J.L. GOWARI AND D.G. HINGOLE*
Department of Plant Pathology, College of Agriculture, Badnapur Dist. Jalna – 431 202 (MS)
The studies were carried out on collar rot caused by Sclerotium rolfsii Sacc. on betelvine (Piper betle L.) during 2015 at department of plant pathology, College of Agriculture Badnapur, Vasantrao Naik Marathwada Krishi Vidyapeeth, Parbhani. The in-vitro evaluation revealed highest average mycelial growth inhibition with systemic fungicides, carboxin and hexaconazole (100% in both), cymoxanil+ Mancozeb (92.26 %), carbendazim (90.64%) and least inhibition rate was recorded in thiophanate methyl (70.69%). While mancozeb recorded maximum growth inhibition (100%) that differed significantly with other non-systemic fungicides. Trichoderma viridae among the bio agents and Allium sativum among the botanicals significantly inhibited the myceial growth of the pathogen to the tune of 82.47 per cent and 100 per cent respectively. In the integrated management of disease study, significantly highest percentage of successful cuttings were recorded with treatment carbendazim + mancozeb + garlic extract + T. harzianum (80.17%) as compared to rest of treatments.
Sclerotium rolfsii, fungicides, plant extracts, bio-agents, betelvine.
Betelvine (Piper betle L.) is the Neglected Green Gold of India. It belongs to the family Piperaceae. It is perennial dioecious creeper. It is usually grown under shade by farmers. The plant is a climber having heart shaped deep green leaves and in India it is locally called as “Paan” in Hindi and “Nagveliche pan” in Marathi. The leaves of betelvine are regarded as excellent mouth freshener and routinely served on the social, cultural and religious occasions such as marriages, puja, sradha, ceremony etc.,. The leaves also helps to improve the digestive capacity when used with lime besides acting as blood purifier. The leaves of betelvine contains vitamins, enzymes thiamin, riboflavin, tannin, iodine, iron, calcium, minerals, proteins, essential oils and medicine for liver, brain, and heart diseases (Khanna, 1997).
Betelvine subjected to a number of diseases viz., root rot, foot rot, collar rot, leaf spot, powdery mildew, anthracnose, bacterial stem canker and root knot nematode. Among these diseases collar rot caused by Sclerotium rolfsii Sacc. is consider as serious disease responsible for causing huge loss of 25 -40 per cent in west Bengal (Maiti and Sen, 1982). The disease caused by this pathogen on different crops are difficult to control because of the production of sclerotia by pathogen. These sclerotia are considered to be extremely hard structures and relatively resistant survival structures.
The pathogen Sclerotium rolfsii Sacc. infests the stem and produce symptoms of darkening of the stem at the collar region of the plant near ground level, above or occasionally higher up in different vines. The leaves soon turn yellow and become flaccid and drop off, eventually whole vine wilts and dries up. The darkened portion of stem tends to shrink and becomes soft and slimy and the bark peels off easily. The colour of the darkened portion ultimately becomes black. White, ropy, fan- shaped mycelial strands creeps over stem portion, developing small light brown to dark brown sclerotia on the infected portion. The sclerotial initials are white at first, later turn brown.
Keeping in view economic importance, and yield losses caused by Sclerotium rolfsii in betelvine the present investigations were undertaken.
In-vitro evaluation of fungicides
Eleven different fungicides (seven systemic and four non systemic) viz., carboxin (Vitavax 75% WP), tridemefon (Bayleton 25% EC), carbendazim (Bavistin 50% WP), hexaconazole (Contaf 5% EC), thiophanate methyl (Topsin M 70% WP), cymoxynil+ mancozeb (Curzet70% WP), metalaxyl (Ridomyl 75% WP), captan (Captan 50% WP), copper-oxy-chloride (Blitox 50 % WP)
, bordeaux mixture (1% bordeaux mixture) and mancozeb (Dithane M -45 50WP) were tested each at a concentration of 500, 1000ppm (systemic) 2000 and 2500 ppm (non-systemic) against Sclerotium rolfsii by poisoned food technique (Nene and Thapliyal, 1993). Potato dextrose agar (PDA) medium amended with test concentration of fungicide was poured in Petri plates and inoculated at the centre with mycelial disc (5mm) from actively growing 5 days old culture of Sclerotium rolfsii. Un amended PDA medium served as control and each treatment was replicated thrice. The inoculated Petri plates were incubated at 27±1oc. Observations on radial mycelial growth/ colony diameters were recorded at an interval of 24 h and continued till untreated control plates were fully covered with growth of test pathogen. The per cent mycelial growth inhibition of test pathogen with test fungicides over untreated control was calculated by applying the formula given by Vincent (1927).
Per cent inhibition (I) = C T u100
C = Growth (mm) of test fungus in control plate.
T = Growth (mm) of test fungus in treatment plate
In vitro efficacy of bio-agents:
Six fungal antagonists viz., Trichoderma viride, T. harzianum, T. koningii, T. virens, T. hamatum, T. lignorum and two bacterial antagonist Pseudomonas fluorescens and Bacillus subtilis were evaluated in- vitro against Sclerotium rolfsii, by dual culture technique (Dennis and Webster, 1971). Seven days old cultures of the bio-agents and test fungus (Sclerotium rolfsii) grown on agar media were used for evaluation. Discs (5 mm) of PDA along with culture growth of the test fungus and bio-agents were taken with sterilized cork borer. The two culture discs, one of the test fungus and the other of the bioagent were placed at equidistance and exactly opposite with each other on solidified PDA medium in Petri plates under aseptic conditions and plates were incubated at 27+10C. Plates inoculated with culture disc of test fungus were maintained as untreated control. Observations on linear mycelial growth of test fungus and bio-agents were recorded at an interval of 24 hours and continued till untreated control plate was fully covered with mycelial growth of the test fungus. Per cent inhibition of the test
fungus over untreated control was calculated by applying the formula given by Arora and Upadhyay (1978) as follows.
Per cent growth inhibition=
Colony growth in Colony growth in
control plate intersecting plate u100
Colony growth in control plate
In- vitro evaluation of botanicals / plant extracts:
Plant extracts of eight botanicals viz. onion (Allium cepa), garlic (Allium sativum), neem (Azardirachta indica), tulsi (Ociumum sanctum), periwinkle (Catharanthus roseus), bougainvillea (Bougainvillea spectabilis), giripushpa (Gliricidia maculata), and nilgiri (Eucalyptus spp.) were evaluated in-vitro against S. rolfssi. Leaf extracts were prepared by grinding with mixer-cum grinder, the 100 g washed leaves, ginger rhizomes and garlic bulbs of each plant species in 100 ml distilled water and filtered through double layered muslin cloth. The filtrates obtained were further filtered through Whatman No. I filter paper using funnel and volumetric flasks (100 ml capacity). The final clear extracts filtrates obtained formed the standard plant extracts of 100 per cent concentration, which were evaluated @10 per cent and 20 per cent in- vitro against S. rolfsii applying poisoned food technique (Nene and Thapliyal, 1993) using potato dextrose agar as basal culture medium.
Observations on radial mycelial growth/colony diameter of the test pathogen were recorded treatment wise at 24 hours interval and continued till mycelial growth of the test fungus was fully covered in the untreated control plates. Per cent inhibition of mycelial growth over untreated control was calculated by applying the formula given by Vincent (1927) as follows
Per cent inhibition (I) = C T u100
C = Growth (mm) of test fungus in control plate.
T = Growth (mm) of test fungus in treatment plate.
Integrated evaluation of fungicides, botanicals, bio-agents and organic amendments
The identified effective fungicides, botanicals, bio-agents and amendments against S. rolfsii during in –vitro studies were selected for integrated management of collar rot (S. rolfsii) of betelvine in pot culture under screen house conditions. The earthen pots (30cm dia.) disinfected with 5 per cent solution of copper sulphate were filled with autoclaved potting mixture of soil, sand and FYM (2:1:1) The Mass multiplied (sand maize medium) inoculums of S. rolfsii was inoculated @ 50g/kg potting mixture, to the potting mixture in pots, mixed thoroughly watered adequately and incubate for two weeks in the screen house to proliferate the pathogen and make the soil/ potting mixture sick. The experiment comprised nine treatments (Table 5). All the treatments were replicated thrice. The most effective test fungicides and talc based formulations of bio-agents were applied (alone and in combinations) as pre- planting treatment (preventative)
to healthy betelvine cuttings and were planted (10 cuttings/ pot) in the earthen pots containing S. rolfsii sick soil/ potting mixture.
The powdered test organic amendment was pre amended (100 g/kg soil or potting mixture) in the earthen pots containing S. rolfsii sick soil mixed thoroughly, watered adequately and maintained in screen house. After 72 h, these pots were planted (10 cuttings of betelvine / pot) with the surface sterilized with Hgcl2 (0.01%), healthy cuttings of cv. local were planted. After 72 h of planting, crude extracts @ 20 per cent concentration of test botanical was drenched @ 50 ml/kg soil as curative treatment.
Surface sterilized (0.01% Hgcl2) healthy betelvine cuttings were planted (10 cuttings/pot) in sick soil/potting mixture. All these pots (treated and untreated) were watered regularly and maintained in the screen house for further observation The observations on per cent successful cuttings, mortality percentage at 15, 30 and 45
days after planting and per cent disease incidence were calculated.
The results indicated that all the systemic fungicides tested at 500 and 1000 ppm concentrations inhibited mycelial growth of S. rolfsii significantly over the control and the inhibition increased with increase in concentration of fungicide tested (Table 1).
At 500 ppm, radial mycelial growth ranged from 00.00 mm (carboxin and hexaconazole) to 28.39 mm (thiophanate methyl) as against 90.00 mm in untreated control. Highest mycelial growth was recorded with the fungicide thiophanate methyl (24.36 mm) followed by tridimefon (17.27mm) and metalaxyl (18.24 mm). Less mycelial growth was found in carbendazim (08.77 mm) and cymoxanil+ mancozeb (07.78 mm). Whereas, nil mycelial growth was found with carboxin and hexaconazole.
At 1000 ppm systemic trend of mycelial growth was observed and it ranged from 0.00 (carboxin and hexaconazole) to 24.36 mm (thiophinate methyl ), as against 90.00 mm in untreated control. Significantly highest mycelial growth was recorded with fungicides thiophanate methyl (24.36 mm) followed by tridemefon (17.27 mm) and metalaxyl (16.72 mm). While nil mycelial growth was recorded with carboxin and hexaconazole (00.00 mm in both), followed by cymoxanil+ mancozeb (06.13 mm) and carbendazim (08.07 mm) (Table 1).
At 500 ppm, per cent mycelial growth inhibition ranged from 68.45 (thiophanate methyl to 100 per cent (carboxin and hexaconazole). Among the tested fungicides, carboxin and hexaconazole were proved the most effective as it gave completely inhibited colony growth (100%). It was followed by fungicides viz., cymoxanil + Mancozeb (91.35%), carbendazim (90.26%) and metalaxyl (79.73%) . Thophanate methyl proved less effective and inhibited colony growth of 68.45 per cent.
At 1000 ppm, similar trend in mycelial growth inhibition was observed and it ranged from 72.93 per cent in thiophanate methyl to 100 per cent (carboxin and hexaconazole) Amongst the tested systemic fungicides
completely inhibited mycelial growth inhibition were recorded with fungicides carboxin and hexaconazole (100%) followed by other fungicides viz., cymoxanil + mancozb (93.18%), carbendazim (91.02%), metalaxyl (81.41%) while thiophanate methyl proved less effective among all the fungicides tested in inhibition of mycelial growth of S rolfsii (72.93%) (Table 1).
All non systemic fungicides tested at both concentrations significantly inhibited mycelial growth of S. rolfsii over untreated control and it increased with increase in concentrations (Table 2).
At 2000 ppm, radial mycelial growth of the test pathogen ranged from 55.85 (bordeaux mixture) to 00.00
mm (mancozeb) as against 90 mm in untreated control. However, significantly highest mycelial growth was recorded with the fungicide Bordeaux mixture (55.85 mm), followed by copper- oxy-chloride (38.17 mm) and captian (23.09 mm), while no mycelial growth (00.00 mm) was recorded with mancozeb treatment.
At 2500 ppm radial mycelial growth of the test pathogen ranged from 50.57 mm (bordeaux mixture) to 00.00 mm (mancozeb). Significantly highest radial mycelial growth was recorded with bordeaux mixture (50.57), followed by copper- oxy-chloride (30.96 mm) and captan (21.37 mm). While no (00.00 mm) mycelial growth was recorded with (mancozeb) (Table 2).
The results indicated that all the non-systemic fungicides tested at both the concentrations inhibited mycelial growth of S. rolfsii significantly over the control and its mycelial inhibition was increased with increase in concentration of the fungicides tested (Table 2).
At 2000 ppm, percentage mycelial growth inhibition ranged from 37.94 (bordeaux mixture) to 100 per cent (mancozeb). However, significantly highest mycelial growth inhibition was recorded with mancozeb (100 %), followed by, captan (74.34 %), and copper oxy-chloride (57.58%). Whereas, least inhibition was found in bordeaux mixture (37.94%).
At 2500 ppm, similar trend of mycelial growth inhibition with the test fungicides was recorded and it
ranged from 43.80 (bordeaux mixture) to 100 per cent (mancozeb). However, significantly highest of mycelial growth inhibition was recorded with mancozeb (100%). Followed by captan (76.25 %) and copper oxy-chloride (65.60 %) and bordeaux mixture (43.80%).
Similar fungistatic effects of fungicides (systemic and non systemic) against S. rolfsii infecting betelvine along with many other crops have been reported (Bhat and Shrivastav, 2003; Mundhe, 2005; Patil and Raut, 2008; Bindu and Bhattiprolu, 2011; Rather et al., 2012; Mahato et al., 2014 and Suryawanshi et al., 2015).
All the bio-agents tested exhibited fungistatic activity against S. rolfsii and significantly inhibited the mycelial growth of test pathogen over control.
Of all the treatments significantly lowest mycelial growth (15.75 mm) and highest mycelial inhibition (82.47 %) were recorded with Trichoderma viride (Table 3).The second and third best treatment found were T. harzianum (19.36 mm and 78.48%) and T. virens (20.50 mm and 77.22%)with mycelial growth and mycelial growth inhibition per cent. These were followed by the treatments viz., T. lignorum (20.90 mm and 76.77 %), T. koningii (25.54 mm and 71.62 %), Pseudomonas fluorescens (28.78 mm and 68.01 %), T. hamatam (33.58 mm and
62.69 %) and B. subtilis (59.51 mm and 33.87 %) with mycelial growth and mycelial inhibition, respectively.
Similar antagonist effect of bio-control agents were reported earlier against S. rolfsii and other pathogens (Agrawal et al.,1977); Henis et al. 1983; Tribhuvanmala et al.1999; Dutta and Das,2002 ; Tripathi and Khare, 2005; Banyal et.al. 2008; Patil and Raut, 2008; Bindu and Bhattiprolu, 2011; Kumar et al. 2011; Manu et al., 2012 and Suryawanshi et al., 2015).
Aqueous extract of all the eight botanicals at both the concentrations significantly inhibited mycelial growth of S. rolfsii over the control and it was found to increase with increase in concentration of the botanicals tested (Table 4).
At 10 per cent concentration, radial mycelial growth of S. rolfsii ranged from 00.00 (Allium sativum) to 72.77
mm (Gliricidia maculata L) as against 90 mm in untreated control.
It was significantly maximum with Gilricidia maculance (72.77mm),this was followed by the botanicals viz., Ocimum santum (68.00 mm), Bougainvillea spectabilis (64.89 mm), Catharanthus roseus (64.56
mm) and Allium cepa (61.33 mm). Comparatively minimum mycelia growth was recorded with the botanicals viz., Eucalyptus spp. (22.63 mm) and Azardirachta indica (18.65 mm).While no colony diameter of test pathogen was recorded with Allium sativum.
At 20 per cent, radial mycelial growth recorded with test botanicals was ranged from 0 0.00 mm (Allium sativum) to 71.37 mm (Gliricida maculatum). However, significantly highest mycelial growth was recorded with Gliricida maculatum (71.37 mm), this was followed by Catharanthus roseus (63.24 mm), Bougainvillea spectabilis (62.63 mm), Allium cepa (58.32 mm) and Ociumum sanctum. (56.48 mm). Comparatively minimum mycelial growth was recorded with the botanicals viz., Eucalyptus spp. (20.97 mm) and Azardirachta indica (16.81 mm). Whereas, nil mycelial growth of test pathogen was found with Allium sativum.
Results obtained on mycelial growth inhibition of S. rolfsii with the aqueous extracts of the test botanicals tested are presented in the (Table 4) revealed that all the botanicals tested (@10 and 20 % each) significantly inhibited mycelial growth of the test pathogen, over untreated control and it was increased with increase in concentration of the botanicals tested.
At 10 per cent, mycelial growth inhibition of S. rolfsii ranged from 19.13 (Gliricidia maculata) to 100 per cent (Allium sativum). However, significantly highest mycelial growth inhibition was found with A. sativum(100%). This was followed by the botanicals viz., Azardirchta indica (79.13%) and Catharanthus roseus (74.85%). While the botanicals viz., Allium cepa (31.84 %), Catharanthus roseus (28.25%) Ociuum sanctum. (24.43 %) and Gliricidia maculata (19.13 %) were found comparatively less effective in inhibiting mycelial growth of test pathogen.
At 20 per cent, mycelial growth inhibition of S. rolfsii ranged from 20.69 (Gliricidia maculata) 100 per cent (Allium sativum). However, significantly highest mycelial inhibition was recorded with A. sativum (100%).This was followed by the botanicals viz., Azardirachta indica (81.32 %) and Eucalyptus spp. (76.70 %). While minimum mycelial inhibition was recorded with Ocimum santum (37.24 %), Allium cepa (35.18 %), Bougainvillea spectabilis (30.40 %), Catharanthus roseus (29.73 %), and Gliricidia maculata (20.69 %).
Aqueous extract of botanical Allium sativum was reported earlier as antifungal against S. rolfsii and other pathogens (Shivapuri et al., 1997; Suryawanshi et.al., 2007; Patil and Raut, 2008 and Sultana et al., 2012).
The results revealed that there were significant differences in per cent disease incidence for management of collar rot of betel vine caused by Sclerotium rolfsii by utilizing fungal bio-agents, organic amendments, botanicals and fungicides. All the treatments significantly enhanced the percentage of successful cuttings and found effective in reducing mortality or percent disease incidence over untreated control (Table 5)
Significantly highest percentage of successful cuttings was recorded with the treatment carbendazim12 per cent
+ mancozeb 63 per cent + Garlic extract+ T. harzianum (80.17 %). The Second and third highest percentage of successful cuttings was recorded with the treatments viz., carboxin+ hexaconazole 5 EC + T. viride + neem seed cake and carbendazim 12 per cent + Mancozeb 63 per cent) (each 75.25% and 70.60% respectively). This was followed by Garlic extract (69.32%), T. viride (68.63%),
hexaconazole 5 EC (67.97%) and Neem seed cake (66.23%) which were at par with each other.
The collar rot disease recorded with all the treatments ranged from 8.95-16.54 per cent as against 23.58 per cent in untreated control. However the treatments, viz., carbendazim (12%) + mancozeb (63%) + Garlic extract+ T. harzianum, carboxin + hexaconazole 5 EC + T. viride were found with significantly lowest stem cutting mortality/ per cent disease incidence of 8.95, 10.77 per cent respectively. The treatments viz., garlic extract (15.44%), hexaconazole 5 EC (15.58%) and neem seed cake (16.54%) were found comparatively less effective with highest stem cutting mortality.
The percentage reduction in cutting mortality/ per cent disease incidence recorded with all the treatments and ranged from 27.58 to 60.44 per cent. However, carbendazim (12%) + mancozeb (63%) + Garlic extract+ T. harzianum, carboxin + hexaconazole5 EC + T. viride+ neem seed cake, carbendazim 12 per cent + mancozeb (63%) , carboxin and T.viride found most effective with significantly highest reduction in cutting mortality/ per cent disease incidence of 60.44, 52.77,49.54,48.29 and 43.01 per cent respectively. Garlic extract (28.83%), hexaconazole 5 EC (28.48%) and neem seed cake (27.85%) were found least effective with minimum reduction in cuttings mortality /percent disease incidence over untreated control. The results of present study obtained on the integrated bio efficacy of the fungicides, botanicals, bio agents and organic amendments against collar rot of betelvine and in other crops are in conformity with those reported earlier by several workers. Integration of fungigicides, botanicals with Trichoderma spp. and organic amendments were effective against S. rolfsii as reported earlier by Suryawanshi et al., 2005; Khosla and Gupta, 2005; Khode and Raut, 2010; Rather et al., 2012; and Tripathi, 2015.