Evaluation of fluorescence-activated cell sorting technology in agrobacterium biocontrol

Volume 10, Issue 2
June 2021
Pages 391-399

Document Type : Original Research

Authors

1 Department of Plant Disease Research, Iranian Research Institute of Plant Protection, Agricultural Research, Education and Extension, Tehran, Iran.

2 Department of Plant Pathology, Faculty of Agriculture, Tarbiat Modares University, Tehran, Iran.

Abstract
Fluorescence-activated cell sorting (FACS) as a novel and sensitive technology was used to evaluate the biocontrol efficiency of Bacillus subtilis against Agrobacterium tumefaciens, a very destructive plant pathogen. The combination of two methods of culturing and cell sorting by FACS technology was used to distinguish a rapid and accurate method in monitoring the biocontrol effect of Bacillus (ATCC21332) on Agrobacterium (IBRC-M10701 and AGL1),. The culture method indicated that the B. subtilis could suppress A. tumefaciens in vitro and in vivo. We used a green fluorescent protein (GFP), reporter, to flow cytometric analysis using FACS. The mean of GFP expression levels was significantly reduced to 17.98, 16.48, and 11.27% in treatments 24, 48, and 72 h post-treatment; however, it was 31.57, 26.06, and 23.98% in the nontreated ones. The experiments demonstrated a positive biocontrol effect of Bacillus against Agrobacterium. Overall, our findings may provide a basis for improving the new rapid biocontrol agent detection method based on FACS.

Keywords

Subjects
Alvarez-Barrientos A, Arroyo J, Canton R, Nombela C, and Sanchez-Perez M .2000. Applications of flow cytometry to clinical microbiology. Clinical Microbiology Reviews, 13: 167–195.
Arima K, Kakinuma A and Tamura G .1968. Surfactin, a crystalline peptide lipid surfactant produced by Bacillus subtilis: isolation, characterization, and its inhibition of fibrin clot formation. Biochem. Biochemical and biophysical research communications, 31:488-494.
Asad A, Ikramullah M, Asif S, Ahmad A, Jamal A and Jaffri A .2010. Evaluation of lipopeptide (surfactin) production by Bacillus subtilis. Biomedical, 26: 34-3.
Baker C J, J R Stavely, C A Thomas, M Sasser and J S Macfall .1983. Inhibitory effect of Basillus subtilis on Uromyces phaseoli and on development of rust pustules on bean leaves. Phytopathology, 73: 1148—1152.
Berney M, Vital M, Hulshoff I, Weilenmann H U, Egli T and Hammes F .2008. Rapid, cultivation-independent assessment of microbial viability in drinking water. Water Research, 42: 4010–4018.
Broadbent P, K F Baker and Y Waterworth .1971. Bacteria and actinomycetes antagonistic to fungal root pathogens in Australian soils. The Australian journal of experimental biology and medical science, 24: 925-944.
Burr TJ, Bazzi C, Süle S, Otten L .1998. Crown gall of grape: biology of Agrobacterium vitis and the development of disease control strategies. Plant Disease, 82: 1288e1297.
Cao X H, Zhao S S, Liu D Y, Wang Z, Niu L L, Hou LH and Wang CL .2011. ROS-Ca2+ is associated with mitochondria permeability transition pore involved in surfactin-induced MCF-7 cells apoptosis. Chemico-biological interactions, 190: 16-27.
Chang I P and T Kommedahl .1968. Biological control of seedling blight of corn by coating kernels with antagonistic microorganisms. Phytopathology, 58: 1395-1401.
Cubeta M A, G L Hartman and J B Sinclair .1985. Interaction between Basillus subtilis and fungi associated with soybean seeds. Plant Disease, 69: 506-509.
Czechowska K, Johnson D R and van der Meer J R .2008. Use of flow cytometric methods for single-cell analysis in environmental microbiology. Current Opinion in Microbiology, 11:1-8.
De Werra P, Baehler E, Huser A, Keel C and Maurhofer M .2008. Detection of plant-modulated alterations in antifungal gene expression in Pseudomonas fluorescens CHA0 on roots by flow cytometry. Applied and environmental microbiology, 74:1339-1349.
Fravel DR .2005. Commercialization and implementation of biocontrol. Annual Review of Phytopathology, 43: 337e359.
Gamalero E, Lingua G, Capri F G, Fusconi A, Berta G and Lemanceau P .2004. Colonization pattern of primary tomato roots by Pseudomonas fluorescens A6RI characterized by dilution plating, flow cytometry, fluorescence, confocal and scanning electron microscopy. Federation of European Biochemical Societies Microbiological Ecology, 48:79-87.
Hakkila K, Maksimow M, Rosengren A, Karp M, and Virta M .2003. Monitoring promoter activity in a single bacterial cell by using green and red fluorescent proteins. Journal of Microbiological Methods, 54:75-79.
Hassanein F M and EL-Goorani M A .1991. The Effect of Bacillus suhtilis on in vitro Growth and Pathogenicity of Agrohacterium tumehciens. Journal of Phytopathology, 133: 239-246.

Henry G, Deleu M, Jourdan E, Thonart P h and Ongena M .2011. The bacterial lipopeptide surfactin targets the lipid fraction of the plant plasma membrane to trigger immune-related defense responses. Cellular microbiology,13: 1824-1837.
Kerr A .1980. Biological control of crown gall through production of agrocin 84. Plant Disease, 64: 24-30.
Kim K, Jung S, Lee S Y, Jung D K, Park J K, Kim D K.and Lee C H .1998. Suppression of inflammatory responses by surfactin, a selective inhibitor of platelet cytosolic phospolipase A2. Biochemical Pharmacology ,55 (7): 975-985.
Loeffler W J S M, Tschen N, Vanittanakom M, Kugler E, Knorpp T F, Hsieh and T G Wu .1986. Andfungal effects of bacilysin and fengymycin from Basilus subtilis F-29-3, A comparison with activities of other Bacillus antibiotics. Journal of Phytopathology, 115:204-213.
Lazo GR, Stein PA, Ludwig RA .1991. A DNA transformation-competent Arabidopsis genomic library in Agrobacterium. Biotechnology, 9:963–967.
Mersereau M, Pazour GJ, Das A .1990. Efficient transformation of Agrobacterium tumefaciens by electroporation. Gene, 90:149–151.

New P B and A Kerr .1972. Biological control of crown gall: Field measurements and glasshouse experiments. Journal of Applied Bacteriology , 35: 279—287.
Pruss G J, Nester E W and Vance V .2008. Infiltration with Agrobacterium tumefaciens Induces Host Defense and Development-Dependent Responses in the Infiltrated Zone. Molecular Plant Microbe Interaction, 21 (12): 1528–1538.
Rochat L, Péchy-Tarr M, Baehler E, Maurhofer M and Keel C .2010. Combination of Fluorescent Reporters for Simultaneous Monitoring of Root Colonization and Antifungal Gene Expression by a Biocontrol Pseudomonad on Cereals with Flow Cytometry. Molecular Plant Microbe Interaction, 23 (7): 949-961.
Schroth MN, McCain AH, Foott JH, Huisman OC .1988. Reduction in yield and vigor of grapevine caused by crown gall disease. Plant Disease, 72: 241e246.
Schmidt M, Hourfar M K, Nicol SB, Spengler HP, Montag T and Seifried E .2006. FACS technology used in a new rapid bacterial detection method. Transfusion Medicine, 16: 355–361.
Sörensen M, Lippuner C, Kaiser T, Misslitz A, Aebischer T and Bumann D .2003. Rapidly maturing red fluorescent protein variants with strongly enhanced brightness in bacteria. Federation of European Biochemical Societies Letter, 552:110-114.
Stein T .2005. Bacillus subtilis antibiotics: structures, syntheses and specific functions. Molecular Microbiology, 56(4): 845–857.
Unge A, Tombolini R, Molbak L and Jansson J K .1999. Simultaneous monitoring of cell number and metabolic activity of specific bacterial populations with a dual gfp-luxAB marker system. Applied and environmental microbiology , 65:813-821.
Utkhede R S and J E Rahe .1980. Biological control of onion white rot. Soil Biology and Biochemistry, 12: 101-104.
Weinhold A R and T Bowman .1968. Selective inhibition of the potato scab pathogen by antagonistic bacteria and substrate influence on antibiotic production. Soil Science, 28: 12-23.
Waksman S A .1969. Successes and failures in the research for antibiotics. advances in Applied Microbiology ,11: 1-16.