Changes in Soil Microbial Activity and Community Composition as a Result of Selected Agricultural Practices

For a constantly growing human population, healthy and productive soil is critical for sustainable delivery of agricultural products. The soil microorganisms play a crucial role in soil structure and functioning. They are responsible for soil formation, ecosystem biogeochemistry, cycling of nutrients and degradation of plant residues and xenobiotics. Certain agricultural treatments, such as fertilizers and pesticides applications, crop rotation, or soil amendment addition, influence the composition, abundance and function of bacteria and fungi in the soil ecosystems. Some of these practices have rather negative effects;others can help soil microorganisms by creating a friendlier habitat or providing nutrients. The changes in microbial community structure cannot be fully captured with traditional methods that are limited only to culturable organisms, which represent less than 1% of the whole population. The use of new molecular techniques such as metagenomics offers the possibility to better understand how agriculture affects soil microbiota. Therefore, the main goal of this review is to discuss how common farming practices influence microbial activity in the soil, with a special focus on pesticides, fertilizers, heavy metals and crop rotation. Furthermore, potential practices to mitigate the negative effects of some treatments are suggested and treatments that can beneficially influence soil microbiota are pointed out. Finally, application of metagenomics technique in agriculture and perspectives of developing efficient molecular tools in order to assess soil condition in the context of microbial activities are underlined.

Occurrence and characterization of toxigenic Bacillus cereus in food and infant feces

Objective: To investigate the true incidence of Bacillus cereus(B. cereus) in food and children diarrhea cases. Methods: A total of 110 samples of various dairy products such as raw milk, long life pasteurized milk, yoghurt and infant powdered milk formulas, raw rice, and feces were examined for the presence of B. cereus by selective plating on mannitol-egg-yolk-polymyxin agar. Confirmation of B. cereus was carried out by biochemical tests and PCR. Identification of non-B. cereus isolates was carried out by 16 S r DNA sequencing. Antimicrobial susceptibility was done by disk diffusion method.Results: Overall 35 samples(31.8%, n = 110) yielded Bacillus-like growth. Of which 19 samples(54.28%) were positive for B. cereus. All isolates were positive for enterotoxin production. No psychrotolerant B. cereus strains were detected in all samples. All B. cereus isolates were resistant to penicillin G, but susceptible to vancomycin, erythromycin and clindamycin. Conclusions: The results of this study confirm the importance of including B. cereus in disease control and prevention programs, as well as in routine clinical and food quality control laboratories in both Saudi Arabia and Egypt.

Biofilm Development, Plant Growth Promoting Traits and Rhizosphere Colonization by <i>Pseudomonas entomophila</i>FAP1: A Promising PGPR

Among the diverse soil bacteria, plant growth promoting rhizobacteria (PGPR) mark an important role in enhancing plant growth through a range of beneficial functions. This is mainly achieved by effective rhizosphere colonization by PGPR. Biofilm development by PGPR is considered as a survival strategy over the planktonic mode of growth under stress and natural conditions. Since the performance of microbial inoculants under field conditions is not always consistent due to various biotic and abiotic factors affecting survival, colonization and functions. Therefore, the rhizobacteria with efficient colonization ability and exhibiting multiple PGP traits are expected to perform better. We hypothesized that the biofilm forming ability of PGPR on plant root will be an added advantage to rhizosphere colonization. Therefore, we have selected a promising isolate of PGPR through random screening programme from rhizoplane of wheat (Triticum aestivum). The selection was based on biofilm development ability, multifarious PGP activities (production of indole acetic acid, sidero-phore, phosphate solubilization, hydrogen cyanide, ammonia production and biocontrol activity) and tolerance to salinity and heavy metals. The selected isolate was identified by 16 s rRNA partial gene sequencing as Pseudomonas entomophila-FAP1. The strain FAP1 formed strong biofilm in microtitre plate, glass surface as well as on the roots of wheat seedlings. Biofilm forming capacity of the FAP1 was characterized by scanning electron microscopy and confocal laser scanning microscopy. FAP1 exhibited biofilm-related traits such as the production of exopolysaccharides, EPS (1501.33 ± 1.08 μg ml-1), alginate (212.81 ± 1.09 μg ml-1), swarming motility (22 ± 1.36 mm), swimming motility (31 ± 2.12 mm) and cell surface hydrophobicity (63%). Rhizosphere colonization by FAP1 was found 7.5 Log CFU g-1 of soil comparable with rhizoplane colonization (7.2 Log CFU g-1 of root). Therefore, biofilm formation on plant roots by promising PGPR may be included as an additional criterion to select a better rhizosphere colonizer. Further, study with mutant deficient in biofilm should be developed for comparative study to explore the exact contribution of biofilm in root colonization under natural soil-plant system.

Mutagenicity of Wastewater Extracts from Pulp and Paper Industry

Wastewater samples were collected from pulp and paper mill located in Kashipur (India) and were extracted using dichloromethane (DCM), chloroform and hexane solvents (all were HPLC-grade). The extracts were assayed for their mutagenic potential using Ames Salmonella mutagenicity assay. TA98 strain was found to be the most responsive, in terms of induction factor (Mi) and slope (m) of the initial linear dose-response curve as determined by linear regression analysis up to the increasing doses indicating the presence of frame shift mutagens in the test samples. Mutagenicity of different extracts is arranged as follows: dichloromethane extracted water samples > hexane extracted water samples > chloroform extracted water samples. Hexane extract exhibited maximum mutagenic index of 13.0 and induction factor (Mi) 2.48 with TA98. The order of responsiveness based on the mutagenic index and induction factor for the test samples was in the following order: TA98 > TA97a > TA100 > TA102 > TA104. Our findings suggest that TA97a, TA98, TA100, TA102, TA104 were sensitive towards the wastewater extracts and showed considerable mutagenicity.

Isolation and Characterization of Novel Chlorpyrifos Degrading Fungus Isaria Farinosa

Chlorpyrifos is a well known organophosphorus pesticide used worldwide. Microorganisms including bacteria, fungi and actinomycetes have been reported to be efficient degraders of chlorpyrifos. The present study was successful in isolating a novel fungus that could degrade chlorpyrifos effectively upto 800 ppm concentration. Morphological and molecular characterization studies revealed the identity of the fungus as Isariafarinosa.

Effect of Varied Irrigation Scheduling with Levels and Times of Nitrogen Application on Yield and Water Use Efficiency of Aerobic Rice

More rice needs to be produced with lesser water to feed the increasing human population. Judicious water management practices and appropriate water saving technologies in rice cultivation are in need in the coming decades. Aerobic rice is one of water saving method of rice cultivation. The field experiment was conducted during Summer season of February 2018 to May 2018 at Tamil Nadu Agricultural University, Agricultural College and Research Institute, Madurai, to find out the effect of irrigation schedules with varied doses and time of nitrogen application on yield of aerobic rice. Irrigation scheduling of IW/CPE (Irrigation Water/Cumulative Pan Evaporation) 1.0 up to panicle initiation stage and thereafter IW/CPE 1.2 up to dough stage recorded higher yield attributes viz., number of panicles hill-1 (9.1), number of filled grains panicle-1 (87.9), test weight (15.3 g), grain yield (4462 kg·ha-1), straw yield (5977 kg·ha-1). However, the highest water use efficiency (6.8 kg·ha-1·mm-1) was recorded in the treatment of IW/CPE 1.0 throughout the crop growth period. Lower yield attributes, yield and water use efficiency were recorded with irrigation scheduling of IW/CPE 0.8 throughout the growth stage. Application of nitrogen at 150 kg·ha-1 in 5 equal splits at 20, 35, 50, 65 and 80 DAS (Days after sowing) recorded higher yield attributes viz., number of panicles hill-1 (9.3), number of filled grains panicle-1 (90.5), test weight (15.4 g), grain yield (4746 kg·ha-1), straw yield (6258 kg·ha-1) and WUE (7.5 kg·ha-1·mm-1). Application of nitrogen 100 kg·ha-1 in 4 equal splits at 20, 40, 60 and 80 DAS recorded lower yield attributes, yield and water use efficiency. The interaction effect between irrigation scheduling and nitrogen management on yield was significant. The combination of IW/CPE 1.0 up to panicle initiation stage and thereafter IW/CPE 1.2 up to dough stage along with application of nitrogen at 150 kg·ha-1 in 5 equal splits at 20, 35, 50, 65 and 80 DAS significantly produced higher number of panicles hill-1 (10.7), grain yield of 5419 kg·ha-1 and straw yield of 6906 kg·ha-1. However, IW/CPE 1.0 throughout the growth period along with application of nitrogen at 150 kg·ha-1 in 5 equal splits at 20, 35, 50, 65 and 80 DAS registered the highest water use efficiency (8.4 kg·ha-1·mm-1) in aerobic rice.

<i>In Vitro</i>Efficacy of Clove Oil and Eugenol against <i>Staphylococcus</i>spp and <i>Streptococcus mutans</i>on Hydrophobicity, Hemolysin Production and Biofilms and their Synergy with Antibiotics

The present study aimed to evaluate Syzygium aromaticum (clove) plant extract, clove oil and eugenol for their antibacterial activity and their potential to eradicate bacterial biofilms alone and in combination with antibiotics. Anti-bacterial efficacy of S. aromaticum extract, clove oil and eugenol was evaluated as minimum inhibitory concentration (MIC) and subsequently sub-MICs was selected for inhibition of virulence factors against test bacterial strains. Biofilm cultivation and eradication was assayed using XTT reduction in 96-well microtiter plate. Checkerboard method was used to study the interaction between essential oils and antibiotics. Staphylococcus aureus MTCC3160, Staphylococcus epidermidis MTCC435, Staphylococcus sciuri (SC-01), Staphylococcus auricularis (SU-01) and Streptococcus mutans MTCC497 were found strong biofilm former among all the test bacterial strains. The potency of test agents was found in the order of eugenol > clove oil > S. aromaticum methanolic extract. Sub-MIC (0.5 × MIC) of clove oil and eugenol showed a significant reduction in cell surface hydrophobicity (p < 0.05) and hemolysin production in the test bacterial strains. Eugenol showed no increase in sessile MIC (SMIC) against S. auricularis (SU-01), S. epidermidis MTCC435 and S. mutans MTCC497 compared to planktonic MIC (PMIC). Antibiotics (vancomycin and azithromycin) exhibited upto 1000-folds increased in SMIC compared to PMIC against all the test bacterial strains. Synergy was observed between eugenol and antibiotics (vancomycin/azithromycin) against all the test bacterial strains in both planktonic and sessile mode. Highest synergy was exhibited between eugenol and azithromycin in planktonic mode (FICI value 0.141). Further, microscopy also confirmed the spectacular effect of combination treatment on pre-formed S. aureus MTCC3160 and S. mutans MTCC497 biofilms. These findings highlighted the promising role of clove oil and eugenol alone and in combination on pathogenic bacterial biofilms.

Evidence of Fungicides Degradation by Rhizobia

Fungicides which are not easily degradable have the greatest adverse effects on soil microbes. These pesticides negatively affect the growth and multiplication of fungi and bacteria and consequently cause the disturbance of the natural soil microbial balance. In this study two fungicide tolerant isolates of rhizobia;clover isolate (TA1) and peanut isolate (8) were assessed in their capacity to degrade Vitavax and Rizolex. The performance of these isolates in fungicides degradation was tested using the colorimetric assay for Rizolex and the HPLC analysis for Vitavax to detect the degradation products. Using HPLC analyses, the control sample showed specific peak indicating the Vitavax presence in the medium. The specific peak did not change in the control samples throughout the experiment.With the strainTA1 the specific peak of the Vitavax fungicides started to reduce as the incubation time goes on. The Vitavax fungicide did not degrade completely after 240 hours of incubation with rhizobial isolate. The Rizolex used in this study contained blend of Thiram (active ingredient of Rizolex) and Tolcofs methyl fungicides in 1:1 ratio. The biodegradation of Rizolex in the liquid media showed the formation of two new intermediates which were released into the medium indicating the degradation of the tested fungicide by peanut rhizobial isolate No. 8 in 48 hrs of incubation 45% of this compound was degraded. This work shows that the selection of fungicides tolerant rhizobial strains is important to protect the rhizobial inoculants from the toxic effect of the pesticides.

Cloning of a New Truncated <i>cry</i>1<i>Ac</i>Gene from an Indian Isolate of <i>Bacillus thuringiensis</i>

Transgenic Bt crops producing insecticidal crystal proteins from Bacillus thuringiensis (Bt), so-called Cry toxins, have proved useful in controlling insect pests. Among the cry toxins, Cry1A toxins are important because of high toxicity to lepidopteran pests and their widespread distribution among Bt strains. In Cry1A proteins, toxin fragment is comprised of about 620 amino acids of N-terminal region and C-terminal half is not required for toxicity. Four indigenous isolates of Bt viz., T15, T16, T20 and T31 were screened by PCR-RFLP for 3’-truncated cry1A gene(s) corresponding to toxin fragment. RFLP analysis of cry1A amplicons obtained from the four isolates of Bt showed presence of cry1Ac-type gene alone in three isolates. One of the cry1Ac-postive isolates, T15 which showed 100 percent mortality in Helicoverpa armigera, was selected for cloning of DNA fragment of about 2.1 kb containing 3’-truncated cry1Ac gene. Nucleotide sequence data generated for 3’-truncated cry1Ac gene of T15 showed 98 to 99 percent homology with 1958 bp of already reported sequences of all cry1Ac genes (cry1Ac1 to cry1Ac24). Deduced amino acid sequence of cry1Ac of Bt strain, T15 showed one to four percent variation in comparison to all reported Cry1Ac holotypes (Cry1Ac1 to Cry1Ac24) by differing at 5 to 19 positions. This suggests that the cry1Ac toxin of Bt isolate, T15 is a new kind of its group.

Enhancing laccase stability and activity for dyes decolorization using ZIF-8@MWCNT nanocomposite

The continuous use of chemical dyes in various industries,and their discharge into industrial effluents,results in severe problems to human life and water pollution.Laccases have the ability to decolorize dyes and toxic chemicals in industrial effluents as green biocatalysts.Their possible industrial applications have been limited by poor reusability,low stability,and loss of free laccase action.In this research,lac-case was immobilized on zeolitic imidazolate framework coated multi-walled carbon nanotubes(Laccase@ZIF-8@MWCNTs)via metal affinity adsorption to develop an easy separable and stable enzyme.The optimum reaction conditions for immobilized laccase are at a pH of 3.0 and a temperature of 60℃.The immobilized laccase was enhanced in storage and thermal stability.The results indicated that Laccase@ZIF-8@MWCNTs still maintained 68%of its original activity after 10 times of repeated use.Most importantly,the biocatalytic system was applied for decolorization of different dyes(20 mg·L^(-1))without a mediator,and up to 97.4%for Eriochrome black T and 95.6%Acid red 88 was achieved in 25 min.Biocatalysts with these properties may be used in a variety of environmental and industrial applications.

Short-Term Effect of Nitrogen Intensification on Aggregate Size Distribution, Microbial Biomass and Enzyme Activities in a Semi-Arid Soil Under Different Crop Types

There is a lack of quantitative assessments available on the effect of agricultural intensification on soil aggregate distribution and microbial properties. Here, we investigated how short-term nitrogen(N) intensification induced changes in aggregate size distribution and microbial properties in a soil of a hot moist semi-arid region(Bangalore, India). We hypothesised that N intensification would increase the accumulation of macroaggregates > 2 mm and soil microbial biomass and activity, and that the specific crop plant sowed would influence the level of this increase. In November 2016, surface(0–10 cm) and subsurface(10–20 cm) soil samples were taken from three N fertilisation treatments, low N(50 kg N ha-1), medium N(75 and 100 kg N ha-1 for finger millet and maize, respectively),and high N(100 and 150 kg N ha-1 for finger millet and maize, respectively). Distribution of water-stable aggregate concentrations,carbon(C) and N dynamics within aggregate size class, and soil microbial biomass and activity were evaluated. The high-N treatment significantly increased the concentration of large macroaggregates in the subsurface soil of the maize crop treatment, presumably due to an increased C input from root growth. Different N fertilisation levels did not significantly affect C and N concentrations in different aggregate size classes or the bulk soil. High-N applications significantly increased dehydrogenase activity in both the surface soil and the subsurface soil and urease activity in the surface soil, likely because of increased accumulation of enzymes stabilised by soil colloids in dry soils. Dehydrogenase activity was significantly affected by the type of crop, but urease activity not. Overall, our results showed that high N application rates alter large macroaggregates and enzyme activities in surface and subsurface soils through an increased aboveground and corresponding belowground biomass input in the maize crop.

Identification of Arbuscular mycorrhizal multiplicity in the saline-sodic soils

This study focused on the Arbuscular mycorrhizal(AM)fungal diversity in the saline-sodic soils based on native spore density and most probable number(MPN)assay.Identification through spore morphology showed existence of five genera in the various crop rhizospheres.The physico-chemical analysis of the native soils revealed that they were saline-sodic with pH ranging from(8.7±0.5)to(9.5±0.6)and habituated five different genera of AM fungi including Glomus,Scutellospora,Acaulospora,Sclerocystis and Gigaspora.Each location revealed presence of varied species of AM fungus namely Acaulospora and Glomus in rhizosphere of maize;Scutellospora and Glomus in tulsi;four isolates of Glomus in onion;Glomus and Sclerocystis in guava;three isolates of Glomus in rice;Glomus in neem and Gigaspora and Glomus in bamboo.The molecular identification through nested PCR analysis showed amplification of 600 bp size in SSU rDNA gene in samples A and C(predominated by Acaulospora and Glomus mosseae respectively).

Genotoxicity of ferric oxide nanoparticles in Raphanus sativus：Deciphering the role of signaling factors,oxidative stress and cell death

We have studied the genotoxic and apoptotic potential of ferric oxide nanoparticles（Fe_2O_3-NPs） in Raphanus sativus（radish）.Fe_2O_3-NPs retarded the root length and seed germination in radish.Ultrathin sections of treated roots showed subcellular localization of Fe_2O_3-NPs,along with the appearance of damaged mitochondria and excessive vacuolization.Flow cytometric analysis of Fe_2O_3-NPs（1.0 mg/m L） treated groups exhibited 219.5%,161%,120.4% and 161.4% increase in intracellular reactive oxygen species（ROS）,mitochondrial membrane potential（ΔΨm）,nitric oxide（NO） and Ca2＋influx in radish protoplasts.A concentration dependent increase in the antioxidative enzymes glutathione（GSH）,catalase（CAT）,superoxide dismutase（SOD） and lipid peroxidation（LPO） has been recorded.Comet assay showed a concentration dependent increase in deoxyribonucleic acid（DNA） strand breaks in Fe_2O_3-NPs treated groups.Cell cycle analysis revealed 88.4% of cells in sub-G1 apoptotic phase,suggesting cell death in Fe_2O_3-NPs（2.0 mg/m L） treated group.Taking together,the genotoxicity induced by Fe_2O_3-NPs highlights the importance of environmental risk associated with improper disposal of nanoparticles（NPs） and radish can serve as a good indicator for measuring the phytotoxicity of NPs grown in NP-polluted environment.

Biological silicon nanoparticles improve Phaseolus vulgaris L. yield and minimize its contaminant contents on a heavy metals-contaminated saline soil

The synthesis of biological silicon nano-particles(Bio-Si-NPs)is an eco-friendly and lowcost method.There is no study focusing on the effect of Bio-Si-NPs on the plants grown on saline soil contaminated with heavy metals.In this study,an attempt was made to synthesis Bio-Si-NPs using potassium silica florid substrate,and the identified Aspergillus tubingensis AM11 isolate that separated from distribution systems of the potable water.A twoyear field trial was conducted to compare the protective effects of Bio-Si-NPs(2.5 and 5.0 mmol/L)and potassium silicate(10 mmol/L)as a foliar spray on the antioxidant defense system,physio-biochemical components,and the contaminants contents of Phaseolus vulgaris L.grown on saline soil contaminated with heavy metals.Our findings showed that all treatments of Bio-Si-NPs and potassium silicate significantly improved plant growth and production,chlorophylls,carotenoids,transpiration rate,net photosynthetic rate,stomatal conductance,membrane stability index,relative water content,free proline,total soluble sugars,N,P,K,Ca2+,K+/Na+,and the activities of peroxidase,catalase,ascorbic peroxidase and superoxide oxide dismutase.Application of Bio-Si-NPs and potassium silicate significantly decreased electrolyte leakage,malondialdehyde,H2 O2,O2·-,Na+,Pb,Cd,and Ni in leaves and pods of Phaseolus vulgaris L.compared to control.Bio-Si-NPs were more effective compared to potassium silicate.Application of Bio-Si-NPs at the rate of 5 mmol/L was the recommended treatment to enhance the performance and reduce heavy metals content on plants grown on contaminated saline soils.

Fungicide tolerant Bradyrhizobium japonicum mitigate toxicity and enhance greengram production under hexaconazole stress

Bacterial strain RV9 recovered from greengram nodules tolerated 2400 μg/mL of hexaconazole and was identified by 16 S rDNA sequence analysis as Bradyrhizobium japonicum(KY940048). Strain RV9 produced IAA(61.6 μg/mL), ACC deaminase(51.7 mg/(protein·hr)), solubilized TCP(105 μg/mL), secreted 337.6 μg/mL EPS, and produced SA(52.2 μg/mL) and 2,3-DHBA(28.3 μg/mL). Exopolysaccharides produced by strain RV9 was quantified and characterized by SEM, AFM, EDX and FTIR. Beyond tolerance limit,hexaconazole caused cellular impairment and reduced the viability of strain RV9 revealed by SEM and CLSM. Hexaconazole distorted the root tips and altered nodule structure leading thereby to reduction in the performance of greengram. Also, the level of antioxidant enzymes, proline, TBARS, ROS and cell death was increased in hexaconazole treated plants.CLSM images revealed a concentration dependent increase in the characteristic green and blue fluorescence of hexaconazole treated roots. The application of B. japonicum strain RV9 alleviated the fungicide toxicity and improved the measured plant characteristics. Also,rhizobial cells were localized inside tissues as revealed by CLSM. Colonization of B.japonicum strain RV9 decreased the levels of CAT, POD, APX, GPX and TBARS by 80%, 5%,13%, 13% and 19%, respectively over plants grown at 80 μg/(hexaconazole·kg) soil. The ability to detoxify hexaconazole, colonize plant tissues, secrete PGP bioactive molecules even under fungicide pressure and its unique ability to diminish oxidative stress make B.japonicum an attractive choice for remediation of fungicide polluted soils and to concurrently enhance greengram production under stressed environment.