New Penicillium and Talaromyces Species from Honey, Pollen and Nests of Stingless Bees
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Antonie van Leeuwenhoek https://doi.org/10.1007/s10482-018-1081-1 ORIGINAL PAPER New Penicillium and Talaromyces species from honey, pollen and nests of stingless bees Renan N. Barbosa . Jadson D. P. Bezerra . Cristina M. Souza-Motta . Jens C. Frisvad . Robert A. Samson . Neiva T. Oliveira . Jos Houbraken Received: 15 January 2018 / Accepted: 31 March 2018 Ó The Author(s) 2018 Abstract Penicillium and Talaromyces species have and six in Talaromyces, including seven new species. a worldwide distribution and are isolated from various These new species were studied in detail using a materials and hosts, including insects and their polyphasic approach combining phenotypic, molecu- substrates. The aim of this study was to characterize lar and extrolite data. The four new Penicillium the Penicillium and Talaromyces species obtained species belong to sections Sclerotiora (Penicillium during a survey of honey, pollen and the inside of nests fernandesiae sp. nov., Penicillium mellis sp. nov., of Melipona scutellaris. A total of 100 isolates were Penicillium meliponae sp. nov.) and Gracilenta obtained during the survey and 82% of those strains (Penicillium apimei sp. nov.) and the three new belonged to Penicillium and 18% to Talaromyces. Talaromyces species to sections Helici (Talaromyces Identification of these isolates was performed based on pigmentosus sp. nov.), Talaromyces (Talaromyces phenotypic characters and b-tubulin and ITS sequenc- mycothecae sp. nov.) and Trachyspermi (Talaromyces ing. Twenty-one species were identified in Penicillium brasiliensis sp. nov.). The invalidly described species Penicillium echinulonalgiovense sp. nov. was also isolated during the survey and this species is validated Electronic supplementary material The online version of here. this article (https://doi.org/10.1007/s10482-018-1081-1) con- tains supplementary material, which is available to authorized Keywords Aspergillaceae users. 8 new taxa Á Á Fungal ecology Á Polyphasic approach Á Taxonomy Á R. N. Barbosa Á R. A. Samson Á J. Houbraken (&) Trichocomaceae Westerdijk Fungal Biodiversity Institute, Uppsalalaan 8, 3584 CT Utrecht, The Netherlands e-mail: [email protected] R. N. Barbosa Á J. D. P. Bezerra Á C. M. Souza-Motta Á Introduction N. T. Oliveira Departamento de Micologia Prof. Chaves Batista, Stingless bees comprise a diverse group of highly Universidade Federal de Pernambuco, Av. Prof. Moraes Rego, s/n, Centro de Biocieˆncias, Cidade Universita´ria, eusocial insects occurring throughout the tropical CEP: 50670-901 Recife, PE, Brazil regions in the world. They are important honey producers and pollinators of several plants (Ramı´rez J. C. Frisvad et al. 2010; Brown and Oliveira 2014). An example of Department of Biotechnology and Biomedicine, Technical University of Denmark, 2800 Kongens Lyngby, a stingless bee species is Melipona scutellaris Denmark 123 Antonie van Leeuwenhoek (Hymenoptera: Apidae: Meliponini), an indigenous 2014). The genera Aspergillus, Penicillium, Mon- species occurring in the North-eastern part of Brazil ascus and Mucor are commonly associated with bees and considered to be one of the first species to be or their products (Egorova 1971; Gilliam et al. 1989; domesticated in the Americas (Kerr 1996; Silva et al. Eltz et al. 2002;Ferrazetal.2008; Barbosa et al. 2013). In this part of Brazil, M. scutellaris is the main 2017). Most fungi associated with bees and nests bee species in meliponiculture (stingless beekeeping). have a saprophytic lifestyle, but fungi can also have a Meliponiculture in the rural areas is a sustainable mutualistic relationship with bees (Menezes et al. activity and the honey from these bees is widely 2015). On the other hand, fungi are also reported to be appreciated as a food source. The composition of the pathogenic to many bee species and cause serious honey of the stingless bees differs from that of bees of problems in honey bee (Apis mellifera) brood. the genus Apis (honey bees) (Vit et al. 2004). The Aspergillus flavus is the primary species responsible honey of stingless bees contains, in comparison to for stonebrood, a disease where dead and mummified honey of honey bees, a more complex mixture of larvae are present in the brood cells, but also other carbohydrates and contains other types of organic Aspergilli such as Aspergillus fumigatus and Asper- acids, proteins, minerals, vitamins, pollen grains and gillus niger are reported as aetiological agents of this enzymes (Almeida-Muradian et al. 2013). Recently, disease (Gilliam and Vandenberg 1988; Foley et al. the interest in honey produced by stingless bees 2014;Lopesetal.2015;Sarwar2016). Though it is increased. Besides being a food source, also several generally accepted that infection only occurs in other functionalities are linked to this type of honey, weakened colonies, the specific conditions predis- such as antiseptic, antimicrobial, anti-inflammatory posing the onset of disease are not fully understood and wound-healing properties (Silva et al. 2013; Rao (Shoreit and Bagy 1995). et al. 2016). Fungi play an important role in many ecosystems; Penicillium and Talaromyces are fungal genera however, only a limited number of studies dealt with classified in the order Eurotiales. In the dual nomen- the association between stingless bees in Brazil and clature era (pre 2012), Talaromyces was known as a filamentous fungi (e.g. Oliveira and Morato 2000; sexual genus related to Penicillium and other genera. Ferraz et al. 2008;Go´is et al. 2010)andyeasts(e.g. In the last decade, the genera Talaromyces and Teixeira et al. 2003;Rosaetal.2003;Danieletal. Penicillium were re-defined due to new taxonomic 2013; Barbosa et al. 2016). In the present study, we insights and the introduction of single name nomen- analysed three different substrates associated with M. clature (Houbraken and Samson 2011; Samson et al. scutellaris bees: bee pollen, nests and honey. In 2011; McNeill et al. 2012; Yilmaz et al. 2014). nature, the M. scutellaris bee nests are mainly located Currently, Penicillium and Talaromyces are separate in tree hollows, and they are kept by beekeepers in genera that contain both sexual and asexual species. artificial wooden hives. The bees use cerumen (a Visagie et al. (2014) accepted 354 Penicillium species mixture of wax and floral resins) for the construction and Yilmaz et al. (2014)88Talaromyces species, and of their nests and this material is also used inside these numbers are rapidly increasing (Houbraken et al. nests in storage pots, brood cells and entrance 2016a). Several of the new species that are being openings (Cortopassi-Laurino et al. 2006;Pianaro discovered are found during ecology and biodiversity et al. 2007). The floral pollen is collected, packed into studies of specific substrates or habitats (Houbraken pollen pellets, and subsequently stored inside the nest et al. 2016a). Describing new species from poorly by worker bees. This stored pollen is referred to as explored substrates and habitats, like those related to ‘bee bread’. The pollen spectrum has been studied in meliponiculture, will add to our knowledge on biodi- the past to get insight in the bee colony’s food versity. With this information, future studies will also requirements, pollinating functions and the plant be able to better understand the ecology of fungi in species visited by the bees (Cortopassi-Laurino et al. these type of environments. 2007). Fungi, such as Penicillium and Talaromyces,can In this paper, we focus on the identification of have a strong association to a specific substrate Penicillium and Talaromyces species isolated from (Peterson et al. 2003; Kobayashi et al. 2008; Visagie three different substrates (bee pollen, nests and honey) 2012,Lietal.2012;Riveraetal.2012;Yilmazetal. associated to M. scutellaris in the Atlantic Rainforest 123 Antonie van Leeuwenhoek in Brazil. Phenotypic characters, combined with ITS Petri dishes were incubated at 25 °C for 7 days and and partial b-tubulin (BenA) sequences were applied additional CYA and MEA plates were incubated at 15, to identify the isolates. Four Penicillium and three 30 and 37 °C. Media preparation, inoculation and Talaromyces species could not be assigned to any incubation were performed as described in Samson known species and are described here as new. Those et al. (2010). Colony diameters were measured after species are described using a polyphasic approach 7 days of incubation and colony characteristics including morphology, ITS, BenA, calmodulin (CaM) recorded (e.g. presence of soluble pigments, exudates, and/or RNA polymerase II second largest subunit obverse and reverse colony colours, colour of (RPB2) sequences and extrolites profiles. mycelium). Microscopic observations of the asexual stage were made from colonies grown on MEA. The presence of a sexual stage was determined from Materials and methods cultures incubated on CYA, MEA and OA for at least 40 days at 25 °C. Lactic acid (60%) was used as Strains mounting fluid and 96% ethanol was used to remove excess conidia. A Zeiss Stereo Discovery V20 Six collections were performed between January and dissecting microscope and a Zeiss AX10 Imager A2 June 2014 in the tropical forest in Pernambuco, Brazil light microscope, both equipped with Nikon DS-Ri2 (8°703000S, 34°5203000W and 8°403600S, 34°5703400W). cameras, were used to capture digital images using the During each collection, four hives were sampled. software NIS-Elements D v4.50. The size, shape and Stingless bees process honey and pollen in cerumen pigmentation of microscopic features were recorded. pots. Per hive, four samples of the honey pots and four of the pollen pots were collected and combined, DNA isolation, PCR and sequencing resulting in one mixed sample of each substrate. In the same hives, also the surface of brood cells and the Genomic DNA extractions were made from 7 days old pollen and honey pots were sampled using sterile colonies grown on MEA using the UltraClean Micro- cotton swabs (in total 48 swabs). Analysis of the bial DNA kit (MoBio Laboratories, Solana Beach, samples was performed using dichloran 18% glycerol CA, USA).