Citrus canker is a disease that affects the major types of commercial citrus crops. Xanthomonas citri subsp. citri, the etiological agent, reaches to mesophyll tissue through the stomata and afterward induces cell hyperplasia. Disease management has been based on both tree eradication and copper spray treatment. Overuse of copper for control of bacterial citrus canker has led to the development and prevalence of copper-resistant strains of Xcc. Several genera of both soil- and plant-associated bacteria became powerful tools in sustainable agriculture for control of Xcc and reduction of citrus canker disease severity. In this chapter we present bacteria able to interfere with quorum sensing as well to display antibacterial activity against Xcc by production of secondary metabolite. These bacteria may represent a highly valuable tool in the process of biological control and offer an alternative to the traditional copper treatment currently used for the treatment of citrus canker disease, with significant environmental, economic, and health implications worldwide.
- quorum quenching
- secondary metabolites
The steady increase in global overpopulation has forced the agricultural producer to introduce environmentally aggressive practices (e.g., undiscriminating use of pesticides and chemical fertilizers), in order to respond to the rising request of cultivated crops for food. The growing breach between supply and request and the negative impact on the environment have stimulated researchers to develop alternative strategies, pursuing to promote a sustainable agriculture.
The interactions between plants and their associated microorganisms have generated a huge interest. A deep understanding of these processes allows the implementation of innovative agricultural applications. Plants produce an extensive collection of organic compounds comprising sugars, organic acids, and vitamins, which can be served as nutrients or signals for microbial communities. On the other hand, microorganisms release phytohormones, small molecules, or volatile compounds, which may act directly or indirectly reducing disease severity caused by phytopathogenic agents. Some of these actions are nutrient competition, antibiotic activity, plant immunity activation or plant growth, and morphogenesis activation .
Prokaryotes and mainly the bacterial domain are the numerically dominant component of most microbial communities in plants. Numerous genera of both soil- and plant-associated bacteria turn out to be powerful tools in sustainable agriculture, because these bacteria display extremely versatile secondary metabolisms with valuable biological activities, including quorum quenching and antibiotic activity. The aim of this chapter is to present two different approaches for biological control of bacterial citrus canker. This antagonism specifically focus in a quorum quenching of DSF pathway and antibacterial activity by
2. Citrus canker disease
One of the most important diseases of citrus is citrus canker, affecting almost all commercial varieties. Bacterium
Traditional control of citrus canker disease centered on the application of copper-based products seeks the reduction of bacterial population in leaf surfaces. However, multiple applications are needed in order to obtain a significant reduction in bacterial burden on phyllosphere. Weather conditions, i.e., wind and rain, decrease drastically the effectiveness of copper applications. The drawbacks of the long-term use of copper compounds to control plant pathogens in the field include selection of copper resistance and horizontal transfer in bacterial populations .
2.1 Disease cycle and transmission mechanisms
Invasion and colonization of the citrus host by
2.2 Types of disease
There are three different types of citrus canker caused by two species of
The diseased plants are characterized by the occurrence of conspicuous raised necrotic lesions that develop on leaves, branches, and fruits. In the leaves, the first appearance is circular patches of 2–10 mm in diameter; their appearance is oily and usually appears on the abaxial surface reflecting stomatal entrance
2.4 Management and treatment
Bacterial citrus canker management involves different approaches ranging from strict quarantine measures to chemical control. Quarantining is a practical usually used in Brazil and United States of America. Extinction of infected and adjacent trees is one of the major prophylactic measures against citrus canker in commercial citrus crops. Once a symptomatic tree is identified, it is uprooted, stacked, and burned, as prophylactic measure surrounding trees is destroyed as mentioned before .
Prevention of primary infection in the new sprouts perhaps is the major effective approach to reduce citrus canker spread. The eradication methodology comprises conducting periodic surveys of the orchard, identifying and eliminating the outbreaks of the disease before its proliferation. Brazilian regulation stipulates that any field that has a number of diseased trees greater than 0.5% of the total orchard must be eliminated. After eradication, the contaminated field should be sprayed with copper fungicide based on 1.5 kg of metallic copper per 1 mL of water (0.15% of metallic copper). The contaminated plantations are prohibited and are forbidden from marketing the production until eradication works are completed.
The use of bactericidal products based on copper by spray application is a practice widely used for more than two decades for the bacterial citrus canker control. The prolonged exposure of bacterial strains to copper has led to the rise of resistant strains in endemic areas. Behlau et al. reported that the genes
Xanthomonas citrisubsp. citri
3.1 Isolation and identification
3.2 Determinants of virulence in
Xanthomonas citrisubsp. citri
An essential stage in bacterial host colonization is its attachment ability. Adhesins are bacterial surface structures that facilitate the attachment to host tissues. The nature of these structures is mainly polysaccharidic, e.g., lipopolysaccharides and exopolysaccharides. However, some of these structures share a proteinaceous nature (type IV pili, chaperone/usher pili, two-partner secretion) .
3.2.2 Protein secretion systems and their effectors
Bacteria inside in
Protein transport from bacterial periplasm to the extracellular environment occurs mainly by T2SS secretion system. Extracellular enzymes as lipases, proteases, and cell wall-degrading enzymes are translocated using this secretion system. Possibly the major enzymes responsible for the degradation of the plant cell wall are secreted by T2SS. T2SS translocator apparatus is composed of up to 12–15 constituents, most of which are linked to the bacterial inner membrane .
The T3SS secretion system also known as “needle” delivers effectors directly into host cells. These act as virulence factors influencing cell host activities . In the Xcc genome, 24 effectors have been identified . One of the main effectors delivered by the T3SS in
T4SS secretion system is an important virulence factor in a wide range of bacterial pathogens. This secretion system involves the secretion of protein or DNA into the host cells . Xcc harbors two gene arrays encoding for T4SS components ; one of them has chromosomal location, and the other one is located at the plasmid pXAC64. Proteins VirB1–VirB11 and VirD4 make up the T4SS translocator apparatus. Nowadays, the structural disposition is well established:
Three ATPases (VirB4, VirB11, and VirD4) located at the cytoplasm. These enzymes have been involved in the process of providing the necessary energy for the secretion process.
Fourteen repetitions of VirB7-VirB9-virB10 trimer. These repetitions form the periplasmic core. It is noteworthy that VirB10 is anchored on both inner and outer membranes; on the other hand, VirB7 is a lipoprotein located at the outer membrane.
An inner membrane complex formed by VirB3, VirB6, and VirB8.
An extracellular pili formed by VirB2 and VirB5.
VirB1 which is a periplasmic transglycosylase .
A recent study has shown that T4SS in Xcc displays the ability to secrete toxins; these toxins are known as VirD4-interacting proteins (XVIPs), and they are recruited by VirD4. The biological role of XVIPs is targeting and destabilizing the peptidoglycan layer in the cell wall of bacterial contenders in the ecological niche. This feature is distinctive in
4. Biological control of Xcc approaches
4.1 Biological control based on DSF quorum quencher pathway
A wide majority of bacterial genera have developed a cell-to-cell communication system known as quorum sensing (QS). This communication system is based on a signal translation mechanism whose objective is to coordinate the expression of genes at the population level in order to respond and fit to environmental changes. The cell-to-cell communication system is based on the production, secretion, and perception of small molecules known as autoinducers. A basal quantity of autoinducers are produced by every single cell, subsequently, which is secreted to extracellular milieu reflecting the bacterial population density. At high population density, the autoinducers reach a critical concentration and enable to cognate receptor to sense them. Consequently, this biological event results in triggering a cascade of diverse cell functions . In the
Since quorum sensing helps to coordinate community-based bacterial behavior, it is not essential for bacterial survival; therefore, the inhibition of QS interrupts only the desired phenotype, i.e., virulence, biofilm formation, and bacterial resistance to different antibiotics. Interference with QS can provide a route for disease control. This interference may involve signal degradation (quorum quenching) or excess signal production (pathogen confusion) . Quorum quenching is a mechanism adopted by a number of bacteria to break the QS signaling of competitors, giving these organisms an advantage within a particular habitat . It is rational that microorganisms can develop mechanisms to disarm the QS systems of competing organisms in order to increase their competitive strength in an ecosystem .
We have conducted a recent study that allows the isolation and identification of bacteria isolated from citrus leaves belonging to plant of field crops with and without citrus canker symptoms. From a total of 114 isolates recovered, 7 bacteria able to disrupt DSF quorum sensing pathway in
Virulence assays were conducted under controlled growth conditions, and canker lesions were quantified at 21 days post inoculation. These assays demonstrated that, when citrus leaves were inoculated with mixtures of Xcc and quorum quencher bacteria, the number of cancer lesions decreased significantly reducing the severity disease (Figure 2).
Quorum quencher bacteria impaired the attachment and biofilm formation of Xcc to leave the surface. These are essential steps in the maintenance, survival, and initial establishment of tissue pathogenicity in citrus canker. In fact, it is completely accepted that QS plays an important, if not an essential, role in the formation of bacterial biofilm . Studies of scanning electron microscopy SEM confirmed the substantial reduction in the adherence ability of Xcc after 10 hours when it was co-infected with quorum quencher bacteria relative to the control used, i.e., the leaves infected with Xcc alone. After 7 days post-infection with Xcc and the inhibitory bacteria of DSF, SEM has shown the absence of biofilm formation on the surface of leaves co-inoculated with
A possible mechanism for explaining the modification or degradation of DSF molecule produced by Xcc could be the quorum quencher bacteria using the DSF molecule as a possible substrate for the UDP-sugar transferase enzyme. The addition of one unit of sugar (from UDP-sugars, i.e., UDP-glucose or UDP-galactose to the short chain of fatty acid impossible the recognition of this version of modified DSF molecule by sensor RpfC. These UDP-sugar pools are produced by the activity of carbamoyl phosphate synthetase enzyme, which is encoded by
4.2 Biological control based on antibacterial activity of
In recent study (in press), we have isolated and identified from soil samples added with a compost five
Quorum sensing is an important target for prophylactic and therapeutic interventions. Identification of new bacteria species as ABC could be a new alternative for the treatment of copper traditionally used for the treatment of citrus canker disease, thus reducing selection pressure for copper resistance. We believe that the search for microorganisms that act as inhibitors of quorum sensing in phytopathogenic bacteria also as antagonist agent could be an effective strategy in a broader context. Since the organisms characterized here were originally isolated from the citrus phylloplane, the present study also contributes to an understanding of the potential interactions of bacteria on leaf surfaces
The authors thank Professor Jesus A Ferro from the Technology Department, Faculdade de Ciencias Agrarias e Veterinarias, Universidade estadual Paulista, UNESP, Jaboticabal, SP, Brasil, and to CREBIO Centro de Recursos Biologicos e Biologia Genomica Univ. Estadual Paulista, Jaboticabal SP, Brazil, for the DNA sequencing.
Conflict of interest
The authors declare no conflict of interest.
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