Molecular Biotechnology: Principles and Applications - Glick B., Pasternak J. 2002

Molecular Biotechnology of Microbial Systems
Plant Growth-Promoting Bacteria
Biocontrol of Pathogenic Microorganisms

Plant Growth-Promoting Bacteria can exert their effects either directly or indirectly. Direct stimulation typically involves providing the plant with a compound synthesized by the bacterium (such as fixed nitrogen) or a plant hormone. Additionally, bacteria can facilitate the uptake of certain nutrients, such as iron or phosphorus, from the environment. Indirect stimulation occurs when bacteria reduce or prevent the harmful effects of one or more phytopathogenic organisms, such as Fungi or bacteria. Phytopathogens can reduce crop yields by 25–100%, causing enormous damage. Chemical agents are commonly used to control them. Unfortunately, in most cases, disease symptoms in plants do not manifest for a long time, until environmental changes trigger bacterial proliferation, leading to rapid disease development and total crop destruction. Controlling such widespread epidemics is difficult and highly costly.

Many chemicals used to control phytopathogens pose a hazard to animals and humans; they accumulate in natural ecosystems and persist in them for a long time. Therefore, it would be advisable to replace Chemical Methods of suppressing pathogenic microorganisms with biological ones that are more environmentally friendly. One biological approach to controlling phytopathogens involves developing Transgenic Plants resistant to one or more pathogens (this approach is discussed in Chapter 18). Attempts have also been made to use plant growth-promoting bacteria as biocontrol agents. These bacteria synthesize compounds that can be used to reduce the damage caused to plants by phytopathogens. These include siderophores, Antibiotics, and various Enzymes. However, despite the promise of this approach, almost all research has so far been conducted under laboratory conditions, in growth chambers, or in greenhouses. A definitive Conclusion regarding the utility of any strategy based on a specific mechanism can only be made after field trials.

Siderophores

Iron is one of the most abundant elements on Earth and is absolutely essential for living organisms. However, in the form in which iron is present in the soil, it cannot be directly utilized by microorganisms. This is because its predominant natural form is trivalent ions. Their solubility is extremely low—at pH 7.4, it is approximately 10^-18 M, which is completely insufficient to support Microbial growth. To survive under these conditions, soil microorganisms synthesize and secrete small, low-molecular-weight iron-binding compounds with a molecular mass of approximately 400–1000 Da, known as siderophores (Fig. 14.8). They efficiently bind Fe(III) and transport it to microbial Cells, where it binds to Cell receptors and enters the cells. Once inside, the iron is released and can be utilized by the microorganism.

Plant growth-promoting bacteria suppress the proliferation of phytopathogenic fungi by synthesizing siderophores that bind most of the Fe(III) present in the soil layer immediately adjacent to the plant ROOT (the rhizosphere). Phytopathogenic fungi also synthesize siderophores, but these typically have a lower affinity for iron than the siderophores synthesized by plant growth-promoting bacteria. This allows the latter to outcompete phytopathogenic fungi for the available iron.

Unlike phytopathogenic microorganisms, plants generally do not suffer from local soil iron depletion resulting from its uptake by plant growth-promoting bacteria. Most plants can grow at significantly lower iron concentrations than microorganisms. Furthermore, evidence suggests that iron bound by bacterial siderophores can be assimilated by plants and used for their own needs. Since the binding of iron by bacterial siderophores can simultaneously suppress the proliferation of A wide variety of phytopathogenic microorganisms, the possibility of using them to develop more effective biocontrol systems is being investigated.

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Fig. 14.8. Structure OF THE pseudobactin siderophore produced by Pseudomonas strain B10. One Fe(III) ion is bound to one siderophore molecule.

Many plant growth-promoting fluorescent pseudomonads secrete a siderophore that is a linear hexapeptide consisting of alternating L- and D-Amino Acids and an attached fluorescent chromophore (Fig. 14.8). One such siderophore, known as pseudobactin, has an affinity for Fe(III) of 10²⁵ L mol^-1. Similar siderophores are synthesized by all fluorescent pseudomonads.

Initial attempts have also been made to investigate pseudobactin synthesis in the plant growth-promoting bacterium Pseudomonas putida WCS358. Using mutagenesis, 28 mutants of this microorganism unable to synthesize the siderophore were obtained. They were selected based on: 1) lack of fluorescence under UV light; 2) inability to grow in the presence of dipyridyl, a substance that binds most of the iron in the culture medium. At very low concentrations of iron ions, only cells that synthesize siderophores can grow. A DNA clone library of P. putida WCS358 was constructed using the broad-host-range cosmid vector pLAFR1, and all 28 mutant forms were transformed via conjugation. The transformants were screened for UV fluorescence and/or The ability to grow in the presence of dipyridyl. Thirteen different complementing cosmid clones with an average insert size of 26 kb were identified. Detailed studies showed that these clones correspond to at least five distinct Gene clusters.

One of these clusters was investigated in greater detail. Its minimum length was approximately 33.5 kb, and it contained five operons with at least seven distinct genes. Thus, like Nitrogen Fixation and Nodulation, siderophore Biosynthesis is a complex process. Since each siderophore is encoded by multiple genes, obtaining recombinant bacteria capable of synthesizing a modified siderophore is no easy task. Fortunately, there are other ways to increase the efficiency of using plant growth-promoting bacteria as biocontrol tools. For example, the range of iron-siderophore complexes recognized by a single bacterial strain can be expanded, allowing a single recombinant strain to recognize and utilize siderophores synthesized by other soil microorganisms, thereby increasing its competitiveness. To achieve this, the genes for the iron-siderophore complex receptors from one plant growth-promoting bacterium were cloned and introduced into other strains.

Antibiotics

One of the most effective mechanisms used by plant growth-promoting bacteria to suppress the proliferation of phytopathogens is the synthesis of antibiotics. For instance, pseudomonads used for biocontrol synthesize antibiotics such as agrocin 84, agrocin 434, 2,4-diacetylphloroglucinol, herbicolin, oomycin, phenazines, pyoluteorin, and pyrrolnitrin.

The ability of plant growth-promoting bacteria to suppress phytopathogen proliferation can be enhanced by introducing genes encoding The biosynthesis of antibiotics typically produced by other bacteria. This would expand THE SPECTRUM OF phytopathogens that a single bacterium can inhibit. Moreover, by limiting the reproduction of other soil microorganisms, antibiotic-secreting plant growth-promoting bacteria facilitate their own proliferation by reducing the number of competitors for limited nutrient resources. Introduction/32.html">Genetic Engineering techniques may eventually make it possible to increase the yield of bacterial antibiotics.

The synthesis of most antifungal metabolites produced by pseudomonads appears to be controlled by a protein that acts as a global transcriptional regulator; therefore, the level of antibiotic synthesis can be increased by altering global regulation. For example, in the case of Pseudomonas fluorescens CHA0, this was achieved by transforming the microorganism with a vector carrying the housekeeping gene rpoD, which encodes the σ⁷⁰ subunit of RNA polymerase. The recombinant strain provided better protection for cucumber plant roots against Damage caused by the fungus Pythium ultimum (Table 14.7). In another study, pyoluteorin synthesis by the same microorganism was stimulated by inactivating the pqq genes involved in the biosynthesis of pyrroloquinoline quinone, a cofactor for various dehydrogenases. The Mechanism of this stimulation is not fully understood; it is possible that the mutation diverts the metabolic flux from other biosynthetic pathways toward pyoluteorin biosynthesis.

Currently, only one recombinant microorganism is commercially available for biocontrol: Agrobacterium radiobacter K84. This strain has been sold in Australia since 1989 to control crown gall, a disease caused by Agrobacterium tumefaciens. Almonds and stone fruit trees, such as peaches, are susceptible to this disease. A. radiobacter synthesizes the antibiotic agrocin 84, which is toxic to A. tumefaciens. However, if a plasmid from A. radiobacter containing the agrocin 84 biosynthetic genes is accidentally transferred to A. tumefaciens, agrocin-resistant strains of A. tumefaciens may arise. To prevent this, the region responsible for plasmid transfer was deleted from the plasmid pAgK84, which carries the agrocin 84 biosynthetic genes (Fig. 14.9). As a result, the A. radiobacter strain could no longer transfer the recombinant agrocin plasmid to pathogenic agrobacteria, while retaining its biocontrol capability.

Table 14.7. Effect of additional copies of the rpoD gene of Pseudomonas fluorescens CHA0 on the ability of this bacterium to protect cucumber plant roots from damage caused by the pathogenic fungus Pythium ultimum¹⁾

¹⁾ From Schnider et al., J. Bacteriol. 177: 5387–5392, 1995.

²⁾ In the absence of P. fluorescens CHA0, a significant inhibition of root growth is observed due to The Effect of the pathogenic fungus P. ultimum. When the plasmid vector used to transform P. fluorescens CHA0 contained the rpoD gene, the protective effect of the bacterium was much greater. Root weight was determined after growing the plants under specific conditions for 2 weeks.

Enzymes

Some plant growth-promoting bacteria synthesize enzymes such as chitinase, β-1,3-glucanase, protease, and lipase, which degrade the fungal Cell wall. In one experiment, the incidence of diseases caused by the phytopathogenic fungi Rhizoctonia solani, Sclerotium rolfsii, and Pythium ultimum was reduced using a Pseudomonas cepacia strain that synthesizes the enzyme β-1,3-glucanase, which degraded the fungal mycelium. Other studies have shown that the antifungal activity of three plant growth-promoting strains of Enterobacter agglomerans is due to the presence of a complex of four different Polypeptides that act synergistically to degrade Chitin in the fungal cell wall. These bacteria effectively protected cotton plants from infection by Rhizoctonia solani. In contrast, Tn5 mutants of E. agglomerans that did not produce active chitinase were unable to protect plants from pathogenic fungi.

Fig. 14.9. Construction of a transfer-deficient (Tra^-) Modification of the A. radiobacter plasmid pAgK84, which encodes agrocin 84 biosynthetic genes and resistance genes. Using the restriction map of pAgK84, a fragment containing the Tra region responsible for transfer, along with its flanking sequences, was excised (1) and inserted into an E. coli plasmid (2). By restriction enzyme Digestion, approximately 80% of the Tra region and part of its flanking sequences (totaling about 6 kb) were removed from the cloned fragment (3). Homologous Recombination was performed between the E. coli plasmid containing the truncated Tra region and the pAgK84 plasmid containing the Tn5 transposon with a kanamycin resistance gene inserted into the Tra region (4). Several derivatives of plasmid pAgK84 with a partially deleted Tra region were obtained (5). The Tra^- plasmid pAgK84 could no longer be transferred to another agrobacterium during conjugation, but was still capable of determining agrocin 84 synthesis and providing resistance to it. The diagram is not drawn to scale.

Many bacterial enzymes that degrade fungal cell walls, including chitinase and β-glucanase, are encoded by a single gene. It would be reasonable to isolate these genes and introduce them into plant growth-promoting bacteria to obtain strains that synthesize, for example, both antibiotics and fungal cell wall-degrading enzymes. Experiments have been conducted in which the chitinase gene isolated from the bacterium Serratia marcescens was transferred into Trichoderma harzianum and R. meliloti cells. Both transformed microorganisms synthesized chitinase and exhibited enhanced antifungal activity. When the S. marcescens chitinase gene was introduced into a plant growth-promoting P. fluorescens strain, a transformant was obtained that stably secreted chitinase and effectively suppressed the proliferation of the phytopathogenic fungus Rhizoctonia solani.

Ice crystal formation and antifreeze Proteins

Certain pathogenic, leaf-infecting bacteria such as Pseudomonas syringae synthesize specific proteins at low temperatures that serve as ice nucleation centers on the leaf surface at sub-zero temperatures. As they grow, these crystals pierce plant cells, causing irreversible damage to the plant, while the bacteria gain access to nutrients released from the ruptured plant cells. In the absence of these ice-nucleating proteins on the leaf surface, brief overnight frosts may not harm the plant, as ice crystal formation in The plant cell Cytoplasm typically begins at temperatures several degrees below the freezing point (i.e., supercooling occurs). To prevent crystallization on the leaves of crops such as strawberries, mutant P. syringae bacteria incapable of synthesizing ice-nucleating Proteins can be sprayed over the plants prior to a frost. Such mutant strains can be developed using Recombinant DNA technology or conventional mutagenesis followed by Selection, and at sufficient concentrations, they will outcompete the wild-type bacteria.

One of the key requirements for the effective biocontrol of pathogens using plant growth-promoting bacteria is the ability of these bacteria to disperse under natural conditions. In Canada, the Scandinavian countries, and the northern United States, they must remain viable through long, cold winters and proliferate in the spring at relatively low soil temperatures (-5—10 °С). Since microorganisms employ various adaptive survival strategies under adverse conditions, genetic engineering could be used to construct recombinant bacteria optimally adapted to low temperatures. It has recently been shown that certain soil bacteria (including some plant growth-promoting strains) can proliferate at 5 °С and secrete antifreeze proteins into the environment at low temperatures. These proteins regulate ice crystal formation within the bacterial cell. Although crystals still form in their presence, they do not grow large enough to damage the cells. Once the genes encoding bacterial antifreeze proteins are identified, they can be transferred into plant growth-promoting bacteria to yield transformed, cold-resistant strains. Currently, there is no data establishing a link between bacterial antifreeze activity and the mechanisms ensuring their survival at low temperatures. It would be highly interesting to investigate whether antifreeze Protein Synthesis is part of an adaptive strategy employed by certain bacteria to confer cold tolerance.