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Seed biopriming with Trichoderma improves yield of Rajmash (Phaseolus vulgaris C.V. HUR-137) in Varanasi region of Uttar Pradesh

Plants of red kidney bean (Phaseolus vulgaris) were treated with different grades of recommended dose of fertilizer along with biopriming with Trichoderma harzianum. The treatment along with the inorganic fertilizer treatment was applied as an attempt to enhance the yield through the microbial agent Trichoderma. Treatment of red kidney bean seeds were treated with the Trichoderma isolates using powder (talc) formulation to promote growth and development of the bean plants in greenhouse conditions. Among the treatments the plants treated with full dose of fertilizer without any bio-treatments was found the best as per growth and development but the bean plants treated with inorganic fertilizers at 90% dose combined with biopriming was comparable and suggests that the use of bio-agents can be used significantly to improve the yield and growth of the crops which is reduced as a part of the treatments. Also, the bean plants treated solely with the bio-agent represented a very good rhizospheric growth without the use of any kind of inorganic inputs. The treatments bioprimed with Trichoderma showed a good growth despite a lower dose of nutrient applied.


Introduction
Common bean (Phaseolus vulgaris L.; 2n=2x=22) is a predominantly self-pollinated crop originated mainly in Latin America and spread to other parts of world and now it is widely cultivated in the tropics and subtropics as well as in temperate regions of the world (Gepts and Bliss 1988;Zeven 1997Zeven , 1999Kumar et al, 2008) [4,15,6] . In India, common bean is known by the names like 'Rajmash' and 'Frash bean (green bean)' and grows in certain parts of the country. Common bean production in India is 4,340 million tones as compared 18,943 million tons in the world. (Anonymous, 2003;Kumar et al, 2008) [1,6] . Bio-priming is a new technique of seed treatment that integrates biological (inoculation of seed with beneficial organism to protect seed) and physiological aspects (seed hydration) of disease control. Biopriming using naturally occurring soil microorganisms is a safe, non-polluting, and environmentally sound disease control measure and an effective pre-sowing seed treatment. There are some known beneficial bacteria and fungi which, if applied to seed prior to planting, may enhance plant growth and development. Biopriming allows rapid colonization of the beneficial organism on the seed, and often results in a more uniform coverage of the seed surface compared to other priming techniques (Smith, 1996;Warren and Bennett, 1997) [10,12] . Biopriming is a common practice in the seed industry worldwide. During the seed coating process, inoculants, fungicides, or insecticides are added together with other substances, which provide a protective barrier on the seed. Biopriming has been shown to be more effective than regular chemical treatment for disease control (Ashraf and Foolad, 2005) [2] . Seed are coated with bacteria and soaked in warm water until their moisture content rises to 35-40%, the term ''biopriming'' is used. The stem diameter, root and foliar dry weight parameters suggested a positive effect of T. koningiopsis Th003 on tomato growth response after transplant as well. These results are in agreement with an early report in cucumber plants inoculated with T. harzianum T-203 (Moreno et al, 2009). The shoot fresh and dry weight as well as yield was enhanced by nonautoclaved Trichoderma WT inocula applied at 1% and produced with a fermentation time of 6 or 12 days in lettuce (Ousley et al, 2013). Bioagents Trichoderma enhance the seed treatment on germination and vigour in chick pea (Kumar et al, 2014) [5] . Single inoculations of common dry bean with selected Trichoderma species have been reported to increase plant dry growth and height (Yobo et al, 2009) [14] . Biopriming of chickpea and rajma seeds with Trichoderma, Pseudomonas and Rhizobium showed increased seed germination and accumulation of biomass in both the crops under both pot and field conditions (Yadav et al, 2013) [13] . Trichoderma isolates increases the height, dry weight and yield of plants as it is known to produce plant hormones like auxin, gibberellins and other volatile compounds (Contreras-Cornejo et al, 2009;Vinale et al, 2008) [3,11] .

Materials and Methods
The pot experiment was conducted during Rabi season of 2016-2017 in the net house of Department of Soil Science and Agricultural Chemistry, Institute of Agricultural Sciences, B.H.U. Varanasi, U.P. To conduct the pot experiment, bulk surface (0-15cm) soil was collected from the Agricultural Research Farm, Institute of Agricultural Sciences, Banaras Hindu University, and Varanasi. The soils of Varanasi are formed on alluvium deposited by the river Ganges and have predominance of illite, quartz and feldspars minerals. The alluvial soil is characterized by higher silt content. Illicit nature of alluvial sediments is clearly demonstrated by higher K 2 O content. The cropping history of the site from where the soil was collected is the rice-wheat. The fertility status of the soil can be included as low to medium with a slightly alkaline reaction. After collecting the soil, it was sieved and 10kg of soil was filled in each polythene lined earthern pot. Seeds of red kidney beans were first soaked in sodium hypochlorite solution for one minute. Rinse with sterilized water 4-5 times and kept for air drying or in laminar air flow. Spores of Trichoderma harzianum were collected in vials by adding 4-5ml of 0.85% saline water in the petri plate. In petri plate, autoclaved filter paper were spread and dried. Per treatment 50 seeds were taken in the petri plates. Then the microbial suspension were spread uniformly over seeds and kept for drying.

Experimental Details
The experiment has been under taken on alluvial soil at the Institute of Agricultural Sciences, Banaras Hindu University, during November 2016 to March 2017, under net-house conditions, bioprimed seed of kidney bean with microbial consortium along with different levels recommended fertilizer doses was grown. Weeding was done as soon as weeds emerge. Irrigation is given as per requirement of crop that keep soil moist throughout growth period. Fertilizers applied in recommended dose N: P: K @ 120: 60: 60 kg/ha, 1/2th N, Full P & K given at time of sowing and rest N given in 2 split dose at 25 DAS and 45 DAS. Pods were harvested after they were fully matured. The experimental design was an experiment under completely randomized block design with three replications (CRD).  It is clearly evident that application of RDF of N: P: K @ 100: 60: 25 kg/ha (T 2 ) significantly produced highest plant height at all growth stages of red kidney bean in comparison to lower doses of N:P:K addition. Among the treatments at 30 DAS, T 2 caused significantly higher plant height and this treatment was significantly superior over rest of the treatments. At 60 DAS significantly higher plant height was recorded with treatment T 2 (36.825cm, RDF of N: P: K @100: 60: 25 kg/ha) which is at par with T 5 and T 6 followed by T 5 (seed treatment with T. harzianum +90% RDF of N:P: K), T 4 (seed treatment with T. harzianum +80% RDF of N:P: K), T 3 (seed treatment with T. harzianum +70% RDF of N:P: K), T 6 (seed treatment with T. harzianum +RDF of N: P: K @ 0:0:0 kg/ha) and T 1 (control N: P: K @ 0:0:0 kg/ha). However, at 90 DAS significantly plant height was recorded with the treatment T 2 (35.675 cm) which is at par with T 5 , this was significantly superior than rest of the treatments. Significantly lower plant height was recorded with T 1 (control N: P: K @0:0:0 kg/ha).
At 90 DAS plant height ranged between, 36.33 to 63.00 and 44.50 to 47.12 cm in different treatments. In case of effect of T. harzianum and different graded dose of NPK application, plant height was recorded in following order: T 2 >T 5 >T 4 >T 3 >T 6 >T 1 in all the growth stages of red kidney bean. Result clearly illustrated that plant height of red kidney bean was boosted by the combined use of Trichoderma harzianum and N: P: K. The increase in plant height may be due to increased nutrient availability by root elongation and plant growth promoting activities by Trichoderma harzianum. It may also be ascribed due to adequate supply of nutrients due to mineralization of nutrients by increased population of Trichoderma harzianum. Enhanced plant height by Trichoderma may be due to production of secondary metabolites which may act as an auxin compound and other secondary metabolites such as harzianolide and, anthraquinone. Similar results also reported by Vinale et al. (2008) [11] in wheat, Molla et al. (2012) [7] .

Yield of red kidney bean
The effects of Trichoderma harzianum and soils type with graded dose of nitrogen application significantly influenced grain yield and are shown in table-4 and graphically represented in figure-2. Significant yield increases due to different treatment combinations were observed. All the treatments with graded NPK application along with seed biopriming with Trichoderma harzianum significantly increased grain yield of red kidney bean. Significantly maximum grain yield was recorded with T 2 (86.48 g pot -1 ) (RDF of N: P: K @ 100: 60: 25 kg/ha) followed by the treatments T 5 (55.39 g pot -1 ), T 4 (49.31 g pot -1 ), T 3 (40.92 g pot -1 ) , T 6 (32.87 g pot -1 ) and T 1 (23.98 g pot -1 ) i.e. control. Grain yield of rajmash ranged from 23.98 to 86.48 g plant -1 . Increase in grain yield by 62.50 g plant -1 in T 2 (RDF of N: P: K @ 100: 60: 25 kg/ha) over T 1 (Control 0:0:0 N and RDF of P: K) have been demonstrated. T 2 with maximum yield is statistically different from rest all the treatments. One of the good yielding treatment i.e. T 5 is statistically different from T 1 , similar to T 4 while at par with T 3 and T 6 . The increase in grain yield may be due to steady and slow release of nutrients to the plants (Figure 2). The overall result revealed that priming of red kidney bean seeds with inoculants of Trichoderma along with 90% RDF application gave better performance in all respect of yield and bio-control potential as compared to control and others and emerged as an alternative to full dose of RDF. These findings are in accordance with number of researchers (Geraldine et al., 2013;Verma et al., 2014) [16,17] observations: under varied agro-ecological niche.

Dry matter production
The data on dry matter production (DMP) are given in table 5 and depicted graphically in figure-3. The experimental results revealed that almost a consistent increase in the DMP occurred with the advancement of the crop growth stages and reaching the maximum at the maturity. The rate of increase in DMP was enhanced rapidly between 30 to 60 DAS. It is clearly evident that application of RDF of N: P: K @ 100: 60: 25 kg/ha (T 2 ) produced lucidly higher DMP/plant than lower doses at all the growth stages. Among the treatments at 30 DAS, T 2 caused significantly higher dry matter production (0.225g plant -1) and this was significant over rest other treatments.T 2 had maximum dry matter production which is significantly different from T 6 and at par with treatments T 5 ,T 4 and T 3. T 1 had minimum dry matter production with 0.122 g plant -1 .
At 60 DAS significantly higher dry matter production was recorded in treatment T 2 (0.3067g plant -1 , RDF of N: P: K @ 100: 50: 25 kg/ha) which is significantly different from T 6 and T 3 while at par with T 4 and T 5 . Dry matter production followed by T 5 (seed treatment with T. harzianum +90% N and RDF of P: K), T 4 (seed treatment with T. harzianum +80%N and RDF of P: K), T 3 (seed treatment with T. harzianum +70% N and RDF of P: K),T 6 seed treatment with T. harzianum+ N: P: K @ 0:0:0 kg/ha and T 1 (control N: P: K @ 0:0:0 kg/ha). At 90 DAS the dry matter production increased as compared to 30 to 60 DAS and the significantly higher dry matter production was recorded with the treatment T 2 (1.425 g plant -1 ) which is statistically different from all other treatments but at par with T 5 (1.277 g plant -1 ) and was significantly higher than rest of the treatments. Lowest dry matter production was recorded with T 1 (control N: P: K @ 0:0:0 kg/ha). At 90 DAS dry matter production ranged between 1.0175, 1.425, 1.1275, 1.21, 1.2775 and 1.21 g plant -1 respectively in different treatments. Result clearly illustrated that dry matter production of red kidney bean plant was boosted by the combined use of Trichoderma harzianum and N: P: K. The increase in dry matter of shoot may be due to increased root dry matter enabling large volume of soil exploitation of the plant which could increase the chance for nutrients uptake through maximum access to use mineral nutrients. It may also be ascribed due to adequate supply of nutrients due to mineralization of nutrients by increased population of Trichoderma harzianum. Significant increase in dry matter of tomato plant by the combined use of biofertilizer and N: P: K (Molla et al., 2012) [7] . None of the interaction was found significant in this respect during the present experimentation. Trichoderma spp. enhancing plant growth has been reported in several crop plants and has been attributed to auxin (Contreras-Cornejo et al., 2009) [3] . Besides, nutrient acquisition is improved enhancing indirect growth promotion.

Conclusion
The research data supports the points that biopriming of the crop with Trichoderma harzianum improved the yield, plant height and dry matter production of red kidney bean. This happens because of the fact that the fungus produces plant growth regulators such as auxin and other volatile organic compounds which results in.