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Open Access
Composting of paddy straw using additives along with microbial consortia
(CCSHAU, Hisar, 2022-07) Vijay Kumar; Saharan, Baljeet Singh
In the present investigation, about 26 isolates (10 bacterial and 16 fungal isolates) available in Soil Microbiology laboratory, Department of Microbiology CCS HAU Hisar, were screened for enzyme activities including cellulase, xylanase, laccase and biosurfactant production. In case of bacterial isolates, maximum cellulose hydrolysis index (5.33) was exhibited by RC13.1 and RC3.5 isolate on CMC agar plate. Isolate RC13.1 has also shown maximum xylan hydrolysis index (4.43) laccase and biosurfactant production. In case of fungal isolates, (maximum cellulase (3.28), xylanase (2.32), laccase (qualitatively) was exhibited by H2, H3 and H16 isolates, respectively. H16 isolate also exhibited maximum biosurfactant production confirmed by oil displacement. The bacterial isolate, M2 showed maximum CMCase (0.33 IU/ml), Fpase activity (0.52 IU/ml) activity and second highest xylanase (7.63 IU/ml) and followed by laccase activity (5.2 IU/ml). In case of fungal isolates, maximum CMCase (0.31 IU/ml), and Fpase activity (0.42 IU/ml) were exhibited by H2 isolate. While isolates H3 and H16 showed maximum xylanase (16.11 IU/ml) and laccase (8.4 IU/ml) activities. Based on these activities and compatibility to each other, two bacterial (M2 and RC13.1) and three fungal (H2, H3 and H16) isolates were used for microbial consortium. Microbial consortium along with additives including FYM, cattle dung, poultry manure and mustard cake was analyzed for paddy straw degradation in flasks, trays, and pits. Treatment T5 having paddy straw + microbial consortium + poultry manure was recorded with lowest total organic carbon and maximum potassium content. Maximum total nitrogen and phosphorus content was observed in treatment T6 having paddy straw + microbial consortium along mustard cake after 90 days of composting. All the treatments having additives along with microbial consortium, had significantly higher microbial activity (dehydrogenase), humic substances and better pH, EC and germination index. On the basis of selected morphological and biochemical characters, the isolates RC13.1 and M2 were identified as Pseudomonas sp. and Bacillus sp., respectively. Based on morphological characteristics, fungal isolates H2, H3, H16 were identified as Aspergillus sp., Aspergillus sp. and Phanerochaete sp., respectively.
Open Access
Impact of co- inoculation of rhizobium and trichoderma on nodulation efficiency, plant growth parameters and biocontrol efficacy in mung bean crop (Vigna radiata L.)
(CCS HAU, Hisar, 2022-08) Pallaganti Sowmya; Ajay Kumar
Mung bean (Vigna radiate L.) belongs to the Leguminosae family and one of the most popular short duration pulse crops of India. It is one of the essential food legumes cultivated worldwide. is an excellent source of high-quality protein and contains about 22-25% protein, 1.0-1.5% oil, 3.5-4.5% fibre, 4.8% ash and 62-65% carbohydrates on dry weight basis. It is easily digestible and hence it is suitable for all age group of people and green fodder to feed animals. Rhizobia are Gram-negative bacteria that can form root nodules in leguminous plants and provides the nitrogen through fixation process which can limit the use of chemical fertilizer. Trichoderma belongs to the Deuteromycetes class of fungi and has been exploited as biocontrol agents against a diversity of plant pathogenic fungi. In the present investigation, mung bean cultures and Trichoderma isolates were screened for their compatibility. Trichoderma asperellum and Rhizobium MB 17a were found compatiblemwere then optimized for their growth interaction. The coculture was found to be more effective when tested for their antagonistic activity against Macrophomina phaseolina. The coculture recorded higher plant growth promoting traits including IAA production, ammonia excretion phosphate solubilization, , zinc solubilization, siderophore production, HCN production and ACC utilization. were evaluated for growth promotion of mung bean under pot culture conditions during 2020. Rhizobium and Trichoderma coculture gave best results in improving growth and seed yield of mung crop.
Open Access
Antioxidative and antimicrobial activity of bioactive peptide from Quinoa (Chenopodium quinoa) fermentation
(CCSHAU Hisar, 2022-08) Khan, Sameer; Sindhu, Meena
Quinoa (Chenopodium quinoa Wild) also known as Inca rice is a pseudocereal having immense nutritional benefits (Dakhili et al., 2019). It is also called perfect food because of its nutritional content which contains proteins, vitamins, minerals (manganese, phosphorous, iron, zinc), dietary fibre, and the dietary minerals. It is gluten free, so can be used as food for people having gluten allergy (Galante et al., 2020). The proteins present in the quinoa can be broken down in to smaller peptide of 2-20 amino acid chain having beneficial effect on human health. Present investigation targeted bioactive peptide production by fermenting quinoa using three probiotic bacterial strains i.e. Lactobacillus plantarum, Lactobacillus rhamnosus and Lactobacillus fermentum. Submerged fermentation was performed using these probiotic bacterial cultures for different time intervals (24h, 48h and 72h). Maximum decrease in pH 6.72 to 3.72 was observed during fermentation of Quinoa with Lactobacillus fermentum. Maximum peptide concentration (13.6 mg/mL) was observed during 48 h of Quinoa by L. fermentum using OPA (o-phthaldialdehyde) spectrophotometric assay and it was selected for further analysis. SDS –PAGE segregated the peptides as per molecular weight and maximum peptides obtained were belong to size range 3-20 kDa. Peptides were separated from the supernatant of fermented quinoa using TCA (tri chloro acetic acid) precipitation and categorized on the basis of different size using membrane filter having cut off size 30kDa, 20kDa and 5kDa. The antioxidant scavenging activity were checked using DPPH (2,2-diphenyl picrylhydrazyl) radical assay (Yen and Duh 1994), ABTS (2,2 azino-bis (3 ethylbenzthiazoline-6sulphonic acid) assay (Joshi et al., 2015) and ferrous ion chelating ability (Do et al., 2014). Maximum DPPH radical scavenging ability (16 %) and maximum ABTS (26.2%) scavenging ability was observed in peptide below 5 kDa produced by Lactobacillus fermentum as compare to control. Ferrous ion chelating ability were maximum (26.83 mg AE/gmDW) in <5 kDa peptides formed by L. plantarum as compare to control (14.69 mg AE/DW). Antimicrobial activity of these different size peptide were also observed against Staphylococcus aureus, E.coli, Fusarium oxysporum and Candida sp.. Maximum zone of inhibition was observed in 5-20 kDa peptide (27mm) produced by Lactobacillus plantarum against Staphylococcus aureus followed by below 5 kDa peptides (26mm) by Lactobacillus fermentum. Maximum antimicrobial activity against E.coli (26mm) and Candida sp. (24 mm) was observed in peptides below 5 kDa produced by Lactobacillus fermentum. No antifungal activity was shown by peptides against Fusarium oxysporum. Significant antioxidant and antimicrobial activity were observed in peptides produced by submerged fermentation using Lactobacillus fermentum and Lactobacillus planatrum hence their application can be explored in fat rich food to prevent rancidity and also in post harvest disease management to enhance shelf life of perishable vegetables/fruits.
Open Access
Bioconversion of microbially hydrolysed Water hyacinth (Eichhornia crassipes) into ethanol using Saccharomyces cerevisiae HAU-1
(Chaudhary Charan Singh Haryana Agricultural University hisar, 2022-12) Sonia; Mehta, Shikha
Bioethanol is considered as a potential liquid fuel due to limited natural resources. Lignocellulosic biomass is investigated as a potential substrate for bioethanol production. Water hyacinth (Eichhornia crassipes) is one of the most abundant lignocellulosic materials, which consist of about 35% hemicellulose, 26% cellulose and 11 % lignin. A pre-treatment process is essential to remove lignin, reduce crystallinity and increase the porosity of material making cellulose desirable to hydrolysis. To hydrolyse the cellulose, cellulases are required which increase the cost of ethanol production. Therefore, to make the process cost-effective, there is need to utilize microbial cellulase. In the present study, a total 43 bacterial and 16 fungal isolates were retrieved from 3 different samples such as soil from CCS HAU, Hisar, decaying woody material from village Mirchpur, Hisar and cattle dung from CCS HAU, Hisar. All the bacterial and fungal isolates were screened for cellulase production on the basis of zone of hydrolysis on carboxymethyl cellulose (CMC) agar medium. Out of 43 bacterial isolates, 13 bacterial isolates showed zone formation on CMC agar medium and three isolates i.e. SB2, SB4 and SB10 showed large zone. Out of 16 fungal isolates, 10 fungi showed the zone formation on CMC agar medium and WF1 isolate showed large zone of hydrolysis. Cellulase activity of SB4 and WF1 was determined and found to be 276.83 IU/mL and 230.62 IU/mL, respectively. Bacterial isolate SB4 was biochemically characterized and stained using Gram’s staining. SB4 was found to be Gram positive. Pre-treatment of water hyacinth was done using acid (1% H2SO4) as well as alkali (2% NaOH). Acid pre-treated water hyacinth consisted of 30.23% cellulose, 23.50% hemicellulose and 5.83% lignin and alkali pre-treated water hyacinth consisted of 29.86% cellulose, 24.30% hemicellulose and 6.13% lignin. Crude enzymes of bacterial isolate SB4 and fungal isolate WF1 were used for enzymatic hydrolysis. Hydrolysis of acid pre-treated water hyacinth with crude enzyme obtained from SB4 isolate released 65.74 mg/g total reducing sugars after 120 min of incubation. Fermentation of hydrolysate was carried out using Saccharomyces cerevisiae HAU-1. Different conditions such as pitching rate, slurry concentration and nitrogen sources were optimized for maximizing ethanol production. Fermentation of hydrolysate of acid pre-treated water hyacinth supplemented with 0.15% urea resulted in production of 1.77% (v/v) ethanol after 72 h of incubation using crude enzyme obtained from SB4 isolate.
Open Access
Microbial valorization and fortification of agricultural wastes through composting process
(Chaudhary Charan Singh Haryana Agricultural University, 2022-12) Amisha; Malik, Kamla
Worldwide, agricultural residue burning has become a serious environmental issue that has a very bad effect on human and animal health. Burning of agri-residues not only disturbs the soil structure but causes of huge loss of plant nutrients, effects microflora and causes greenhouse gas emissions (Sarkar et al., 2020). Paddy straw, Sugarcane bagasse, and saw dust are agricultural waste and are composed of lignocellulose compounds that cannot be degraded easily (Perez et al., 2002). Their direct use causes the immobilization of nutrients, so composting arises as a safe option, resulting in the reusability of nutrients contained in these residues. So, the present investigation was planned with the objectives of managing paddy straw, sugarcane bagasse, and sawdust along with cattle dung and microbial consortia in different ratios using pits with an initial C/N ratio of these wastes (Raj and Antil, 2011). In log (CFU/ml), the treatment with paddy straw + sugarcane bagasse + sawdust + cattle dung + Microbial consortia (4:1:1:1) (T6) had the highest bacterial and fungal counts, while the treatment with sugarcane bagasse + sawdust + cattle dung + Microbial consortia (4:1:1:1) (T9) had the highest actinomycetes count. Alkaline phosphatase, cellulase, dehydrogenase (DHA), and protease were maximum at 30 days of composting in T6 followed by T9. The pH value is also neutral in the mature compost after 90 days. EC values are minimum with T9 followed by T6. Total organic carbon of the compostable material in different treatments decreased more in T6 followed by T9 due to losses of C and total N increased due to accumulation of nitrogen up to 90 days. The maximum reduction in C/N ratio was in T6 - 26.50 followed by T9. Total nitrogen (%) increased from 0.65 to 1.48. The same pattern was observed in total phosphorus, potassium in T6 was followed by T9. The temperature of compostable material increased from 300C to 55°C after 40 to 45 days of composting and then decreased. The quality of compost was tested by C: N ratio, CO2 evolution, humic substances, and germination index. The C/N ratio of compostable materials ranged from 78.20 to 80.05 initially and dropped down to 23.25 to 31.97 after 90 days of composting. The amount of humic substances was highest in treatment T6 compared to all other treatments. The germination index of wheat and mung bean at different treatments are as follows: T1-71-74%, T2-72-75%, T3-70-71%, T4-80-82, T5- 57-65, T6- 90-91, T7- 83-81, T8-83-85, T9-87-89, T10-83-85, T11-84-85 and T12-88-89% and germination index of mustard is less. Among all treatments, the germination index was highest in T6 followed by T9. These two treatments (T6 and T9) were selected for further fortification with poultry and vegetable waste. Among the fortified treatments, T6 + Poultry showed Organic carbon -30.40%, C/N ratio-18.54, humic acid of 142 mg/gm, CO2 evolution -101 mg per 100 gram of compost and germination index - 90 to 100% leachate of compost extract, which indicates it doesn’t have any phytotoxic effect on seed
Open Access
Development of biocontrol agent against phytopathogenic fungi of tomato (Lycopersicon esculentum Mill.) crop.
(CCSHAU, Hisar, 2020-07) Kapoor, Divya; Rakesh Kumar
In the present study, seventy-eight bacterial isolates were retrieved from the tomato rhizosphere soil and screened for their antagonistic activity against Fusarium oxysporum f. sp. lycopersici, Alternaria solani and Rhizoctonia solani under in vitro conditions. Thirteen isolates, NAU5, NAU10, NAU13, NAU20, NAU21, NAU23, NAU29, NAU32, NAU36, NAA59, NAA60, KBA43 and KBA44 showed broad inhibition zones against A. solani measuring more than 20 mm. Six isolates, NAU19, NAU20, NAU23, NAU24, NAU27 and NAU30 resulted in broad inhibition zones against F. oxysporum f. sp. lycopersici measuring 10.1-20 mm. Six isolates, NAU20, NAU30, NAU35, LBV58, NAV66 and NAV73 resulted in broad inhibition zones against R. solani measuring 15.1-25 mm. Among the retrieved 78 isolates, 28% of rhizobacteria produced high amount of IAA and seven isolates, NAU4, NAU5, NAU10, NAU20, NAU28, KBA43 andKBV47 produced maximum IAA. The amount of IAA production by different isolates varied from 3.62μg/ml (NAV69) to 26.96μg/ml (NAU5). Among 78 isolates tested for chitinase assay, two isolates, NAU13 and NAV68 showed maximum activity. Out of 78 isolates, siderophore production was observed in 42% of retrieved bacteria and five isolates, NAU19, KBV45, KBV46, LBV56 and NAV75 produced maximum orange halo zone. HCN was produced by 10% of isolates and highest HCN was observed by 6 isolates, NAU4, NAU6, NAU20, NAU29, NAU41 and NAA59, qualitatively. Seven isolates, NAU5, NAU20, NAU30, NAU35, NAA59, NAV68 and NAV75 were selected and total of 27 treatments were prepared on the basis of antagonistic and plant growth promoting attributes tested in vitro for further evaluating under pot house conditions. Isolate NAU5 showed maximum shoot length, shoot weight, root weight, fruit yield and disease control (100%) against F. oxysporum f. sp. lycopesici. Isolate NAV75 showed maximum shoot length, shoot weight, root weight, fruit yield and disease control (87%) against A. solani. Isolate NAU35 showed maximum shoot length, shoot weight, root weight, fruit yield and disease control (100%) against R. solani. Irrespective of the fungi, isolate NAU5 showed biocontrol activity against all the three phytopathogens. The seven promising isolates showed the PGPR activities in the order NAV75> NAV68> NAA59> NAU5> NAU35> NAU20> NAU30 irrespective of the fungi in pot house studies. The isolates NAU5 against all the three fungi, NAU35 against R. solani and NAV75 against A. solani can be further tested on the tomato fields as potent biocontrol agents. On the basis of biochemical characterization isolate NAU5 was found to belong to Alcaligenes, NAU35 to Bacillus and NAV75 to Burkholderia genus.
Open Access
Decolourization and Detoxification of Direct Blue Textile azo dye Using Bacteria
(CCSHAU, Hisar, 2021-09) Yogita; Ranga, Poonam
In India industrialization resulted in discharge of large amount of effluent to the environment leads to pollution. There are severely harmful impacts of dyes discharged into wastewater as well as their decomposition products are poisonous, cancerous or mutagenic to human beings & other causing health concerns (Roy DC et al., 2018). Due to their simplicity, cost-effectiveness in synthesis, firmness and range of colors of synthetic dyes are commonly used in a various industries. (Kurade et al., 2016; Ajaz et al., 2019). There are various methods to treat the industrial effluent such a physical, chemical and biological method for decolourization of azo dyes. Microorganisms play a crucial role in minerali- zation and decomposition of contaminants. Therefore, microbial decolourization of dyes has gained popularity in recent years due to their ease of rapid growth of cells and exponential rise. A total of 52 bacterial isolates were retrieved from different soil and sludge samples collected from various industrial sites. Screening of bacterial isolates was done on the basis of their ability to de- colourize Direct blue dye. In primary screening six bacterial isolates (YPR 5, YPR 13, YPR 25, YPR 34, YPR 36 and YPR 44) were selected on the basis of their decolourization potential for direct blue dye (10mgL-1). In secondary screening, decolourization efficiency of selected bacterial isolates was determined with respect to time and dye concentration. Maximum decolourization was shown by bacterial isolates YPR 36 (66.9%) and YPR 25 (62.5%) at dye concentration of 200 mgL-1 and YPR 5 (54.4%) at 100 mgL-1 after incubation period of 72 h. Optimization of cultural conditions to maximize decolourization was carried out using different parameters viz. temperature, pH and shaking/static cul- ture conditions. Under optimized conditions, maximum deolourization was observed using bacterial isolates YPR 36 (74.8%), YPR 25 (66.9%) and YPR 5 (61.6%) after 72 h. To determine the phenome- na of decolourization either by biodegradation or adsorption, both living as were as autoclaved cells of isolates were used. Selected bacterial isolates were checked for their decolourization efficiency in tex- tile effluent at different concentration (25-100%). Maximum decolourization was shown by YPR 36 (48.5%) using textile effluent concentration of 25%. Toxic effect of treated and untreated effluents was tested on Chick pea (Cicer arietinum), Green gram (Vigna radiata) and Black gram (Vigna mungo). Germination percentage, plumule and radical size were measured. Treated effluents were found to be less inhibitory to seed germination and seedling growth in comparison to untreated effluents. Based on molecular characteristics (16s rRNA gene sequencing) the promising isolates i.e. YPR 5, YPR 25 and YPR 36 were identified as Pseudochrobactrum kiredjianiae strain YPR 5, Brevundimo- nas diminuta YPR 25 and Alcaligenes faecalis YPR 36, respectively
Open Access
Impact of co-inoculation of plant growth promoting actinomycetes and biofertilizers on mustard (Brassica juncea L.) crop
(CCSHAU, Hisar, 2020) Umang; Leela Wati
Mustard (Brassica juncea L.) a major oilseed crop and the second largest agriculture commodity after cereals in India, plays a significant role in Indian economy. Due to inconsistent climatic changes, occurrence of diseases and implementation of unsuitable dosage of chemical fertilizers and pesticides, overall production of mustard has been comparatively static for last two decades regardless of the use of numerous traditional and breeding approaches. Actinomycetes, a class of actinobacteria are Gram-positive bacteria found in soil and possessing various plant growth traits can play significant role in improving growth and yield of mustard crop. In the present study, two biofertilizer cultures viz., Azotobacter chroococcum and phosphate solubilizing bacterium (PSB) along with seven available actinomycetes isolates AK1, AK3, AK6, AK11, AK17, AK20 and AK37 were screened for different plant growth promoting traits including IAA production, phosphate solubilization, potassium solubilization, zinc solubilization, siderophore production, HCN production, cellulose and chitin degradation under in vitro conditions. The actinomycetes isolates exhibited three or more plant growth promoting traits and were compatible with standard biofertilizer cultures. Actinomycete isolates exhibiting multiple plant growth promoting traits along with biofertilizer cultures were evaluated for growth promotion of mustard under pot culture conditions during 2019-2020. Actinomycete isolate AK17 was adjudged best in improving growth andseed yield of mustard crop when coinoculated with Azotobacter chroococcum and PSB. The isolates AK20 & AK37 were found promising in improving vegetative growth only. The actinomycete isolate AK17 identified by 16S rDNA sequencing showing 99% similiarity with Streptomyces pseudgriseolus, could further be explored as bioinoculant in agricultural practices.
Open Access
Impact of chitinase producing bacteria on thrips infestation in citrus plantation
(CCSHAU,HiSAR, 2020) Sharma, Nidhi; Pathak, D.V.
The studies on chitinase activity of bacterial isolates against thrips on Kinnow were carried out at citrus orchard with the objective Impact of chitinase producing bacteria on thrips infestation in citrus plantation. Plant growth promoting rhizobacteria (PGPR) are root associated bacteria and benefit plants by providing growth promotion through a variety of mechanism. The public concern over the harmful effects of chemical pesticides on the environment and human health has enhanced the search for safer, environmentally friendly control alternatives. Due to the importance of chitinolytic enzymes in insect, nematode, and fungal growth and development, they are receiving attention in regard to their use as biopesticides and microbial biocontrol agents. Therefore, the present study was aimed to isolate and characterize the chitinolytic bacterial isolates and also evaluate their efficacy. A total of eighty morphologically distinct bacterial isolates were retrieved from soil samples of different microbiomes on King‘s B media and these bacterial isolates were screened for chitinase production out of these twenty isolates was showed zone of clearance on the collodial chitin agar plates where zone of clearance and enzyme activity index were used for initial screening of chitinolytic bacteria. Twenty out of eighty bacterial isolates were selected as chitinase producers on the basis of zone size ranging from 12.40 mm to 38.50 mm and were further screened for quantitative chitinase production. Quantitatively it was screened for chitinase enzyme activity on the basis of release of N-acetylglucosamine units (U) by action of chitinase, maximum enzyme activity was recorded for isolate B1 (3.905) followed by B5 (3.52) and D2 (1.35). A total of twenty bacterial isolates, were studied on the basis of their morphological and microscopic characterstics which showed a variation in margins, elevation, shape, pigmentation, Gram‘s reaction and cell shape. These bacterial isolates was biochemically characterized, isolate B5 was found positive for various biochemical tests such as citrate utilization test, Voges Proskauer (VP) test and negative for methyl red test. All the twenty isolates was screened for multifarious plant growth promoting traits viz. IAA production and HCN production. Three bacterial isolates which exhibited maximum chitinase activity were further evaluated for their biocontrol potential against thrips of citrus under field conditions. Kinnow were sprayed with three different bacterial isolates B1, B5, D2 (106 cfu/ml), dimethoate phosphamide spray and nimbicide at different concentrations (1.5ml/L, 2ml/L respectively). One isolate B1 showed maximum biocontrol efficacy in reduction of thrips population -79.66 percent against thrips in kinnow in comparison to control where -37.70 percent of reduction of thrips population was recorded. The percent infestation recorded for B1 isolate was 20 percent as compared to control where infestation was 36 percent. The mean population density recorded for B1 was 0.4 as compared to control where density of thrips was 0.96. The identification of B1 was tentatively confirmed by biochemical characterization and it was identified as strain belonging to Bacillus sp.

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