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Bioactive Constituents, Metabolites, and Functions
Antifungal Metabolite p-Aminobenzoic Acid (pABA): Mechanism of Action and Efficacy for the Biocontrol of Pear Bitter Rot Disease Pedro Laborda, Chaohui Li, Yangyang Zhao, Bao Tang, Jun Ling, Feng He, and Fengquan Liu J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b05618 • Publication Date (Web): 08 Feb 2019 Downloaded from http://pubs.acs.org on February 11, 2019
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Journal of Agricultural and Food Chemistry
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Antifungal Metabolite p-Aminobenzoic Acid (pABA): Mechanism of
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Action and Efficacy for the Biocontrol of Pear Bitter Rot Disease
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Pedro Laborda,#,†,‡ Chaohui Li,#,† Yangyang Zhao,† Bao Tang,† Jun Ling,† Feng He,† and
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Fengquan Liu†,*
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†
Institute of Plant Protection, Jiangsu Academy of Agricultural Sciences, Jiangsu Key
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Laboratory for Food Quality and Safety-State Key Laboratory Cultivation Base of
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Ministry of Science and Technology, Nanjing 210014, People’s Republic of China
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‡
School of Life Sciences, Nantong University, Nantong, 226019, People’s Republic of
China
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#
These authors contributed equally to this work.
*
Corresponding author: Prof. Fengquan Liu; email:
[email protected] 15
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ABSTRACT
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Colletotrichum fructicola, a fungal pathogen that causes bitter rot disease in pears, has
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recently emerged in Eastern Asia and caused enormous economic losses and crop
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penalties. For this reason, new strategies for the management of bitter rot disease are
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severely necessary, and can have a great impact on the field. In this regard, our research
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group recently reported that p-aminobenzoic acid (pABA), which was found in the
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secretions of rhizobacterium Lysobacter antibioticus OH13, showed a broad spectrum
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of antifungal activities. Following this project, the antifungal mode of action of pABA
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has been elucidated in this work indicating that pABA affects the fungal cell cycle of C.
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fructicola by inhibiting septation during cell division. pABA stability and diffusion
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screening revealed that pABA degrades after 15 days and is able to cross the pear skin
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into the external parts of the mesocarp. In vivo studies demonstrated that pABA shows
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high curative ability against the infection of C. fructicola in pears. To show the efficacy
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of OH13 for the biocontrol of bitter rot disease, cultures of OH13 containing 379.4
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mg/L pABA were sprayed on inoculated pears, significantly reducing the symptoms of
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the pathogen.
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Key
words:
Biocontrol,
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Colletotrichum fructicola.
Lysobacter,
pear
diseases,
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p-aminobenzoic
acid,
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Journal of Agricultural and Food Chemistry
INTRODUCTION
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Pears are the third most consumed fruit after apples and grapes, reaching 27.3 million
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tons in world production in 2016.1 Pears are extensively cultivated in Asia, America and
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Europe, with major production areas in China, the USA, Italy, Argentina and Spain.1
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However, pears, before or after the harvest phase, are highly susceptible to a wide range
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of devastating fungal pathogens.2,3 Pear fungal pathogens usually secrete pectinolytic
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enzymes that are able to degrade tissues and produce fast-growing black spots easily
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observed with the naked eye.4,5 Among pear pathogens, Colletotrichum fructicola,
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which is responsible for pear bitter rot disease, is an extremely destructive pathogen that
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currently causes serious economic losses and crop penalties in eastern Asia.6,7 Bitter rot
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disease has been reported to produce black spots on young pears, followed by severe
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soft rot on the matured fruits.8,9 Brutal occurrences of this disease have taken place in
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areas of Dangshan County of Anhui Province in China, achieving losses of 60-90%
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even with the application of fungicides.7 In 2008, a bitter rot disease spread affected
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more than 40,000 ha of pear plantations in China and caused economic damages of
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approximately 150 million dollars.9
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The main defense against fungal pear pathogens consists of the use of synthetic
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fungicides, such as carbendazim or thiabendazole, which arrest the fungal cell cycle,
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and thus pathogen growth.10 Fungi habitually produce asexual spores, called conidia,
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which germinate in the presence of favorable substrates, such as sugars and amino
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acids.11,12 Then, formation of the germ tube, DNA replication and septation steps take
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place, allowing for the formation of new cells and thus fungal expansion.13-15 The
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increase in environmental and public health-related social concerns and legal
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requirements regarding the poisonous residues of traditional pesticides, together with
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the appearance of numerous resistant pathogens, such as C. fructicola, are stimulating
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the development of new environmentally friendly and robust strategies for the
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management of pear diseases.16-21 In this sense, Lysobacter species are emerging as a
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promising green source of new bioactive products for the biocontrol of plant diseases
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and plant growth promotion.22-24 Among Lysobacter species, Lysobacter antibioticus
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OH13 was initially isolated from the rhizosphere soil of rice and exhibited potent
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antibacterial and antifungal activities. Six different N-oxide phenazines were found to
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be responsible for the antibacterial activity, whereas p-aminobenzoic acid, pABA, was
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shown to be the metabolite involved in the antifungal activity.25,26 pABA is a benzoic
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acid derivative that contains an amino group in position 4 of the benzene ring. Activity
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screening revealed that pABA shows antifungal activity against a number of fungal
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plant pathogens, including Fusarium graminearum, Magnaporthe oryzae, Rhizoctonia
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solani, Sclerotinia sclerotiorum and Valsa ambiens var. pyri, and against the oomycete
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Phytophthora capsici. When 3 mM pABA was used, more than 50% inhibitory activity
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was observed for all pathogens, achieving 71% and 75% inhibitory activities in the
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screening of Phytophthora capsici and Rhizoctonia solani, respectively. Following this
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project, after proposing the fungal cell cycle of C. fructicola for the first time, the
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antifungal mode of action of pABA has been elucidated in this work. Further, the
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stability and diffusion of pABA in the skin and mesocarp of pears were studied. To
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demonstrate the worth of pABA as a biocontrol agent for pear diseases, the efficacy of
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L. antibioticus OH13 cultures to reduce C. fructicola symptoms was examined.
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MATERIALS AND METHODS
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General information and bacterial strains. All reagents and chemicals were used as
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received from commercial suppliers without further purification or modification.
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Lysobacter antibioticus OH13 (CGMCC No.7561) was grown in nutrient broth (NB; 3 g
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beef extract paste, 1 g yeast extract, 5 g peptone, and 10 g sucrose at pH 7.0-7.2 in 1 L
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of distilled water). Fungi were grown on potato dextrose agar medium (PDA medium).
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M3S medium was used for conidium germination (2.5 g magnesium sulfate
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heptahydrate, 2.7 g potassium dihydrogenphosphate, 1 g peptone, 1 g yeast extract and
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10 g glucose per 1 L of distilled water), and potato dextrose broth (PDB) was employed
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to induce the formation of conidia.
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Data analysis. The statistical analyses were performed using SPSS (Statistical
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Package, Version 20.0). The variables were subjected to student’s t-test and were tested
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for significance at P
