Soil Biotechnology and Sustainable Agricultural

Indian J. Fert., Vol. 11 (10), pp.87-105 (19 pages)

Soil Biotechnology and Sustainable Agricultural Intensification D.L.N. Rao1, D. Balachandar2 and D. Thakuria3 1

ICAR-Indian Institute of Soil Science, Bhopal, Madhya Pradesh 2 Tamil Nadu Agricultural University, Coimbatore, Tamil Nadu 3 Central Agricultural University, Barapani, Meghalaya email: [email protected]

Unsustainable agricultural intensification including intensive tillage, imbalanced and excessive use of chemical fertilizers, excessive pesticides, etc., have both short and long-term adverse impacts on soil microbial diversity and functioning. Identification of sustainable levels of intensification of production requires knowledge of thresholds of the soil functions trade-offs in different agro-ecosystems and production regions. Recent advances in microbiology and biotechnology provide a) deeper insights into changes in soil microbiome structure and function through molecular tools, enabling better judgement of the consequences of agricultural intensification and b) technologies like improved microbial inoculants, composts, transgenic crops that help reduce the use of industrial inputs, thus reducing their imprint on the soil microbiome. These technologies maximise the potential of soil microbial resources in meeting the nutrient demand, improve soil health, impart resistance to abiotic and biotic stresses and minimize the risks due to soil degradation and pollution. The best options currently available for sustainable intensification are integrated farming systems (IFS) involving practices like: residue recycling, conservation agriculture, legume rotations, composts, biofertilisers and biocontrol agents. A distant goal is to exploit biotechnology to tailor the crops for various environments for achieving high productivity and resistance to stresses and disease through suitably manipulating the soil microbiome by rhizosphere engineering.

Introduction Sustainable agriculture is based on ecological principles which ensure that production systems are not only economically viable but also do not degrade the environment and at the same time bring about social equity in the long run. Agriculture also contributes to the sustainability of societies; by helping urban areas to manage wastes, e.g., by recycling urban sewage sludge, waste waters and rural areas by improving employment opportunities and contributing to agro-industrial sector. The Food and Agriculture Organization of the United Nations is set to adopt a set of 17 sustainable development goals by end of 2015 which instils a greater sense of urgency, a task in which soil scientists have a very major role to play. The exploitation of a diversity of crops and varieties and maintenance of soil biodiversity is one of the ways for optimizing ecosystem processes and functions to sustain agroecosystems. This is especially crucial for farming systems practiced by small-scale farmers and rural communities in marginal

lands. Meeting the food, fibre, biomass and energy needs of an additional 3.5 billion people that will reside in developing countries in future will mean utilizing marginal soils or other barren lands to improve productivity; ameliorating degraded or desertified soils to improve ecological functions; improve water use efficiency; minimize risks of water pollution; create reserves for species preservation, recreation and enhancing aesthetic value of soil resources (95). Over the centuries ever increasing human needs have been exerting continued pressure on soil, as a result, biodiversity is declining a thousand times faster now than at rates found in the fossil record (118). This requires us to identify properties, processes and practices that affect the sustainable management and ensure that they are tailored and practiced in a way as to enable greater production but in a sustainable manner. This will require determination of the threshold levels of soil degradation (e.g., soil organic matter, soil biota and its functions) that can be tolerated to attain a Indian Journal of Fertilisers, October 2015 87

certain production level. Lal (2009) listed ten governing principles for arresting soil degradation and restoring soils which require inter-disciplinary approaches with close linkages between soil scientists and others including chemists, physicists, geologists, hydrologists, climatologists, biologists especially the molecular geneticists dealing with human, animal and microbial processes, system engineers, information technologists, economists, and social scientists. Despite the fact that soils are home to a quarter of all living species on Earth, its importance is not understood and often neglected. How some of our agricultural management practices are impacting soil biodiversity and ecosystem processes and in turn soil health? How application of biotechnology in soil science can help in sustainable agricultural intensification? Some of these crucial concerns and opportunities are highlighted in this review. How Do Function?

Soil

Ecosystems

The ecosystem provides four kinds

of services: i) food, fresh water, fuel, fibre and wood, ii) regulation of gas and water, climate, floods, erosion, biological processes such as pollination and diseases, iii) provision of aesthetic, spiritual, educational and recreational services and iv) supporting services including nutrient cycling, production, habitat, and biodiversity (118). Soils are three dimensional, spatially and temporally heterogenous, with several trophic levels and complex physico-chemical and biological interactions, of several orders of magnitude, all of which serve to regulate the multitude of functions called “soil functions” which ultimately deliver the ecosystem services. Thus soils are one of the most complex biomaterials on the earth and least understood among the systems that provide ecosystem services (164). The type of soil, vegetation and its management impacts these functions significantly. Relationships between plant diversity and soil biological properties are very complex in nature and of key significance for ecosystem function (65). Biodiversity plays a direct key role in regulating and modulating ecosystem ‘processes’ and ‘functioning’ that underpin the delivery of ecosystem services. Only a few studies have linked the soil properties and management to ecosystem services. By linking the soil biodiversity and functions to the ecosystem services, the soil ecosystem can be better conserved and managed for sustainable agricultural intensification (1, 7, 105). Soil Biology Drives Agricultural Sustainability Agricultural intensification has depended on increased use of synthetic fertilizers, herbicides and pesticides; improved mechanization; efficient crop production systems and development of global markets (84). However, the soil biological responses to these developments were not recognized or were overlooked resulting in

unintended consequences on soil health and long-term agricultural sustainability (96). Soil biota refers to the collective biomass and activities of soil-dwelling organisms from an array of trophic levels. Soil organisms control the decomposition of plant and animal materials; are involved in biogeochemical cycling of elements, including nitrogen fixation; contribute to the formation of soil structure; transform the organics and inorganics applied to soils and regulate the production and consumption of greenhouse gases in soils. It is estimated that there are one billion bacterial cells per gram of soil comprised of thousands to millions of individual species (62). The biomass of fungi is often more than bacterial biomass in most soils. The total microbial biomass existing in the underground may be equal to the sum of all living biomass on the earth surface. A good “quality” soil or “healthy” soil is recognized as one that could accept, hold and release nutrients, water and other chemical constituents; promote and sustain root growth; maintain suitable soil biotic habitat; respond to management and resist degradation (50). All these attributes of good soil health are directly a reflection of how well is biology functioning. Hence, the current emphasis is on optimizing the solutions for sustaining the soil health and reversing the soil degradation by improving the soil’s biology (91, 138). The scales of assessment of soil biodiversity ranges from pico- and nano-scale studies for example on structure and chemical composition of organic substances and microorganisms; interactions between the biota and humic substances. Micro-scale investigation refers to soil aggregates or on microhabitats characterised by high turnover of organic materials (e.g. the rhizosphere, drilosphere and soillitter interface). High-activity areas are heterogeneously distributed within the soil Indian Journal of Fertilisers, October 2015 88

matrix. Biologically active hotspots may make up less than 10% of the total soil volume, yet may represent more than 90% of the total biological activity (16). In terms of above-ground vegetation, soil biodiversity studies can be categorized under forest land, grassland and agriculture lands. Soil Microbiome Soil Microbiome Complexity Unravelled by Molecular Tools Soil microbiome is a sensitive indicator of soil health as it provides an indication of the direction and magnitude of the changes in ecosystem structure and function, earlier and better. Soil microbial biomass, respiration, soil enzyme activities are extensively used to assess the microbiome. However, due to its complexity, less attention has been given to community-level microbial properties. Soil microbial analyses should ideally include the determination of communities (diversity), growth, distribution, functions and also interactions among the species. In the past 15 years, many biotechnological approaches have become available, allowing a better assessment (75, 76, 86, 170). The difficulties to separate the microbial cells from the soil matrix, morphological and biochemical similarities among the microbes and ambiguity in microbial taxonomy are the major challenges and hurdles that remain in understanding microbial diversity. However polyphasic approaches integrating the knowledge from various approaches are now allowing a better appreciation of the structure of the community. Soil microbial diversity describes complexity and variability at different levels, principally the number of species (richness), relative abundance (evenness) and functional groups in communities. It is known that billions of cells with vast diversity (10 4 to 10 6 distinct taxa)

are present in each gram of soil (62) and information about the vast majority is lacking as the routine cultural methods recover only a small fraction of soil microorganisms (for example less than 1% of soil bacteria). Nucleic acid diversity is the most useful in providing new understanding of microbial community structure, yet a few non-nucleic acid methods such as community-level physiological profiling (CLPP) (63) and phospholipid fatty acid (PLFA) analysis (192) are also used despite their limitations. CLPP uses a commercial taxonomical system, BIOLOG ® for analysis of soil microbial communities based on carbon source utilization and is frequently applied to assess the community shifts caused by various environmental factors (59); however, the method is limited by bias encountered in culture plating methods and difficulties in data interpretation. The PLFA are unique signature molecules present only in the cell membranes and metabolised rapidly in the soil after cell death; PLFA analysis serves as a good indicator of active microbial biomass as against the dead microbial cells. However the inadequate information on PLFA profiles of individual taxon in data bases and data interpretation problems hamper the use of this method as a holistic measure of diversity at community level. The determination of 16S rRNA gene sequence in prokaryotes and 18S rRNA gene and intergenic transcribed spacer region between the ribosomal genes (ITS) in eukaryotes are now the standards for estimating microbial community composition and diversity in soil. The ribosomal genes have both conserved and hyper-variable regions, the sequence changes are easily related to the evolution (phylogeny) and also provide secondary structures for mobility based fingerprint analyses; thus it is advantageous to use ribosomal genes for community diversity analysis. Various studies from different ecological niches have shown that

more than 90% of the microorganisms present can be extracted and analysed (134) as compared to culturable methods which recover only 0.1% of the actual population. Numerous studies conducted thereafter have used these techniques to assess the soil microbial communities. More recently, several highly powerful sequencing techniques, referred to as next generation (NG) sequencing methods were developed (146) and among them, 454-based or pyrosequencing and Illumina/ Solexa’s Genome Analyser sequencing are now predominantly used. Soon after these techniques emerged, soil meta-genomes (DNA) and meta-transcriptomes (RNA) were analysed (145,180). The NGS methods bypass three major bottle-necks of classical clonal library based sequencing: they avoid amplification bias in PCR assays, preparation of clonal libraries and template preparation for sequencing. Due to these, the direct sequencing of soil DNA allows one to dissect the system finely and to uncover the mostabundant to rare species (145). However small read lengths and more error-prone sequence data compared to Sanger sequencing are the drawbacks of next-generation sequencing. Although high cost of equipment and procedures limits the use of NG sequencing, the situation is rapidly changing and NG sequencing methods are extensively being used and are now the gold standard for the analyses of soil microbial community structure. Since the 16S rRNA gene may occur in multiple copies per genome, single-copy markers such as rpoA, gyrB and recA have been extensively used. Even though, the analysis of these alternative genes is a promising approach, the limited amount of sequences available for these genes in the databases and primer designing problems hamper this approach. Another alternative to community-based approach (ribosomal genes) is the function-based approach which relies on distribution and abundance of organisms based on Indian Journal of Fertilisers, October 2015 89

their metabolism and assesses the functional genes, i.e., those that code for enzymes involved in the biogeochemical nutrient cycling. Such functional markers that are used include amoA (ammonia oxidation or nitrification), nifH (nitrogen fixation), nir and noz (denitrification), nod (nodulation) genes. Following the sequencing based community profiling (via cloning libraries), a number of fingerprinting methods have been developed to get comparative overview of the composition and diversity of soil microorganisms. The PCR generated amplicon based molecular fingerprinting methods such as denaturing gradient gel electrophoresis (DGGE) (121), temperature gradient gel electrophoresis (TGGE) (121), terminal restriction fragment length polymorphism (T-RFLP) (93), single-strand conformational polymorphism (SSCP) (157), ribosomal internal spacer analysis (RISA) (137) and length heterogeneity-PCR (LH-PCR) (144) have emerged. All these methods enable the direct fingerprinting of soil microbial communities at different level of resolution and have their advantages as well as limitations (178). Even though the molecular tools have widened our knowledge of the soil microbiome structure and diversity during the past 10-15 years, they have not fully helped to explain how soil microbial diversity influences the functioning of soil ecosystem. This is because of the enormous redundancy of microbial functions, in the sense that the loss of one species is compensated by the presence of others that can do the same function. The 16S rRNA gene sequencing of soils have revealed a widespread distribution of mesophilic archaea (members of Crenarchaeota) in soil, which contribute about 5% of total number of prokaryotes (154). Despite the abundance and diversity of archaea detected in soil, no archaeal species from soil have yet been cultured in the laboratory. The metagenomic analysis of soil revealed that crenarchaeotes contribute to the

first step of nitrification, viz., ammonia oxidation by ammonia monooxygenase (98) showing that they may play important role in soil nitrogen cycling (71). Some reports also suggest that the members of Crenarcheota and Euryarchaeota may colonize the rhizosphere of maize akin to bacteria (36). The abundance of methanogenic Euryarchaeota was well-documented in rice fields and anaerobic landfill sites (185). The significance of archaea in terrestrial ecosystems has been recently reviewed (52); more work is warranted in this area to exploit the knowledge of archaea for sustainable intensification of agriculture. Another recent molecular technique commonly applied in soil microbial ecology is Q-PCR or real-time PCR which allows the quantification of the abundance and expression of functional genes within the ecosystem. The technique combines traditional PCR with fluorescent detection technology to record the accumulation of amplicons in real time during each cycle of PCR amplification. Thus it becomes possible to quantify the target gene or its transcripts present as template (163). Using this technique, the abundance of 16S rRNA (eubacteria), nifH (nitrogen fixers), amoA (nitrifiers), nirK/nirS (denitrifiers), nosZ (nitrous oxide reducers), atz (atrazine degraders) genes were quantified in various soil ecosystems (163). The major limitations of qPCR technique are similar to the bias in PCR based soil DNA extraction; inherently biased picture of target gene abundance and also it does not detect any gene of same function whose sequence is different. However, qPCR is well suited at micro-habitat level to assess the impact of local conditions on gene abundance and its expression. Soil microbiome and nutrient cycling The role of soil fauna in regulating nutrient cycling is well known. Soil carbon build-up in a sorghum field was more affected by the quality of organic amendments than the

quantity of carbon inputs in presence of soil fauna (130). Yan et al. (2007), in long-term field experiments in paddy soil of China found that microbial biomass C and N closely correlated with particulate organic matter (POM >53µ fraction) rather than total soil organic matter content. While Cmineralization closely related to the amounts of POM-N, POM-C, microbial biomass C and soil organic C, there was no significant correlation between N mineralization and C or N amounts in soil and its fractions. Relationship between N-cycling communities and ecosystem functioning in a clay loam from a 50-year-old fertilization experiment in Sweden (73) were studied and it was shown that there were significant correlations between community composition and size of all functional guilds: nitrate reducers, denitrifiers and ammonia oxidizers in addition to the total bacterial community. Similarly, high correlation was also reported between denitrification enzyme activity and the size of the denitrifier community estimated by most probable number (133) or by the quantification of nirK genes (168), which suggests that the size of the denitrifier community could predict the corresponding process rate. However, the differences in process rates for the ammoniaoxidizing bacterial community were correlated neither with differences in size nor composition (73). Thus, archaeal ammonia oxidizers may have greater role than the bacterial counterparts in many environments in N-cycling processes (135). In order to assess biological condition of a soil under agro-ecosystems in Australia, Chapman et al. (2012) used an environmental microarray of key functional genes across bacterial and archaeal domains (2934 unique probes from 6420 gene sequences) that facilitate soil processes like nitrogen and phosphorus cycling, one-carbon degradation, biodegradation of complex organic toxins, and the production of antibiotics, etc. In two soil land-use sites Indian Journal of Fertilisers, October 2015 90

(agricultural management and remnant vegetation) from two soil orders, it was shown that the strongest determinants in altering functional microbial community following agricultural management are modification of the soil N cycle. Soils under cropping were associated with higher levels of ammonium (NH4+) and increased frequency of abundance of ammonia monooxygenase genes (amoA). In contrast, soils under grazing management were associated with higher levels of nitrate (NO 3 - ) and increased abundance of nitrogenase genes (nifH). How does agricultural intensification impact the soil microbiome? Removal of natural vegetation followed by crop cultivation results in reduction of soil organic carbon, deterioration of soil structure, decrease of microbial biomass and activity and community composition (32). The changes in above-ground vegetation affect the microbial community composition and functioning (126). However, agricultural practices do not always deplete diversity; the impact of agriculture (including types of crops, agronomic practices, soil management practices) to microbial diversity may be positive, negative or neutral (161). Soils are the habitat of diverse communities of microorganisms with about 10000 to 50000 species present in one gram of soil (155). Ecological processes are driven by microbes which regulate and maintain the balance between the source and sink and these processes themselves affect the distribution, diversity or amount and number of microorganisms across time and space (74). Among the ecological processes, nutrient cycling is one of the most important one that sustains life on planet earth. Factors like land use change, agricultural intensification, quality and quantity of inorganic and organic inputs, synthetic chemicals, etc., have direct impact on the taxonomic and functional diversity of soil biota involved in

Figure 1 – 16S rRNA gene sequence based eubacterial community composition of soils (A- Control, unfertilized control soil; B – OM, organic manures amended soil; C –IC, inorganic chemical fertilizer amended soil) influenced by long-term application of organic manures and chemical fertilizers application. Values of each panel denote the number of similar sequences. [Chinnadurai et al. 2014, http://www.tandfonline.com/doi/full/10.1080/03650340. 2013. 803072, © Taylor & Francis, 2014].

carbon, nitrogen, and phosphorus cycles. The inherent soil properties which affect the community diversity are soil pH (57), texture (68), nitrogen (60) and phosphorus (54). Soil pH is the most sensitive and influential property that drives the bacterial diversity in soil (57, 147). The reason for this relation might be because bacterial cells exhibit tolerance of growth to a relatively narrow pH range. Other inherent properties including altitude and ratio of cation concentrations have been reported to influence soil microbial community (54). The main differences in soil bacterial community structure in Amazon secondary forest land and crop fields under agricultural practices by indigenous people (82) related to changes in the base saturation, Al 3+ , and pH gradients which accounted for 31% of the variability of the bacterial communities. Long-term intensive tillage affects soil organic carbon, water content, temperature, aeration, soil aggregation, etc., (114) which in turn impacts microbial diversity. Several reports showed that zerotillage or minimal tillage favorably improves or sustains the microbial

properties of soil (70). The culture independent 16S rRNA gene sequence diversity analyses revealed that members of phyla Proteobacteria and Acidobacteria are more abundant in no-tillage plots and were lowered by intensive tillage (158). Next to tillage, nutrient management has a significant impact on the soil microbiome. Long-term organic fertility management favours the microbial abundance, diversity and activity of soil (24) by increasing the soil organic carbon. The use of inorganic fertilizers also brings change in soil properties including enzymes and microbiome (81). However, Zhong and Cai (2007) reported that longterm practice of balanced mineral fertilizers (NPK) may cause negligible deleterious effects to the soil biological properties than those from unbalanced fertilization (NP, NK, PK). The investigation in 100-year old permanent nutrient management trial revealed that continuous application of organic manures enhanced the abundance of members of phyla Acidobacteria and Actinobacteria, while the balanced inorganic fertilization had minimal shift in the bacterial Indian Journal of Fertilisers, October 2015 91

community composition of semiarid tropical Alfisol (Figure 1). The consequences of agricultural intensification with fertilizers and pesticides in Vertisols of Guntur, Andhra Pradesh showed differential effects in chilli and black gram. Very high inputs (~5× fertilizers and 1.5× pesticides) in chillis had an adverse effect on soil biological properties but high chemical inputs at ~2.3× fertilizers and pesticides in black gram did not have adverse effect. In chillis, the counts of copiotrophs increased, the counts of fungi and Actinobacteria reduced significantly, glucosidase activity increased and acid phosphatase activity reduced with very high inputs (7). Metagenomic analysis of 16S rRNA gene showed a drastic reduction in the diversity of bacteria that was represented by only three phyla in very high input soils of chilli, whereas in normal there were 12 phyla (105), (Figure 2). The proportion of Actinobacteria reduced from 30% in normal input soils to 14% in very high input soils. The dominant genus was Asticaccaulis which is closely related to Sphingomonads.

potentially by favouring various slow-growing oligotrophic organisms (107).

Figure 2 – Relative distribution of principal eubacterial phyla based on 16S rRNA gene sequences of soils. BG 1 and BG 2 are ‘normal’ and ‘high’ input (~2.3× fertilizers and pesticides ) site of black gram; CH 1 and CH 2 are ‘normal’ and ‘very high’ input ((~5× fertilizers and 1.5× pesticides) site of Chilli (modified from Malhotra et al 2015)

In chilli, the labile carbon mineralization coefficient was comparatively higher in very high input farming compared to normal indicating that the system is under stress (7). Thus there was indeed a relation between reduction in microbial structure and diversity and robust function like carbon mineralization efficiency. In black gram, which is a legume, the labile carbon mineralization coefficient was lesser than that in normal input soils indicating lesser stress on the ecosystem. The counts of copiotrophs, fungi, Actinobacteria, -glucosidase and acid phosphatase activity increased. The phylogenetic diversity of bacteria did not reduce in high input soils of black gram but was altered. No significant reduction in proportion of Actinobacteria was observed. Among the Actinobacteria, the genus Geodermatophilus was the dominant genus in normal input soils of black gram (105). This organism was not observed in normal input soils of chilli, high input soils of black gram and very high input soils of chilli. Another

genus of Actinobacteria, Marmoricola which often shares niche with Geodermatophilus is a coccoid form and was observed in normal input soils of chilli and high input soils of black gram but was absent in very high input soils of chilli. The proportion of rhizobial nifH in blackgram soil was reduced by 46% due to high inputs compared to normal inputs (105). Methods for measurement of soil biological health should therefore emphasize the size of Actinobacteria. The relative proportions of Actinobacteria and Proteobacteria thus serves as a good indicator of soil biological heath (142). Application of organic amendments increased the richness of soil microbiota by promoting copiotrophic organisms whose predominance in turn reduced evenness. In contrast, the absence of such substrate rich organic amendments led to a less eutrophic environment and a likely more variable distribution of nutrients, leading to reduced richness while increasing evenness and dispersion

In organically farmed Aridisols of Rajasthan, there was increase in counts of copiotrophs, oligotrophs, actinobacteria, higher content of glomalin (a measure of vesicular arbuscular mycorrhizal fungi), dehydrogenase activity, acid phosphatase, fluorescein diacetate hydrolysis and - glucosidase. The eubacterial diversity analysis by sequencing of metagenomic DNA using next generation sequencing technologies showed that organic cropping soil contained Actinobacteria, Firmicutes, Bacteroidetes, Chloroflexi and Cyanobacteria in significantly greater proportion compared to conventional cropping. Actinobacteria were 10% higher in organic and Proteobacteria were 20% higher in proportion in conventional management. Shannon diversity index was higher for organic soils (6). The distribution of bacterial species observed in organic cropping was more even. A higher quantity of 16S rRNA gene was found in organic soils compared to conventional soils by quantitative real time PCR analysis of metagenomic DNA. The quantity of bacterial amoA was higher in conventionally cropped soils. Overall, the results demonstrated that organic amendments improved the biological quality through an alteration of the microbial community structure and function and could be included in the group of ‘Ecosystem Engineers’ (186) that selectively modify the environment and make soil ecosystems more sustainable. The differences between organic farming and conventionally managed soils became smaller under integrated fertilization management (183). Bulluck et al. (2002) showed in three organic and three conventional vegetable farms in Virginia and Maryland that organic amendments (cotton-gin trash, composted yard waste or cattle manure) increased the soil organic matter,

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cation exchange capacity, beneficial soil microorganisms and lowered the bulk density and pathogen populations, thus improving soil health. Organics supported higher propagule densities of Trichoderma spp., thermophilic microorganisms and enteric bacteria than NPK fertilizer. The propagule densities of Phytophthora and Pythium species were lower in soils amended with organics than in synthetic fertilizers. Manna et al. (2005) showed in several long-term fertilizer experiments in India that application of NPK + FYM improved soil microbial biomass C and N, labile carbon fractions, overall aggregation and contributed best to sustainable yields. In China, long-term application of organic manure (18 years) supported significantly higher population of organic P mineralizing and inorganic P solubilizing bacteria OPMB and IPSB (77). The OPMB-specific mineralization potential and IPSB-specific solubilization potential was significantly higher in P-deficient NK treatment. Likewise, plants were also more dependent on arbuscular mycorrhiza in Pdeficient soils. Practices like organic farming that promote production in an environmentally safe way have been found to counteract negative impacts of agricultural intensification. The beneficial effects on belowground decomposer diversity might only be evident years after the conversion from conventional farming (103). In contrast, confounding factors like soil types seem to be relatively more important than management practice in shaping soil communities (177). In a study of eight pairs of adjacent commercial organic and conventionally managed strawberry fields for functional gene microarray signal intensity and diversity, the organic soils consistently showed higher values which were correlated to soil

microbial biomass, cellulose, dehydrogenase, ammonium and sulphur content (143). In the Swiss DOK experiment, more than three decades of continuous organic and conventional farming altered soil microbial diversity (74). Though the management-sensitive taxa were heterogeneously distributed across the taxonomic tree under both organic and conventional farming systems, some consistent patterns were often observed especially among members of the Acidobacteria and Firmicutes. Acidobacteria showed the strongest bimodal response indicating favoring more than one factor. Operational taxonomic units (OTUs) assigned to the genus Candidatus Solibacter (and one Candidatus Koribacter) were dominant with systems not receiving FYM (no fertilizers or only mineral fertilizers). Members of this genus are known to be slow-growing oligotrophs adapted to nutrient-limited environments (181). In contrast, OUTs assigned to Chloracidobacteria and RB25 were dominant in FYM-based systems, whose lifestyles are largely unknown. These findings corroborated the past observation that Acidobacteria generally prefer soil environments of low resource availability (56) and higher acidity (83). All OTUs (except Paenibacillus chondroitinus) of Firmicutes showed strong association with systems receiving FYM. In case of fungi, OTUs assigned to coprophilous taxa such as Coprinellus, Coprinopsis, Preussia, Psathyrella and Mortierella, including members of the family Lasiosphaeriaceae such as Cercophora, Cladorrhinum, Podospora, Schizothecium and Zopfiella (23, 92), were tightly associated with FYM-based systems. Apart from nutrient management, cropping pattern and crop growth stages also influence the soil microbial dynamics by altering the temporal and spatial distribution of available substrates from rhizodeposition and root biomass

(108, 165). All the assessments revealed that the active vegetative stage irrespective of crops tested recorded more abundant of diversified rhizo-deposits which significantly favoured the microbial colonization and diversity. In a study in hill ecosystem of Nagaland, microbial diversity and function was compared through metagenomics in soils of a longer fallow cycle of 20 years (F4) versus those of a shorter cycle of 5 years (F1) in jhum cultivation (166). Rice was the main crop and other common crops like maize, vegetables and sesamum were grown as mixed crops during cropping phases in both jhum cycles. After two years cropping phase, jhum farmers abandoned these two crop fields as fallows for a period of 3 years (F1) and 18 years (F4), respectively. At the end of the fallow phases (just before slash and burn operations of regenerated plant biomass during fallow phase), surface soil samples (0 to 10 cm depth) were subject to metagenomic analyses. F1 fallow soils had greater bacterial and archaeal abundance coupled with lesser eukaryotic abundance (compared to F4 fallow soils) which was indicative of an unstabilized ecosystem exposed to environmental stresses. Due to frequent exposure of F1 fallow to slash and burnt events every 3 years interval, the above- and below-ground biological inputs/ resources and associated biological interactions got disrupted. As a result, prokaryotic communities dominated in F1 fallow phase in order to support ecosystem processes. This is also evident from the 1.5x higher metabolism related functional proteins in F1 fallow (60%) than in F4 fallow (38%). The above-ground plant community in F1 is dominated by herbaceous plants, seasonal weeds including grass species indicating N deficit compared to woody plants, perennials and bryophytes in F4 that indicated a more stabilized N-rich ecosystem. Plant inputs of carbon compounds to soil are of the most easily degradable sugars and amino acids in F1, which provide a soluble and

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readily usable resource for microbes. The dominance of woody species in F4 fallow favours lignin decomposer community as well as moderately labile substances such as cellulose and hemicelluloses. This is supported by the occurrence of peroxidases and pectin esterases as the most exclusive enzymes in the F4 system, thus longer length of fallow phase allows ecological succession of plant species towards a stabilized N-sufficient ecosystem. This study demonstrates clearly the role of the regenerated above-ground vegetation during fallow phase in shaping the soil biodiversity and thereby regulating the ecosystem processes for stabilizing the ecosystem. A recent microcosm study on grassland ecosystem has shown that the growth of individual plant species in soil can lead to the selection of specific microbial communities in the root zone (99). Similarly, plant effects on the abundance and structure of rhizosphere microbial communities depends on the soil type (79); bacteria were positively affected by the growth of various grasses and herbs in fertile grassland soil, but the same plants negatively affected these microbes when grown in less fertile soil. The interplay between above-ground and below-ground diversity is of key significance for ecosystem function, and hence needs to be considered for understanding the importance of diversity in terrestrial ecosystems. Plants recruits the soil microbiome It is well known that the layer of soil in the immediate vicinity of roots is much richer in bacteria (101000 times) than the surrounding bulk soil. As 5-20% of the carbon fixed by the plants is secreted as root exudates mostly organic acids and sugars, the rhizosphere microorganisms get the immediate nutritional benefit (10). For effective colonization of the rhizosphere, the bacteria should able to compete well with other rhizosphere microbes for nutrients

secreted by the root and for space that can be colonized on the root (175). The rhizosphere organisms influence the plants by producing regulatory compounds; both positive and negative influences are elicited from the plants (19). Decomposition, nutrient solubilization and cycling, secretion of plant growth hormones, antagonism and pathogenicity and induction of plant immune system are the direct or indirect effects due to rhizosphere microorganisms. The developmental stage of the host plant also has a significant role in the shaping of rhizospheric microbiome. Apart from plant and soil, the other external factors including biotic or abiotic stress, climatic conditions and anthropogenic effects also can impact the rhizosphere microbial dynamics. There are three groups of microbes present in the rhizosphere, commensal, beneficial and pathogenic microbes and their competition for plant nutrition and interactions confer the overall soil suppressiveness against pathogens and insects (19). The interaction between soil microorganisms, edaphic factors and the host plant results in the overall crop productivity. Apart from being a predictor of soil quality, the soil microorganisms also exert increased disease suppressiveness against pathogens and alleviate the abiotic stresses to the crop plants. The microbial community analysis of disease-suppressive and conducive soils (against Rhizoctonia solani) of sugar beet revealed that  and  Proteobacteria ( P se u do m on a da c ea e , Burkholderiaceae, and Xanthomonadales) and Firmicutes (Lactobacillaceae) were the major microbial components of soil suppression. These groups were found more abundant during R. solani infection, implying the possibility of host induced microbiome recruitment to combat the pathogenic attack (117). Similar mechanism operates during drought tolerance of Arabidopsis, in which 14 operational Indian Journal of Fertilisers, October 2015 94

taxonomical units (OTUs) covering species of Micromonospora, Streptomyces, Bacillus, Hyphomicrobium, Rhizobium, Burkholderia, and Azohydromonas spp. were found predominant in the rhizosphere and responsible for inducing drought-tolerant genes (195). Likewise, the soil microbiome alters the metabolome of Arabidopsis in response to herbivory. The phenolics, amino acids, sugars and sugar alcohols were significantly altered in the plant by soil microorganisms in turn influencing the feeding of herbivores (9). In another dimension of soil microbiological interactions, there is strong evidence showing the existence of negative feedback mechanism (Janzen-Connell effect) between the soil microorganisms and plant community dynamics for coexistence of strong and diverse competitors sharing the same ecological niche (22, 58). According to Janzen-Connell hypothesis the pathogens and predators that specifically target a tree species will not allow the survival of seedlings surrounding that parent plant, which produce the seeds. In other words the soil microbiome keeps key plant species rare enough to reduce their competitive ability enough so as to make space available for many other species. This effect is due to the negative impact of soil borne pathogens and predators (87). The positive feedback (implies the establishment of single plant species, i.e., agricultural system) comes from soil mutualists and hence both negative and positive feedback effects originated at the cost of virulence and mutualism, respectively are crucial for establishing plant population structure (58, 87). However, the positive feedback has a great potential role in an agricultural system, where single monoculture crops are used instead of a diverse species population. In contrast, the negative feedback from whole soil microbial communities of native ecosystems can help to achieve the restoration of native plant communities. However, in-depth

studies are required to understand the feedback mechanisms and microbial community structure and function so essential for ecosystem restoration and engineering of agricultural systems for sustainable development particularly in anthropogenically disturbed sites. Practices to Improve Soil Health By definition, sustainable agriculture should manage the natural resources so as to maximize yield output with use of minimal synthetic inputs, whose level should not harm the ecosystem and the management practices should have positive impacts on microbial community. Therefore, the soil microbial communities and the role they play are more valued than they are in conventional agriculture. Sustainable intensification of production should be achieved without any long-term detrimental effect on microbial communities. Any short-term adverse impacts should be of a degree that is tolerable as a tradeoff for the higher production. More research is required for determining the levels of intensification and trade-offs in various production systems, soil types and agro-ecological regions. Some important management practices that impact soil microbial diversity and functioning are discussed below: Reduced Tillage Tillage plays major role in nutrient storage and release from soil organic carbon. A worldwide analysis of 67 long-term experimental results revealed that when the agricultural fields were converted from conventional tillage to no tillage, amount of C sequestered was 57g m -2 yr -1 (182) with peak sequestration occurring 5 to 10 years of conversion. Compare to conventional tillage, no-tillage had 1.5 times slower carbon turn-over (162), higher rates of key enzymes associated with C, N and P cycling

(116) and more richness and abundance of bacteria, especially Proteobacteria and Acidobacteria (158). Chemical Fertilizers A number of studies have reported beneficial effects of applying balanced dose of chemical fertilizers on crop productivity, soil organic matter and biological properties (138). In long-term fertilization trials in cropping systems worldwide, a metaanalysis of 107 datasets from 64 trials (64) showed that mineral fertilizer application led to a 13% increase in organic carbon and 15% increase in the microbial biomass (C mic ) above levels in unfertilized control. While fertilization tended to reduce Cmic in soils with a pH below 5 in the fertilized treatment, it had a significantly positive effect at higher soil pH values. The input of N per se does not seem to negatively affect C mic in cropping systems. Organic Amendments The impact of application of animal manures, sewage sludges, composts and other organic amendments on changes in microbial properties of soil is welldocumented. These amendments increase the size (4), activity (26) and diversity (40) of soil microbial communities. Such enhancements have the benefits for crop productivity through increased nutrient cycling rate (51) and impact climate change (89). In recent years, there is an increasing interest in the application of pyrolysis-derived organic rich biomass called ‘biochar ’ as amendment to improve the nutrient holding and structural characters of soil (112). Increasing soil fertility was reflected in soil characters such as nutrient retention, high water holding capacity, high cation exchange capacity and increase of pH in acid soils (3, 97, 128). Changes in soil microbial communities due to biochar application also offer additional support for the benefits of biochar addition. The T-RFLP

coupled pyrosequencing showed that biochar application enhanced the abundance of members of Bradyrhizobiaceae, H y ph o m i c r o b i a c e a e , Streptosporangineae and Thermomonosporaceae while the Streptomycetaceae and Micromonosporaceae had negative impact due to biochar in sub-tropical soils of Australia and New Zealand (3, 124). Plant Growth Promoting Rhizobacteria Plant growth promoting rhizobacteria, PGPR sub-group of rhizospheric bacteria are characterized by their competitiveness to colonize the roots and promote the growth of plants. Functionally, PGPR have been divided into two groups: those involved in nutrient cycling and phyto-stimulation and those involved in biocontrol of plant pathogens. Previously Pseudomonas and Bacillus were the most commonly described PGPR, but now many other taxa have been reported. PGPR benefit plant growth directly through nutrient mobilization or production of plant growth hormones or indirectly by suppression of soil borne pathogens and improving plant immune systems and through expression of induced systemic tolerance to abiotic stresses. In recent years it has also been postulated that inoculation of PGPR could also alter the rhizosphere microbial communities and thus indirectly promote plant growth (20). The possible mechanisms by which PGPR promote growth are through: symbiotic nitrogen fixation; associative nitrogen fixation; solubilization and mineralization of nutrients such as potassium, calcium, silica, zinc, iron, manganese through organic acid production; ability to produce hormones such as indole acetic acid, abscisic acid, gibberellic acid and cytokinins; production of water-soluble B group vitamins niacin, pantothenic acid, thiamine, riboflavine and biotin; ability to

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produce 1-aminocyclopropane-1carboxylate deaminase to reduce the level of ethylene in the root of developing plants; antagonism against phytopathogens by producing siderophores, antibiotics and through induced systemic resistance; ability to alleviate drought by triggering induced systemic tolerance in plants; other stresses including salinity, water logging and oxidative stress and soil aggregation through biofilm formation (19, 20). The role of PGPR in sustainable agriculture is gaining importance, as its usage reduces the synthetic inputs and enhances soil health and sustainability. PGPR can also indirectly reduce plant disease incidence through induced systemic resistance (ISR). The defensive capacity of the entire plant is enhanced against a broad spectrum of pathogens and insect herbivores; acquired upon local induction by beneficial microbes. A wide variety of root-associated mutualists, including Pseudomonas, Bacillus, Trichoderma and mycorrhiza species sensitize the plant immune system for enhanced defence system. P. aeruginosa 7NSK2 and Serratia plymuthica IC1270 induce resistance against Magnaporthe oryzae in rice, but they enhance disease severity caused by Rhizoctonia solani (43). However, some pseudomonads induce resistance of rice against R. solani. Induction of resistance by a specific strain of PGPR is not restricted to only one plant species: for example, P. aeruginosa 7NSK2 triggers ISR in rice (44) as well as wheat (120). Application of a PGPR mixture enhances the efficacy of resistance induction compared with the use of individual strains in both dicots (42) and monocots (102). Diverse microbial molecules have been identified as ISR elicitors in monocots. Exopolysaccharides produced by Pantoea agglomerans and siderophores and antibiotics produced by Pseudomonas strains, such as pseudobactins and pyocyanin, are important defence elicitors in rice against M. oryzae (45). All these signal transductions

were reported for Arabidopsis, which is a dicot system. However, in monocots like rice, the immune response to PGPR is not yet fully understood. Some PGPR possess certain unique abiotic stress alleviation traits, apart from the regular plant growth promotional traits. These strains induce physical and chemical changes in plants thus enabling them to overcome the illeffects of drought (induced systemic tolerance, IST) (189). In one of the earliest studies, Timmusk and Wagner (1999) reported that inoculation with a PGPR, Paenibacillus polymyxa enhanced the drought tolerance of Arabidopsis thaliana. By using RNA display, they concluded that mRNA transcriptions of a droughtresponse gene: ‘early response to dehydration 15’ (ERD15), were augmented in inoculated plants compared to uninoculated controls. Kohler et al. (2008) demonstrated the higher activity of antioxidant catalase in lettuce plants under severe drought conditions when inoculated with PGPR Pseudomonas mendocina and AMF (Glomus intraradices or G. mosseae) and postulated that they can be used in inoculants to alleviate the oxidative damage elicited by drought. Saravanakumar et al. (2011) reported the ability of P. fluorescensPf1 to increase the activity of catalase and peroxidase in water stressed green gram plants when compared to untreated plants. The bacterized plants were found to tolerate stress better than the uninoculated controls. The ability of the rhizobacterial strain Pseudomonas putida GAP-P45 to improve the plant biomass, relative water content, leaf water potential, proline sugars, and free amino acids of maize plants exposed to drought stress was recently reported by Sandhya et al. (149). Methylobacterium commonly referred as pink-pigmented facultative methylotrophs (PPFM) are phyllosphere dwelling proteobacteria and have the ability to induce the systemic Indian Journal of Fertilisers, October 2015 96

tolerance to drought through ACC deaminase enzyme (39). By spraying this bacteria under large scale (24,000 litres covering 1.25 lakh ha of rice crop), the shortterm drought tolerance (for about 15 days) was induced in rice, which avoided the complete yield loss due to monsoon failure in 2011-12 in Tamil Nadu Cauvery delta area. These findings provide a new dimension in inoculant technology for mitigating drought stress which is so crucial in this era of climate vagaries. Technically, those PGPR strains able to supply nutrients to crop plants are included under “Biofertilizers”, while the strains responsible for controlling insect pests and diseases are called as “Biocontrol agents”. The use of such microbial inoculants in sustainable agriculture has been shown to reduce the inorganic fertilizer inputs (nearly 25%) by helping to explore the natural resources (140). The use of inoculants also improves the nutrient use efficiency of various inorganic fertilizers under integrated nutrient management and also the quality of the produce (141). The global market for inoculants is growing at the rate of 10% per annum (20) had a value of $440 million in 2012 and is expected to reach about $1295 million by 2020 (172). India and China are promoting the use of inoculants through tax incentives and exemptions, grants to support for the manufacture and distribution of inoculants (131). Among the bacterial inoculants, Azospirillum and phosphate solubilizing bacteria (Phosphobacteria) shared nearly half of the total annual production. There is a need to increase the share of Rhizobium in the inoculants production to improve production of pulses. Microbial inoculants not only improve agronomical yield increase, reduce the use of synthetic inputs, but their use also improves the soil health (8, 85). As reviewed by Trabelsi and Mhamdi (2013), microbial inoculation may also cause

tremendous changes in number and composition of taxonomic groups; the effects may be either transient or long-term and may be direct effects due to trophic competition and synergistic or antagonistic interaction of inoculant with native microbial communities, or caused by indirect effect of enhanced root growth and exudation. The microbial inoculant based integrated nutrient management (INM) practice (Azospirillum, Rhizobium and phosphate-solubilizing bacteria and fertilization with rock phosphate, compost, and muriate of potash) on rice-legume–rice rainfed production system in Assam reported significantly better cumulative grain yield, N, P and Zn uptake with positive N balance in soil than farmer ’s practice (167). Increased soil aggregates, better fungal/bacterial biomass C, high number of earthworm casts and high degree of bacterial community diversity are the other significant impacts reported in those soils used with microbial inoculants. In Northeast India, in rice-rapeseed rotation for two years soil health as reflected in soil enzyme activities were highest when fertilizers, composts and biofertilizers were added together (122). Plant Breeding Physiological and morphological changes induced by breeding may affect the community diversity of microorganisms of rhizosphere and bulk soil both directly and indirectly. Breeding genotypes for disease resistance will decrease the growth and survival of particular pathogenic microorganism in the soil and thereby bring a community shift. However, plant breeding may change the soil microbial communities and functions in unpredicted way and the effect may be of transient or often unknown. Due to the apparent variability of effect, it is essential to increase the knowledge about the plant genotype - microbe interactions effect on the diversity and function of soil microbiome (113). There is evidence that

conventional plant breeding efforts have selected for varieties with reduced response to plant pathogens, AM fungi and rhizobia (18). While the first effect provides a benefit, reduced responses to beneficial microorganisms, will compromise crop production in a future scenario of limited availability of chemical fertilizers or altered tillage practices. There are already restrictions on agrochemical usage; P fertilizer availability and also supply of fossil fuels for manufacture of N fertilizers will level off in next twenty years. Therefore, fresh programmes to breed varieties that suit the new soil environment and that have reduced response to pathogens with a concomitantly increased response to VAM and rhizobia are urgently called for (18). Residue Management Recent awareness of organic farming has made the recycling of agro-wastes an essential component of natural resource management in agriculture. Increasing mechanization in agriculture leaves behind more crop residues; burning is environmentally unfriendly, causing soil erosion and air pollution, loss of carbon and nutrients. Direct incorporation of agro-residues in soils incurs labour costs, irrigation and additional tillage along with yield reduction in subsequent crop due to nutrient immobilization. Under wetland condition, methane emission is also getting increased due to direct incorporation (38). In the long-term perspective, direct incorporation improves the soil health significantly (21). Managing these crop residues using microbial technology is beneficial; microorganisms producing hydrolytic enzymes e.g., laccase, peroxidase, polyphenol oxidase, cellulase, xylanase etc. are now being increasingly deployed. Thermophilic lignolytic and cellulolytic fungi, bacteria and actinobacteria such as Trichoderma viride, T. reesei, Phanerochaete chrysosporium, Aspergillus niger, A. Indian Journal of Fertilisers, October 2015 97

nidulans, A. awamori, Streptomyces spp., are being successfully demonstrated as inoculants for rapid decomposition of paddy straw, wheat straw, jowar, pearl millet stover as also urban solid wastes (61; 109). For microbial enrichment of compost, after the thermophilic phase of composting is over, N fixers and P solubilizers are added e.g., Azotobacter, P solubilizing fungi Aspergillus awamori to enrich available N and P. Addition of rock phosphate (12%), pyrite (10%), urea (1%), ZnSO4 (0.1%) were also found useful for hastening the decomposition of crop residues as well as nutrient balancing of organic amendments. Composting Composting and compost based organic amendments are essential components of sustainable soil fertility management strategies. Compost application helps improve soil structure, water holding capacity, cation exchange capacity, soil organic matter quality and quantity, microbial biomass and enzyme activities (55), soil microbial communities (46) and enhances the competitiveness among the soil microorganisms. The ability of compost to create an adverse environment for the occurrence of the plant disease, even though a pathogen might be present and the plant is susceptible to it, is the basis for compost based suppressivity. In the compost based, the suppressiveness refers to all diseases including soil-, aerial- and seed borne diseases, while for soil, it refers to the suppression of soil borne pathogens (115). The continuous use of composts and organic amendments seems to lower the soil borne pathogens by improving soil suppressiveness (132). Sustainable agriculture and development will also require urban areas to contribute to the society. As estimated by United Nations, 77% of the population in low and middle income-countries will live in the cities by 2020. Recycling all the urban municipal solid waste wastes into composts is a virtual imperative to prevent

serious environmental consequences. The landfills of organic wastes are responsible for greenhouse gas emission (27). Understanding the microbial processes, microbial successions, utilization of microbial inoculants for rapid composting and nutrient enrichment are the areas to focus in future for solving the pollutionrelated problems of urban wastes and also provide renewable source of nutrients for agriculture and help maintain clean water resources. Nanotechnology Metal oxides, ceramics, magnetic materials, quantum dots, lipids, polymers, dendrimers, silicates and emulsions are used for making nano particles (136). Due to their size dependent qualities, high surface-to-volume ratio and unique optical properties, NPs have potential application in crop nutrition and plant protection (66). They include slow release fertilizers, micronutrients coated with NPs for improving the useefficiency, nano-formulation of biofertilizers for enhanced shelflife and efficiency, controlled release of nanocides (pesticides encapsulated in NPs) to minimize the environmental risks and stabilization of agro-chemicals with nano-materials. The nanotechnology based biosensors (nano-sensors) enable the early detection of pathogens in the fields. The silica based NP conjugated with an antibody specific to a pathogen and filled with fluorescent dye enables detection of a single bacterial cell (193). Likewise, the NPs can be used in biosensors for detection of pesticide residues in agricultural commodities. The gold-NP based dipstick competitive immunoassay, is able to detect organochloride pesticides such as DDT in very low quantities and can be used for detection of pesticide residues in food samples as well as in situ field monitoring (100). Several other nanosensors have been devised for successful detection of organophosphate pesticides, dimethoate, pyrethroid,

cypermethrin and permethrin (66). Guo et al. (2009) developed portable gold NPs based one-step strip assay allowing simultaneous detection of two pesticides, carbofuran and triazophos within a short time (8–10 min) without any equipment. The advancements in nanotechnology have further expanded to bioremediation of pesticide and heavy metal contamination in the polluted soils as well as ground water (15). The investigations also suggest that addition of NPs (Cu, Mn and nanoclay NPs) can improve the soil structure of soft soils (104). Since nanoparticles spread into the entire ecosystem, eco-toxicological risks and unintentional consequences on the biosphere have not yet been fully understood. Very little data exists on the toxicity of NPs to soil microbial community. No harmful effects of introduction of NPs (fullerene NP) were found on the soil bacterial and anaerobic microbial communities (127). Shah and Belozerova (2009) also reported that the introduction of silica, palladium, gold and copper NPs showed no significant difference among the soil bacterial communities as resolved by FAME and CLPP. Further advancements in agricultural use of nanotechnology would promote “precision farming” by coupling the nanosensors and delivery systems allowing the optimum use of natural resources as well as the off-farm inputs. Threats to soil health Pesticides The global agricultural sector is the primary user of pesticides, consuming over 2.4 million tonnes of pesticides annually. Less than 1% of total applied pesticides generally get to the target pests while most of it remains unused, which causes serious ecological problems (129). High inputs of synthetic chemicals is habitat damaging and leads to loss of diversity and/or function within the soil microbial community. In addition to these, pesticides Indian Journal of Fertilisers, October 2015 98

adversely affect the populations of arthropods (insects, mites, spiders, millipedes and centipedes), molluscs (snails, slugs), annelids (earthworms) and protozoans (Paramecium, Amoeba), etc. Bioremediation approaches for those soil pollutants are a costeffective alternative to physical and chemical methods of waste remediation. Over the past 15 years, use of microorganisms has shown promise in remediation of soil pollutants which include organic compounds, heavy metals, hydrocarbons, poly-chlorinated aromatic compounds and so on. Pseudomonas putida was the first one to be patented for a broad range of organic compounds in 1974 followed by numerous strains thereafter (69). They degrade the organic pollutants aerobically in the presence of oxygen by respiration or under anoxic conditions by specific biochemical processes like denitrification, methanogenesis, sulfidogenesis, etc. The polycyclic aromatic hydrocarbon pollutants are degraded by various bacterial and fungal fauna through the oxidation of aromatic rings (13). Polychlorinated biphenyls are treated by novel bioremediation systems- Phanerochaete (187) dechlorinates the PCB followed by oxidative degradation by Burkholderia LB400 (106). Community-level degradative potential of soil microbiome rather than single strain concept is utilized to benefit from the symbiotic and proto-cooperative synergistic effects among the microorganisms. The recent advances in molecular methods and gene mining allow identification of novel bacterial species and genes for bioremediation. The genome sequence of Pseudomonas revealed that many genes coding for enzymes responsible for metabolism of non-natural substrates were present (123). The functional screening of metagenomic libraries instead of looking for individual organism in the soil have led to discovery of several novel bioremediative genes coding for enzymes like polyphenol

oxidase, ester and glycosyl hydrolase, laccase and so on (80). Presently, the DNA stable isotope probing (DNA-SIP) technique used in conjunction with NGS based metagenomics and metabolic functioning of an ecosystem allows detection of novel enzymes and bioactive compounds from any environment even from those containing a species in low abundance (37). Geneticallyimproved strains are now available with more degradative capability for specific xenobiotic compounds. Though there are several ex situ and in situ techniques available for bioremediation of contaminated water and soil resources, in situ treatments are generally the most desirable options due to lower cost and lesser disturbance. Despite advances in our understanding of m i c r o b i a l - m e d i a t e d bioremediation processes and technologies, newer strategies are still needed for the breakdown of extremely recalcitrant compounds. Metal Pollution Agricultural soils are polluted due to seepage from landfills, discharge of industrial wastes, percolation of contaminated water into the soil, excess application of pesticides and fertilizers and so on which ultimately find their way to the soil and affect the ecological balance and harm human and animal health due to contaminated produce. Of these, the one issue that needs firm attention is the menace of heavy metal pollution because ‘heavy metals are forever’, they do not degrade or disappear and can only be transformed from one oxidation state to another of lower solubility. Indiscriminate release of heavy metals such as cadmium, copper, lead, nickel, zinc, etc. from various sources including fertilizers, urban solid wastes, etc., into soil and aquatic environments has altered their geochemical cycles and biochemical balance. Heavy metals adversely affect soil respiration, microbial activity and soil enzyme activity, inhibit nutrient transformation processes such as ammonification and

nitrification, reduce earthworm population, suppress algal population, crops yields and quality, soil microflora and cause health problems through accumulation in food chain (2). Heavy metal contamination leads to suppression of potentially active microorganisms in soil and decrease substrate induced respiration of soil (25). Metabolic quotient is also influenced by heavy metal contamination of soil (5). The adverse effect of heavy metals on rhizobia resulting from application of sewage solids on nodulation parameters has been reported (35). In soils amended with sewage sludge enriched with varying level of heavy metals, none of the heavy metals at permitted European Union (EU) levels had any adverse effect on soil microbial biomass (33), zinc, copper or cadmium at twice the EU limits decreased the biomass by 20%, whereas nickel at 4 times decreased the biomass by 15%. Toxicity towards nitrogen mineralization was in the order Zn > Ni > Cu > Cd. EU mandatory upper limits are 300 µg total Cu, 75 µg Ni, 3 µg Cd and 140 µg Zn/g soil. Fertiliser quality standards in India include permissible limits of several heavy metals in city compost and vermicompost. Since microorganisms have developed various strategies for their survival in heavy metalpolluted habitats, these organisms are known to develop and adopt different detoxifying mechanisms such as biosorption, bioaccumulation, biotransformation and biomineralization, which can be exploited for bioremediation either ex situ or in situ. AM fungi are involved in bioremediation through phytoremediation, the technique based on the use of plants for soil remediation. This includes phyto-stabilization (174) strategy where AM is involved in immobilization of heavy metals in the soil by the production of chelates or by absorption. This reduces both soil erosion (VAM improves soil aggregates binding) and transfer of the heavy metals to

aquifers, thus avoiding their dispersion by the wind. Alternatively, phyto-extraction takes advantages of the ability of non-mycorrhizal plants (with exceptions) to hyper accumulate metals (174). Functionally compatible rhizobacteria (67) and AM fungi (47) adapted to heavy metal contaminated soil, interact synergistically and contribute to the benefits of phytoremediation. Barea et al. (2005) found that Brevibacillus spp. enhanced the phytoremediation activity in AM plants through (i) improved rooting, and AM formation and functioning; (ii) enhanced microbial activity in the mycorrhizosphere; and (iii) accumulation of metals in the rootsoil environment, thus avoiding their transfer to the trophic chain, or to aquifers. The bacteria showing resistance to heavy metal toxicity posses specific genes for that and there is evidence that organic farming can enhance proportions of those genes in rhizosphere (142). Bacterial inoculation can alleviate metal toxicity (Aluminium) crop plants in polluted soils. (Figure 3). Bioremediation approaches, can play a key role in maintaining the proper proportion of metals in soil using microbes in phytoremediation of heavy metal polluted soils by enhancing plant growth through siderophores (for improving Phytoextraction), Phytostabilization (reduction- Cr, As, Se, U) methylation (Hg, As, Se) followed by volatilization and finally bioreactors for effluent treatment (139). Climate Change The concentration of carbon dioxide (CO2) and other greenhouse gases in the atmosphere is continuously increasing every year with attendant consequences on the environment. Most agricultural soils have lost their original SOC to a tune of 25-75 % (17) and global warming may further decrease the SOC pool. Soil management with extractive farming practices (residue

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Figure 3 – Influence of endophyte colonization and Al levels (mg kg-1 soil) on root morphological features and H2O2 activity in fully expanded 3rd leaf tips determined by diamino-benzidine (DAB) assay at 21 DAT of Kharif rice grown in an acid Inceptisols (D. Thakuria et al. unpublished data).

removal, low or no external input of organic amendments) have also reduced the SOC pool. Most of the microbiological processes such as respiration, methanogenesis, nitrification and denitrification are significantly enhanced due to climatic changes (12). Hence, sustainable practices of maintaining soil health are important for mitigating the effects of climate risk factors. Ensuring a positive budget of SOC (Cinput>Coutput) through recommended sustainable management practices is important. Use of conservational agricultural management practices like crop residue mulching; integrated nutrient management, complex cropping system involving forages and agroforestry are the strategies for improving C input . In addition to carbon sequestration, healthy soils also oxidize methane and reduce the N 2O by moderating nitrification as well as denitrification. While it is relatively welldocumented that elevated CO 2 (eCO 2) changes the belowground carbon allocations, there are fewer reports on the response of microbial communities (191). The biomass and activities of rhizosphere-associated microbial communities such as mycorrhizal fungi and diazotrophs may or may not increase due to eCO2 (173). The community structure also has

shown highly varied response to eCO2 (28, 119). Niklaus et al. (2003) reported no major effect on microbial biomass and community in spite of increase in plant productivity and shift in soil. Marilley et al. (1999) observed an eCO 2 induced community shift, particularly of the pseudomonads, in the rhizosphere microbiome of Lolium perenne. However, the changes in the community composition are unpredictable and the changes are mostly due to the amount and composition of plant material input into the soil. Hence, novel biotechnological approaches such as carbon sequestration through microbial inoculants such as cyanobacteria and photosynthetic bacteria, improved methane oxidation using methanotrophic bacteria, thermostable microbial inoculants for efficient nutrient cycling under elevated CO2 and temperature are to be focused in future for managing the soil health under climatic stress. Under elevated CO 2 , increased immobilization of fertilizer N by soil microbial biomass or stimulation of mineralization of soil organic nitrogen is observed. Thus greater microbial demand for N (>27%) was observed under elevated CO 2 (184). In such scenarios meeting the higher N demand through chemical fertilizers would further aggravate Indian Journal of Fertilisers, October 2015 100

the greenhouse effect. Therefore cultivation of legumes to meet the increased demand through biological nitrogen fixation is the only sustainable option. GMOs Genetically engineered or modified crops (GM crops) give superior yields with less use of pesticides but their effects on non-target organisms including soil microbial communities are the major risks. Root exudates of Bt-maize containing the insecticidal toxin from Bacillus thuringiensis (Bt toxin) showed no significantly different effect on microbial populations compared with those of its non Btcounterpart (153). Differences in functional bacterial population in the rhizosphere of Bt-cotton and non-Bt-cotton were insignificant (148). BIOLOG® profiles of carbonsubstrate utilization and molecular analysis of 16S rRNA genes by DGGE of rhizosphere of transgenic and non-transgenic corn also revealed no difference in the community structure (53). When CryIA(c) and CryIA(b) like Bt-toxins were incorporated into soil at a concentration of 0.05 g toxin per g or the activated insecticidal toxins from Bacillus thuringiensis subspp. kurstaki (antilepidopteran), morrisoni strain tenebrionis (anti-coleopteran), and israelensis (anti-dipteran) were dissolved into liquid media for

dilution and disk-diffusion assays, no significant effects on soil microbial communities or on specific microorganisms were observed (49, 90). Most studies revealed that the Bt-toxins excreted through the root or present in the plant are harmless to most plantassociated microorganisms (29; 53), phosphorus- and potassium solubilizing bacteria, nitrogenfixing bacteria (148) and soil enzymes like urease, phosphatase, dehydrogenase, phenol oxidase and protease (160). The diversity of rhizosphere bacteria of transgenic, herbicide-resistant corn was not different from that of the corresponding non-transgenic variety (156). Balachandar et al. (2008) recorded no significant difference in the community composition of pink-pigmented facultative methylotrophs (PPFMs) between Bt- and non-Bt cotton. Soil respiration, dehydrogenase and mineral nitrogen were reduced in Bt-cotton soils while microbial biomass carbon and nitrogen and phosphatase showed an increased trend compared to non-Bt cotton (151, 152). Few reports revealed that the transgenic crops had different microbial interactions in the rhizosphere when compared to its non-transgenic counterpart. Microbial communities associated with rhizospheres of transgenic cotton (Gossypium hirsutum L.) and alfalfa (Medicago sativa) significantly differed from these of the nontransgenic isogenic lines (48, 49). Cowgill et al. (2002) observed changes in the microbial community profile of rhizospheric microorganisms as revealed by PLFA profiling between transgenic and non-transgenic potato. The herbicide (glyphosate) tolerant transgenic soybean accumulated the herbicide in the nodules and adversely affected the nodulation and nitrogen fixation (190). The root exudates of Bt-corn significantly reduced the presymbiotic hyphal growth of Glomus mosseae (arbuscular mycorrhiza). The development of appressoria was also affected and 36% of them failed to produce

viable infection pegs (31, 176). The Bt varieties of corn, cotton and rice have been assessed for the residue decomposition rates in the soil because of unintended effect of Cry1Ab on the lignin content of the plant (153). Most of the results did not find a difference in decomposition between Bt and Non-Bt varieties (101, 179). Most studies have shown that effects are transient and did not persist into next season. Recent surveys in field have shown that organic carbon content and microbial populations were higher in the rhizosphere of transgenic cotton than nontransgenic cotton in on-farm sites in Maharashtra (78). Soils are always in a state of starvation as far as readily available substrates are concerned and the soil microbes rapidly attack and breakdown even the most difficult to decompose materials. Given the high soil temperatures in the tropics and moisture fluctuations during rapid drying-wetting cycles, the carbon cycling processes are even more intense. Thus any fears of transgenic crops on soil health are not borne out by any rigorous experimentation.

diseases and mitigation of water, temperature and other climate related abiotic stresses. Further, application of microbial resources is now being used for recycling of municipal, industrial and agricultural wastes into compost production, organic amendments for crop nutrition, conditioners for soil health, etc. It is also being extended for remediation of polluted soils. Some areas requiring more attention in future are:

Future soil research should focus on ecosystem services rendered by microbes and how they help meet the sustainable development goals. Such researches would make a valuable contribution to soil security and sustainable environmental policy.  Develop one or two sensitive, compound indicators of soil biological health and sustainability, easily measurable and cost-effective probes based on genes or their transcripts for rapid soil measurement of the soil biological condition.  Even though knowledge of soil

Conclusions and Future Needs The recent molecular and biotechnological tools allow a deeper understanding of the vast potential of soil microbial resources and their role in sustainable agriculture. Improvement of soil biology is the key to improving soil health and ameliorate degraded lands. While the effects of crop and soil management strategies on the soil microbial community and functionality, how microbial colonization and composition is altered due to rhizosphere effect, etc., are well studied, information on how the microbes in turn alter the physiology and immune system of the plant is meagre. Microbial inoculants now play an important role in organic and sustainable agricultural systems. There is now a big repository of microorganisms for enhancing nutrient uptake and growth of crops, management of pests and Indian Journal of Fertilisers, October 2015 101

microbial communities is well advanced, yet functions of many of dominant communities in soil ecosystem are little known, e.g., Acidobacteria.

 Unraveling the role of archaea in the soil health and sustainability and how their activity can be modulated or how they can be deployed.

 More efforts are required for understanding the plant-microbe interactions at molecular level. Exploring the microorganisms as “Engineers” of rhizosphere for plant health manipulation may be useful to mitigate the biotic and abiotic stresses.

 Though the PGPR usage in agricultural system is in increasing trend, the exact mechanisms by which plant growth improvement, fitness to withstand biotic and abiotic stress conditions happen are not yet fully understood.

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