Evaluation of Endophytes for Enhancing the Growth and Seed Yield in Chickpea (Cicer arietinum L.)

Legumes are highly important crops in human and animal nutrition and are grown globally under a wide range of agro-climatic conditions as a cash crop and as a source of nitrogen assimilation via nitrogen fixation. Chickpea is cultivated in nearly 50 countries around the world and accounts for more than 20 per cent of the world pulse production and much of the world chickpea supply (70%) comes from India. India ranks first in area and production of chickpea in the world, with an area of 105.61 lakh ha (70.90% in world), production of 112.29 lakh tonnes (67.41% in world) and productivity of 1063 kg/ha (Anonymous, 2019). Madhya Pradesh is leading state in terms of area and production as it contributes around 34 and 40 per cent share to the total area and production of chickpea in the country (Anonymous, 2018). Karnataka is one of the major chickpea producing states in the country and it is grown mainly in rabi season in black soil areas particularly in the northern districts of the state viz., Vijayapur, Dharwad, Belagavi, Gadag, Bagalkote and Bidar. Even though India is the largest producer of chickpea, it still imports chickpea from Australia and several other countries. At present, India represents the biggest importer of chickpeas accounting for around one-fifth of the total global import volumes. Keeping in view, the ever-increasing demand for this legume crop, it is essential to improve the production and area under cultivation, at the same time minimizing the stress on this crop plant. Slower growth of developing seedlings under various abiotic factors or biotic factors limits the growth and yield of crops, development of techniques for fast and homogeneous growth of seeds could be a sustainable approach for better agricultural productivity (Osburn and Schroth, 1989). In this aspect, improving the quality and germination and establishment of good plant stand through ‘seed priming’ is a sustainable approach to enhance yields and performance of plants (McDonald, 2000).
       
The use of microbes for seed priming is a viable and promising approach in the context of improvement in seed characteristics under changing environmental conditions. Seed priming with the use of endophytic microbial strains appears as more beneficial or stable than rhizospheric microbial strains due to better colonization adaptability and suitability under biotic and abiotic stress conditions (Ajaykumar et al., 2019). Inoculation with plant growth promoting bacteria has been devised as a beneficial strategy for improving the fitness of crop plants especially under harsh environmental conditions.
               
Hence an attempt has been made to examine the effect of endophytes on crop performance in chickpea under pot culture and field condition.

Effect of endophytes on crop performance in chickpea under pot culture
 
The pot culture experiment was conducted in the green house of Agricultural College, Vijayapur during 2020-21. The soil having the properties of pH 6.5, EC 0.30 dsm^-1 available nitrogen 458 Kg/ha, available P₂O₅ 40 Kg/ha and K₂O 190 kg/ha was passed through a 4 mm sieve and mixed along with farmyard manure at 2: l proportion and filled in pots @ 10 kg/pot as detailed below:
 
Experimental details
 
T1:   Control.
T2:   Chickpea seeds treated with Bacillus thuringiensis.
T3:   Chickpea seeds treated with B. cereus strain LPDB2.
T4:   Chickpea seeds treated with B. cereus strain LPDB5.
T5:   Chickpea seeds treated with Rhizobium GV-2.GV-2.
T7:   Chickpea seeds treated with B. cereus strain LPDB2+Rhizobium GV-2.
T8:   Chickpea seeds treated with B. cereus strain LPDB5+Rhizobium GV-2.
T9:   Chickpea seeds treated with B. thuringiensis+B. cereus strain LPDB2+ B. cereus strain LPDB5+Rhizobium GV-2.
Replication: 3.
Design: Completely randomized design.
       
The endophytic bacteria (Bacillus thuringiensis, B. cereus strain LPDB2, B. cereus strain LPDB5) and Rhizobium were grown on nutrient broth for 48 h at room temperature. The bacterial cells harvested by centrifugation at 1000 rpm for 15 minutes and biofertilizer was made using talc. Talc was prepared separately containing four different bacteria along with different combinations. Chickpea seeds were treated accordingly @ 1250 g per hectare and sown in pots as well as in field (Sushma, 2015).
 
Observations recorded
 
Plant height
 
Plant height at 30 days, 60 days and at harvest were recorded and expressed in cm. After harvest, the plants were uprooted carefully and were dipped in water for 30 min to loosen the soil.
 
Number of branches per plant
 
The branches arising from the main stem was counted at harvest of the crop and the mean was worked out and expressed as number of branches per plant.
 
Number of pods per plant
 
Number of filled pods present was counted individually and the average was worked out and expressed as number of pods per plant at harvest.
 
Seed yield per plant
 
The seeds harvested from individual plant were dried, threshed, cleaned, weighed and expressed in grams.
 
Plant dry matter accumulation per plant
 
The plant samples were oven dried at 60°C for 4-6 days and dry weight of samples were recorded and expressed in gram after harvest depicted in Plate 1.


 
Effect of endophytes on crop performance in chickpea under field condition
 
The experiment was conducted in seed farm, Department of Seed Science and Technology Block at College of Agriculture, Vijayapur which is situated in Northern dry zone of Karnataka (Zone 3), It is situated at 160 49’ North latitude, 750 43’ East longitude and at an altitude of 593.8 m above the mean sea level (MSL).
 
Experimental details
 
Title of experiment: Effect of endophytes on crop performance in chickpea (Cicer arietinum L.), crop: chickpea, variety: JG-11, season: Rabi, location: seed farm, College of Agriculture, Vijayapur, design of experiment: RBD, treatments: 9, replications: 3, spacing: 45 × 10 cm, fertilizer: 4N:10P₂O₅ kg ha^-1, plot size (gross plot): 3.15 m × 3 m, net plot size: 2.7 m × 2.4 m, seed rate: 20 kg/acre and seed source: seed unit, RARS, Vijayapur.
 
Treatment details
 
The similar treatments were imposed as detailed in pot culture experiment with three replications laid out in RBD concept.
 
Observations recorded
 
On growth and yield components.

Effect of endophytic bacteria and Rhizobium on growth parameters under pot culture and field condition in chickpea
 
Significant differences were observed in the growth parameters like plant height at 30 DAS, 60 DAS and at harvest, number of branches per plant and plant dry matter accumulation at 30 DAS, 60 DAS and at harvest between the treatments (Table 1 and 2). The highest plant height in pot culture and field condition was recorded in treatment T₉ (15.63 and 17.97 cm) Bacillus thuringiensis+B. cereus strain LPDB2+B. cereus strain LPDB5+Rhizobium GV-2 followed by T₆ (14.93 and 17.27cm) B. thuringiensis+Rhizobium GV-2 and treatment T₇ (14.49 and 16.83 cm) B. cereus strain LPDB2+Rhizobium GV-2 while, least plant height was recorded in Control (12.74 and 15.07 cm) at 30 DAS. At 60 days after sowing, highest plant height in pot culture and field condition was recorded in T₉ (22.53 and 24.07 cm) B. thuringiensis+B. cereus strain LPDB2+B. cereus strain LPDB5+Rhizobium GV-2 followed by T₆ (21.07 and 23.40 cm) B. thuringiensis+Rhizobium GV-2 and treatment T₇ (20.93 and 23.27 cm) B. cereus strain LPDB2+Rhizobium GV-2 while, least plant height was recorded in T₁ (18.60 and 20.93 cm) Control and at harvest, the maximum plant height in pot culture and field condition was seen in T₉ (40.60 and 42.93 cm) B. thuringiensis+B. cereus strain LPDB2+B. cereus strain LPDB5+Rhizobium GV-2 followed by T₆ (39.40 and 41.73 cm) B. thuringiensis+Rhizobium GV-2 and treatment T₇ (38.53 and 41.00 cm) B. cereus strain LPDB2+Rhizobium GV-2 while, least plant height was recorded in T₁ (36.27 and 38.60 cm) Control. Similar work was also carried out by Verma et al., (2013) who revealed that mutual inoculation improves the plant height as compared to uninoculated control. It may be because of the growth promoting hormones and nutrient uptake ability. Increase in the vegetative and reproductive growth in all the combination of isolates could have been lead to the better solubilization of phosphorous, development of disease resistance and production of plant growth hormones (Sturz et al., 1997). The increased plant height could be attributed to increased cell elongation and multiplication because of enhanced nutrient content in plants following inoculation of Rhizobium sp. and endophytic bacteria. Similar results were also reported earlier by Tomar et al., (1996).
 

 

       
The maximum number of branches per plant in pot culture and field condition at harvest was observed in T₉ (7.87 and 8.90) Bacillus thuringiensis+B. cereus strain LPDB2+B. cereus strain LPDB5+Rhizobium GV-2 followed by T₆ (7.50 and 8.53) B. thuringiensis+Rhizobium GV-2 and T₇ (7.27 and 8.30) B. cereus strain LPDB2+Rhizobium GV-2 while, least number of branches per plant was recorded in T₁ (5.97 and 7.00) Control. Similarly, maximum dry matter accumulation of chickpea seeds from field condition at 30 DAS was observed in T₉ (1.64 gm) B. thuringiensis+B. cereus strain LPDB2+B. cereus strain LPDB5+Rhizobium GV-2 followed by treatment T₆ (1.63 gm) B. thuringiensis+Rhizobium GV-2 and treatment T₇ (1.62 gm) B. cereus strain LPDB2+ Rhizobium GV-2 while, least dry matter accumulation was recorded in T₁ (1.54 gm) Control. At 60 DAS, the maximum dry matter accumulation of chickpea seeds from field condition was observed in treatment T₉ (10.76 gm) B. thuringiensis+B. cereus strain LPDB2+B. cereus strain LPDB5+Rhizobium GV-2 followed by treatment T₆ (10.61 gm) B. thuringiensis+Rhizobium GV-2 and treatment T₇ (10.29 gm) B. cereus strain LPDB2+ Rhizobium GV-2 while, least dry matter accumulation was recorded in T₁ (9.97 gm) Control. At harvest, the maximum dry matter accumulation of chickpea seeds from pot culture and field condition was observed in treatment T₉ (24.66 and 26.70 gm) B. thuringiensis+B. cereus strain LPDB2+B. cereus strain LPDB5+Rhizobium GV-2 followed by treatment T₆ (24.13 and 26.17 gm) B. thuringiensis+Rhizobium GV-2 and treatment T₇ (23.93 and 25.96 gm) B. cereus strain LPDB2+ Rhizobium GV-2 while, least dry matter accumulation was recorded in T₁ (22.13 and 24.17 gm) Control. Coinoculation of these rhizobial and nonrhizobial endophytic isolates resulted in increase in plant biomass, plant height and number of branches per plant in chickpea crop under pot culture and field condition when compared with single inoculation and uninoculated control. Zhao et al., (2011) reported that the legume Sophora alopecuroides on co-inoculation with Zong 1 P. chlororaphi (endophyte)+SQ1 Mesorhizobium sp. showed high growth under greenhouse conditions than those of single inoculation and showed a significant difference (p < 0.05) when compared to a negative control, suggesting that strains of Zong 1 and Mesorhizobium sp. SQ1 have better synergistic or addictive effect. Increase in plant growth parameters might be due to the production of phytohormones like IAA which was stated by Liao et al., (2014) which invigour the cellular division fostering root growth or it may be due to the siderophores production, which can amend the bioavailability of plant nutrients and thereby bracing growth (Joseph et al., 2012).
 
Effect of endophytic bacteria and Rhizobium on yield parameters under pot culture and field condition in chickpea
 
Significant differences were observed in the yield parameters like number of pods per plant, seed yield per plant, seed yield per net plot and seed yield per hectare between the treatments and depicted in in figure. At harvest, the maximum number of pods per plant in pot culture and field condition was observed in T₉ (59.33 and 61.67) Bacillus thuringiensis+B. cereus strain LPDB2+B. cereus strain LPDB5+Rhizobium GV-2 followed by T₆ (58.67 and 61.33) and T₇ (57.00 and 60.00) compared to T₁ (52.67 and 55.33) Control and their superiority was to an extent of 11.22 and 10.28, 10.22 and 9.78, 7.59 and 7.78 per cent respectively. The maximum seed yield per plant in pot condition was observed in T₉ (20.65 gm) B. thuringiensis+B. cereus strain LPDB2+B. cereus strain LPDB5+Rhizobium GV-2 followed by T₆ (20.44 gm) and T₇ (19.97 gm) compared to T₁ (18.53 gm) Control and their superiority was to an extent of 10.26, 9.34 and 7.21 per cent respectively. The maximum seed yield per net plot in field condition was recorded in T₉ (1.46 kg/plot) B. thuringiensis+B. cereus strain LPDB2+B. cereus strain LPDB5+Rhizobium GV-2 followed by T₆ (1.43 kg/plot) and T₇ (1.40 kg/plot) compared to T₁ (1.21 kg/plot) Control and their superiority was to an extent of 17.12, 15.38 and 13.57 per cent respectively. The maximum seed yield per hectare in field condition was observed in T₉ (1545 kg/ha) B. thuringiensis+B. cereus strain LPDB2+B. cereus strain LPDB5+Rhizobium GV-2 followed by T₆ (1515 kg/ha) and T₇ (1485 kg/ha) compared to T₁ (1281 kg/ha) Control and their superiority was to an extent of 17.08, 15.44 and 13.73 per cent respectively Fig 1.
 

               
The increase in seed yield is due to the higher number of pods, branches and seed size in treated seeds than control. Several other reasons which contribute to high yield are better seedling emergence and vigour, increased nutrient supply, increased photosynthetic pigments and phytohormone production with reduced disease and pest incidence. Liao et al., (2014) stated that the impact of yield might be due to early vegetative growth that help the plants to outpass the biotic and abiotic stress condition. This outcome is in accordance with Kabaluk and Ericsson (2007) who reported that stand density and yield of corn increased by controlling the wireworm. Oyetunji et al., (2019) reported that increase in grain number was observed in rice treated with M. anisopliae and B. bassiana along with reduction in adults of African gall midge. As seed yield depends on various environmental variables like plant density (Diepenbrock, 2000), increase in seed yield might be due to better field emergence and plant density.

Seed treatment  with B. thuringiensis+B. cereus strain LPDB2+B. cereus strain LPDB5+Rhizobium GV-2 were treated @ 1250 g per  hectare and sown in pots as well as in field has recorded higher growth and seed yield attributing parameters followed by B. thuringiensis+Rhizobium GV-2 as compared to untreated under pot and field conditions.

None.