Fungal Biology 123 (2019) 364e372 Contents lists available at ScienceDirect Fungal Biology journal homepage: www.elsevier.com/locate/funbio Natural occurrence in Argentina of a new fungal pathogen of cockroaches, Metarhizium argentinense sp. nov. * Alejandra C. Gutierrez a, , Andreas Leclerque b, Romina G. Manfrino a, Christian Luz c, Walter A.O. Ferrari a, Jorge Barneche a, Juan J. García a, d, Claudia C. Lopez Lastra a a Centro de Estudios Parasitologicos y de Vectores e CEPAVE (CONICET, Consejo Nacional de Investigaciones Científicas; UNLP, Universidad Nacional de La Plata), La Plata, 1900, Buenos Aires, Argentina b Institute for Microbiology and Biochemistry, Geisenheim University, Geisenheim, D-65366, Germany c Instituto de Patologia Tropical e Saúde Pública (IPTSP), Universidade Federal de Goias (UFG), Goiania,^ 74605-050, Goias, Brazil d Comision de Investigaciones Científicas (CIC), La Plata, 1900, Buenos Aires, Argentina article info abstract Article history: The aim of this study was to search for entomopathogenic fungi that infect wild cockroaches in forest Received 18 October 2017 ecosystems in two protected natural areas of Argentina. Two isolates of Metarhizium argentinense were Received in revised form obtained and identified from wild cockroaches (Blaberidae: Epilamprinae) through the use of morpho- 16 January 2019 logical characteristics and molecular phylogenetic analyses. This novel species was found in Argentina Accepted 12 February 2019 and is a member of the Metarhizium flavoviride species complex. Phylogenetic analyses, based on Available online 21 February 2019 sequence similarity analysis using internal transcribed spacer (ITS) and a set of four protein-coding Corresponding Editor: Gustavo Henrique marker sequences (EF1A, RPB1, RPB2 and BTUB), supported the status of this fungus as a new species. Goldman In addition, we tested the biological activity of the new species through assays against Blattella germanica nymphs and found that the two evaluated isolates were pathogenic. However, isolate CEP424 was more Keywords: virulent and caused a confirmed mortality of 76 % with a median lethal time of 7.2 d. This study reports Blattodea the southernmost worldwide location of a Metarhizium species that infects cockroaches and will help Clavicipitaceae expand the knowledge of the biodiversity of pathogenic fungi of Argentine cockroaches. DNA sequencing © 2019 British Mycological Society. Published by Elsevier Ltd. All rights reserved. Entomopathogenic fungi Hypocreales 1. Introduction Hamilton, 1990). The control of these urban pests in large cities is difficult due to prevalent insecticide (pyrethroids, carbamates and Approximately 4000 cockroach species have been described others) resistance (Zhu et al., 2016; Wu and Appel, 2017). In addi- worldwide, and about 1 % of them are considered pests (Cochran, tion, several studies have shown that the exclusive use of in- 2003). Urban cockroaches act as mechanical vectors of pathogenic secticides does not promote satisfactory long-term control when microorganisms for humans and contribute to allergic processes compared to an integrated pest management approach (Wang and and exacerbation of asthma (Pai et al., 2004; Fu et al., 2009; Pomes Bennett, 2010). et al., 2017). There are no significant natural enemies of synan- Natural infection records of Blattodea pathogens and parasites thropic cockroaches in human environments, and this factor con- are scare and fragmented (Roth and Willis, 1960; Suiter, 1997). The tributes to the high levels of home infestations (Schal and few recorded fungal pathogens of cockroaches are Hymenostilbe ventricosa, described on nymphs that attached to the underside of leaves of forest plants in Thailand (Hywel-Jones, 1995); Ophio- cordyceps blattae (Petch), found on cockroaches in Sri Lankan gar- * Corresponding author. Centro de Estudios Parasitologicos y de Vectores e dens (Petch, 1931); Ophiocordyceps blattarioides, described on wild þ CEPAVE (CONICET-UNLP), La Plata, 1900, Buenos Aires, Argentina. Fax: 54 0221 adult cockroaches from Colombia (Sanjuan et al., 2015). Recently, 4232140. E-mail addresses: [email protected], [email protected] Montalva et al., (2016) described the novel species Metarhizium (A.C. Gutierrez), [email protected] (A. Leclerque), manfrino@cepave. blattodeae as a natural fungal pathogen of a sylvatic cockroach in edu.ar (R.G. Manfrino), [email protected] (C. Luz), [email protected] Brazil and evaluated its pathogenicity against Periplaneta americana (W.A.O. Ferrari), [email protected] (J. Barneche), [email protected] nymphs. (J.J. García), [email protected] (C.C. Lopez Lastra). https://doi.org/10.1016/j.funbio.2019.02.005 1878-6146/© 2019 British Mycological Society. Published by Elsevier Ltd. All rights reserved. A.C. Gutierrez et al. / Fungal Biology 123 (2019) 364e372 365 Genus Metarhizium (Metschn.) Sorokin was originally described Emergence of hyphae was monitored daily, and the fungus was based on its anamorphic stage. Liang et al. (1991) were the first to reisolated in fresh culture medium, SDAY 1 % and potato dextrose confirm the relationship between Metarhizium and its sexual form, agar (PDA), without antibiotics to obtain the pure fungal culture. which has been demonstrated by culturing. This genus level rela- tionship between anamorph-teleomorph was supported by 2.2.1. Morphological characterisation phylogenetic studies conducted by Liu et al. (2002) and Huang et al. Fungal species were identified based on macroscopic and (2005). The teleomorphic stages through sexual forms were microscopic features. Macroscopic features included the aspect, included in the Genera Cordyceps or Metacordyceps (Sung et al., colour and mycelium texture of the fungal colonies. Microscopic 2007; Kepler et al., 2012). Phylogenetic analysis of nuclear ribo- characteristics were obtained from material mounted in lactophe- somal RNA operon internal transcriber spacer (ITS) sequences nol/cotton blue (0.01 % w/v) and observed under brightfield optics demonstrated that the Metarhizium anisopliae species complex is microscopy (Olympus CH3). The observed features were the shape monophyletic (Driver et al., 2000). Subsequently, molecular taxo- and size of the conidia, conidiogenous cells and mycelium. These nomic studies of the fungal genus Metarhizium Sorokin (Hypo- characteristics were photographed using a digital camera (Sony creales; Clavicipitaceae) have increased considerably in recent DSCP73). Measurements were based on 50 objects per micro- years. However, species delineation within this genus, as in many structure and were used to calculate the mean, standard error of other entomopathogenic fungal genera, has remained a difficult the mean (SEM) and range (minimum and maximum values), all in task on the basis of morphological characteristics and ITS sequence micrometer (mm). Semi-permanent slides were mounted according data alone (Crous et al., 2005; Rehner and Buckley, 2005; Tsui et al., to Humber (2012a). Fungi were initially identified according to 2006). A more sensitive and robust basis for molecular taxonomic taxonomic keys of Humber (2012a) and for molecular taxonomic studies of Metarhizium fungi was achieved by the introduction studies were used Bischoff et al. (2006, 2009), Kepler et al. (2014) (Bischoff et al., 2006, 2009) and successful application (Kepler et al., and Rehner and Kepler (2017). Conidial germination percentage 2014; Montalva et al., 2016; Rehner and Kepler, 2017) of an addi- was calculated according to Lane et al. (1988). Isolates were pre- tional Multilocus Sequence Analysis (MLSA) scheme. This analysis served by cryopreservation at À20 C and subsequent lyophilisa- utilises partial sequences of four gene markers: translation elon- tion (Humber, 2012b); preserved samples were deposited at the gation factor 1-alpha (EF1A), RNA polymerase II subunit 1 (RPB1), RNA Entomopathogenic Fungal Culture Collection of CEPAVE (La Plata, polymerase II subunit 2 (RPB2) and beta tubulin (BTUB). Argentina) and the USDA-ARS Collection of Entomopathogenic Since previous records of natural infections of cockroaches by Fungal Cultures (ARSEF, Ithaca, New York, USA). entomopathogenic fungi are scarce, the aim of this study was to search for entomopathogenic fungi that infect wild cockroaches in 2.2.2. DNA extraction, PCR amplification and sequencing forest protected natural areas of Argentina. We describe a novel The fungal strains were cultured on potato dextrose agar (PDA) species of Metarhizium and characterise its pathogenicity against medium and incubated at 25 ± 1 C with a 12 h light and 12 h dark the urban pest Blatella germanica. cycle. A small portion of mycelium was harvested with a sterile loop from five-day-old cultures and placed in a 1.5 mL microcentrifuge 2. Material and methods tube. DNA was extracted using the ionic exchange resin in the Insta- Gene Matrix kit (Bio-Rad) according to the manufacturer's in- 2.1. Collection of infected cockroaches structions, and the supernatants that contained the genetic mate- rial were transferred to a 1.5 mL microcentrifuge tube. DNA samples Cockroaches with signs of mycoses were collected between Jun were stored at À20 C until use. Partial sequences of four nuclear 2013 and Mar 2015 in two natural protected areas of Argentina: El protein coding genes were amplified as follows: TUBB, using PCR Palmar National Park (Entre Ríos province; 31 510 1100 S, 58 190 2100 primers T1 and T22 (O'Donnell and Cigelnik, 1997); a large exon W) the predominant