Impact of Microbial Cultures on Soil Biological Quality and Growth of Spinach Grown in Polluted Soils

In this present studied poly bag experiment was conducted following complete randomized block design with 12 treatments and three replications. Polluted Soil with supply of fresh water, Unpolluted soil with supply of fresh water, Unpolluted soil with supply of polluted water. The results of pot culture were reveals that the Influence of microbial cultures on biological quality and microbial population of polluted soil and spinach yield at 30 and 60 DAS was estimated. Significantly highest bacterial population was recorded in treatments T12 (124.21 ×10 7 CFU g-1 soil) at 30 DAS and treatment T8 (88.68 ×107 CFU g-1 soil) at 60 DAS. The highest molds population was observed in treatment T3 (17.91, 11.32 ×103 CFU g-1 soil) at 30 and 60 DAS respectively. The treatment T6 showed significantly highest rhizobial population at 30 and 60 DAS (29.23, 20.71 ×103CFU g-1 soil) respectively. The VAM population was highest in the treatment T7 (24.13 ×10 g -1 soil) at 30 DAS and T12 (9.0) at 60 DAS. The highest leaf fresh weight was recorded in T8 (41.63 g plant-1) at 30 DAS and (70.03 g plant-1) at 60 DAS and the lowest fresh weight was recorded in T3, T9 at 30, 60 DAS respectively. The highest leaf dry weight was recorded in T8 (6.62 g plant -1), (4.17 g plant-1) at 30 and 60 DAS respectively and the lowest values were found in T3 at 30 and 60 DAS.

Soil fertility is a complex concept that involves many interacting parameters.Cultivated plants may suffer nutritional stresses when the amount or availability of soil nutrients is lower than that required for sustaining metabolic processes in each growth stage .Thus, restoring of nutrients and enhancing their availability by improving soil characteristics and efficiency of plants, are the main objectives of the modern agriculture.Due to the increasing sensitivity to environmental and economic issues, researchers and consumers are more and more aware of the impact of agriculture on the environment.Pollutants such as heavy metals and chemicals in the soil, water and air are affected by various physicochemical, biological, and environmental factors.Bioremediation is a biological process by which environmental pollutants are removed or transformed to less toxic substances.Soil amendments including fertilizer and lime, appropriate moisture levels, and periodic tilling can maximize or improve bioremediation (Brigmon et al. 2002).Phytoremediation specifically utilizes plants for contaminant control and has been combined with soil amendments for increasing or reducing metals uptake (Wilde et al. 2005).
Plants tolerant to heavy metals are able to immobilize metals by accumulation in the roots, adsorption in/onto the roots, and/or precipitation in the rhizosphere.Currently, most of what is known about aided phytostabilization of heavy metalcontaminated soils focuses on analysis of physicochemical soil properties, especially concentration of bioavailable forms of metals/ metalloids and their accumulation in plant tissues (Wilde et al. 2005).
Bioindicators are the most important criterion of soil quality (Alkorta et al. 2010;Markert et al. 2003).Many definitions of soil quality have been suggested.However, a short and comprehensive definition is given by Doran and Parkin (1994) who have defined soil quality/soil health as "The capacity of a soil to function within ecosystem boundaries to sustain biological productivity, maintain environmental quality and promote plant and animal health".Phytoremediation, the use of plants to extract, sequester, and/or detoxify pollutants through physical, chemical, and biological processes (Saxena et al., 1999) has been reported to be an effective, in situ, non-intrusive, low-cost, aesthetically pleasing, ecologically benign, socially accepted technology to remediate polluted soils (Alkorta and Garbisu, 2001;Garbisu et al., 2002;Weber et al., 2001).It also helps prevent landscape destruction and enhances activity and diversity of soil microorganisms to maintain healthy ecosystems, which is consequently considered to be a more attractive alternative than traditional methods to the approaches that are currently in use for dealing with heavy metal contamination.
The objective of this work was to use traditional microbiological methods based on culture techniques to evaluate the biological quality of heavy metal-contaminated soil that has been remediated with aided phytostabilization

Soil Samples and Soil Characteristics
Soil samples of polluted and unpolluted soils were collected before sowing and analysed for the physical(pH, EC, and particle size and chemical characters like N,P,K and organic carbon parameters) and microbiological properties by adopting standard procedures at Department of Agricultural Microbiology and Bio-energy and Department of Soil Science and Agricultural Chemistry, College of Agriculture, Rajendranagar, PJTSAU, Hyderabad.Water samples were also analyzed before sowing of crop in polluted and unpolluted soils.(table 1).

Crop details
The pot culture experiment was conducted at Department of Agricultural Microbiology and Bioenergy during 2014-15.For this investigation leafy vegetable crop, spinach beet, Pusa Jyothi variety was sown in pot experiments followed completely randomized block design with four treatments and three replications.Microbial cultures (Pseudomonas, VAM) collected from our laboratory.The treatments for poly bag experiment were fixed as twelve treatments each treatment with three replications were designed.
All three replications were used to record observations on yield, quality parameters of spinach around 30 and 60 days after sowing.
In this context of pot culture experiment having twelve treatments and followed statistical design .inthis treatment subdivided into three parts: polluted soil with supply of fresh water, unpolluted soil with supply of fresh water and unpolluted soil with supply of polluted water.Polluted soil with supply of fresh water have T1: SF Soil+FYM@12 t/ha, T2: SF Soil + FYM + VAM + Pseudomonas, T3: SF Soil + RDF, T4: SF Soil + RDF + FYM + VAM + Pseudomonas.Unpolluted soil with supply of fresh water, have T5: Soil + FYM, T6: Soil + FYM + VAM + Pseudomonas, T7: Soil + RDF, T8 : Soil + RDF + FYM + VAM + Pseudomonas.

Chemical fertilizers
Phosphorus and potassium @ 0.24 g poly bag -1 P 2 O 5 and 0.37 g poly bag -1 K 2 O were applied through Di Ammonium Phosphate and Muriate of Potash respectively as basal application.Nitrogen was applied in the form of Urea @ 0.24 g poly bag - 1 after germination and after 30 and 60 days after sowing.Farmyard manure was applied @ 78.75 g poly bag -1 which was mixed with soil according to the treatments requirement.EC and pH of FYM were 0.95 dS/m and 7.59 respectively and Ni, Co, Cd content in FYM was 0.91, 0.20, 0.01-0.02respectively.

Seed Sowing and maintenance
The poly bags were sown with Pusa Jyothi variety of spinach beet at the rate of 20 seeds per poly bag.After germination, thinning was done and routine care was taken to protect the plants from pest and diseases.

Microbial population (CFU g -1 of Soil)
Influence of microbial cultures on biological quality of polluted soil and spinach yield at 30 DAS and 60 DAS on the microbial population in soil was estimated viz., bacteria, Rhizobium, Azospirillum, Azotobacter, actinomycetes, Pseudomonas, molds and VAM show the data presented in the Table and .

Bacterial population (× 10 7 CFU g -1 of Soil)
Bacterial population in soil differed significantly on application of microbial cultures on biological quality of polluted soil and spinach yield .Initial bacterial population in the polluted soil was 30×10 7 CFU g -1 of soil and in unpolluted soil was 40×10 7 CFU g -1 of soil.At 30 DAS, treatment T 12 recorded significantly higher bacterial population (124.21) as compared to all other treatments but was on par with treatment T 2 (117.85) and T 8 (123.85).The significantly lowest (82.98) bacterial population was found with the treatment T 7 .At 60 DAS, treatment T 8 recorded significantly higher (88.68) bacterial population than all other treatments and treatment T 5 (83.38) and T 12 (84.21)was on par with T 8.The significantly lowest (46.46) bacterial population recorded in the treatment T 9 .Rhizobium population (× 10 3 CFU g -1 of Soil) Rhizobial population differed significantly as influenced by application of microbial cultures on biological quality of polluted soil and spinach yield.Initial rhizobial population in polluted soil was 12.4×10 3 CFU g -1 of soil and in unpolluted soil was 18.4×10 3 CFU g -1 of soil.At 30 DAS, treatment T 6 recorded significantly higher (29.23) rhizobial population as compared to all other treatments and was on par with treatment T 5 (26.23),T 7 (26.58),T 8 (28.00) and T 11 (27.20).The significantly lowest (16.32) rhizobial population recorded in the treatment T 4 .At 60 DAS, treatment T 6 recorded significantly higher (20.71) rhizobial population as compared to all other treatments and was on par with the treatment T 5 (18.66),T 7 (17.90)and T 9 (17.00).The significantly lowest rhizobial Azotobacter population (× 10 3 CFU g -1 of Soil) Azotobacter population differed significantly as influenced by application of microbial cultures on biological quality of polluted soil and spinach yield.Initial Azotobacter population in polluted soil was 3.1×10 3 CFU g -1 of soil and in unpolluted soil was 4.9×10 3 CFU g -1 of soil.At 30 DAS, treatment T 7 recorded significantly higher (15.13)Azotobacter population among all other treatments and was on par with T 6 (12.7),T 9 (14.26) and T 11 (15.00).The significantly lowest (9.7) Azotobacter population found in the treatment T 4 .At 60 DAS, treatment T 5 recorded significantly higher (12.46)Azotobacter population as compared to all other treatments.The significantly lowest Azotobacter population recorded in the treatment T 3 (2.43).Azospirillum population (× 10 3 CFU g -1 of Soil) The population of Azospirillum differed significantly as influenced by application of microbial cultures on biological quality of polluted soil and spinach yield (Table & ) Initial Azospirillum population in polluted soil was 4.2×10 3 CFU g -1 of soil and in unpolluted soil was 6.5×10 3 CFU g -1 of soil.At 30 DAS, treatment T 12 recorded significantly higher (12.1)Azospirillum population among all other treatments and was on par with T 8 (10.5),T 9 (10.9),T 10 (11.4) and T 11 (12.00).The significantly lowest (8.36) Azospirillum population found in the treatment T 1 and T 5 .At 60 DAS, treatment T 12 recorded significantly higher (9.4) Azospirillum population as compared to all other treatments and was on par with treatment T 4 (8.53).The significantly lowest Azospirillum population recorded in the treatment T 11 (4.26).

Actinomycetes population (× 10 4 CFU g -1 of Soil)
Actinomycetes population differed significantly as influenced by application of microbial cultures on biological quality of polluted soil and spinach yield (Table &).Initial Actinomycetes population in polluted soil was 8.2×10 4 CFU g -1 of soil and in unpolluted soil was 6.2×10 3 CFU g -1 of soil.At 30 DAS, treatment T 7 recorded significantly higher (41.77) actinomycetes population among all other treatments.The significantly lowest (21.48)Actinomycetes population found in the treatment T 9 .At 60 DAS, treatment T 7 recorded significantly higher (34.73) actinomycetes population as compared to all other treatments and was on par with treatment T 5 (34.27).The significantly lowest actinomycetes population recorded in the treatment T 9 (15.67).

Pseudomonas population (× 10 4 CFU g -1 of Soil)
Pseudomonas population differed significantly as influenced by application of Initial Pseudomonas population in polluted soil was 18.2×10 4 CFU g -1 of soil and in unpolluted soil was 28×10 4 CFU g -1 of soil.At 30 DAS, treatment T 10 recorded significantly higher (96.10)Pseudomonas population as compared to all other treatments and was on par with treatment T 12 (96.00).The significantly lowest Pseudomonas population recorded in the treatment T 1 (29.16).At 60 DAS, treatment T 12 recorded significantly higher (60.25)Pseudomonas population as compared to all other treatments and was on par with T 7 (58.68).The significantly lowest (30.89)Pseudomonas population was recorded in the treatment T 11 .

Molds population (× 10 4 CFU g -1 of Soil)
Molds population differed significantly as influenced by application of microbial cultures on biological quality of polluted soil and spinach yield .Initial molds population in the polluted soil was 15×10 4 CFU g -1 of soil and in unpolluted soil was 29×10 4 CFU g -1 of soil.At 30 DAS, treatment T 3 recorded significantly higher (17.91) molds population as compared to all other treatments.The significantly lowest (4.33) molds population was recorded in the treatment T 10 .At 60 DAS, higher molds population (11.32) was observed with the treatment T 3 as compared to all other treatments and was on par with treatment T 1 (10.04).The significantly lowest (1.95) fungal population recorded in the treatment with T 8 .VAM population (× 10 3 CFU g -1 of Soil) VAM population differed significantly as influenced by application of microbial cultures on biological quality of polluted soil and spinach yield (Table 4. 1& 4.2,Fig 4.8).Initial VAM population in polluted soil was 4.8×10 0 CFU g -1 of soil and in unpolluted soil was 7.2×10 3 CFU g -1 of soil.At 30 DAS, treatment T 7 recorded significantly higher (24.13)VAM population among all other treatments and was on par with T 6 (20.80).The significantly lowest (7.49) VAM population found in the treatment T 3 .At 60 DAS, treatment T 12 recorded significantly higher (9.00)VAM population as compared to all other treatments and was on par with treatment T 2 (8.04), T 6 (8.99), T 9 (8.34),T 10 (7.88).The significantly lowest VAM population recorded in the treatment T 7 (4.14).
Microbial population recorded in the rhizosphere soil at flowering stage and harvesting stage indicated significant increase due to application of microbial cultures.Population was significantly higher under the treatment T 8 : SF Soil + RDF + FYM + VAM + Psuedomonas and T 12 = Soil + RDF + FYM + VAM + Psuedomonas.This is due to application of VAM and Pseudomonas along with FYM will enhance high number of cells in the rhizosphere which will compete with the nature genera.

Microbial Biomass Carbon
Microbial biomass carbon differed significantly at harvesting stage (60 DAS) as influenced by application of microbial cultures on biological quality of polluted soil and spinach yield.At 60 DAS, treatment T 12 recorded significantly higher (129.99)microbial biomass carbon as compared to all other treatments and treatment T 8 (123.68) was on par withT 12 .The significantly lowest microbial biomass carbon recorded in the treatment T 1 (83.3).The MBC was higher in unpolluted soil compared to polluted soil individually that the microbial activity and mass is reduced in polluted soil due to the accumulated pollutants.

Leaf fresh weight (g plant -1 )
The data presented revealed that the leaf fresh weight was significantly affected by different treatments with RDF, combination of inorganic, organic manures (FYM, and biofertilizer ) at 30 DAS and 60 DAS of crop.The highest leaf fresh weight plant -1 was recorded in treatment T 8 (41.63 g plant - 1 ) than the rest of treatments at 30 DAS in unpolluted soils.The lowest leaf fresh weight per plant was showed in T 3 (23.02g plant -1 ) at 30 DAS in polluted soils.The highest leaf fresh weight was observed in T 8 (70.03 g plant -1 ) and the lowest value observed in T 9 (38.12 g plant -1 ) at 60 DAS in unpolluted soil.It was observed that the treatment T 8 (70.03 g plant -1 ) comprising RDF + FYM + VAM and Pseudomonas showed highest values at 30 DAS, 60 DAS in unpolluted soils over other treatments.

Leaf dry weight (g plant -1 )
The data presented revealed that the leaf dry weight was significantly influenced by recommended dose of fertilizers, combination of inorganic, organic manures (FYM) and biofertilizers (VAM and Pseudomonas ) at 30DAS and 60 DAS.The highest leaf dry weight plant -1 was observed in T 8 (6.62 g plant -1 ) and lowest value in T 3 (3.16g plant -1 ) was observed at 30 DAS .The highest leaf dry weight was observed in T 8 (4.17 g plant -1 ) and the lowest in T 3 (2.22 g plant -1 ) at 60 DAS.Among all the treatments, T 8 comprising RDF, FYM, VAM and Pseudomonas was showed highest dry weight of leaf per plant at 30 DAS & 60 DAS in unpolluted soils.In same way, the lowest dry weight of leaf was found in T 3 at 30 and 60 DAS in polluted soils.Similar results were reported by Madhvi et al. (2014).It was reported that increased leaf area and leaf dry weight in spinach was due to application of chemical fertilizers along with organic manures and biofertilizers.

CONCLUSIONS
Taken together the results obtained in the present study clearly indicate that use of the polluted soil or polluted water for raising spinach beet crop gave reduced yield in terms of leaf fresh weight and dry weight.The yield was significantly highest in the treatment (T 8 ) with FYM, RDF and microbial cultures VAM & Pseudomonas in a normal soil irrigated with fresh water.The microbial populations viz.bacteria, molds were more influenced by the application of FYM and chemical fertilizers irrespective of the soil or water pollution.

Table 1 .
Effect SF soil = Student Farm Soil, RDF = Recommended dose of fertilizers, FYM = Farm Yard Manure population (10.7) recorded in the treatment T 4 .

Table 2 .
Effect of microbial cultures on fresh weight at 30 and 60 DAS in polluted and unpolluted soils of spinach beet

Table 3 .
Effect of microbial cultures on dry weight at 30 and 60 DAS in polluted and unpolluted soils of spinach beet

Table 4 .
Influence of different treatments on microbial population in polluted and unpolluted soils of spinach beet at 30DAS

Table 5 .
Influence of different treatments on microbial population in polluted and unpolluted soils of spinach beet at harvesting stage (60DAS)