Management of Root Rot of Fenugreek Caused by Rhizoctonia solani using Plant Extracts and Bio-agents

Fenugreek (Trigonella foenum-graecum L.) vernacularly known as ‘Methi’ has tremendous significance as a food, medicine and belongs to family Fabaceae. It is commercially cultivated for seeds, which are used as condiments and as a flavoring agent for relishing food preparations. During 2017-18, India produced 2,02,000 tonnes of fenugreek from 1,49,000 ha area (Anonymous, 2018), whereas Rajasthan produced 1,13,432 tonnes from 97,866 ha area with an average productivity of 1,159 kg/ha (Anonymous, 2017-18).  The seeds of fenugreek contain essential steroid ‘diosgenin’ which is used in preparation of contraceptives. In general, fenugreek stimulates the digestive process as well as metabolism. In industry, seeds are utilized for mining of alkaloids and steroids while dried leaves and flowers are used for flavoring vegetable curries in Indian cuisine (Arya, 2000) and also used for manufecturing of pickles, soap and cosmetics (Choudhary et al., 2013). Fenugreek leaves and seeds are consumed in different regions of the world in various forms and has been recognized for treating several ailments known to man (Laila et al., 2013) and its oil have 16 fatty acids including linoleic acid and linolenic acid and lipids which are advantageous for health (Rathore et al., 2017).
               
Fenugreek is attacked by many fungal diseases including root rot (Godara et al., 2010). Among them, root rot caused by Rhizoctonia solani has become an important constraint to the cultivators of fenugreek in Rajasthan as well as India. It also known as damping off of fenugreek is one of the most significant diseases which reduce the yield of the crop significantly. Damping off is the typical symptoms of the disease at early stage of the crop. The newly emerged seedlings fall over and die, as post- emergence mortality in severely infected fields. Symptoms may also develop as foot rot and reddish brown cankers on root and stem near the ground level. The pathogen chiefly attacks the root and underground parts, but it is also capable of infecting to green foliage, seeds and hypocotyls (Acharya et al., 2014). The development of roots of infected plants is either poor or not formed or rotted. Plants show yellowing and stunted growth and can easily be pulled out. In field of Rajasthan, the incidence of root rot of fenugreek was noted up to 20.00 per cent in Jaipur district (Anonymous, 2007-08). Singh and Rao (2015) observed 34.67 per cent incidence of root rot of fenugreek caused by Rhizoctonia solani with yield loss of 55.26 per cent from Chhattisgarh region of India. In Karnataka, Rani and Hedge (2017) recorded 48.35 per cent root rot incidence in fenugreek incited by Rhizoctonia solani.
               
Earlier, Haque and Ghaffar (1992) tried some bio-control agents (T. harzianum, T. pseudokoningi and T. hamatum) by applying through seeds and soil for controlling Rhizoctonia root rot of fenugreek and observed positive response in reducing disease. Patole et al., (2016) tested the efficacy of plant extracts against R. solani causing root rot disease of soybean and minimum incidence was recorded in seed treatment with botanical extracts viz., Zingiber officinale (31.48) followed by Allium sativum (35.18) and Azadirachta indica (40.18).
               
Exclusive dependency on fungicides and their indiscriminate use for managing diseases of various crops has resulted in residue, environmental hazards and development of resistance in pathogens. Due to increasing consciousness in consumers about organic produce, particularly medicinal products, efforts are being diverted to manage the diseases through eco-friendly approaches as a tool for integrated disease management because they do not cause bio-accumulation in eatables and environmental pollutions. Involvement of plant extracts and bio-agents in disease management system is also an important segment of disease control, looking to the hazards caused by toxic chemicals or in a situation where pathogens develop resistance to fungicides. In lieu of this, the present investigation was undertaken to evaluated different plant extracts and bio-agents alone and in combination in field for two consecutive years (2016-17 and 2017-18) for managing the disease and increasing yield recovery for the profit of growers and consumers.

The experiments were conducted in laboratory and field during 2016-17 and 2017-18 at Department of Plant Pathology, SKN College of Agriculture, Jobner, Jaipur (Rajasthan) Jobner, is situated at latitude 26°5” N, longitude of 75°20” E and altitude of 427 meters above MSL (mean sea level). This region falls under semi-arid eastern plain (Agro Climatic Zone- lll A) of Rajasthan.
 
Efficacy of plant extracts in vitro
 
In recent years, many phyto-extracts are being used as fungitoxicants for the management of various plant diseases. The present investigation was carried out using six different natural phyto-extracts to see their antimycotic behaviour on the growth of Rhizoctonia solani in vitro and in vivo. The crude extracts of neem leaves (Azadirachta indica), Alstonia leaves (Alstonia scholaris), garlic cloves (Allium sativum), datura leaves (Datura stramonium), tulsi leaves (Ocimum tenuiflorum) and aak leaves (Calotropis gigantea) were screened at 5, 10 and 15 per cent concentration through poisoned food technique.
Preparation of the plant extract
 
Plant materials such as fresh leaves/cloves of botanicals under test were harvested and thoroughly washed with tap water. Hundred grams from each plant was collected and washed 2-3 times with water and allowed to dry at room temperature (25±1°C) for six hours. Before extraction, leaves of each plant (100g) were crushed separately with 100 ml sterilized distilled water. The extract was filtered through muslin cloth and centrifuged at 5000 rpm for 15 min. The extracts were then sterilized by passing them through a Millipore filter using a swimming filter adapter. The supernatant obtained was considered as 100 per cent and diluted accordingly and stored at 5°C for further use.The extract of each plant species was diluted in order to achieve three concentrations viz., 5, 10 and 15 per cet. Petri plates containing PDA supplemented with different phyto-extracts, each with three concentrations and replicated three times. Plates were inoculated with 7 days old culture (5 mm dia. disc). A suitable check was also maintained. Fungal colony was measured after 7 days of incubation at 25±1°C. The linear growth of test fungus was recorded and per cent growth inhibition was calculated by Vincent’s (1947) formula:
                                 
Whereas,
C= Diameter of the colony in check (average of both diagonals), T = Diameter of colony in treatment (average of both diagonals).
 
Effect of plant extracts on disease incidence and seed yield (in vivo)
 
The field trial was conducted during winter season of 2016-17 and 2017-18 in randomized block design with three replications in 2 m × 2 m plots. The seeds of fenugreek (var. RMT-305) were soaked for 30 minutes and dried under shade. Untreated seeds sown under similar conditions served as control. Inoculum, multiplied on sterilized sorghum grains, was added @ 20 g/m row length at the time of sowing in the field. All the recommended agronomic practices were followed to raise the crop. In both the years, the experiment was sown in the first week of November. Disease incidence was recorded at 60 days after sowing (DAS) by counting diseased plants and seed yield per plot was also recorded and calculated in kg/ha at harvesting. Per cent disease incidence (PDI) and per cent disease control was calculated as per following formulae.
 
Effect of bio agents on disease incidence and seed yield (in vivo)
 
The field trial was conducted during winter season of 2016-17 and 2017-18 in randomized block design in 2 m × 2 m plots. The seeds of fenugreek (var. RMT-305) were treated with commercial formulations of three bio-agents alone and in combination by applying as seed treatment. Four replications of each bio-agent were maintained. Before applying bio-agent on the surface of the seeds, seeds were moistened with five per cent gum solution applied at 10 ml/kg of seeds. The bio-agents were used as seed treatment @ 6 g/kg seeds alone and 3+3 g/kg seeds with combinations. In both the years, crop was sown in the first week of November. Disease incidence was recorded at 60 DAS by counting diseased plants and seed yield per plot was also recorded and calculated in kg/ha at harvesting.

Effect of plant extracts on mycelial growth under in vitro conditions
 
Six plant extracts were tested at 5, 10 and 15 per cent concentration on mycelial growth of Rhizoctonia solani through poisoned food technique. All the plant extracts significantly inhibited the radial growth of the fungus at the tested three concentrations and statistically differ to each other and also over control (Table 1). Plant extracts of garlic, aak, neem and datura were found highly fungitoxic than others. The garlic clove extract was found significantly superior in inhibition of mycelial growth (79.52%) followed by aak extract (62.48%), neem extract (53.37%), datura extract (40.89%) and tulsi (23.40%). Alstonia leaves extract was found least effective (17.37%) in inhibition of mycelial growth. Our results are in agreement with the findings of Sinha and Tripathi (2009) and Srinivas et al., (2013) who have recorded garlic and neem as an effective plant extract in maximum inhibition R. solani of sheath blight of rice. Sharma et al., (2018) also observed maximum inhibition of mycelia growth (71.85 %) with clove extract of garlic against Rhizoctonia solani causing sheath blight of rice.
 

 
Effect of plant extracts on disease incidence and seed yield (in vivo)
 
Different plant extracts was tested by seed soaking method (10%) for 30 minutes against root rot of fenugreek under field conditions. All the tested plant extracts caused significant reduction in incidence of root rot disease and increased seed yield of fenugreek as compared with untreated check (Table 2). Results of two years pooled analysis showed maximum reduction in disease incidence (62.02%) with garlic clove extract followed by aak extract (56.56%), neem extract (52.46%), datura extract (44.20%) and tulsi extract (38.90%) over control. The least effective was Alstonia leaves extract (33.19%). Statistically, garlic with aak, aak with neem, datura with tulsi and tulsi with Alstonia were found at par to each other.
 

               
Two years pooled analysis of seed yield data indicated that highest yield was obtained with garlic extract (1696.00 kg/ha) followed by aak extract (1639.00 kg/ha), neem extract (1597.50 kg/ha), datura extract (1511.00 kg/ha) and tulsi extract (1446.50 kg/ha) with increased seed yield (65.38, 59.82, 55.78, 55.78 and 47.34%, respectively) over control. Our results are in accordance with the findings of Patole et al., (2016), who tested the efficacy of some plant extracts against R. solani causing root rot disease of soybean and minimum incidence was recorded in seed treatment with botanical extracts viz., Zingiber officinale (31.48%) followed by Allium sativum (35.18%) and Azadirachta indica (40.18%).
 
Effect of bio-agents on disease incidence and seed yield (in vivo)
 
Treatment with antagonistic agents, alone and/or in combination, caused significant reduction in root rot incidence and increment in seed yield of fenugreek compared with untreated control. Results of two years pooled analysis (Table 3) showed that maximum disease reduction (66.81%) was obtained by combined application of Trichoderma harzianum + Pseudomonas fluorescens (3+3 g/kg) followed by T. viride + P. fluorescens (64.17%), T. harzianum (56.14%) and T. viride (52.25%) over control. Statistically, T. harzianum + P. fluorescens and T. viride + P. fluorescens were found at par to each other. Two years pooled analysis of seed yield data also indicated that highest yield was recorded with T. harzianum + P. fluorescens (1741.00 kg/ha) followed by Trichoderma viride+ P. fluorescens (1706.00 kg/ha) and T. harzianum (1627.50 kg/ha) with increased seed yield (73.06, 69.58 and 61.78%, respectively) over control. P. fluorescens was found least effective (1504.50 kg/ha). Research findings of earlier workers with bio-control agents have also been proved to be very effective especially against soil borne pathogens. Our observations are in slight authentication with the findings of Haque and Ghaffar (1992), who observed effectiveness of bio-agents in controlling root rot of fenugreek caused by Rhizoctonia solani with T. harzianum, T. pseudokoningi and T. hamatum by applying through seed and soil. Jetiyanon and Kloepper (2002) proposed a combinational use of different bio-control agents for improved and stable bio-control agents against a complex of diseases. Manjula et al., (2004) have also been reported improved bio-control activity of P. fluorescens GB 27 + T. viride pq1 against stem rot disease in groundnut in field conditions.
 

In conclusion, seed soaking with garlic clove extract (@ 10%) for 30 minutes or seed treatment with Trichoderma harzianum + Pseudomonas fluorescens (@ 3+3 g/kg seeds) can be utilized to manage the disease effectively and also to obtain organic produce.

The authors are highly grateful to The Dean, SKN College of Agriculture, Jobner and The Head, Department of Plant Pathology, SKN College of Agriculture, Jobner, (Jaipur), Rajasthan for providing all the essential facilities to conduct experiments successfully.