Bactericidal Effect of Entomopathogenic Bacterium Pseudomonas entomophila Against Xanthomonas citri Reduces Citrus Canker Disease Severity

Abstract

The bacterium Pseudomonas entomophila has been recognized as an exceptional species within the Pseudomonas genus, capable of naturally infecting and killing insects from at least three different orders. P. entomophila ingestion leads to irreversible gut damage resulting from a global blockage of translation, which impairs both immune and tissue repair systems in the insect intestine. In this study we isolated a P. entomophila bacterial strain from soil samples which displayed a strong activity against Xanthomonas citri subsp, citri (Xcc), the etiological agent of citrus canker disease. The antagonism potential of isolated bacteria against Xcc and its ability to reduce citrus canker severity was assessed both ex planta and in planta. Our findings show that pathogenicity assays in Citrus x limonia by pressure infiltration and spray with a mixture of P. entomophila and Xcc leaded to a significant reduction in the number of canker lesions in high susceptible citrus leaves, at 21 days post-infection. To the best of our knowledge this is the first report of antibacterial activity of P. entomophila against a phytopathogenic bacterium. Collective action of P. entomophila factors such as diketopiperazine production and the type 6 secretion system (T6SS) may be involved in this type of biological control of citrus canker. The results suggest that the P. entomophila strain could be a promising biocontrol agent acting directly against Xcc.

Leaf infiltration in plant science: old method, new possibilities

“…Investigation of plant defense mechanisms Leaf infiltration with suspension of phytopathogen cells as method for controlled induction of plant infection Suspension of phytopathogens or non-pathogen endophyte cells in water or properly selected infiltration medium, most often composed of MES (10 mM) and magnesium salt (MgCl 2 or MgSO 4 , 10mM). Spontaneous infiltration, forced infiltration/whole leaves on the mother plant, whole harvested leaves, leaf fragments Citrus × limonia — Xanthomonas citri subsp, citri [ 66 ] A. thaliana — P. syringae pv. tomato DC3000 [ 4 ] A. thaliana, Solanum lycopersicum var.…”

“…cerasiforme cv. ‘Tenten— Bacillus cereus (GU982920.1), Variovorax paradoxus (JN990697.1), Rhodococcus kyotonensis (AB920569.1) , R. corynebacteriodies (AY438619.1) [ 11 ] Applied microbiology, agricultural microbiology Design and testing of plant protection products containing microorganism with an antagonistic effect towards phytopathogens Citrus × limonia — Pseudomonas entomophila, Xanthomonas citri subsp , citri ; Bacillus amyloliquefaciens LE109 [ 66 ] S. lycopersicum var. cerasiforme cv.…”

“…Leaf infiltration is widely used in the research of plant disease mechanisms, where it has been developed for the controlled induction of plant infections, for example with the model prokaryotic pathogen Pseudomonas syringae , under standard laboratory conditions [ 4 , 5 , 64 ]. Although various bacteria and not only phytopathogens can actively penetrate the internal leaf tissues via stomata or tissue wounds, applying the inoculum to the leaf surface for spontaneous infiltration of microorganisms is a less efficient method than forced infiltration [ 20 , 47 , 65 , 66 ]. Due to the need for spatially precise application of the inoculum and control of microbial load introduced into the plant tissue, it is preferable to use syringe infiltration for this purpose rather than vacuum infiltration [ 4 , 5 , 12 ].…”

“…Leaf intercellular spaces, due to the presence of many nutritional compounds, optimal humidity and protection against the direct impact of external environmental conditions, provide unique habitats for various microorganisms, many of which are endophytes that are not considered pathogenic [ 4 , 66 ]. The colonization of plant organisms by bacterial or fungal endophytes can enhance resistance to biotic and abiotic stress, supporting the healthy growth and development of the plant hosts [ 12 , 67 ].…”

“…Spontaneous and forced infiltration methods can be useful in the design and testing of bio-protection products containing microbes with an antagonistic effect on phytopathogens. These two infiltration techniques were used, for example, to test the protective effect of the insect pathogen Pseudomonas entomophila naturally inhabiting the soil, as it displayed a strong activity against the phytopathogenic bacteria Xanthomonas citri subsp citri ( Xcc ), the etiological agent of citrus canker disease [ 66 ]. Applying the mixture of P. entomophila and Xcc to the leaves using forced and spontaneous infiltration led to a significant reduction in the number of canker lesions in highly susceptible citrus leaves [ 66 ].…”