Source of resistant against Fusarium wilt and Stemphylium blight in lentil (Lens culinaris Medikus)

The crop yield of lentil is below attainable levels which are mainly attributed to pathological factors especially lentil wilt caused by Fusarium oxysporum f.sp. lentis and Stemphylium blight caused by Stemphylium botryosum .Fusarium wilt is a potential threat to letil production not only in Nepal but worldwide that can cause complete crop failure. While stemphylium blight is recently being serious problem to Bangladesh, Nepal and India. In Nepal, multiple disease resistance work was not reported in lentil before the present study. Two trials separately using 185 genotypes were conducted to study the wilt and stem phylium blight were conducted. For wilt screening one hundred eighty five lentil lines including resistant check ILL7164 and susceptible check sindur, were sown in a previously developed wilt sick bed, while for stem phylium blight serarate trial was conducted in the research area of Regional Agricultural Research Station Nepalgunj using augmented design. Natural inoculums were also relied upon. Disease severity data were recorded. Results revealed that incase of Fusarium wilt sixteen genotypes were found resistant and twenty-three moderately resistant while in case of Stemphylium blight 87 resistant and 36 moderately resistant in field condition. It also indicated that 9 genotypes i.e RL-13,RL-21,ILL6468,ILL9996,\ILL6024,ILL6811,ILL7164,Arun,Maheswar bharti showed combined resistant to both the diseases. Keyword: Lentil; Fusarium wilt; Stemphylium blight; Source of resistant; Combine resistant.


Introduction
Lentil (Lens culinaris fsp. culinaris Medikus) is affordable source of dietary proteins (22-35%), minerals, fiber, and carbohydrates to poor people and plays a vital role in alleviating malnutrition and micronutrient deficiencies in developing countries. It is highly recommended by physicians for the people suffering from diabetes, obesity, and cardiovascular diseases (Srivastava and Vasishtha, 2012). In fact, vegetable protein is gaining preference over the animal protein for consumption by the health conscious people in the present day. This could be one of the reasons for increased per capita consumption (Vandenberg, 2009) and fivefold increase in global lentil production (from 0.85 to 4.43 Mt) during the last five decades, through a 155% increase in cultivated area and the doubling of average yields from 528 to 1068 kg ha -1 (FAOSTAT, 2014) Lentil ranks first among pulse crops in Nepal. Its area and production is 2,05,939 ha and 2,26,830 metric ton respectively with productivity of 1,101 kg per hectare (MoAD, 2014). Its production seems adequate but current status of lentil is prone to a number of pathological threats including lentil wilt, stemphilium blight, collar rot and root rot in Nepal (Yadav, 2004) Among these diseases, lentil wilt caused by Fusarium oxysporum f.sp. lentis (Fol) is believed to be the most important constraint in lentil production worldwide (Taylor et al., 2007) . Lentil wilt is characterized by a sudden drooping of the leaves (more like wilting and damping off), followed by the leaves drying and the eventual death of the seedling. Apparently, the root system seems healthy but with a reduced proliferation and nodulation rate. Other symptoms at the seedling stage include seed rot (Kraft et al., 1981).) It is soil born disease causing 5-10% yield losses lentil worldwide but sometimes severe damage may result complete crop failure under favorable conditions of disease development (Chaudhary and Kaur, 2002), especially in a warm spring and dry / hot summer. Temperature and soil moisture are the main factors in determining fungal growth rates and symptom expression (Falahati et al., 2010).

Research Article
N.K. Yadav et al. (2017) Int. J. Appl. Sci. Biotechnol. Vol 5(1): 102-107 This paper can be downloaded online at http://ijasbt.org&http://nepjol.info/index.php/IJASBT Fusarium oxisporum f.sp lentis (Fol) is considered as warm-weather pathogen generally found in sandy and acidic soil. It can remain in the soil for up to 10 years. Fusariium oxysporum attacks plants and is most severe at air and soil temperatures of 24°C to 32° or 35°C (75° to 90° or 95°F. The optimal soil and air temperature for the pathogen is about 28°C (Eagling, 2009). The management of pathogen is difficult, because of its wide host range and ability to grow saprophytically or survive for extended periods in the form of thick walled chlamydospores in absence of a susceptible crop. Excessive use of chemical fungicides may apparently lower the density of pathogens in soil for short period of time but might give rise to mutant strains of pathogens with altered pathogenicity; making the previously resistant varieties of crops susceptible. Selection of durable resistant germplasm and effective breeding programs are needed to employ. Use of bio-control methods, crop rotations and genetically modified crops are some of the methods of disease management to curb down extreme use of chemicals on field to control pathogens Stemphylium blight cause by Stemphylium botryosum is a serious problem in some parts of the world especially in West Asia and South Asia, North Africa and widely distributed in Saskatchewan, Canada where it was considered to be a minor disease of lentil. The disease has been first time reported in Bangladesh by Erskine and Sarker (1997) and it was mentioned that it can cause 70% yield loss up or even total crop failure in epidemic years. It is reportedly spread by air borne conidia in the field. It overwinters on seed and as mycelium on dead stems and leaves in many crops left in the soil. Limited information is available on whether the pathogen is seed-borne in lentil or not (Bayaa and Erskine, 1998). In alfalfa, it spreads by airborne and water-borne conidia (conidia and ascospore) and by sowing infected seed. Airborne conidia of S. botryosum land on host tissue and germinate when conditions are favorable. Generally penetration occurs through stomata but Stemphylium spp. also produce the toxin stemphol that may aid host infection (Solfrizzo et al., 1994). Environment plays a major role in stemphylium blight disease development and that is why understanding the environmental role in disease development is important for effectively controlling the disease. The diverse host range S. botryosum that includes leguminous and non leguminous crops in different parts of the world indicates the adaptability of the pathogen to different environmental conditions. Most of the research on infection by Stemphylium spp. of different hosts has confirmed that temperature and moisture are the two most important environmental factors. In South Asia, temperatures of 18°-20°C and relative humidity over 85% have been reported to favor the development of disease (Erskine and Sarker, 1997). The pathogen requires at least 8 hr of wetness at low temperatures (10°C) for successful infection and infection increases with increased leaf wetness for 24h (Mwakutuya, 2006) The pathogen causes a leaf blight, plant defoliation and death. The disease is poorly understood but apparently there is some resistance available in the germplasm.
Use of resistant varieties is the only practical measure for controlling the disease in the field (Tamietti and Valentino, 2006). The search for sources of resistance to diseases is a primary and most eminent research for most of the work carried out in the past and also is continuing presently (Shankar et al., 2013).

Materials and Methods
The present investigation was carried out during winter growing season in 2012/13 at the crop field of Nepalgunj,The Latitude, Longitude and altitude of Nepalgunj is 28° 05' N, 81° 61' E and 181 masl respectively with1111mm annual rain fall. Two trials using 185 genotypes were conducted to study the wilt and Stemphylium blight separately. The experimental materials used in the experiment were collected from International Centre for Agricultural Research in the Dry Areas (ICARDA) and National Grain Legumes Research Program, Rampur (NGLRP ). Each germplasm line as one treatment was planted in road row design with 4 block.

Fusarium Wilt Screening Nursery
One hundred eighty five lentil genotypes/varieties including resistant check ILL7715 and susceptible check Sindur, were sown in a previously developed wilt sick plot, Debris of previous crops were incorporated to the soil to develop sick plot. Sindur (a highly susceptible line) was repeatedly planted after every two-test entries to increase the inoculums pressure of Fol under natural conditions. Plot size was of 1 row of 2 meter length. Crop was planted at 25 cm row to row and 5cm plant to plant spacing. Fertilizer was applied at the rate of 20:40:20 kg NPK /ha. . No insecticide and fungicide were applied. The weather conditions were highly favorable for disease development. Disease severity data were recorded. Disease reaction, on individual seedlings basis, was estimated using a 1-9 scale. 1=no symptoms (Highly resistant); 3=yellowing of the basal leaves only (resistant); 5=yellowing on 50% of the foliage (moderately susceptible); 7=complete yellowing of the foliage; 9=flaccidity of the top leaves, & partial drying (susceptible); 9=the whole plant or a unilateral shoot is wilted and/or dry (highly susceptible).
Observations of infected/wilted plant on plot basis were recorded at vegetative, and maturity stages following 1-9 scoring scale (Bayaa et al., 1997) Yadav et al. (2017) Int. J. Appl. Sci. Biotechnol. Vol 5(1): 102-107 This paper can be downloaded online at http://ijasbt.org&http://nepjol.info/index.php/IJASBT Stemphylium blight screening nursery One hundred eighty five lentil genotypes/varieties including Bari masur-4 as a resistant check and Shital as a susceptible checks were planted .Plot size was of 1 row of 2 meter length. Crop was planted at 25 cm row to row and 5cm plant to plant spacing. Fertilizer was applied at the rate of 20:40:20 kg NPK/ha. Debris of previous crops were incorporated to the soil to develop sick plot, Shital (susceptible check of SB) were repeatedly planted after two test entries. The lentil crop was raised by following recommended agronomic practice No insecticide and fungicide were applied. The weather conditions were highly favorable for disease development, particularly for Stemphylium blight.
Horsfall-Barrat's logarithmic scale had unequal intervals in disease scores and is difficult to use for quantitatively inherited traits. To overcome this problem Hashemi (2005) modified this scale to a 0-10 linear semi-quantitative scale. This scale considered disease development pattern consisting of the appearance of chlorotic spots followed by gradual defoliation of plants. 0=free of disease, 1=a few tiny tan spots, 2=few small to large chlorotic spots, 3=expanding lesions on leaves to defoliation started, 4=20% nodes on main stem showing necrotic symptoms and defoliation, 5=40% nodes on main stem showing necrotic symptoms and defoliation, 6= 60% nodes on main stem showing necrotic symptoms and defoliation, 7=80% nodes on main stem showing necrotic symptoms and defoliation, 8=100% leaves defoliate but small green tip recovering, 9=100% leaves defoliate but stem still green, 10= Completely dead). Kumar (2007) also used this scale (0-10) for stemphylium blight screening. Observations of infected plant on plot basis were recorded at vegetative, and maturity stages following 1-9 scoring scale suggested by Bayaa et al. 1997 for foliar diseases.

Conclusion
Based on findings of the present study, it is concluded that 9 genotypes i.e RL-13,RL-21, ILL6468, ILL9996, ILL6024, ILL6811, ILL7164, Arun, Maheswarbharti were found combine resistant to both the diseases. While DPL-62, Sagun resistant to wilt and moderately resistant to SB. Genotypes ILL6021, ILL6256, ILL9949, ILL8132, LN0137, ILL10045 were found resistant to S.B and moderately resistant to FW, while genotype ILL3490and ILL7980 exhibits moderately resistant to both the diseases. These genotypes could be used in future breeding program to get stable and higher yield of lentil in the country.