atholog P y & nt a M l i P c f r o o b l i Journal of a o l n o r g u Chen, et al., J Plant Pathol Microbiol 2016, 7:3 y o J DOI: 10.4172/2157-7471.1000339 ISSN: 2157-7471 Plant Pathology & Microbiology Research Article Open Access Inhibition of Penicillium digitatum and Citrus Green Mold by Volatile Compounds Produced by Enterobacter cloacae Po-Sung Chen1, Yu-Hsiang Peng1, Wen-Chuan Chung2, Kuang-Ren Chung1, Hung-Chang Huang3 and Jenn-Wen Huang1* 1Department of Plant Pathology, College of Agriculture and Natural Resources, National Chung-Hsing University, Taichung 40227, Taiwan 2Section of Biotechnology, Seed Improvement and Propagation Station, Hsinshe, Taichung 42642, Taiwan 3Plant Pathology Division, Agricultural Research Institute, Wufeng, Taichung 41362, Taiwan *Corresponding author: Jenn-Wen Huang, Department of Plant Pathology, National Chung-Hsing University, Taichung, Taiwan, 40227, Tel: 8864228403569; E-mail: [email protected] Received date: March 18, 2016; Accessed date: March 29, 2016; Published date: March 31, 2016 Copyright: © 2016 Chen PS, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Abstract Penicillium digitatum causes green mold decay on citrus fruit, resulting in severe economic losses to citrus growers and packers worldwide. The present study is to evaluate the control of citrus green mold by volatiles produced by Enterobacter cloacae. An E. cloacae strain isolated from plant rhizospheres was able to produce three volatile organic compounds, which were identified as butyl acetate, phenylethyl alcohol, and 4,5-dimethyl-1-hexene by GC/MS chromatography. The volatile compounds produced by E. cloacae inhibited conidial germination and hyphal elongation of P. digitatum and reduced green mold severity. E. cloacae cultured at temperatures ranging from 16°C to 28°C, at pH values ≤6, or in a substrate carrier (sphagnum moss, vermiculite, or perlite) provided superior control against P. digitatum. A laboratory formulation using E. cloacae and perlite protected citrus fruit from green mold up to 22 days and its effectiveness outperformed fungicide application at room temperature (~25°C). The results implicate practical application of E. cloacae as a biofumigant for controlling citrus postharvest decay caused by P. digitatum. Significantly, the study provides a model for future research on how to formulate an effective biocontrol agent for disease management. Keywords: Biocontrol; Biofumigant; Fungi; Gas-producing metabolites and inorganic volatile substances such as ammonia, all of bacterium; Postharvest disease; Orange which might contribute to biocontrol activity [19,24]. Enterobacter cloacae has been shown to inhibit Pythium ultimum sporangium Introduction germination and damping-off by competing for fatty acids [25]. Biological control using volatile compounds-producing Green mold, caused by Penicillium digitatum (Pers.: Fr.) Sacc., is a microorganisms has been reported with increasing success on the noxious postharvest disease of citrus. Penicillium digitatum is an various fruit crops and diseases [26-34]. However, little is known about opportunistic pathogen that resides on healthy citrus fruit and attacks volatile compounds produced by E. cloacae. The objectives of this citrus fruit through injuries caused by rough handling during study were to examine whether or not E. cloacae will emit volatile harvesting, transportation and storage. Infection of P. digitatum in compounds and to assess their antimicrobial activity against P. citrus often results in tissue maceration and fruit decay. Economic digitatum and control of green mold of citrus fruit in storage. losses caused by green mold decay could be enormous for citrus growers and packers worldwide. Application of fungicides, such as Materials and Methods imazalil and thiabendazole, is a common practice to control P. digitatum induced fruit decay in the packinghouse [1-4]. However, repeated use of toxic chemical compounds could induce the emergence Biological materials and culturing conditions of fungicide resistant strains of the pathogen and also could increase The P-51 and DOB-2 strains of P. digitatum (Pers. Fr.) Sacc. were human health risks. single spore isolated from diseased orange fruit grown in Fongyuan To lessen the above-mentioned problems, the use of biological and Dalin (Taiwan), respectively. DOB-2 strain has been characterized control is a promising alternative. Various microorganisms, including elsewhere [35]. The affected fruit were incubated in plastic boxes until Bacillus subtilis, B. pumilus, Burkholderia cepacia, Pseudomonas conidia were produced. Conidia were picked and transferred onto 2% syringae, P. glathei, Pantoea agglomerans, Candida famata, C. sucrose water agar. Fungal strains were cultured on potato dextrose oleophila, Trichoderma viride, and Myrothecium roridum have been agar (PDA; Difco) in a growth chamber with 12 h daily illumination at shown to have fungistatic or fungicidal effects to P. digitatum and have 25°C to 26°C. Fungal strains were identified as P. digitatum based on been explored, with some success, for reducing postharvest green mold the distinct characteristics of conidia and colony morphologies formed decay in citrus [5-15]. on malt extract agar (MEA), Czapek yeast autolysate agar (CYA), and glycerol nitrate agar (GN25N) as described by Pitt [36]. The identity of Enterobacter cloacae is a rod-shaped, gram-negative bacterium P. digitatum was further confirmed by sequence analysis of the 5.8S commonly found in plants and has been shown to control a wide range ribosomal internal transcribed spacers (ITS). of plant pathogens [16-22] and insect pests [23]. Strains of E. cloacae have been known to produce hydroxamate siderophore, non-volatile J Plant Pathol Microbiol Volume 7 • Issue 3 • 1000339 ISSN:2157-7471 JPPM, an open access journal Citation: Chen PS, Peng YH, Chung WC, Chung KR, Huang HC, et al. (2016) Inhibition of Penicillium digitatum and Citrus Green Mold by Volatile Compounds Produced by Enterobacter cloacae. J Plant Pathol Microbiol 7: 339. doi:10.4172/2157-7471.1000339 Page 2 of 8 Bacterial strains were single-colony isolated from rhizospheres of SUPELCO) fibers. After 15 min, the HS-SPME device was removed Yardlong bean (Vigna sesquipedalis (L.) Fruwith). Bacterial cells were and inserted into the injector (200°C) of a gas chromatograph (GC)- grown on King’s medium B (KB) plate at 30°C for 2 days, harvested by mass spectrophotometer (MS) equipped with a Model CP-3800 GC low speed centrifugation (4000× rpm), suspended in 20% glycerol and a Saturn 2000 MS (Varian, USA) connecting to an electron capture water, and stored at -80°C. Bacterial strains were identified as E. detector (Model 902B, ECD, splitless mode). Nitrogen was used as a cloacae by sequence analysis of an rpoB gene encoding a RNA gas carrier and flowed at 0.2 mL/min through a VF-5MS capillary polymerase β-subunit. column (30.0 m × 0.25 mm ID, 0.25 μm film thickness, Agilent, Santa Clara, CA, USA). Analytical temperatures modified from Kai et al., Identification of and fungal bacterial strains [41] were set as following: Initial column temperature at 40°C for 2 min, followed by a ramp of 5°C/min up to 170°C with a final hold for 1 Microbial genomic DNA was extracted with phenol/chloroform min at 170°C. KB medium without bacteria was used as controls. The partition and precipitated with isopropanol. Fungal 5.8S rDNA was identities of volatile compounds were verified by comparing them with amplified by PCR with the primers ITS1 (5’- chemical databases deposited in the GC-mass spectra (GC-MS) library TCCGTAGGTGAACCTGCGG-3’) and ITS4 (5’- (Saturn 2000, USA). Phenylethyl alcohol (PEA) and butyl acetate (BA) TCCTCCGCTTATTGATATGC-3’) as described by White and were purchased from Sigma-Aldrich (St. Luis, MO, USA). The toxicity colleagues [37]. Bacterial DNA fragment was amplified with colony of commercially available chemicals individually or in combination on PCR as described by Fukui and Sawabe [38] using a PCR Master Mix the growth of P. digitatum also was assessed by modified plate-to-plate kit (GeneMark, Taichung, Taiwan). Bacterial rpoB gene was amplified assays by placing a test compound onto a filter paper disc (0.5 cm in by PCR with the primers CM7 (5’- diameter). AACCAGTTCCGCGTTGGCCTGG-3’) and CM31b (5’- CCTGAACAACACGCTCGGA-3’) as described by Mollet and Fungal inoculation and assays for disease severity colleagues [39]. PCR fragments were directly sequenced. Sequences were searched against the databases at the National Center for Fruit of ‘Gold Seal’ orange (Citrus sinensis Osbeck), showing no Biotechnology Information (NCBI) using the BLAST network service visible lesions or injuries, were collected from an orchard in Gukeng to determine the similarity of the amplified fragments. Sequence data (Yunlin county, Taiwan) and used throughout the experiment. Citrus reported in this article have been deposited in the EMBL/GenBank fruit stored at 11°C for 5 to 7 days were washed by immersing in Data Libraries under accession numbers AJ543728 (E006 rpoB) and distilled water, surface sterilized with 70% alcohol, and dried in a AJ543682 (E010 rpoB). sterile hood. Conidia of P. digitatum were harvested by flooding with sterile water containing 0.01% Tween 20 and the concentration was Volatile antimicrobial assays adjusted to 104 conidia per milliliter with the use of a haemocytometer. Orange fruit were wounded with 2 mm long by 1 mm wide tips (10) In-vitro volatile antimicrobial activity was assessed using a plate-to- mounted on a steel rode as described [30] prior to inoculation. The plate method as described [27,40]. Bacterial cells grown on KB agar at wounded fruit were immersed in conidial suspensions or water for 10 24°C for 24 h were used for antifungal activity assays. Petri dish lips s, dried, and placed in plastic boxes (Lock&Lock, HanaCobi, Korea) were removed and the plate was turned upside down and attached onto for lesion development with or without E.