Southernmost records of Entomophtoromycotina. Updated review of Entomophthoralean fungal insect pathogens of Argentina Registros más australes de Entomophthoromycotina. Revisión de los hongos Entomophthorales patógenos de insectos de la Argentina

The objective of this work is to further update the records of Argentinean Entomophthoralean fungal species and their geographical distribution. Our survey includes five new records of Zoophthora radicans infecting insect hosts belonging to the orders Diptera, Hemiptera, and Lepidoptera from Pampasic and Littoral regions of Argentina. These new records not only increase the host range of Entomophthoralean fungi but also their geographical distribution around the world.


INTRODUCTION
Entomophthoromycota (Phylum Zoopagomycota) is a fungal subphylum, which includes a great diversity of species that predominantly infects insects. The previous reports and articles on the subject recorded for Argentina were focused on taxonomy, biodiversity, epizootiology and some laboratory evaluations of biological activity of species Batkoa sp., Conidiobolus coronatus ( Entomophthoralean fungi infecting insects have been studied and reported from all over the world (Eilenberg et al. 1987, Keller 1987, Feng et al. 1990, Eilenberg et al. 1992, Bałazy 1993, Santamaría and Girbal 1996, Hatting et al. 1999, Nielsen et al. 2001, Pell et al. 2001, Steinkraus and Boys 2005, Jensen et al. 2009, Hajek et al. 2012, Humber 2012, while this fungal group was poorly studied in South America (Aruta et al. 1974, 1984, Delalibera et al. 1992, Alzugaray et al. 1999). The first Argentinean reference was published by Fresa (1979) but in the last decade, the number of publications about this fungal group has increased (Méndez-Sánchez et al. 2001, 2002a, b, 2009, Delalibera et al. 2004, Toledo et al. 2007, 2008a, b, Jensen et al. 2009, Alzugaray et al. 2010, Scorsetti et al. 2010, 2012, Sosa Gómez et al. 2010, Manfrino et al. 2013, 2014a, b, c, Montalva et al. 2016a. The overall goal of this work is to follow this endeavor and further update the knowledge of Argentinean Entomophthoralean fungal species with new records that expanded their insect host range and their geographical distribution.

MATERIALS AND METHODS
Survey of Entomophthoralean fungi were performed collecting dead and alive insects in different types of environments as poultry houses (houseflies), crops (Lepidoptera and Hemiptera) and natural preserved areas (Diptera). Insects infected with Entomophthoralean fungi were collected from under plant leaves, walls, and leaf litter in several localities from Argentina ( Fig. 1) during 2004-2014.
Fungal infected insects were used for actively discharging conidia in a typical "descendent discharge" according to the methodology described by Eilenberg et al. (1992), Keller (2006Keller ( , 2007. Conidia were cultured on Sabouraud dextrose agar with yeast extract + egg yolk + skimed milk (Hajek et al. 2012) and incubated at 20 ºC in darkness. Fungal structures were stained with aceto-orcein 1 %. Observations were performed by using an Optic Microscope Olympus CH30 and photographs with a differential interference contrast fitted with a Power Shot A80 camera. The taxonomic identification was based on the morphology of conidiophores, morphology, and sizes of conidia, rhizoids and cystidia and presence or absence of resting spores, using the keys and monographs of Bałazy (1993), Keller (2007) and Humber (2012).

RESULTS
Entomophthoralean species recorded up to date in Argentina are listed below. This information includes a brief description of the five new records of Zoophthora radicans (Bref.) Batko, infecting insect hosts belonging to the orders Diptera, Hemiptera, and Lepidoptera. These new records, marked with an asterisk in the checklist, not only increase the host range of Entomophthoralean fungi but also their geographical distribution.
As a result of this study, it was possible to obtain and preserved three fungal isolates of Z. radicans, which were deposited in the Agricultural Research Service Entomopathogenic Fungi collection (ARSEF) (Ithaca, New York, USA) under the accession numbers ARSEF 5814, ARSEF 6917 and ARSEF 8466. The other records reported as new in this study are kept as reference dried material (exicata) or as permanent microscopic slides (mounted in LPAO Lactophenol-aceto-orcein and sealed with nail polish) at CEPAVE Fungarium Collection (Centro de Estudios Parasitológicos y de Vectores) (La Plata, Buenos Aires, Argentina). Hosts: Orthoptera Acrididae, several species.

DISCUSSION
Knowledge about distribution and diversity of Entomopthoralean species infecting arthropods in Argentina is scarce and spread out.
As result of our study, the insect host range of these fungi was extended, being Hemiptera the order with the highest number of species infected with Entomophthoralean fungi, and followed by Lepidoptera and Diptera species, even though there are a lot of other insect groups that up to date there were never surveyed in order to look for Entomophthoralean fungi in Argentina yet.
Molecular taxonomic identification and characterization of fungal species are being to be submitted for publication soon, and these studies will contribute to reinforce and strongly support the present records (Manfrino, com. Pers.). We hope that as a result of future researches the number of species recorded in Argentina will increase in accordance with the diversity of habitats that owns the country.

AUTHOR'S CONTRIBUTION
RM and AT collected the samples; CL identified the specimens; CL and AT wrote the text. AG made the map and figures.

CONFLICT OF INTEREST
The authors declare that there are no conflicts of interest.

ACKNOWLEDGMENTS
This work was partially financed by CONICET (National Research Council of Argentina) and FCNyM-UNLP (Natural Sciences College University of La Plata). We thanks to Rich Humber of ARSEF Culture Collection, for preserving specific cultures and confirmed some species identification, and to Ms. M.L. Morote for collaborating with illustrations.