An efficient protocol for isolating melanised

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(rocks, plants, and animals) and modes of life (sapro- phytes, plant and animal mutualists, and plant pathogens) [1–4]. Recently, new members of the Chaeto-.
Environment  Health  Techniques 98

Mario X. Ruiz-González et al.

Method Paper An efficient protocol for isolating melanised chaetothyrialean anamorphic fungi associated with plant-ants Mario X. Ruiz-González1, 2, 3, Jérémie Lauth4, Céline Leroy5, Alain Jauneau6, Hervé Gryta1, 2, Patricia Jargeat1, 2, Alain Dejean5, 7 and Jérôme Orivel5 1

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Université de Toulouse, UPS, UMR5174 EDB (Laboratoire Evolution et Diversité Biologique), Toulouse, France CNRS, UMR5174 EDB (Laboratoire Evolution et Diversité Biologique), Toulouse, France Abiotic Stress Department, Integrative and Systems Biology Group. Instituto de Biología Molecular y Celular de Plantas, C.S.I.C.–U.P.V. C/Ingeniero Fausto Elio s/n., Valencia, Spain Université des Antilles et de la Guyane; UMR Ecologie des forêts de Guyane, Campus Agronomique, Kourou, France CNRS, UMR Ecologie des Forêts de Guyane, Campus Agronomique, Kourou, France CNRS, IFR40 Pôle de Biotechnologie Végétale, Plateforme d’imagerie cellulaire de Toulouse, Auzeville, Castanet-Tolosan, France Université de Toulouse, ECOLAB, Toulouse, France

Because of their ecological characteristics, slow growth rates and the presence of contaminants, Chaetothyriales fungi associated with structures built by tropical plant-ants can be difficult to isolate with standard procedures. Here, we describe an easy-to-use protocol for obtaining pure cultures by using cotton as a first substrate. We have further found by means of fluorescent stains that nuclei concentrate either in young hyphae or in the tips of the hyphae. Keywords: Allomerus ants / Black fungi / Chaetothyriales / Fluorescent staining / Isolation method Received: October 28, 2011; accepted: December 3, 2011 DOI 10.1002/jobm.201100539

Black fungi are a polyphyletic group of fungi (Chaetothyriales and Dothideales) with remarkable ecological adaptations to extreme conditions (high and low temperatures, drought, osmotic stress, irradiation), substrates (rocks, plants, and animals) and modes of life (saprophytes, plant and animal mutualists, and plant pathogens) [1–4]. Recently, new members of the Chaetothyriales group have been found associated with tropical arboreal ants [5–8]. Standard procedures for isolating fungi involve washing and diluting the samples and then culturing many plates at a time that must then be monitored on a daily basis. The isolation of some of these fungi by using standard procedures can, however, become a challenging task due to two main constraints. First, sometimes the symbionts do not grow despite

Correspondence: Dr. M. X. Ruiz-González, Abiotic Stress Department, Integrative and Systems Biology Group. Instituto de Biología Molecular y Celular de Plantas, C.S.I.C. – U.P.V. C/Ingeniero Fausto Elio s/n. 46022 Valencia, Spain E-mail: [email protected] Phone: þ34 963879934 Fax: þ34 963877859 ß 2012 WILEYVCH Verlag GmbH & Co. KGaA,Weinheim

repeated attempts to grow them [8]. Second, many black fungi are slow-growing organisms, and their cultures can be rapidly overwhelmed by the many contaminant fungi present as spores in the original samples [7]. Here we describe a general method to easily grow and isolate various saprophytic anamorphic fungi including the melanised fungi associated with ants. We focused on the fungal symbiont, Trimmatostroma sp., found on the galleries built by Allomerus plant-ants on the stems of their host plants (Fig. 1A, B). These ants manipulate the fungal mycelium to strengthen their galleries which they later use to ambush for prey [9]. We first qualitatively investigated whether the hyphae present in young and old galleries could grow or if they were dead. The hyphae were placed on glass slides and the nuclei stained with two fluorescent stains, diaminophenylindole (DAPI), and bisbenzimide (Hoechst 33342) at 1 mg/ml. DNA stained with either DAPI or Hoechst emits a blue fluorescence (461 nm) at around 350 nm excitation. Observations were made with an inverted wide-field fluorescence microscope (Leica, Rueil-Malmaison, France) using a 40 long-distance objective lens with

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J. Basic Microbiol. 2013, 53, 98–100

Isolation of black fungi associated with plant-ants

Figure 1. Fungal symbiont. (A) The anamorphic fungi growing in the ants galleries. (B) The hyphae present at the entrance of a domatium. (C) A sample of the symbiotic fungus growing in the cotton with many fresh hyphae expanding radially (arrows). (D) A detail of a hypha removed from the cotton and cultured in a YMG agar plate; the arrow points to the newly grown part of the hypha. (E) Hyphae growing from the fungal plug pasted by the ants to repair a wounded domatium. Scale bars: (A, C, E) 1 mm; (B) 100 µm; (D) 20 µm.

a numerical aperture of 0.55. We used the excitation filters 340–380 and emission filters LP425. Images were acquired with a CCD camera (Color Coolview, Photonic Science, Robertsbridge, UK). We analyzed the nuclei localized in the first 100 mm behind the hyphal tips and the density of the nuclei in the distal hyphae with Image Pro Plus software (Media Cybernetics, Silver Spring, MD, USA). The staining of fresh trap samples with two fluorescent stains revealed an unbalanced distribution of nuclei across hyphae (Fig. 2). The nuclei were most likely to be present in young rather than in old hyphae, while most old cells were devoid of nuclei. Such a concentration of nuclei in young cells can explain, at least partly, the isolation failures experienced with several of these fungi. Consequently, the galleries of Allomerus ants were sampled from areas under construction where the associated fungus is expected to be prospering and contaminant spores are less likely to be present. The samples were immersed in sterile, distilled water, broken into smaller pieces and washed to rid them of as many exogenous and highly prevalent spores as possible. As this fungus grows on a plant substrate, we pre-cultured small pieces of loose mycelium by placing them in wet cotton where any fast-growing contaminants were easily detected. The major contaminants that sometimes grew on the cotton associated to the fungal symbiont were ß 2012 WILEYVCH Verlag GmbH & Co. KGaA,Weinheim

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Figure 2. Hoechst fluorescent stain. (A, C, E, G) Hyphae under normal light. (B, D, F, H) The same hyphae under UV light. Not all hyphae have nuclei. (A, B, E, F) Only less melanised hyphae show nuclei. In many samples, the nuclei were found only in the young parts of pure mycelium cultured in plates. (C, D) Nuclei migrate to cells in division (the tips of the hyphae) in new parts of the trap where the mycelium is expanding. White and black arrows point to hyphae with stained nuclei. Scale bars: 20 mm.

identified through the sequencing of the ITS region of their nuclear rDNA as Botryosphaeria sp., Hypocrea sp., Ophiocordyceps sp., Penicillium sp., and Trichoderma aureoviride. All of the latter genera, except Ophiocordyceps sp., are known because of their cellulose-degrading activity. After 5 to 20 days under these conditions at laboratory temperature, the melanised hyphae expanded, leaving behind the old mycelia with all of the persistent contaminant spores attached to their walls but unable to sprout in the cotton (Fig. 1C, D). The pure cultures were obtained by harvesting between 15 and 30 new hyphae with sterile forceps. These hyphae were washed in a drop of sterile, distilled water and plated on solid YMG medium (4 g yeast extract, 10 g malt extract, 4 g glucose, and 15 g of agar in 1 l of distilled water) [10] including a cocktail of five antibiotics (100 mg/l Na-ampicillin, 120 mg/l SO4-streptomycin, 15 mg/l tetracycline, 30 mg/l chloramphenicol, and 30 mg/l SO4-kanamycin). The plates were kept at 25 °C for 6 to 20 days and monitored daily to assess growth. All potential contaminants were immediately removed, and, if necessary, growing hyphae were transferred to new plates. With this protocol, we have successfully isolated pure mycelia from the main symbiont present in 213 colonies of two Allomerus ant species from different areas in French Guiana with a success rate of 77%. A modification to this protocol results from the ability of the ants to repair damages to the plant structures with the fungus where they nest. We artificially damaged plants in the field by cutting out a circular piece of ca.

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2 mm in diameter in the wall of plant leaf-domatia with a scalpel. The wound was entirely filled up by the ants with pieces of mycelium within 1 day if the domatium sheltered the queens or within four days otherwise with a mixture of plant trichomes and mycelium. We then collected these fungal plugs with sterile forceps and stored them in sterile Eppendorf tubes. Thereafter, the isolation procedure was the same as described above. This technique is particularly useful in isolating the fungal symbiont associated with A. octoarticulatus because young hyphae are scarce even in galleries under construction. The protocol described here introduces the use of wet cotton as a selective medium to stimulate the growth of the saprophytic anamorphic melanised fungi while minimising the growth of contaminants. This protocol highly enhances the isolation success of a few hundreds of symbiotic fungi in less than 1 month.

Conflict of interest statement The authors have no competing financial interests.

References [1] de Hoog, G.S., Zalar, P., Urzì, C., de Leo, F., Yurlova, N.A., Sterflinger, K., et al. 1999, Relationships of dothideaceous black yeasts and meristematic fungi base don 5.8.S and ITS2 rDNA sequence comparison. Stud. Mycol., 43, 31–37. [2] Gueidan, C., 2008, Chaetothyriales. Version 29 January 2008 (under construction). http://tolweb.org/Chaetothyriales/29305/2008.01.29 in The Tree of Life Web Project, http://tolweb.org/. [3] Gueidan, C., Ruibal Villaseñor, C., de Hoog, G.S., Gorbushina, A.A., et al., 2008, A rock-inhabiting ancestor for mutualistic and pathogen-rich fungal lineages. Stud. Mycol., 61, 111–119. [4] Hughes, S.J., 1976, Sooty moulds. Mycologia, 68, 693–820.

Acknowledgements

[5] Defossez, E., Selosse, M.A., Dubois, M.P., Mondolot, L., et al., 2009, Ants-plants and fungi: a new threeway symbiosis. New Phytol., 182, 942–949.

We are grateful to Nathalie Séjalon-Delmas for insightful advice and to A. Yockey-Dejean for editing the manuscript. We would also like to thank the Laboratoire Environnement de Petit Saut and the Nouragues Research Station for furnishing logistical help. Financial support was provided by a research program of the French Agence Nationale de la Recherche (research agreement n°ANR-06-JCJC-0109-01), by the ESF-EUROCORES/TECT/BIOCONTRACT program (06-TECT-FP-007), by a fellowship from the Fondation pour la Recherche sur la Biodiversité (research agreement n°AAP-IN-2009-050), by the Programme Interface Physique, Chimie, Biologie of the French Centre National de la Recherche Scientifique (CNRS) and by a Nouragues research grant from the CNRS. J.L. was supported by a doctoral fellowship provided by the FSE (Fond Social Européen).

[6] Mayer, V.E., Voglmayr, H., 2009, Mycelial carton galleries of Azteca brevis (Formicidae) as a multi-species network. P.R. Soc. London, 276, 3265–3273.

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[7] Ruiz-González, M.X., Malé, P.-J.G., Leroy, C., Dejean, A., et al., 2011, Specific, non-nutritional association between an ascomycete fungus and Allomerus plant-ants. Biol. Lett., 7, 475–479. [8] Voglmayr, H., Mayer, V., Maschwitz, U., Moog, J., et al., 2011, The diversity of ant-associated black yeasts: insights into a newly discovered world of symbiotic interactions. Fungal Biol. 115, 1077–1091. DOI: 10.1016/ j.funbio.2010.11.006. [9] Dejean, A., Solano, P.J., Ayroles, J., Corbara, B., Orivel, J., 2005, Arboreal ants build traps to capture prey. Nature, 434, 973. [10] Rao, P.S., Niederpruem, D.J., 1969, Carbohydrate metabolism during morphogenesis of Coprinus lagopus (sensu Buller). J. Bacteriol., 100, 1222–1228.

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