J. Gen. Appl. Microbiol., 62, 233–239 (2016) doi 10.2323/jgam.2016.04.004 „2016 Applied Microbiology, Molecular and Cellular Biosciences Research Foundation Full Paper Antifungal activity of rhamnolipids against dimorphic fungi (Received March 22, 2016; Accepted April 13, 2016; J-STAGE Advance publication date: September 21, 2016) Ruyi Sha1,2 and Qin Meng2,* 1 School of Biological and Chemical Engineering/School of Light Industry, Zhejiang University of Science and Technology, 318 Liuhe Road, Hangzhou, Zhejiang, 310023, P.R. China 2 Department of Chemical and Biological Engineering, Zhejiang University, Hangzhou, 38 Zheda Road, Hangzhou, Zhejiang, 310027, P.R. China Introduction In this paper, rhamnolipids are investigated, for the first time, for their feasibility for inhibiting di- Biocontrol agents of food with high sustainability can morphic fungi. Rhamnolipids were found to effec- be applied either together with chemical pesticides to re- tively inhibit a dimorphic fungus isolated from to- duce the possible development of fungicide resistance, or mato plants which was identified as Mucor directly as agrochemicals to help prevent health and envi- circinelloides according to characterizations by ronmental problems (Ongena and Jacques, 2008; Velho et morphologies as well as 28S rDNA sequences. al., 2011). Rhamnolipids markedly reduced growth of this Rhamnolipids, as biodegradable and low toxic fungus in both the yeast-like form and the biosurfactants produced by P. aeruginosa, are one of the filamentous form. Such an inhibitive effect was most extensively-studied biocontrol agents (Banat et al., similarly obtained with Verticillium dahliae, a rep- 2010; Rufino et al., 2014; Santana-Filho et al., 2014). They resentative member of dimorphic fungi, confirm- show potential applications in the biocontrol of Gram- ing the effectiveness of rhamnolipids in the two positive and Gram-negative bacteria, yeast, and fungi growth forms of dimorphic fungi. Interestingly, (Abalos et al., 2001; Kahraman and Erenler, 2012), and rhamnolipids showed a greater inhibitive function also insects as well (Kim et al., 2011). In particular, in the case of the pathogenic growth mode of di- rhamnolipids can effectively control some plant pathogens morphic fungi, such as the mycelium growth for such as Phytium aphanidermatum, Pythium splendens, M. circinelloides and the yeast-like growth for V. Pythium myriotylum, Phythophthora capsici, Plasmopara dahliae, than their non-pathogenic modes. The use lactucae-radicis and Colletotrichum orbiculare (Kim et of rhamnolipids might greatly reduce the fre- al., 2000; Perneel et al., 2008; Stanghellini and Miller, quently-reported drugresistance to the common 1997). Furthermore, they can induce plants to generate anti-fungal agents by deterring the possible switch defense responses against fungal infections (Varnier et al., between the two modes of dimorphic fungi. Over- 2009). all, rhamnolipids as environment-friendly Synthetic fungicides, such as members of the azole fam- biocontrol agents have a potential use in protect- ily, though of widespread use in controlling food and plant ing plants from dimorphic fungi infections, and pathogens and postharvest diseases, are being used in a could also offer guidance toward future research limited way due to the emergence of fungal resistance over into controlling dimorphic disease infection in hu- long-term use (Makovitzki et al., 2007; Savory et al., mans. 2011). For example, most zygomycota species, including Mucor species, are resistant to most typical antifungal Key Words: antifungal; dimorphism; Mucor agents (Gomez-Lopez et al., 2003; Khan et al., 2009), circinelloides; rhamnolipids; Verticillium dahliae among which M. circinelloides has even shown resistance to broad-spectrum posaconazole and itraconazole (Gomez- *Corresponding author: Qin Meng, Department of Chemical and Biological Engineering, Zhejiang University, Hangzhou, 38 Zheda Road, Hangzhou, Zhejiang, 310027, P.R. China. Tel: +86-571-87953193 Fax: +86-571-87951227 E-mail: [email protected] None of the authors of this manuscript has any financial or personal relationship with other people or organizations that could inappropriately influence their work. 234 SHA and MENG Lopez et al., 2003; Khan et al., 2009). M. circinelloides, electron microscopy (SEM) were used for the observa- like V. dahliae, is a dimorphic microorganism which can tion of the fungal morphologies. Light microscopy was simply switch between filamentous branching hyphae and routinely used to examine the spore generation and myc- multipolar budding yeasts by adapting to changing envi- elium. For an observation with high resolution, samples ronmental cues (Nadal et al., 2008). The pathogen mode were fixed by immersion in 5% (v/v) glutaraldehyde for of dimorphic fungi occurs either in mycelia form 24 h at 4∞C and post-fixed with 1% (w/v) osmium tetrox- (Fingeroth et al., 1994; Mahlert et al., 2006), yeast-like ide for 2 h at 4∞C before assay with SEM. The fixed sam- growth (Neumann and Dobinson, 2003), or switching be- ples were dehydrated in a graded acetone series and cov- tween the two forms (Nemecek et al., 2006). Usually, ered with gold prior to examination in a Scanning Elec- chemically synthesized fungicide has exhibited differen- tron Microscope (TM-1000, Hitachi, Tokyo). tial inhibitions against the two growth modes, as well as a Molecular testing and phylogenetic analysis. DNA ex- differential resistance, of the dimorphic fungi (Zwiers and traction and PCR amplification of the isolated strain were De Waard, 2000). conducted in Guangdong Detection Center of Microbiol- Besides the known antifungal activities, however, ogy (Guangzhou, China) following previous protocols rhamnolipids have never been investigated for their anti- (Sambrook et al., 2000), and the forward primer NL1 (5 - fungal activities against the dimorphic growth of fungi. ¢ GCATATCAATAAGCGGA GGAAAAG-3 ) was used. There is an obvious need to search for alternative ¢ The amplified fragment was sequenced by Invitrogen Bio- biocontrol agents that are nontoxic to animals and technology Co., Ltd. (Shanghai, China). Its 28S rDNA nonpolluting environmentally to control both the yeast- sequence having a length of 660 bp was then submitted like and filamentous growth forms of dimorphic fungi for for a BLAST search at the GenBank database (National agricultural applications. The present study, presents, for Center for Biotechnology Information, Washington, DC, the first time, an examination of the possibilities of using http://www.ncbi.nlm.nih.gov). This sequence was subse- rhamnolipids to control dimorphic plant pathogens by con- quently assigned the accession number JF823517 by sidering an isolated dimorphic fungus from a severely in- GenBank. fected tomato field, and another devastating fungus V. The above sequence was aligned using the CLUSTALX dahliae ATCC 7611. program (version 2) (Yoon et al., 2011) and adjusted us- ing the BioEdit program (version 7.0.9), while ambigu- Materials and Methods ous bases and alignment gaps were not taken into consid- eration. The obtained sequence was further analyzed by Fungal strain isolate and cultivation. Samples of patho- the MEGA4 software. The evolutionary distances were gen-infected tomato plants from a severely contaminated determined according to the Kimura two-parameter model tomato field (Wenzhou, China) were collected in a steri- using bootstrap values based on 1000 replications. The lized polythene bag and delivered to the laboratory. The similarity values were calculated and the neighbor-join- tomato plants were swabbed in 75% ethanol for 2 min, ing tree was generated using the same software. rinsed several times with distilled water and then blotted dry with sterile filter papers. Lesions were aseptically cut Preparation of rhamnolipids. Pseudomonas aeruginosa and plated on sterile potato dextrose agar (PDA) in 9 cm strain ZJU211 (CCTCC M209237) was isolated from heav- Petri Plates for incubation at 28∞C. Two days later, the ily oil-contaminated soil as described previously (Tang et mycelium was transferred onto fresh agar plates (PDA, al., 2007) and applied to produce rhamnolipids using colza Czapek yeast extract agar and Czapek’s agar) to determine oil (6%, v/v) as the sole carbon source. 60 mL of medium the most appropriate agar plates at 28∞C. According to (Tang et al., 2007) contained in a 250 mL Erlenmeyer flask morphological observations, this fungus was found to grow was inoculated with 3% (v/v) of bacteria, then cultured in fast and well on PDA and Czapek yeast extract agar, but a reciprocating shaker (ZHWY-1102C, ZHICHENG, not on Czapek’s agar. Hence, the convenient and effective Shanghai, China) at 180 rpm at 37∞C. After incubation for PDA was used for subculture every 7 days. During the 2 days, the complete culture volume was used as inoculums investigation of spore generation using a light microscope for a 10-L bioreactor (GUJS-10C, Eastbiotech, Zhenjiang, (XSS-2A, Nanjing, China), it was found that spore gen- China) with a working volume of 5 L which was aerated eration was of quite different morphology in shaking tubes by a sparger at 5 L air min–1 and stirred at a speed of 300 than that in statically cultured tubes. Spores generated into rpm at 37∞C. At the end of fermentation (96 h), the cul- germ tubes in a shaking (aerobic) culture following the ture broth was heated to 121∞C for 20 min, centrifuged at majority of mycelia, while showing a yeast-like mode of 5000 rpm for 20 min to obtain a cell-free culture