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Int.J.Curr.Microbiol.App.Sci (2017) 6(8): 843-854 International Journal of Current Microbiology and Applied Sciences ISSN: 2319-7706 Volume 6 Number 8 (2017) pp. 843-854 Journal homepage: http://www.ijcmas.com Original Research Article https://doi.org/10.20546/ijcmas.2017.608.107 Effect of Biological Seed Coating on Pigeonpea Seedling Vigour V. Jagadeesh1, Sujatha Patta1*, S. Triveni2, K. Keshavulu1, K. Jhansi Rani1 and Kavali Raghavendra3 1 2 Department of Seed Science and Technology, PJTSAU, Rajendranagar, Hyderabad 30, India Department of Agriculture Microbiology and Bio-energy, PJTSAU, Rajendranagar, Hyderabad 3 Centor India, Hyderabad, Telangana, India *Corresponding author ABSTRACT Keywords Pigeonpea, Seedling vigour, Biocoating, Biocontrol, Polymers, Biofertilizers. Article Info Accepted: 14 June 2017 Available Online: 10 August 2017 The present investigation was carried out to study the effect of biological seed coating with bioprotectants and biofertilizers using polymer on the seed quality and storability of pigeonpea. The pigeonpea seed was coated with Trichoderma viride and Pseudomonas fluorescens with combinations of Rhizobium or Phosphorus Solubilizing Bacteria using two adjuvants like polymer and sugar syrup. The seedling vigour parameters were studied at monthly intervals during storage period of six months. Six months after treatment, seed combinely inoculated with Trichoderma and Rhizobium (1314 and 69, respectively for SVI1 and as SVI 2 and Pseudomonas and Phosphorus Solubilizing Bacteria 1178 and 69, respectively for SVI1 and as SVI 2 showed high seedling vigour. Among adjuvants, biopolymer recorded good vigour compared to farmer practice of sugar syrup. Seed combinely coated with bioprotectants and biofertilizers recorded more vigour compared to single organism inoculation. The results of the study thus indicated the possibility of coating seed with biological agents with effective biofriendly polymer as an adjuvant immediately after processing before packaging without affecting the seed quality. Thus, the farmers can be supplied biologically coated seed in advance and the general practice of treating the seed one day prior to sowing can be avoided. Introduction Pigeon pea [Cajanus cajan (L.)] is an important staple food pulse crop rich in protein content of 22%, which is almost three times that of cereals. In India, pigeonpea is cultivated in an area of 3.90 mha with a production of 3.17 Mt and productivity of 813 kg ha-1 (IIPR, 2014 and FAO, 2016). The main constraints in obtaining potential yield of the pigeon pea are incidence of diseases, insects and other physiological stresses in the field. It is known to be affected by more than hundred pathogens, among them, pigeonpea wilt caused by Fusarium udum is the major disease in India. The disease may appear during early stages of plant growth (4-6 week old plant) period and essentially affects yield. This disease drastically influences the crop yield by poor field emergence, seedling establishment and plant stand in the field finally leading to the reduction in the productivity and production. Moreover, the seed performance can be enhanced by the way of application of fungicides, insecticides and other protecting agents on to the seed surface by means of 843 Int.J.Curr.Microbiol.App.Sci (2017) 6(8): 843-854 coating to protect the seed from pathogens. Biological seed coating is a new technique of seed treatment through which biological agent will be coated over seed surface by mixing in a polymer that is sprayed on the seeds as they fall through a specialized machine. The polymer is rapidly dried, so that the seeds emerge with a complete, dry polymeric coating containing biological control agent. By encasing the biological control agent within a thin film of biodegradable polymer, the adherence of seed treatment to the seed is improved, ensures dust free handling, making treated seed both useful and ecofriendly. It is recently used as an alternative method for controlling many seed and soil-borne pathogens. It is an ecological approach using selected fungal antagonists against the soil and seed-borne pathogens and it has a potential to provide an alternative to chemical control. Considering the importance of the biological agents in the eco-friendly control of pests and diseases, it is very important to treat the seed before taking up of sowing. Even though seed treatment with biological agents is not new, the main constraint is that the seed treatment can only do just before the sowing. Pseudomonas fluorescens, Rhizobium and Phosphorus solubilising bacteria and its effect on seed quality and storability of pigeon pea. Materials and Methods The present investigation was out during Kharif, 2015 and Rabi, 2016 at Department of Seed Science and Technology and Department of Agricultural Microbiology and Bio-energy, College of Agriculture, PJTSAU, Rajendranagar, Hyderabad, Telangana. Materials Freshly harvested seeds of Pigeonpea variety LRG - 41 were collected from Regional Agricultural Research Station, Lam, Guntur, Andhra Pradesh. The seed possessed initial germination of 85 %, electrical conductivity of seed leachates as 12.12 µmhos/cm and seed moisture content of 8.1%. Biological agents like Trichoderma viride, Pseudomonas fluorescens, Rhizobium spp and Phosphorous Solubilizing Bacteria were obtained from Agri Biotech Foundation (ABF), Rajendranagar, Hyderabad and Telangana. Bio friendly polymer was collected from Centor India, Bliss Paradise; Hitex, Secunderabad, Telangana. In India, there are meager or no studies to encourage seed treatment with biological agents considerably after seed processing. In this connection, the adjuvants play a key role to improve the shelf life of bio agents and seed storability. Addition of certain polymers offers a ray of hope in this direction. The present study is planned to carry out biological seed treatment with polymer as an adjuvant well in advance of sowing i.e., immediately after seed processing or before packaging and to study the viability and shelf life of biological agent and its effect on seed storability. In this context, this research study is planned with the objective to study the effect of bio friendly polymer as an adjuvant on the viability and shelf life parameters of Methodology For biological seed coating, two different adjuvants like bio friendly polymer or sugar syrup were used @ 6 g kg-1 seed. Biological agents like Pseudomonas fluorescens, Rhizobium and Phosphorous Solubilizing Bacteria were used @ 12 g kg-1 seed. These biological agents were used for coating either individually or in combination of two. For each treatment, 600 g pigeon pea seed was weighed separately and placed into seed coating machine. Biological coating material was prepared by weighing 6 g of adjuvant (either bio friendly polymer or sugar syrup) 844 Int.J.Curr.Microbiol.App.Sci (2017) 6(8): 843-854 into a beaker and to that 3.6 g of distilled water was added and mixed thoroughly. To this diluted adjuvant, 12 g of biological agent was added and mixed thoroughly. For combined inoculation of two biological agents 6 g of each organism was used for preparing slurry. Uniformly coated seed was removed from the coating machine and was shade dried for 2 hr. This treated seed was then made into 3 replications @ 200 g each and packed in zip lock polythene covers and kept for storage under ambient conditions. T4: Seed + Pseudomonas fluorescens+ Rhizobium + Sugar syrup T5: Seed + Pseudomonas fluorescens+ Phosphorus Solubilizing Bacteria (PSB) + polymer T6: Seed + Pseudomonas fluorescens+ Phosphorus Solubilizing Bacteria (PSB) + Sugar syrup T7*: Seed + Pseudomonas fluorescens+ polymer T8: Seed + Thiram T9: Seed (Untreated Control) Pigeonpea biological seed coating with Trichoderma viride, Rhizobium and phosphorus solubilizing bacteria using polymer *Note: Farmer practice of seed inoculation as one day before sowing (fresh treatment every month). Testing of seed quality and storability of biologically coated pigeonpea seed T1: Seed + Trichoderma viride + polymer T2: Seed + Trichoderma viride + Sugar syrup T3: Seed + Trichoderma viride + Rhizobium + polymer T4: Seed + Trichoderma viride + Rhizobium + Sugar syrup T5: Seed + Trichoderma viride + Phosphorus Solubilizing Bacteria (PSB) + polymer T6: Seed + Trichoderma viride + Phosphorus Solubilizing Bacteria (PSB) + Sugar syrup T7*: Seed + Trichoderma viride + polymer T8: Seed + Thiram T9: Seed (Untreated Control) Seed germination (Percent) The laboratory test for germination was conducted as per the ISTA rules (ISTA, 2009) by adopting between paper method. Three replications of 100 seeds each were taken from each treatment and uniformly place on germination paper. The rolled towel was kept in the seed germinator and maintained a constant temperature of 25± 0.50c and 95 per cent relative humidity. Evaluation of normal seedlings, abnormal seedlings, fresh ungerminated seeds and dead seeds were done on sixth day. Germination percentage is expressed on the number of normal seedlings by following *Note: Farmer practice of seed inoculation as one day before sowing (fresh treatment every month). Pigeonpea biological seed coating with Pseudomonas fluorescens, Rhizobium and phosphorus solubilizing bacteria using polymer Germination % = Number of normal seedlings ------------------------------------ × 100 Total number of seeds planted T1: Seed + Pseudomonas fluorescens+ polymer T2: Seed + Pseudomonas fluorescens+ Sugar syrup T3: Seed + Pseudomonas fluorescens+ Rhizobium + polymer Seedling length (cm) Ten normal seedlings were selected randomly per replication in each treatment on 6th day of germination test. The root length was 845 Int.J.Curr.Microbiol.App.Sci (2017) 6(8): 843-854 measured from the tip of the primary root to base of the hypocotyl with the help of a scale and the mean root length was expressed in centimeters. in 50 ml of distilled water at 25 ± 1⁰ C temperature, for 24hrs, with frequent stirring. The leachate was decanted and conductance was estimated by using a conductivity meter. The reading obtained was multiplied with cell constant, after deducting the conductance of distilled water, and the data was expressed in dSm-1. Ten normal seedlings used for root length measurement, were also used for the measurement of shoot length. The shoot length was measured from the tip of the primary leaf to the base of the hypocotyl and mean shoot length was expressed in centimeters. Statistical analysis The data recorded were analyzed statistically by adopting Completely Randomized Design (CRD), as described by Panse and Sukhatma (1985) and the standard error of difference was calculated at 5% probability level to compare the mean difference among the treatments. The data recorded as percentage were transformed to the respective angular (arc sin) values before subjecting them to statistical analysis. Seedling dry weight (g) Ten seedlings in each replication of germination test were oven dried in hot air oven at 80⁰ ± 1⁰ C for one day and dry weight per seedling was expressed in grams (g). Seedling vigour index I Results and Discussion Seedling vigour index was calculated using the formula given by Abdul Baki and Anderson (1973) and expressed in whole number. Effect of biological seed coating with bioprotectants and biofertilizers on pigeonpea seedling vigour index-1 Seedling vigour index = Germination percentage (%) × Seedling length (cm) The data on the effect of biological seed coating with Trichoderma and bio fertilizers using polymer on seedling vigour index 1 was computed based on seedling length and seed germination percentage and were presented in table 1. The mean seedling vigour index 1 recorded at initial and 6 month of storage period was 2042 and 977, respectively. At the end of six months of storage, there was about 52 % reduction in the mean SVI-I. The highest seedling vigour index 1 (1314) was recorded in seeds treated with Trichoderma, Rhizobium using polymer (T3) and it is significantly differed from all other treatments. The lowest seedling vigour index 1(640) recorded in seeds treated with Trichoderma with sugar syrup. Similar trend was also observed with regard to biological Seedling vigour index II Seedling vigour index II was calculated as per the method suggested by Abdul-Baki and Anderson (1973) and expressed in whole number. Seedling vigour index II = Germination percentage (%) × Seedling dry weight (g) Electrical conductivity (µmhos 50 seed-1) Fifty seeds, in two replicates, were subjected to 3-4 washings in distilled water, with various shaking. The seeds were then soaked 846 Int.J.Curr.Microbiol.App.Sci (2017) 6(8): 843-854 seed coating with Pseudomonas (Table 1). The highest seedling vigour index 1 (1178) was recorded in the seeds treated with Pseudomonas, PSB using polymer (T5) and lowest (806) was recorded in the seeds treated with Pseudomonas and PSB with sugar syrup. Pseudomonas treatments. Six months after storage, seed treated with Pseudomonas and Phosphorus Solubilizing Bacteria (PSB) using polymer (T5) was found superior for seedling vigour index-2 (69) and it was differed significantly from that of all other treatments. The effect of adjuvants on the seedling vigour index 1 of biologically coated seed pigeonpea was presented in figures 1 and 2. Polymer as an adjuvant showed slightly higher vigour (1077 with Trichoderma and 1040 with Pseudomonas) but sugar syrup reported lower vigour (814). The effect of adjuvants on the seedling vigour index-2 of biologically coated seed pigeonpea was presented in figures 5 and 6. Polymer showed slightly on par vigour index-2 (64) compared to seed coated with fungicide and untreated control (65) and sugar syrup reported lower vigour index-2 (56). The effect of type of inoculation on seedling vigour index 1 of biologically coated pigeonpea seed was presented in figures 3 and 4. Combined inoculation of Trichoderma viride (1143) or Pseudomonas (1111) with Rhizobium or PSB and showed higher vigour compared to single inoculation of Trichoderma (1010) or Pseudomonas (969). These findings are in conformity with the findings of Verma and Verma (2014) in soybean, Kaushik et al., (2014) in maize, Ambika et al., (2014) in hybrid rice and Shakuntala et al., (2014) in sunflower who have reported good Vigour index-2 in polymer coated seed. In a similar study Vinod et al (2013) reported that all the seed quality parameters viz., 100 seed weight, germination percentage, seedling length, seedling dry weight, vigor index and electrical conductivity were found to higher in seeds obtained from seeds treated with deltamethrin 2.8 EC at 0.3 ml/kg seeds+vitavax power at 3 g/kg seed+polymer seed coating at 5 ml/kg seeds. Effect of biological seed coating with bioprotectants and biofertilizers on pigeonpea seedling vigour index-2 A gradual decrease in the seedling vigour index II was also observed with the advancement of storage period (Table 2). The mean seedling vigour index -2 recorded at initial and 6 month of storage period was 88 and 62, respectively. At the end of six months of storage, there was 30 % reduction in the mean SVI 2. Highest seedling vigour index- 2 (69) was recorded in seeds treated with Trichoderma and Rhizobium using polymer (T3) and it was significantly differed from that of control. The lowest vigour index-2 (54) was recorded in seeds treated with Trichoderma using sugar syrup (T2). Except for sugar syrup (T2, T4 and T6), all other treatments were found to be at par with each other with regard to good SVI II and seed storability at 6 months storage. Similar trend with regard to SVI 2 was also observed with The effect of type of inoculation on seedling vigour index-2 of biologically coated pigeonpea seed was presented in the figures 7 and 8. Combined inoculation showed higher seedling vigour index-2 (69) compared to single inoculation. Effect of biological seed coating with Trichoderma viride, Rhizobium, PSB using polymer on electrical conductivity (µmhos 50 seed-1) The data presented in table 3 showed a significant effect of biological seed coating of bioprotectants, biofertilizers with adjuvants 847 Int.J.Curr.Microbiol.App.Sci (2017) 6(8): 843-854 on electrical conductivity (µmhos 50 seed-1). Electrical conductivity was increased progressively with the advancement of storage period (18.69 to 37.61 with Trichoderma and 15.99 to 30.93 with Pseudomonas). Effect of biological seed coating with bioprotectants and biofertilizers on pigeonpea seedling vigour index-1 and 2 The highest seedling vigour index 1 was recorded in seeds treated with Trichoderma, Rhizobium using polymer and Pseudomonas, PSB using polymer. The lowest seedling vigour index 1 recorded in seeds treated with sugar syrup. These results revealed that polymer coating improved the seed storability by maintaining seed vigour. These findings are in conformity with the findings of Yadav et al., (2010) in chick pea, Singh et al., (2013) in Trigonella foenumgraecum, Kaushik et al., (2014) in maize, Ambika et al., (2014) in hybrid rice, Shakuntala et al., (2014) in sunflower, who have reported an increase in seed germination and seedling vigour in the polymer coated seeds compared to uncoated seeds. Similarly, Kaushik and Kumar (2014) reported that the seed treatment with polymer @ 9 ml + thiram 2 g per kg of seed observed significant shoot length, root length, seedling length, viability, fresh weight and higher vigor index and high germination rate was observed for seed coated with polymer @6 ml+ thiram 2 g per kg of seed after 6 month of storage. At the end of six months of storage period, lowest electrical conductivity was recorded with polymer (28.81 with Trichoderma viride and 22.62 with Pseudomonas) compared to untreated control (35.41). And highest electrical conductivity (43.06) was recorded in seed, treated with Trichoderma and PSB using sugar syrup. And highest electrical conductivity (35.43) was recorded in seed, treated with Pseudomonas and Rhizobium with sugar syrup. There was a significant difference among the treatments with regard to electrical conductivity. The effect of adjuvants on the electrical conductivity was presented in the figures 9 and 10. Throughout the storage period, polymer as an adjuvant reported lower leachates compared to untreated control. Six months after storage, sugar syrup reported higher electrical conductivity (41.81). These findings are in line with Chachalis and Smith (2001) who have reported that seed coating with hydrophobic polymer Vinamul 3650 could lower the solute leakage in aged soybean seeds compared to sugar syrup. Among adjuvants, polymer recorded more seedling vigour indices compared to farmer practice of sugar syrup. This might be due to the fact that polymer might have reduced the rate of deterioration and might have supplied sufficient nutrients to the seedling. The effect of type of inoculation on electrical conductivity of biologically coated pigeonpea seed was presented in the figures 11 and 12. Combined inoculation of bioprotectants and biofertilizers also recorded good seedling vigour than single inoculation. This might be due to the fact that bioprotectants might give protection during seed storage and biofertilizers might have supplied sufficient nutrients to the young seedling. Seed coating with single biological organism, showed more electrical conductivity (32.96 with Trichoderma, and 33.03 with Pseudomonas) compared to combined inoculation of bioprotectant and biofertilizers. 848 Int.J.Curr.Microbiol.App.Sci (2017) 6(8): 843-854 Table.1 Effect of biological seed coating with bio-protectants and bio-fertilizers on seedling vigour index 1 and storability of pigeon pea Treatment T1 T2 T3 T4 T5 T6 T7 T8 T9 Mean S.Em S.Ed CD (0.05) C.V Seed coating with Trichoderma viride, Rhizobium and PSB 0MAS 2MAS 4MAS 6MAS 2104 1722 1373 1043 2092 2044 1477 898 2113 1793 1315 1314 2049 1715 1328 640 2094 1587 1306 972 2013 1725 1414 904 2078 1817 1155 1051 2283 1650 1227 999 2060 1690 1311 969 2098 1764 1340 977 70.17 41.56 41.25 31.35 99.24 58.77 58.34 44.34 210.12 124.43 123.53 93.87 5.95 4.08 5.33 6.04 Seed coating with Pseudomonas fluorescens, Rhizobium and PSB 0MAS 2MAS 4MAS 6MAS 2032 1721 1240 1125 2163 1982 1360 966 2010 1771 1215 1044 2033 1563 1083 852 2105 1860 1391 1178 2097 1646 1340 806 2120 1679 1022 910 2108 1690 1376 1110 2007 1689 1339 1028 2075 1734 1263 902 31.98 64.33 42.24 19.52 45.22 90.97 59.73 27.61 95.75 192.62 126.47 58.47 2.83 6.42 5.79 3.41 Table.2 Effect of biological seed coating with bio-protectants and bio-fertilizers on seedling vigour index 2 and storability of pigeon pea Treatment T1 T2 T3 T4 T5 T6 T7 T8 T9 Mean S.Em S.Ed CD (0.05) C.V Seed coating with Trichoderma viride, Rhizobium and PSB 0MAS 2MAS 4MAS 6MAS 88 78 71 65 82 77 70 54 85 78 71 68 82 82 71 57 88 80 73 63 85 79 71 58 84 78 68 65 81 76 68 65 87 79 72 66 88 79 71 62 2.14 2.14 2.24 1.99 3.03 3.03 3.17 2.81 N.S. N.S. N.S. 5.96 4.73 4.73 5.51 5.62 849 Seed coating with Pseudomonas fluorescens, Rhizobium and PSB 0MAS 2MAS 4MAS 6MAS 79 76 75 64 81 78 73 60 77 75 70 64 77 73 66 56 79 78 73 69 77 75 70 56 80 74 68 65 80 77 75 65 77 76 72 65 80 76 71 63 1.91 2.52 1.99 1.15 2.71 3.56 2.82 1.63 5.74 N.S. 5.97 3.45 4.09 5.70 4.82 3.15 Int.J.Curr.Microbiol.App.Sci (2017) 6(8): 843-854 Table.3 Effect of biological seed coating with bioprotectants and biofertilizers on Electrical conductivity (µmhos 50 seed-1) and storability of pigeon pea Treatment T1 T2 T3 T4 T5 T6 T7 T8 T9 Mean S.Em S.Ed CD (0.05) C.V Seed coating with Trichoderma viride, Rhizobium and PSB 0MAS 2MAS 4MAS 6MAS 19.18 34.18 36.40 37.12 19.21 33.48 41.07 42.94 17.29 22.44 31.14 42.07 19.61 36.55 41.04 41.22 19.12 29.24 37.47 38.60 17.10 21.58 42.17 43.06 16.48 26.30 28.81 29.10 14.30 23.21 31.25 31.34 13.53 20.41 34.54 35.41 17.31 26.99 37.61 38.14 0.558 0.797 0.767 0.518 0.789 1.128 1.085 0.732 NS 2.387 2.298 1.550 5.172 5.117 3.485 2.384 Figure 1. Effect of seed coating material and Trichoderma, Rhizobium and PSB on SVI-1 Seed coating with Pseudomonas fluorescens, Rhizobium and PSB 0MAS 2MAS 4MAS 6MAS 17.28 24.80 29.10 33.03 20.51 28.84 35.10 36.90 17.91 24.08 30.57 32.80 16.14 16.48 33.80 35.43 13.66 16.88 21.70 22.62 11.81 20.84 33.70 33.91 17.62 30.94 31.40 33.71 13.65 20.64 29.80 33.76 12.30 20.74 25.50 26.70 15.99 23.42 30.50 30.93 0.578 0.699 0.996 0.954 0.818 0.989 1.408 1.349 1.732 2.093 2.981 2.855 6.266 5.171 5.651 5.340 Figure 2. Effect of seed coating material and Pseudomons, Rhizobium and PSB on SVI-1 Note: MAS-Months After Storage 850 Int.J.Curr.Microbiol.App.Sci (2017) 6(8): 843-854 Figure 3. Effect of type of inoculation with Trichoderma and biofertilizers on SVI-1 Figure 4. Effect of type of inoculation with Pseudomonas and biofertilizers on SVI-1 Note: MAS-Months After Storage Figure 5. Effect of seed coating material and Trichoderma, Rhizobium and PSB on SVI-2 Figure 6. Effect of seed coating material and Pseudomons, Rhizobium and PSB on SVI-2 Note: MAS-Months After Storage 851 Int.J.Curr.Microbiol.App.Sci (2017) 6(8): 843-854 Figure 7. Effect of type of inoculation with Trichoderma & Biofertilizers on SVI-2 Figure 8. Effect of type of inoculation with Pseudomonas & Biofertilizers on SVI-2 Note: MAS-Months After Storage Figure 10. Effect of seed coating material and Pseudomonas, Rhizobium and PSB on EC Figure 9. Effect of seed coating material and Trichoderma, Rhizobium and PSB on EC Note: MAS-Months After Storage 852