Antifungal Activity of Botanicals and Biological agents against Sclerotium rolfsii Causing Foot and Root Rot Disease of Lentil

Lentil (Lens culinaris) is the second most important pulse crop in Bangladesh (Ali et al., 2003). Pulses are the cheapest source of proteins and Indians fulfill 20 to 30 per cent of their protein requirement from pulses (Grover and Singh, 2015).

Lentil suffers from the attack of a number of seed borne diseases such as vascular wilt, collar rot, root rot, stem rot, foot and root rot, rust, powdery mildew and downy mildew caused by Fusarium oxysporum f. sp. lentis, Sclerotium rolfsii, Rhizoctonia solani, Uromyces fabae, Erysiphe polygoni and Peronos poralentis, respectively (Khare et al., 1979). The foot and root rot of lentil caused by S. rolfsii is very destructive disease of the crop causes reduction in plant stand and ultimately seed yield. Due to attack of S. rolfsii develops necrotic lesions on the stem base of lentil plants at any growth stage. The lesion girdles stem causing upper plant parts to become chlorotic and wilted; plants become necrotic after they die; disease often causes a patchwork of symptomatic plants throughout a field with plants initially becoming chlorotic and finally dying; fungus causes characteristic white lesions on stems which may be covered in a fluffy white growth during periods of wet weather.

Management of fungal disease using synthetic fungicides is not so effective because fungus displays strong ability to survive in soil through the formation of spherical sclerotia that have strong resistance to chemicals. Synthetic fungicides have potential harmful effects on human health and the environment. Therefore, search for alternative disease control strategies like plant based formulations are considered as promising alternates to synthetic fungicides (Amin et al., 2013 and Darvin, 2013).

Biocontrol agents like Trichoderma harzianum, T. viride  and T. asperellum may be effective to control S. rolfsii the causal fungus of foot and root rot of pulse crops.  Trichoderma is the most widely known biocontrol agent in agriculture and several species have success-fully been used against important fungal plant pathogens. Trichoderma have gained considerable recognition as biological agent. Several strains of Trichoderma have been found to be effective as bio-control agents against various soil borne plant pathogenic fungi such as Fusarium, Sclerotium and  Rhizoctonia (Chet and Invar, 1994).

The present study was conducted to investigate the inhibitory effect of botanicals and biological agents on in vitro mycelial growth of Sclerotium rolfsii, the causal fungus of foot and root rot of lentil. The specific objectives of the investigation were as follows:
(a) To isolate, purify and identify S. rolfsii causing foot and root rot of lentil isolated from infected plants.
(b) To evaluate the efficacy of selected botanical extracts and compost and liquid formulations of T. harzianum and T. viride to inhibit mycelium growth of S. rolfsii.

Isolation, purification and identification of Sclerotium rolfsii
 
Isolation of S. rolfsii was done following tissue planting method (Tuite, 1969). Lentil plant specimens having typical symptoms of foot and root rot disease were collected from the experimental fields of Regional Agricultural Research Station (RARS), Ishurdi, Pabna, Bangladesh during rabi season of 2015. The collected samples were put in polyethylene bags and brought to the laboratory of Pulse Research Center, RARS, Ishurdi, Pabna, Bangladesh. Later the specimens were washed with tap water to make free from soil particles. Diseased specimens were cut into small pieces (0.5 cm) along with a portion of healthy tissues and surface sterilized with chlorox (1.0% sodium hypochloride solution) for one minute. Surface sterilized plant pieces were rinsed with sterilized water thrice and placed on filter paper to remove excess water. Three pieces of diseased specimens were plated on solidified PDA in Petri dishes maintaining equal distances under sterile conditions. The plates were incubated at room temperature and observation was made regularly to record growth of S. rolfsii from the plant tissue. The fungus was transferred to fresh PDA plates. The fungus was purified following hyphal tip culture method and identified using appropriate Key Book (Booth, 1971). Pure culture of the fungus was multiplied on PDA in test tube slants and preserved at 5±1°C for future use.
 
Comparison of in vitro colony growth of isolates of Sclerotium rolfsii
 
Seven isolates of S. rolfsii were isolated from infected lentil plants. The isolates of S. rolfsii were designated as Sc-7, Sc-12, Sc-13, Sc-27, Sc-33, SC-35 and Sc-38 where Sc represents first two letters of the genera Sclerotium and number represents serial number of isolates. Colony growth of the isolates of S. rolfsii was compared on fresh PDA plates incubated at 25°C. The colony diameter was measured using a ruler two days after incubation. In this study, three replicates used for each isolate (Punja and Damiani, 1996). The fast growing isolate (Sc-38) was selected as a test fungus for in vitro test of plant extracts as well as Trichoderma spp.
 
Collection of botanicals and Trichoderma spp.
 
Leaves of Allamanda (Allamanda carthertica),Tulsi (Ocimum basilicum), Creat (Andrographis paniculata), Khokhssha (Ficus glumerata), Henna (Lawsonia inermis), Neem (Azadirata indica), Tobacco (Nicotiana tabacum) and Bashok (Justicia adhatoda), seeds of Black cumin (Nigella sativa), Jute (Corchorus capsularis) and Mahogoni (Sweteria macrophylla), rhizome of Zinger (Zinger officinalis) and cloves of Garlic (Allium sativum) were collected from Pabna and Rajshahi districts of Bangladesh. The plant materials was chopped into small pieces, 50 ml of sterilized water was added and ground in a wiring blender. The sap thus extracted was first passed through four layers of muslin cloth and then filtered through Whatman filter paper no. 2 into 250 conical flasks. The mouth of the flask was closed with aluminum foil until use. The extracts were used as crude aqueous extract for evaluating their antifungal activity.

Both compost and liquid formulations of T. harzianum and T. viride were collected from Natore Development Society, Natore, Bangladesh. To prepare 15% liquid formulation of T.  harzianum and T. viride, 15 ml liquid was mixed with 85 ml distilled water in 100 ml sterilized conical flask and shaken well. To prepare 15% extract of compost, 15 g compost of T.  harzianum and T. viride were mixed with 85 ml distilled water in 100 ml sterilized conical flask and shaken well. The mixtures were first passed through four layers of muslin cloth and then filtered through Whatman filter paper no.2 into 250 conical flasks. The mouth of the flask containing diluted liquid or extract of compost of T.  harzianum and T. viride was closed with aluminum foil until use.
 
Amendment of PDA with botanicals and biocontrol agents
 
Potato Dextrose Agar (PDA) medium was prepared by mixing extract of 200 g peeled potato slices, 15 g dextrose, 20 g agar and distilled water to make the volume 1000 ml. The mixture was cooked on a hot plate for melting the agar properly.  To prepare 15% (v/v) plant extracts medium, 150 ml liquid PDA was poured into 250 ml sterilized conical flask and 27 ml of plant extract was poured into each conical flask with the help of pipette. After thorough mixing with the extract, the medium was sterilized at 121°C under 1.1 kg/cm2 pressure for 20 minutes in an autoclave. After sterilization, PDA amended with plant extracts was poured into 90 mm Petri dishes at approximately 20 ml/dish. The pH of the medium was adjusted to 6.5 using pH meter with the help of 1N HCl and 1N NaOH. The amended PDA was poured into Petri dishes (9 cm) at 20 ml per dish. The Petri dishes containing PDA were autoclaves. After solidification, 15% liquid or 15% compost extract of T.  harzianum and T. viride were poured onto surface of dishes containing PDA for absorption. After full saturation of PDA with bio-control agents, the plates were used for the antifungal test. The plates having unamended PDA served as control.
 
Antifungal test of plant extract and biocontrol agents
 
After solidification of PDA, the plates were inoculated with 5 mm mycelial disc of S. rolfsii. One disc of test fungus (Sc-38) was placed upside down in agar media at the center of the petriplates and incubated at room temperature. The plates were arranged on the laboratory desks following completely randomized design (CRD) with three replications. Mycelial growth of the fungus was measured by taking the diameter in two directions and the average was computed. The growth reading was recorded at 3, 6, 9, 12 and 15 days after incubation.
 
Statistical analysis
 
The data were analyzed statistically. Analysis of variance was computed following CRD according to Gomez and Gomez (1984). Analysis was performed using MSTAT-C computer package. Mean differences were compared following Duncan´s Multiple Range Test.

Comparative growth of the isolates of Sclerotium rolfsii
 
Comparative radial mycelium growth of seven isolates of S. rolfsii on fresh PDA is shown in (Table 1). The maximum colony diameter was found for Sc-38 at all stages of data collection. At 3 and 6 DAI, the colony diameter ranged 1.70-5.57 and 4.37-8.53 cm, respectively. Significantly the highest colony diameter was recorded from Sc-38 at these two stages compared to other isolates. At 9 DAI, the colony diameter of different isolates varied from 5.67 to 9.00 cm. The radial colony diameter of seven isolates ranged 6.83-9.00 and 8.00-9.00 cm at 12 and 15 DAI, respectively. The lowest growth was found in Sc-33 which was statistically similar to Sc-13 at 15 DAI. Banakar et al., (2017) observed the maximum colony diameter (9.0 cm) of S. rolfsii in Potato dextrose agar (PDA) within 48 hours of incubation. Hussain et al., (2003) found the maximum radial growth (8.5 cm) of S. rolfsii within 7 days of inoculation in cornmeal agar media. 



From the results of this experiment, it was evident that isolate Sc-38 of S. rolfsii was a fast growing fungus, which was selected as the test fungus to study efficacy of botanical extracts and biocontrol agents on radial colony growth of S. rolfsii (Table 1).
 
Efficacy of botanical and biocontrol agents to inhibit in vitro growth of Sclerotium rolfsii
 
In control (PDA without any amendment), the radial colony diameter of S. rolfsii was 3.83, 7.43, 9.00, 9.00 and 9.00 cm at 3, 6, 9, 12 and 15 DAI, respectively. The radial colony diameter on PDA amended with thirteen botanical extracts ranged 0.00-8.67, 0.83-9.00, 4.27-9.00, 5.50-9.00 and 6.00-9.00 cm at 3, 6, 9, 12 and 15 DAI, respectively. Among the botanicals only Black cumin significantly inhibited colony growth of S. rolfsii at all stages and significant inhibition was also obtained with Tulsi extract at 3, 6 and 9 DAI compared to control. Amendment of PDA with extracts of Garlic, Henna and Zinger reduced the colony growth to 0.67, 1.47 and 2.82 cm at 3 DAI which were significant compared to respective control. On the contrary, the radial mycelial growth was increased to 5.22, 5.17, 67.67, 8.00 and 8.67 cm due to amendment of PDA with extracts of Allamanda, Creat, Jute seed, Mahogani and Basak compare to control. Diameter of S. rolfsii under control and other treatments was statistically similar. At 12 and 15 DAI, colony diameter under control and treatments with all botanical extracts was also statistically similar except only Black cumin (Table 2). Amin et al., (2013) tested different plants viz., rhizome ginger, neem leaf, tobacco leaf and rhizome of turmeric. All plant extracts inhibited the growth of S. rolfsii at higher concentration, while rhizome of turmeric inhibited the growth at a low level. Bangladesh is enriched with high plant diversity so many desirable antifungal compounds are isolated from various parts like root, leaf and stem (Jahan and Rahman, 2022).

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Amendment of PDA with compost and liquid formulations of T. harziznum and T. viride significantly reduced the colony diameter of S. rolfsii compared to control at all stages of measurement. The colony diameter ranged 1.50-2.97, 1.50-3.08, 1.33-2.95 1.33-2.92 and 1.33-2.92 cm under different amendment with two biocontrol agents at 3, 6, 9, 12 and 15 DAI, respectively. Efficacy of four treatments biocontrol agents was better than all treatments with botanicals (Table 2). Radwan et al., (2006) reported that T. harzianum and T. hamatum were most effective against S. rolfsii. Patro and Madhur (2013) observed that T. harzianum inhibits in-vitro mycelial growth of S. rolfsii,which cause foot rot in finger millet. Khalid (2013) isolated four bioagents viz, Bacillus subtilis, Pseudomonas, yeast and T. viride which inhibited damping-off disease of bean caused by S. rolfsii. Hooda et al., (2008) and Darvin et al., (2013) selected three species of Trichoderma (T. viride, T. harzianum, T. longibrachiatum) for inhibition of radial growth of S.rolfsii. Trichoderma viride and T. harzianum have highest radial growth inhibition and T. longibrachiatum has lowest radial growth inhibition of S. rolfsii using dual culture technique in vitro. Basumatary et al., (2015) and Swathi et al., (2015) reported that T. harzianum and T. virens were more active against S. rolfsii with 100% inhibition under in vitro condition.

Based on the results of present experiment, the botanical extracts of Black cumin seed and Tulsi leaf were found effective against growth of S. rolfsii. Liquid and compost formulations of T. harziznum and T. viride were found prospective bio-control against S. rolfsi. The extracts of botanicals and bio-control agents may be effective to control foot and root rot disease of lentil.