6 Chapter 11. Microbial Biotechnology in Agriculture

Chapter 12

Microbial biotechnology applications in agriculture

Learning outcome

• Understand the use of microorganisms in crop plant growth
• Explain the microbial function in plant biotic and abiotic stress-resistant

Section – 1: Plant growth and challenges to agriculture

The Role of Plants and Agriculture

Plants are products of domestication, with gradual long-term changes in their qualitative and quantitative traits because of continuous natural and human-directed selection. Whereas some of the first improvements in plants and animals could have resulted from chance alone, the eyes and brains of the “primitive” scientist-farmer were crucial in selecting the good from the bad and the productive and quality crop from the less worthy. Thus, advancements in agriculture and plant science are primarily the result of scientific discoveries, judgments, and historical innovations and are sometimes revolutionary, such as food products, the use of inorganic fertilizers, and, more recently, plant genetic transformation.

Man-made technologies and biotechnologies, including food and fiber, have shaped human life since immemorial. Fermented plant and animal products (e.g., bread, cheese, and wine), conventional crop breeding since the birth of agricultural communities, the “Green Revolution” of later years, molecular marker-assisted selection, and recombinant DNA techniques are biotechnologies. The urgent need to look for alternative biotechnologies and the actual accelerated rate of adopting plant molecular biotechnologies since the breakthrough report of the first transgenic plant is due to four major causes:

1. Increase in world population and the need for more food
2. Recognition that human health is affected by disease-causing pathogenic organisms and by the nutritional quality of foods, especially vitamins and minerals
3. Adverse global climatic changes accompanied by detrimental biotic and abiotic hazards (stresses) to crops and ecosystems
4. Human societies search for novel, non-food plant products such as biomaterials, therapeutics, and biofuels.

Thus, agricultural and plant biotechnologies must be swiftly implemented where population growth is outstripping food production (both for quantity and quality).

Climate Change: A Debacle Against Sustainable Plant Biomass Production

Climate changes (increasing or decreasing temperature, lack or abundance of water) and changes in the chemical composition of terrestrial and aquatic ecosystems have hindered the desired natural productivity of plants and threatened food security (Ahmad & Prasad, 2011). For example, higher temperature stress has been recognized as a significant limiting factor affecting plant growth but has also influenced soil rainfall patterns and moisture flux. It has been estimated that an increase of 3°C to 4°C would cause a reduction in plant productivity by up to 15 to 35% by the end of the 21st century (Tayade et al., 2018). Various types of abiotic stress (flooding, salinity, and heavy metals) have been estimated to reduce plant productivity by 51–82% (Cooke & Leishman, 2016; Mittler & Blumwald, 2010). With the increasing human population, food security can be easily disrupted. Among these stress factors, flooding has been considered a major limiting factor for plant growth and production (Nanjo et al., 2014). Climate-induced changes in the precipitation pattern (Sasidharan et al., 2017) and increased rate of submergence create excessive hypoxia (Lee et al., 2011), and subsidiary stresses such as pathogenesis and herbivory (Hsu & Shih, 2013). Continuous submergence can influence soil nutrient balance, leading to high salinity and/or alkalinity, where the plant utilizes a higher amount of energy to defend its existence (Valliyodan et al., 2016).

According to one estimate, up to 831×106 ha of the earth’s land is saline. Of that, 434×106 ha is affected by soil alkalinity in more than 100 countries, causing severe damage to plant growth and loss of agricultural productivity (Jin et al., 2006; Xu et al., 2013). Alkaline stress hinders plant growth compared with salinity stress (Guo et al., 2010). Despite this, the tolerance mechanisms of plants in response to alkaline stress have received less attention than the adaptive mechanisms of the salinity stress (Degenhardt et al., 2000; Hurkman, 1992; Yang et al., 2008; Zhu, 2001). Plant molecular response pattern to abiotic stress triggers the gene expression profile and biosynthetic pathways and enables signal transduction to produce biochemical metabolites and enzymes that increase the defense responses of plants (Ahuja et al., 2010; Godoy et al., 2021; Razi & Muneer, 2021). However, these adaptive mechanisms at molecular, biochemical, and metabolite levels are variable across different species of plants, their growth conditions, and exposure to stress factors.

Figure 12.1 Climate change affects plants and health, influencing yield and food security.

Agriculture Crop’s Response to Climatic Change

Physiologically, ESF initiates oxidative stress by producing reactive oxygen species (ROS), superoxide (O2–), singlet oxygen (1O2), hydrogen peroxide (H2O2) that damages functional proteins, lipids, carbohydrates, and nucleic acid (Boyarshinov & Asafova, 2011) (Boogar et al., 2014). Resulting in cellular and tissues injuries are regulated by the activation of a defense mechanism by the production of antioxidant enzymes such as superoxide dismutase (SOD) (Tunc-Ozdemir et al.), peroxidase (PPO), ascorbate peroxidase (APX), polyphenol oxidase (POD), and catalase (CAT) (Cui et al., 2017; Wang et al., 2016) and osmolytes (proline, trehalose, and polyphenols (Sharma et al., 2019). Maintaining a high antioxidant capability to scavenge toxic ROS molecules is associated with enhanced plant tolerance to harsh conditions (Chen et al., 2011; Zaefyzadeh et al., 2009). Reoxygenation during flooding further enhances the post-submergence damage (Fukao et al., 2011). For example, exposure to atmospheric oxygen after 7–10 days of submergence also induced leaf dehydration in rice (Fukao et al., 2011; Setter et al., 2010). Flooding increased ethylene accumulation and a drop in ABA level in AR primordia tissue. ABA treatment inhibited activation of AR primordia by flooding and blocking ABA to reactivate AR primordia in the absence of flooding (Dawood et al., 2016).

One of the significant submergence tolerance regulators in silicious plants, e.g., rice, is SUB1A, which confers tolerance to oxidative stress and dehydration through activation of ROS detoxification and abscisic acid (ABA) responsiveness (Fukao et al., 2011). SUB1A is the master regulator of submergence tolerance, allowing plants to endure complete submergence for 14–16 days (Fukao et al., 2006). Ethylene response factors (ERF-VIIs) have been found to regulate several gene expressions to low oxygen and flooding (Laurentius & Julia, 2015). For example, Five ERF-VII genes (RAP2.12, RAP2.2, RAP2.3, HRE1, and HRE2) are critical regulators for flooding and low-oxygen tolerance in Arabidopsis (Bui et al., 2015; Gasch et al., 2016). Other rice genes, such as ERF-VII, SNORKEL1 and SNORKEL2, have been explicitly found in deep water rice (Hattori et al., 2009). The importance of ERF-VIIs in flooding responses and tolerance is also indicated in Rumex and Rorippa, dicot species from flood-prone environments (van Veen et al., 2014). The hypoxia-induced group-VII ERFs promote adventitious root (AR) elongation, while ethylene inhibits the adventitious root formation (Eysholdt‐Derzsó & Sauter, 2019).

Similarly, high rhizosphere pH significantly affects plant phenotype and genotype under alkaline conditions. Recent studies described the mechanism of alkaline soil tolerance, focusing on the ability of plants to acidify the rhizosphere via plasma membrane H+-ATPase-mediated proton secretion (Fuglsang et al., 2007; Li et al., 2015; Xu et al., 2012; Xu et al., 2013; Yang et al., 2010). Several factors are known to regulate the activity of H+-ATPase. For example, DNAJ HOMOLOG3 (J3) and PROTEIN KINASE5 (PKS5) play critical roles in proton secretion by regulating the interaction between 14-3-3 proteins and the plant plasma membrane H+-ATPase (Fuglsang et al., 2007; Yang et al., 2010). Another study reported that PIN-FORMED2 (PIN2, an auxin efflux transporter) is required to tolerate alkaline stress conditions by regulating proton efflux in the roots (Xu et al., 2012). However, other adaptive mechanisms by those plants that can tolerate alkaline stress and how microbial symbionts can intervene have yet to be explored. Overall, genes involved in cell-wall modification, calcium signaling, ethylene, and reactive oxygen species (ROS) change during cell division and regeneration under conditions of low oxygen (Rajhi et al., 2011; Yamauchi et al., 2018). However, these concurrent molecular signaling and the role of genomics level responses have seldomly been studied and known across economically important plants during ESF and microbiome functions.

Figure 12.2      Plants exposed to abiotic stress conditions and cellular responses to growth and development.

Section – II: Microbial utilization in agriculture development

Agrobacterium tumefaciens use in agriculture

Agrobacterium radiobacter (more commonly known as Agrobacterium tumefaciens) is the causal agent of crown gall disease (the formation of tumors) in over 140 species of eudicots (Young et al., 2001). It is a rod-shaped, Gram-negative soil bacterium (Smith & Townsend, 1907). Symptoms are caused by the insertion of a small segment of DNA (known as the T-DNA, for ‘transfer DNA,’ not to be confused with tRNA that transfers amino acids during protein synthesis) from a plasmid into the plant cell, which is incorporated at a semi-random location into the plant genome. Plant genomes can be engineered using Agrobacterium to deliver sequences hosted in T-DNA binary vectors. Agrobacterium tumefaciens is an Alphaproteobacterium of the family Rhizobiaceae, which includes the nitrogen-fixing legume symbionts.

Unlike the nitrogen-fixing symbionts, tumor-producing Agrobacterium species are pathogenic and do not benefit the plant. The variety of plants affected by agrobacterium makes it of great concern to the agriculture industry (Moore et al., 1997). Economically, A. tumefaciens is a severe pathogen of walnuts, grape vines, stone fruits, nut trees, sugar beets, horse radish, and rhubarb, and the persistent nature of the tumors or galls caused by the disease makes it particularly harmful for perennial crops (Morton & Fuqua, 2012). Agrobacterium tumefaciens grows optimally at 28 °C (82 °F). The doubling time can range from 2.5 to 4 hours depending on the media, culture format, and level of aeration. At temperatures above 30 °C, A. tumefaciens begins to experience heat shock, likely to result in errors in cell division (Weisberg et al., 2020).

Gene transfer through A. tumefaciens

The A. tumefaciens-mediated plant genetic transformation process requires two genetic components on the bacterial Ti-plasmid. The first essential component is the T-DNA, defined by conserved 25-base pair imperfect repeats at the ends of the T-region called border sequences. The second is the virulence (vir) region, composed of at least seven significant loci (virA, virB, virC, virD, virE, virF, and virG) encoding components of the bacterial protein machinery mediating T-DNA processing and transfer. The VirA and VirG proteins are two-component regulators that activate the expression of other vir genes on the Ti-plasmid. The VirB, VirC, VirD, VirE, and VirF are involved in the processing, transferring, and integrating the T-DNA from A. tumefaciens into a plant cell. Fig 12.3 shows the significant steps of the Agrobacterium-mediated plant transformation process.

Figure 12.3     A. tumefaciens mediated transformation of Ti plasmid. This is a natural process. This figure has been adopted from “(Hwang et al., 2017)

The overall process of Ti plasmid is as follows:

1. T-DNA tumor-causing genes are deleted and replaced with genes of interest (GOI) driven by plant-expressed promoters.
2. Genes for DNA transfer and insertion into plant genome are retained (vir genes)
3. Selectable marker genes are added to track transformed plant cells. Ex. Hygromycin (antibiotic), Bialaphos (herbicide)
4. The modified Ti plasmid is constructed first in E. coli and then transformed into A. tumefaciens
5. A. tumefaciens, with GOI, is co-cultured with plant leaf disks with hormone conditions favoring callus development (undifferentiated)
6. Antibacterial agents are added to kill A. tumefaciens
7. Hygromycin or bialaphos is added to kill non-transgenic plant cells
8. Surviving cells = transgenic plant cells

Figure 12.4     Agrobacterium-Mediated Gene Transfer (Transformation) in Plants. Created with BioRender.com

Genetically modified crops using microbial biotechnology approaches

1. a) Tomato:

• Flavr Savr, a genetically modified tomato, was the first commercially grown genetically engineered food to be granted a license for human consumption. Through genetic engineering, Calgene hoped to slow down the tomato’s ripening process and thus prevent it from softening while still allowing it to retain its natural color and flavor.
• The tomato was made more resistant to rotting by adding an antisense gene that interferes with the production of the enzyme polygalacturonase (PG). The enzyme typically degrades pectin in the cell walls and results in the softening of fruit, which makes them more susceptible to being damaged by fungal infections.
• The intended effect of slowing down the softening of Flavr Savr tomatoes would allow the vine-ripe fruits to be harvested like green tomatoes without more significant damage to the tomato itself.
• The Flavr Savr disappointed researchers in that respect, as the antisense PG gene had a positive effect on shelf life, but not on the fruit’s firmness, so the tomatoes still had to be harvested like any other unmodified vine-ripe tomatoes.

1. b) Herbicides

• Glyphosate is a herbicide used in agriculture and non-crop situations to control many weeds.
• Chemically, the active ingredient glyphosate (N-phosphonomethyl-glycine) is a glycine derivative, the most minor amino acid found in proteins. In the glyphosate molecule, one of the amino hydrogen atoms of glycine is replaced with a phosphonomethyl group.
• Once absorbed by the plant, glyphosate binds to and blocks the activity of the enzyme enolpyruvylshikimate-3-phosphate synthase (EPSPS).
• Structural similarities to phosphoenol pyruvate enable glyphosate to bind to the substrate binding site of the EPSPS, inhibiting its activity and blocking its import into the chloroplast.
• Inhibiting the function of the shikimic acid pathway causes a deficiency in aromatic amino acids, eventually leading to the plant’s death by starvation.

1. c) Bacillus thuringensis crops

• Protection against insect pests in crops. BT cotton is one of the famous examples.
• Engineered with the gene for the toxic Cry (crystal; Cry1Ac!) protein from the bacterium Bacillus thuringensis
• The BT proteins are absorbed by receptors in the lining of the insect’s gut. The activated protein forms a complex that bores into the gut of an insect, causing a rupture in the gut lipid layer – eventually killing the insect.

1. d) other examples of crops

Golden rice, iron-enriched rice, and antioxidant-enriched tomatoes are some of the recent examples of genetically modified crops to produce high-quality food products.

Figure 12.5     Photo credits: Golden Rice Humanitarian Board © 2007; Credit: ETH Zurich / Christof Sautter; Reprinted by permission from Macmillan Publishers, Ltd: Butelli, E., et al., Nature Biotechnology 26, 1301 – 1308 copyright (2008).

Plant growth-promoting microbes and their use in agriculture

Plant growth-promoting rhizobacteria (PGPR) can significantly facilitate the growth of many cereals and other vital crops. Different types of (PGPR) in soil suppress many plant pathogens and promote plant growth by different methods, such as direct and indirect production of different phytohormones, mineralization, and decomposition of organic matter, and improving the bioavailability of different mineral nutrients like iron and phosphorous. PGPR inhabit in the rhizosphere belongs to Achromobacter, Arthrobacter, Azotobacter, Azospirillum, Bacillus, Burkholderia, Enterobacter, Klebsiella, Microbacterium, Paenibacillus, Pantoea, Pseudomonas, Serratia, Streptomyces, etc. These PGPRs are generally used as inoculants for biostimulation, biocontrol, and biofertilization (Waqas et al., 2012).

These bacteria and other microbes improve plant growth under different environmental stress conditions. The PGPR is a naturally available source for mitigating biotic and abiotic stresses. PGPR usually improves plant growth promotion by triggering plant growth hormones and antioxidant system, producing siderophore, and enhancing the nutritional capacity of the plants. The effects of PGPR on plants include promoting growth and increased plant productivity. There are various mechanisms through which PGPR can be used for plant growth promotion, such as increased root and shoot growth by producing different phytohormones like auxins and cytokinins (Numan et al., 2018).

Indole acetic acid is an active form of auxin and an essential plant growth regulator. IAA has a crucial role in plant growth and development through its life cycle. IAA is mainly produced in actinobacteria. The strains Kitasatospora sp., Nocardia sp., and Streptomyces genus have been identified to produce IAA. The radicular system is activated by IAA root elongation, derived by apical meristem and lateral root development, increasing plant access to soil nutrients. IAA is the main auxin that promotes plant growth (Kang et al., 2014).

In addition to being a reservoir of bioactive secondary metabolites, endophytic fungi have recently been known to produce plant growth regulators. Such regulators increase plant growth and development and improve plant health by increasing tolerance against diverse environmental stresses. Plant growth regulators such as indole 3-acetic acid (IAA) and gibberellins (GAs) can stimulate rapid responses of cell elongation, cell division, and differentiation in plants(Khan et al., 2014). Some of the strains of rhizobacteria, viz., Rhizobium phaseoli, Acetobacter diazotrophicus, and Herbaspirillum seropedicae, Bacillus pumilus and B. licheniformis, B. cereus, B. macroides, and B. pumilus, Azotobacter chroococcum SE370, and Burkholderia cepacia SE4, have been known to produce GAs. Some strains of bacteria also produce IAA, which can extend growth-promoting effects during symbiosis (Verma et al., 2001). Genera such as Bacillus, Microbacterium, Methylophaga, Agromyces, and Paenibacillus have been found to produce IAA (Weyens et al., 2014).

Figure 12.6     Impact of PGPR application on plant growth and development with or without stress conditions.

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Questions:

Q1: What is climate change, and what factors are contributing to climate change for crops?

Q2:  What are PGP microbes?

Q3:  PGP microbes produce _______________ to improve plant growth.

1. Indole acetic acid (IAA)
2. Gibberellins (GAs)
3. Enzymes
4. None of the above

Q4: What is the flavor savor tomato, and how are these developed?

Q5: To improve agricultural productivity, ___________ is a key microbe to develop improved qualities in crop-based food production.

1. Bacillus
2. Agrobacterium
3. Sphingomonas
4. Streptococcus