C H A P T E R
12 Microbial Technologies for Sustainable Crop Production Chittranjan Bhatia*,a, Prasun K. Mukherjee† ⁎
Mumbai, India †Bhabha Atomic Research Centre, Mumbai, India
1 INTRODUCTION In the evolutionary history, microbes evolved before the plants on the planet earth. It is believed that both evolved from the last universal common ancestor. Since the arrival of plants, in the Paleozoic era, some 360–480 million years back, microbes and plants have coexisted and coevolved in natural ecosystems. Though crop plants interact with microbes primarily in the rhizosphere, microbes are also abundant in phyllosphere. Only the soil microbes are relatively well investigated and include bacteria, archaea, fungi, oomycetes, mycorrhiza, and protozoa. They could be free living, as symbionts occupying the surface of the roots or forming nodules or as intra- and intercellular endophytes. With the evolution of agroecosystems, managed by humans, 10,000–12,000 years back, microbe and crop plant interactions have been greatly altered in cropping systems and keep changing, with the crops and agronomic practices followed. It is widely recognized now that the present crop production based on high external inputs of chemicals as fertilizers and pesticides, referred to as exploitative agriculture (Swaminathan, 2013), is not sustainable. In the future, for long-term sustainability, microbial technologies can play a much larger role in the evolution of crop production systems. The biologicals, such as the microbials, as referred to in the industry, include biofertilizers, biopesticides, plant growth promoters, and herbicides and often combine more than one functions. Microbes associated with plants represent an enormous biodiversity, of which only a small part has been investigated. Earlier, only those that were amenable to culture in laboratory were investigated. With the advances in molecular and cloning techniques, the available genetic diversity can be exploited even without culturing the organism as such. Further, new diverse types can be created. This chapter focuses on the immense possibilities of microbial technologies, other than the well-known biological nitrogen fixation (BNF) and mycorrhiza, in enhancing crop plant a
Former Secretary, Department of Biotechnology, Government of India, New Delhi
Crop Improvement through Microbial Biotechnology https://doi.org/10.1016/B978-0-444-63987-5.00012-8
© 2018 Elsevier B.V. All rights reserved.
12. Microbial Technologies for Sustainable Crop Production
productivity in the years to come. BNF and mycorrhiza have been investigated extensively and deserve entire chapters. Considering wide diversity of the microbes and genetic diversity within each species, its characterization, and further modifications using the molecular techniques, the potential is very large. However, a better understanding of microbial diversity and its utilization is essential for developing commercially successful products, comparable with the various chemical products widely used at present.
2 ORIGIN OF FARMING Plant-microbe interactions in human-designed farming systems are very recent, only 10,000–12,000 years, since crop plants were domesticated. Humans as food gatherers and hunters discovered that the grains they have been collecting from distant places can grow in their own backyard. They started putting seeds in the ground where they germinated to produced plants and grains. This was the beginning of farming. However, large-scale cultivation of crops was possible only after clearing of the existing natural vegetation, grass lands and forests, with enormous loss of the then existing plant diversity. This, known as slash and burn farming, is still practiced by the forest-dwelling tribal populations in many parts. Since then, more and more of natural vegetation have been cleared to meet the food demand of growing population. Early farmers soon realized that growing cereals, year after year, on the same plot reduced the grain harvest. They shifted to new areas and started keeping fallow land that restored soil fertility. It is well established now that soil microbes play an important role in restoring soil fertility in fallow land. Crop rotations involving leguminous crops, in between the two cereal crops, were practiced even before the role of soil microbes in fixing nitrogen was established. This kind of farming continued till the discovery of synthetic nitrogen fixation by Haber-Bosch process to produce ammonia in the last century. It remains the main source of N fertilizers. Rock phosphates, another finite resource, are used for making the P fertilizers. Potassium needs of crops are met by potassium nitrate. Moderate levels of NPK fertilizers were used for increasing productivity, till the arrival of the “green revolution” (Gaud, 1968) technology in the mid-1960s. The new semidwarf plant types of wheat and rice, the main stay of the technology, almost doubled the yield levels and also the fertilizer and irrigation water needs. The use of pesticides to protect the crops from pests, pathogens, and weeds was also enhanced. Chemical fertilizers and plant protection products have played a major role in the development of high-yield modern crop production.
3 URGENT NEED TO INCREASE SUSTAINABLE CROP PRODUCTIVITY Population growth and increasing affluence will continue to enhance the demand of food in the years to come. “Nutritional security” and “hidden hunger” are now considered more important than the calorie security. According to the FAO estimates, the world will have 9.6 billion humans by the year 2050. The food production must increase by 70% to meet the demand. This increase necessarily must come from the same or even a slightly reduced area of arable land. Water resources for crops would face intense competition from industrial and other human needs. Besides, climate changes are likely to affect crop life cycles and duration, enhancing the overall stress of temperature and water, both drought and floods.
6 Crop Production as an Energy Harvesting Process
4 UNDESIRED EFFECTS OF INCREASED INPUTS OF CHEMICAL FERTILIZERS AND PESTICIDES Increased productivity of the main cereal crops saved the needs of cultivating additional land, thereby saving forests and natural vegetation with associated biodiversity. However, the adverse effects of the new technology of using higher inputs of chemical fertilizers and pesticides became apparent after few years. These include release of nitrogen oxides, the greenhouse gases, into the atmosphere; nitrogen runoff from fields and its accumulation in water bodies; and soil degradation and accumulation of pesticide residues. Emergence of pesticide resistance in insect pests and pathogens and adverse effect on human health are well recognized now, besides contamination of soils and groundwater.
5 RHIZOSPHERE MICROBIAL DIVERSITY Rhizosphere, defined as the narrow zone of soil that is highly influenced by the roots and root exudates of the aboveground plants, is extremely rich in microbial diversity. It includes bacteria actinomycetes, fungi, oomycetes, mycorrhiza, algae, protozoa, and nematodes. Early studies on rhizosphere microbial populations were limited to those that could be cultured in the laboratory. This represented less than 1% of the microbial diversity present. With the molecular techniques based on isolation and characterization of nucleic acids from soil samples, the presence of 6000–10,000 different genomes from 1 g of soil was reported (Torsvik et al., 1990). More recent analyses using the advanced molecular methods suggest the presence of 277,000 genomes in a gram of soil. Soil microbe and plant interactions are extremely complex, depending upon biological and other variables. At best, they are only partially understood (Reinhold-Hurek et al., 2015). Majority of the investigations have been carried out in natural ecologies (Bever et al., 2012). Soil microbes could have a positive or negative effect on plant growth. Positive influence is exerted by biocontrol agents (e.g., Trichoderma spp., Bacillus subtilis, and fluorescent pseudomonads), plant growth promoters (like PGPRs, rhizobia, mycorrhizae, free-living nitrogen fixers, and phosphate-solubilizing fungi and bacteria), and nonpathogenic strains of plant pathogens that outcompete the pathogens (e.g., nonpathogenic fusaria) (Prasad et al., 2015). Pathogens that overcome the natural defense of the crop plants cause the negative response and reduce the yield (e.g., Fusarium spp., Rhizopus, Pythium, Ralstonia solanacearum, and aspergilli).
6 CROP PRODUCTION AS AN ENERGY HARVESTING PROCESS Cropping systems aim to maximize the fixation of solar energy, through photosynthesis, into plant biomass within the constraints of temperature, water, and plant nutrients. Soils provide water and nutrients for enlarging leaf canopy for increased interception of solar radiation. Carbon, hydrogen, oxygen, nitrogen, and sulfur, along with other macro- and micronutrients, are incorporated into organic molecules through light-dependent reactions for construction, maintenance, and turnover of different macromolecules and plant organs such as leaves, roots, and root exudates into rhizosphere.
12. Microbial Technologies for Sustainable Crop Production
7 ROOT EXUDATES SUPPORT MICROBIAL POPULATIONS IN THE RHIZOSPHERE Organic compounds released as root exudates are main source to support the microbial populations in the rhizosphere. These exudates contain many different organic compounds including sugars, amino acids, proteins, and signal peptides (Uren, 2007). The exudates provide the nutrients for the soil microbial population growth and metabolic functions. They increase the growth of soil microbes and their predators. Association of microbial communities with individual plant species and different crops is well established (Bever et al., 2012) and is determined by the root exudates.
8 NEW TECHNIQUES 8.1 Root Phenotyping In their efforts to modify the crop plants, breeders have neglected the hidden half, that is, the phenotype of the roots. The methods used earlier in investigating the roots were not easy. They involved either digging out the plants, washing the roots carefully for recording root architecture, or growing plants in pots with slanted glass sides for observing the root development. Special glass houses were designed for trees where the root growth could be observed at the below ground level. Computed tomography (XRT or X-ray CT), along with information technologies, similar to the CT scan used for medical diagnostics, has been developed recently at the Danforth Center for Plant Research for noninvasive phenotyping of plant roots (http://www.danforthcenter.org/news-me).
8.2 Genomics Currently, we are in the genomics era where an increasing number of whole-genome sequences of plants and microbes are becoming available, including pathogens and beneficial microbes. Population genomics will help in elucidating the genes and the mechanisms involved in plant-microbe interactions including pathogenicity, virulence, and other important areas.
9 USING MICROBIAL DIVERSITY FOR ENHANCED CROP PRODUCTION The story of Gram-positive soil bacterium Bacillus thuringiensis (Bt), in general, provides the road map for the use of other microbes. Bt produces “Cry” proteins that could be toxic to many insect pests. Bt was used as a biopesticide for many years, and appropriate formulations were commercialized before further advancements in knowledge and its applications. Structure of Cry proteins is determined by Cry genes. More than 200 Cry genes have been identified and evaluated for their toxicity to different insects. Some Cry genes are located on the chromosome and others on the plasmids. Amino acid sequences of Cry proteins have been determined. Many Cry genes have been cloned, and some were genetically modified
10 Registration and Commercial Issues
to produce more toxic proteins. Cry genes were transferred to model plant tobacco to obtain the proof of concept and later to cotton that was commercialized in 1995. Subsequently, more than one Cry genes have been stacked in the new cotton hybrids. Cry genes have also been transferred to eggplant (Solanum melongena) and rice. Currently, the new gene editing CRISPR/Cas technology is being used to modify the Cry genes. Essentially, this would be the road map for other microbes, starting from the organism, identifying the genes using genomics and genetic variability by applying population genomics and finally developing modified microbes or the transfer of gene(s) into the crop plants.
10 REGISTRATION AND COMMERCIAL ISSUES The global agricultural microbial market is expected to grow at a CAGR of 15.5% during 2015–20, with the market value expected to reach $4.73 billion by 2020 from an estimated $2.3 billion in 2015 (http://www.researchandmarkets.com/reports/3795475/global-agricultural-microbials-market-growth.htm). While certain microbials are used as biofertilizers (plant growth stimulators), others are used as biopesticides. The border is not often well defined though; certain microbes can act as both plant growth stimulators and biopesticides (like fluorescent pseudomonads and certain strains of Trichoderma). While the registration procedures for biofertilizers are lax, with no toxicological data required, the registration procedure for “biopesticides” is very rigorous and requires extensive toxicological data generation, which is very expensive and is a major deterrent for the entry of novel strains into the market. The international regulation treats biopesticides essentially as chemicals and ensures that they pose minimum or zero risk (Chandler et al., 2011). There is often a demand for relaxing the registration formalities for biopesticides, much like the biofertilizers. This needs to be carefully examined as we are dealing with live organisms and their metabolic products, many of which could be hazardous, for example, mycotoxins produced by certain strains of Trichoderma (Scharf et al., 2016). No generalized rule should be applied to biopesticides, and each species and strain need to be critically examined by subject-matter experts, rather than a general “registration committee.” While the use of microbials should be encouraged for sustaining ecological balance and high productivity, their safe use will have to be ensured. In India, we have a registration process in place very similar to that followed for chemicals (http://www.cibrc.nic.in/guidelines.htm). However, this has not deterred the registration of nearly 500 microbial biopesticide products, which is a very large number compared with 200 in the United States and 60 in Europe (Singh and Keswani, 2015; Kumar and Singh, 2015; Keswani et al., 2016). Of 500, more than 300 registered products are based on Trichoderma, majority carrying only one strain (Trichoderma asperelloides, formerly T. viride, from TNAU, Coimbatore). Thus, the biopesticide/biofertilizer industry has literally turned into a cottage industry due to low initial investment and low-cost production. However, this growth has certainly resulted in compromised quality control. Unlike chemicals, which have a defined shelf life under ambient conditions, and the performance not much affected by the biotic and abiotic environment at the site of application, biopesticides require specialized storage and handling and require education of farmers for proper use. To be successful, it requires a holistic crop management practice, rather than a stand-alone treatment. The talc-based formulation developed about three decades ago is still in vogue, with a little R&D efforts and
12. Microbial Technologies for Sustainable Crop Production
adoption of novel mass multiplication, formulation, and delivery strategies that would improve the performance of microbial products. This also needs automation that in turn requires huge investment. It is a good indication that several multinational “chemical giants” (BASF, Syngenta, Bayer, Monsanto, and Sumitomo) are entering the microbial market (http://www. researchandmarkets.com; http://www.freshplaza.com/article/106578; http://www.agro. basf.co.za). The involvement of big companies in agrimicrobe business in India should be encouraged in order to improve the quality of the product and to grow the faith of farmers in such alternative crop management practices. This will also ensure a sustainable market for agricultural microbes.
11 CHALLENGES OF MICROBIAL PRODUCTS The root problem in widespread and efficient use of microbials lies in the fact that these are living entities and need utmost care in growth, formulation, delivery, and crop management practices, which in turn comes from educating the manufacturers, dealers, and farmers. Compared with chemicals, their bioefficacy is often influenced by the prevailing soil and crop health, and this reduces the faith of the farmers (especially small and marginal farmers) in such products. There are several ways by which the bioefficacy of microbials could be improved. One such approach is combining with a reduced dosage of chemicals (fertilizers and pesticides). For this, the biological and the chemical components must be compatible. There are simple ways to alter the susceptibility of these microbes to chemicals, for example, a fungicide, by induced mutagenesis. Though several R&D efforts have been made successfully, no such strain is available commercially. In many cases, the efficacy is not sustainable over a long period of time, for example, for the entire duration of the crop. In such cases, there may be a need for repeated applications, which complicates the usage as in most cases, there is no standard package of practices available for microbials. Moreover, in many cases, the biologicals work best as protective rather than a curative agent, and application of chemicals becomes imperative in case of severe epidemics. There are certain other issues that also need to be addressed for long-term success of such microbes. What is the fate of such microbes that are being loaded into soil year after year? Is there any shift in population dynamics of the introduced and the resident flora and fauna?
12 CONCLUSIONS AND OUTLOOK FOR THE FUTURE Microbes associated with the plants are found in the rhizosphere and the phyllosphere (Yang et al., 2001; Lindow and Brandl, 2003; Timms-Wilson et al., 2006; Berg and Smalla, 2009). Only a small fraction of the rhizosphere microbes have been studied intensively. They show enormous genetic variability for different characters investigated, so far, and offer immense possibilities for sustainable cropping. The fastest approach would be to develop microbial products for direct application, like the original B. thuringiensis isolated from soil. Many are currently used as organisms, or their genes have been transferred and expressed in crop plants. Ability of the introduced organism to establish themselves in competition with the existing native microbes determines the success. Many applications can be demonstrated
in laboratory experiments. However, innovative approaches will be the key to the success of microbial biotechnologies needed for sustainable crop production to replace the toxic chemicals currently used. Collaborative efforts of individuals, with knowledge in different specialities, microbiology, genetics, molecular biology, plant breeding, soil science, and agronomy, are necessary for developing successful commercial products.
References Berg, G., Smalla, K., 2009. Plant species and soil type cooperatively shape the structure and function of microbial communities in the rhizosphere. FEMS Microbiol. Ecol. 68, 1–13. https://doi.org/10.1111/j.1574-6941.2009.00654.x. Bever, J.D., Platt, T.G., Morton, E.R., 2012. Microbial population and community dynamics on plant roots and their feedbacks on plant communities. Annu. Rev. Microbiol. 66, 265. Chandler, D., Bailey, A.S., Tatchell, G.M., Davidson, G., Greaves, J., Grant, W.P., 2011. The development, regulation and use of biopesticides for integrated pest management. Philos. Trans. R. Soc., Biol. Sci. 366 (1573), 1987–1998. Gaud, W.S., 1968. The green revolution: accomplishments and apprehensions (No. REP-11061.CIMMYT). Keswani, C., Bisen, K., Singh, V., Sarma, B.K., Singh, H.B., 2016. Formulation technology of biocontrol agents: present status and future prospects. In: Arora, N.K., et al. (Eds.), Bioformulations for Sustainable Agriculture. Springer, India. https://doi.org/10.1007/978-81-322-2779-3_2. Kumar, S., Singh, A., 2015. Biopesticides: present status and the future prospects. J. Fertil. Pestic. 6 (2). https://doi. org/10.4172/jbfbp.1000e129. Lindow, S.E., Brandl, M.T., 2003. Microbiology of the phyllosphere. Appl. Environ. Microbiol. 69, 1875–1883. https:// doi.org/10.1128/aem.69.4.1875-1883.2003. Prasad, R., Kumar, M., Varma, A., 2015. Role of PGPR in soil fertility and plant health. In: Egamberdieva, D., Shrivastava, S., Varma, A. (Eds.), Plant Growth-Promoting Rhizobacteria (PGPR) and Medicinal Plants. Springer International Publishing, Switzerland, pp. 247–260. Reinhold-Hurek, B., Bünger, W., Burbano, C.S., Sabale, M., Hurek, T., 2015. Roots shaping their microbiome: global hotspots for microbial activity. Annu. Rev. Phytopathol. 53, 403–424. Scharf, D.H., Brakhage, A.A., Mukherjee, P.K., 2016. Gliotoxin–bane or boon? Environ. Microbiol. 2016 (18), 1096–1109. Singh, H.B., Keswani, C., 2015. Bio-pesticides in India: constraints in regulation, commercialization and IPR issues. Book of abstract, ICCPMI 2015. Swaminathan, M.S., 2013. Genesis and growth of the yield revolution in wheat in India: lessons for shaping our agricultural destiny. Agric. Res. 2, 183–188. Timms-Wilson, T.M., Smalla, K., Goodall, T.I., Houlden, A., Gallego, V., et al., Timms-Wilson, T.M., 2006. Microbial diversity in the phyllosphere and rhizosphere of field grown crop plants: microbial specialisation at the plant surface. In: Bailey, M.J., Lilley, A.K., PTN, S.-P. (Eds.), Microbial Ecology of Aerial Plant Surfaces. CAB International, Wallingford, pp. 21–36. Torsvik, V., Salte, K., Sørheim, R., Goksøyr, J., 1990. Comparison of phenotypic diversity and DNA heterogeneity in a population of soil bacteria. Appl. Environ. Microbiol. 56, 776–781. Uren, N.C., 2007. Types, amounts, and possible functions of compounds released into the rhizosphere by soil-grown plants. In: The Rhizosphere: Biochemistry and Organic Substances at the Soil-Plant Interface. Marcel Dekker, New York, pp. 1–21. Yang, C.H., Crowley, D.E., Borneman, J., Keen, N.T., 2001. Microbial phyllosphere populations are more complex than previously realized. Proc. Natl. Acad. Sci. U. S. A. 98, 3889–3894. https://doi.org/10.1073/pnas.051633898.