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Our Project

Introduction

The chemicals secreted into the soil by roots of plants are broadly referred to as root exudates. Through the exudation of a wide variety of compounds, the roots may regulate the soil microbial community in their immediate vicinity, cope with herbivores, encourage beneficial symbioses, change the chemical and physical properties of the soil, and inhibit the growth of competing plant species. A large amount of literature suggests that root exudates may act as messengers that communicate and initiate biological and physical interactions between roots and soil organisms.^[1-5]

Our project aims to “programme” microbes to detect specific crops by sensing their root exudates. Each plant species has a different set of root exudates ^[6-13], and the aim is to engineer a chassis bacterium, Bacillus subtilis, to respond differently to different exudates and hence sense the plant that is currently being grown in the soil. There are numerous possible applications of this concept, including plant specific nutrition, stimulating disease resistance, and targeted killing of weeds. An application that we decided to work on is solubilization of phosphorous present in soil for easy uptake by plants.​

Plan of Action

The goal is to study the unique response of B. subtilis to different plants' root exudates. For this, four crops that are widely cultivated in India, namely rice, wheat, soybean and tomato, were used as experimental specimens. Seeds were germinated in sterile soil, and saplings were grown under sterile conditions and their root exudates were collected. B. subtilis was grown in presence of these exudates, and RNA was extracted from the bacterial cells. RNA sequencing is being carried out to identify genes that are highly expressed in the presence of the exudates. Promoters of such highly expressed genes will be used to construct a genetic switch that responds to plant exudates of specific crops.

Application in Phosphorous Solubilization

Phosphorous in soil

Phosphorus (P) deficiency in soil is a major constraint for agricultural production worldwide. Many soils throughout the world are P-deficient because the free phosphorus concentration (the available form to plants) even in fertile soils is generally not higher than 10 mM, even at pH 6.5 when it is most soluble. Low levels of P are due to the high reactivity of soluble P with calcium (Ca), iron (Fe) or aluminum (Al), which leads to P precipitation. Inorganic P in acidic soils is associated with Al and Fe compounds, whereas calcium phosphates are the predominant form in calcareous soils.

Despite this, most soils contain significant amounts of total soil P that occurs in inorganic and organic fractions and accumulates with phosphorus fertilization. Organic phosphorus inputs to soil from plants and microbes are mainly phosphodiesters, which must be hydrolysed by phosphodiesterase and phosphomonoesterase prior to the release of free phosphate for biological uptake.

The ability of soil microorganisms to solubilize various forms of insoluble P fractions is well documented. Bacillus spp. and Rhizobium spp. are the most powerful phosphate solubilizers among bacteria.

Role of Engineered B. subtilis

The engineered B. subtilis will perform two functions:

1. Detecting the plant
2. Amplifying its own phosphodiesterase production

Upon detecting the plant, the engineered bacteria will secrete enzymes that convert the phosphorus in the soil into a form that is more easily assimilated by the plants. The production of the enzyme will be amplified by incorporating a gene amplification circuit within the chassis bacteria. Since the circuit will be triggered only in the presence of specific exudates, depending upon the plant of interest, weeds will effectively be prevented from using up most of the phosphate in the soil.

Potential application: Systemic acquired resistance (SAR)

What is SAR?

Systemic acquired resistance is an induced defense mechanism in plants that inherently protects the plant against a variety of diseases. It is analogous to the immune system in animals, as it primes the plant to defend itself against pest attacks and diseases following earlier localized exposure to the pathogen. The limitation of this system is that it is activated only after exposure to the pathogen, i.e., after the pest has attacked the plant once. Further, the effect of SAR is not perpetual and wears off with time.

Synthetic activation of SAR

A number of commercially available chemical activators induce SAR in plants, the most popular of which is salicylic acid (SA). However, a number of problems are reported with the continuous and prolonged usage of such activators, such as low yields and reduced seed sizes. This is mainly due to the large energy deficit caused to plants when they are under constant influence of the activator.

A Solution

A way around this problem is to design a dosage system in such a way that the plant is periodically exposed to the activator such that it has intermediate periods in which it is free of the activators’ influence. For this purpose, an oscillatory circuit can be incorporated within the chassis organism, B. subtilis, such that it releases salicylic acid (which can be synthesized from a metabolic intermediate), in optimized doses.

How will this project benefit Society?

As a part of Integrated Human Practices, the team spoke to multiple experts in the field of agriculture, fertilizers and bio-fertilizers, including farmers and innovators. From the surveys and farm visits, a lot of information was gained which indicated the need for the solutions that this project promises.

The efficiency of phosphate solubilization can be increased with the use of the engineered bacteria, which may improve the popularity of bio-fertilizers among farmers, who currently prefer chemical fertilizers. Thus the larger effect would be to reduce the usage of chemical fertilizers in Indian agriculture. The project also has potential to reduce the usage of weedicides.

Pesticides are currently being used as a curative measure for plants affected by pathogens and pests. However, pesticides have a large number of well-known ill-effects such as persistence in groundwater, neurological disorders, cancer, respiratory illnesses in farmers, and contamination of fruits and vegetables.

Moreover, the use of pesticides is not a preventative measure. If SAR can be continuously primed in the plants, it would eliminate the need for pesticides and ensure that the disease does not affect the plant in the first place.

References

1. Walker, T.S., Bais, H.P., Grotewold, E. and Vivanco, J.M., 2003. Root exudation and rhizosphere biology. Plant physiology, 132(1), pp.44-51.
2. Bais, H.P., Weir, T.L., Perry, L.G., Gilroy, S. and Vivanco, J.M., 2006. The role of root exudates in rhizosphere interactions with plants and other organisms. Annu. Rev. Plant Biol., 57, pp.233-266.
3. Dakora, F.D. and Phillips, D.A., 2002. Root exudates as mediators of mineral acquisition in low-nutrient environments. In Food Security in Nutrient-Stressed Environments: Exploiting Plants’ Genetic Capabilities (pp. 201-213). Springer, Dordrecht.
4. Bertin, C., Yang, X. and Weston, L.A., 2003. The role of root exudates and allelochemicals in the rhizosphere. Plant and soil, 256(1), pp.67-83.
5. Rovira, A.D., 1969. Plant root exudates. The botanical review, 35(1), pp.35-57.
6. Vančura, V. and Hovadik, A., 1965. Root exudates of plants: II. Composition of root exudates of some vegetables. Plant and Soil, pp.21-32.
7. Lugtenberg, B.J., Kravchenko, L.V. and Simons, M., 1999. Tomato seed and root exudate sugars: composition, utilization by Pseudomonas biocontrol strains and role in rhizosphere colonization. Environmental Microbiology, 1(5), pp.439-446.
8. Kato‐Noguchi, H., Ino, T., Sata, N. and Yamamura, S., 2002. Isolation and identification of a potent allelopathic substance in rice root exudates. Physiologia Plantarum, 115(3), pp.401-405.
9. Kato-Noguchi, H., 2004. Allelopathic substance in rice root exudates: rediscovery of momilactone B as an allelochemical. Journal of plant physiology, 161(3), p.271.
10. Bacilio-Jiménez, M., Aguilar-Flores, S., Ventura-Zapata, E., Pérez-Campos, E., Bouquelet, S. and Zenteno, E., 2003. Chemical characterization of root exudates from rice (Oryza sativa) and their effects on the chemotactic response of endophytic bacteria. Plant and Soil, 249(2), pp.271-277.
11. Prikryl, Z. and Vancura, V., 1980. Root exudates of plants. 6. Wheat root exudation as dependent on growth concentration gradient of exudates and the presence of bacteria. Plant and Soil, 57(1), pp.69-83.
12. Graham, T.L., 1991. Flavonoid and isoflavonoid distribution in developing soybean seedling tissues and in seed and root exudates. Plant physiology, 95(2), pp.594-603.
13. Zheng, X.Y. and Sinclair, J.B., 1996. Chemotactic response of Bacillus megateriumstrain B153-2-2 to soybean root and seed exudates. Physiological and Molecular Plant Pathology, 48(1), pp.21-35.

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  Eco-Friendly

  Our project can eliminate the need for chemical activators and excessive fertilizers

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  Smart and Selective

  Identification of unique responses can be extended to plant specific nutrition, disease resistance, etc.

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  Safe for the Community

  Our project involves the use of B. subtilis, a bacterium found in soil and is a generally regarded as safe (GRAS).

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  Plethora of Applications

  Potential applications can be selective growth of plants, provision of different nutrients in different zones of the farm, and much more.