three pots per treatments per replication were maintained and all the treatments replicated thrice.All these pots were watered regularly and maintained in screen house at the Department of Plant Pathology, Parbhani.
Observations on pre-emergence seed rot were recorded at one week after sowing and on post -emergence seedling mortality recorded at interval of 7 days and counted till 35 DAS and averaged finally.Observations were recorded for pre-emergence seed rot, post-emergence seedling mortality and per cent pre-emergence seed rot and per cent post-emergence seedling mortality were calculated by formula devised by Kataria and Grover (1967).

Number of seeds rot per pot %PESR =
x 100 Total number of seeds per pot Number of seedlings affected per pot %POESM = x 100 Total number of seeds per pot PESR is pre emergence seedling mortality and POESM is post emergence seedling mortality.

The efficacy of fungicides under in vitro tests
The results obtained on in vitro bio-efficacy of the nine fungicides (alone and combination) viz., Captan, Thiram, Carbendazim,, Mancozeb, Carbendazim + Mancozeb, Metalaxyl, Benomyl, Carbendazim + Thiram, Captan + Metalaxyl against P. ultimum of the present study are presented in Table 1, Plates 3, 4 and 5 and Figure 1.

Mycelial inhibition
Results (Table 1) revealed that all the fungicides at 500, 1000 and 1500 ppm significantly inhibited mycelial growth of the test fungus over untreated control.Further, it was found that per cent mycelial inhibition was increased with the increase in the fungicides concentrations.

Radial mycelial growth
Results (Table 2) reveal that all the botanicals/plant extracts tested exhibited a wide range of radial mycelial growth of the test pathogen and it was varied with their concentrations used.At 10%, (Plate 6) radial mycelial growth of the test pathogen ranged from 13.93 (Garlic) to 73.86 mm Turmeric).However, it was maximum with turmeric 73.86 mm).This was followed by Tulsi (67.90 mm), Shatawari (66.96 mm), Neem (66.06 mm), ginger (47.46 mm), Parthenium (47.50 mm) both of which were at par, (Datura (37.43 mm) and Mehandi (35.56 mm).
Comparatively, least growth was recorded with Adulsa Plate 7. In vitro efficacy of plant extract/ botanicals at 15% concentration on radial growth of P. ultimum.
Thus, all the plant extracts tested were found as fungistatic/antifungal against P. ultimum and significantly inhibited mycelial growth of the test pathogen over untreated control However, Garlic recorded highest mean mycelial inhibition (94.83%) followed by Adulsa (75.53%) and Datura (60.65%).Result of the present study are in conformity with those reported earlier by several workers (Bhat and Shrivastava, 2003;Bhora et al., 2006;Muthukumar et al., 2010Ambikapathy et al., 2011).
Result (Table 3) reveals that all the bioagents evaluated exhibited fungistatic /antifungal activity against P. ythium ultimum Trow.and significantly inhibited its mycelial growth over untreated control (Plate 9).Of the antagonist tested, T. viride was found most effective and recorded significantly least mycelial growth (27.49mm) with highest mycelial inhibition (69.44%) of the test pathogen over untreated control (00.00%).The second and third best antagonists found were T. koningii and T. hamatum which recorded mycelial growth of 29.40 and 32.40 mm, respectively and inhibition respectively of 67.32 and 63.99%.This was followed by Bacillus subtilis and P. flueroscens with colony growth respectively of 36.25 and 39.35 mm and corresponding growth inhibition of 59.71 and 56.27%.G. virens recorded mycelial growth of 41. and corresponding growth inhibition of 54.14%.T. harzianum was found relatively less effective with 45.35 mm colony diameter and 49.60% inhibition of the test pathogen.
Thus all the fungal and bacterial antagonists/bioagents evaluated in vitro were found fungistatic /antifungal against P. ultimum and caused significant reduction in the linear mycelial growth of the test pathogen over untreated control.
The inhibition effects of Trichoderma spp., P. fluorescens and B. subtilis against P. ultimum may be attributed to the mechanisms viz,.antibiosis, lysis, mycoparasitism, competition and production of volatile substances, by the test bioagents/antagonists.
Results of the present study on inhibitory effects of the test antagonists: Trichoderma spp., B. subtilis, and Plate 9.In vitro efficacy of the bioagents against P. ultimum Trow.P. fluorescens are in conformity with those reported earlier by several workers (Manoranjitham et al., 2000;Chakrabarti et al., 2005;Pandey and Pandey, 2005;Valerie et al., 2005, Abeysinghe, 2009).

Integrated disease management strategies
The results obtained on IDM in vitro studies (pot culture) of 11 treatments against P. ultimum of present study are presented in The Table 4 and depicted in Plate 10 and Figure 4.

Pre-emergence damping off
Results (Table 4) reveals that the percent pre-emergence damping off recorded with all the treatment ranged from 16.66 to 55.55% as against 72.21% in control.The least pre-emergence damping off percentage was recorded with the treatment (T 10 ) Captan + Metalaxyl + Garlic + T. viride (16.66%), followed by the treatment (T 7 ): Captan + Metalaxyl + Garlic (22.21%).The treatments T 2 , T 5 , T 8 , T 9 were found on par with T 10 and T 7 .
The maximum pre-emergence damping off was recorded with the treatment (T 4 , T. viride) (55.55%), followed by the treatment (T 3 , Garlic) (44.44%) and depicted in Plate 10 and Figure 4.

Post emergence damping off
Results (Table 4) revealed that the percent post emergence damping off recorded with all the treatments ranged from 20% to T 4 as against 66.66% in control.
The similar pattern of result was recorded in post emergence damping off as observed in pre-emergence damping off.The least post emergence damping off was recorded with the treatment Captan + Metalaxyl + Garlic + T. viride (T 10 ) (20%) followed by Captan + Metalaxyl + Garlic (T 7 ) (21.66%).The treatments T 2 , T 5 and T 8 were found on par with T 10 and T 7 .The maximum post emergence damping off, was recorded with the treatment T 4 : T. viride (49.99%), followed by the treatment T 3 : Garlic (41.66%) and treatment T 9 : Metalaxyl + Garlic + T. viride (41.66%) and depicted in Plate 10 and Figure 4.

Average percent mortality
Results (Table 4) reveal that the average percent mortality of pre and post emergence damping off with all the treatments ranged from 18.33 to 52.77% as against 69.43% in control.

Reduction in damping off
The results obtained on integrated disease management in vitro studies (pot culture) of 11 treatments against P. ultimum are presented in Table 4.   T 11 Control (untreated) 00.00 (00.00) 00.00 (00.00) 00.00 (00.00)

Percent reduction in pre-emergence damping off
Results (Table 5 and Figure 5) reveal that the percent reduction in pre-emergence damping off recorded with all the treatments ranged from 23.07 to 76.92% as against the control.The maximum percent reduction in pre-emergence damping off was recorded with the treatment T 10 : Captan + Metalaxyl + Garlic + T. viride) (76.92%), followed by the treatment T 7 : Captan + Metalaxyl + Garlic (69.24%).The treatment T 2 , T 5 , T 8 and T 9 were found on par with T 10 and T 7 .

Percent reduction in post emergence damping off
Results (Table 5 and Figure 5) revealed that the percent reduction in post emergence damping off recorded with all the treatments ranged from 25.50 to 69.99%.

Average percent reduction in damping off
Results (Table 5) reveals that the averaged percent reduction in pre and post emergence damping off with all the treatments ranged from 24.28 to 73.45%.

Plate 10 .
Experiment (pot culture) on integrated management of damping off in Brinjal Cv.Hadgaon local

Figure
Figure in parenthesis are angular transformed values.

Figure 5 .
Figure 5. Efficacy of fungicides, bioagents and botanicals in reducing Pre and post emergence damping of in brinjal cv.Hadgaon local.

Table 1 .
Efficacy of the fungicides against Pythium ultimum Trow.
Colony growth in Control Plate -Colony growth in intersecting plate Percent Inhibition (PI) = x 100 Colony growth in control plate *Mean of three replications; figure in parenthesis are angular transformed values.Plate 3.In vitro efficacy of fungicides at 500 ppm concentration on radial growth of P. ultimum.Plate 4. In vitro efficacy of fungicides at 1000 ppm concentration on radial growth of P. ultimum.Plate 5.In vitro efficacy of fungicides at 1000 ppm concentration on radial growth of P. ultimum.Figure 1.In vitro efficacy of fungicides at 500, 1000, 1500 ppm concentrations on radial growth of P. ultimum Trow.
* Mean of three replications.(Figure in parenthesis are angular transformed values).
*Mean of three replications.Figure in parenthesis are angular transformed values.

Table 4 .
Integrated disease management with effective fungicides, botanicals and bioagents in pot culture.

Table 5 .
Efficacy of fungicides, bioagents and botanicals in reduction damping off in brinjal cv.Hadgoan local