Oomycetes ( Oomycota ) from Maranhão State , Brazil 1

(Oomycetes (Oomycota) from Maranhão State, Brazil). This study aimed at enhancing the knowledge on the diversity and distribution of Oomycetes within Parque Natural Municipal Lagoa do Sambico, in Timon municipality, Maranhão State, Brazil as well as their geographical distribution in Brazil as a whole. Of the 98 isolates, 16 were identified as Oomycetes, which belong to six families: Achlyaceae, Leptolegniellaceae s. lat., Pythiaceae s. lat., Olpidiopsidaceae s. lat., Saprolegniaceae s. str. and Verrucalvaceae. We report herewith the first records of Olpidiopsis aphanomycis Cornu and Saprolegnia luxurians (Bhargava & G.C. Srivast) R.L. Seym. for Brazil, as well as Achlya aff. diffusa J.V. Harv. ex T.W. Johnson, Achlya proliferoides Coker, Aphanomyces helicoides Minden, Aplanopsis terrestris Höhnk, Dictyuchus monosporus Leitg., Globisporangium proliferum (Cornu) P.M. Kirk, and Pythiogeton uniforme A. Lund for the State of Maranhão.


Introduction
The phylum Oomycota (Kingdom Straminipila), has two classes (Saprolegniomycetes and Peronosporomycetes). Saprolegniomycetes includes three orders, seven families and 33 genera, whereas Peronosporomycetes has three orders, five families and 40 genera.Further several classes, five orders, ten families and 13 genera as classified as incertae sedis (Beakes et al. 2014).The phylum comprises saprobes and parasites found in aquatic and terrestrial ecosystems; its members play an important role in the decomposition of organic matter and participate in the cycling of nutrients.Important parasites attack plants, algae, fish, crustaceans, fungi, mosquito larvae, nematodes, rotifers, mammals, and even humans (Marano et al. 2008, Beakes et al. 2014).
Oomycota are widely distributed and ubiquitous.Saprobic species have extensive enzymatic capacity, with the ability to degrade a wide range of vegetal and animal substrates such as cellulose (algae and plant debris), keratin (snake skin, hair and feather), chitin (exoskeleton of crustaceans and insects), lignin (dead tissue of woody plants) and sporopollenin (pollen grains).Saprotrophic species are abundantly found in decaying plant material, but their occurrence and frequency are underestimated unless specific techniques are applied for their study (Hawksworth 2004, Marano et al. 2011).
The genera Saprolegnia, Achlya and Aphanomyces are commonly found parasitizing fish as well as their eggs.Pythium and Phytophthora species are highly pathogenic in plants of economic interest (Moore-Landecker 1996, Alexopoulos et al. 1996).Pythium insidiosum stands out as equine parasite, but also parasites other animals such as cattle, goats, dogs, cats, as well as human.Subcutaneus ulcerative lesions in animals caused by this species have been consistently reported (Santurio et al. 1998, Leal et al. 2001, Sallis et al. 2003, Rech et al. 2004), including in humans for the fi rst time in Brazil (Bosco et al. 2008) and in horses in Piauí State (Rocha et al. 2010).However, there have been very few reports of Oomycetes in Maranhão State.A single study by Sales (2009), surveyed species of Oomycetes from groundwater wells in three villages within Timon municipality.The present study aimed at increasing the knowledge on the diversity of Oomycetes within Parque Natural Municipal Lagoa do Sambico, municipality Timon, in Maranhão State, Brazil.Isolation technique -The multiple baiting technique with cellulosic, chitinous and keratinous substrates was applied to isolate Oomycetes from the samples, following Milanez (1989), according to organic material is used as bait for Oomycetes colonization and culture.Water samples were collected from the lake surface with the aid of a plastic cup and then stored in wide-mouth sterile glass fl asks (100 ml Wheaton vials), with perforated plastic cover to allow oxygenation of the water.Prior to collection, nine baits were added to each fl ask: cellulosic substrates (corn straw, sorghum seeds, onion skin, cellophane and paper fi lter) and chitinous (termite wings) as well as keratinous (snake skin and human hair) items, which served as organic colonization substrates.Soil samples were collected with the aid of a sterile metal spatula, and the surface layers were removed and about 250 g of soil were collected at depth of approximately 20 cm, then packed in 500 g polyethylene bags properly labeled with the respective collection points.The collected material was transported to the Mycology Laboratory of Universidade Federal do Piauí (UFPI) for analysis.30 ml from water samples were placed into Petri dishes (100 × 20 mm) with organic substrates.Approximately, 20 g from the soil samples were placed into in Petri dishes (100 × 20 mm) with sterile distilled water to decant.After decanting, cellulosic, chitinous and keratinous substrates were added to the plates.Then the plates were incubated with the samples at room temperature (30-32 °C) for a 7-day period.

Materials and methods
We performed microscopic analysis of the samplings in order to assess the presence of Oomycetes, which were then placed into new Petri plates with the respective substrates to be colonized (cellulosic, chitinous or keratinous).Subsequently, sterile distilled water was added to each plate.The plates were incubated for seven days for the identification of developing fungal structures.The maintenance of the strains was done with the exchange of water and adding new substrates to each plate.
Vegetative and reproductive structures were recorded by using an optical microscope (Olympus BX41 model, Tokyo, Japan), and photographed with a Nikon digital camera (Coolpix -S4100).Taxonomic descriptions of isolates were done in accordance with pertinent publications (Sparrow 1956, Johnson Jr. 1960, Scott 1961, Dick 1990, Johnson et al. 2002).Selected strains were deposited in the mycology culture collection at Universidade Federal do Piauí (UFPI).Beakes et al. (2014) carried out an up-to-date phylogenetic and taxonomic review of the Straminipila (Labyrinthulomycota, Hyphochytridiomycota, and Oomycota) based upon molecular sequence data, biology and evolutionary history.This taxonomic revision was adopted in the present study.We obtained 98 isolates of Oomycota, including 16 species, which belong to Achlyaceae (Achlya aff.diffusa J.V. Harv.ex T.W.  Geographical distribution in Brazil: Piauí: Parque Nacional de Sete Cidades (Rocha 2002).
It formed oogonia with whole oospores (figure 7).Antheridia diclinous, sometimes, absent.The species is described as a second record for Maranhão State.

Aplanopsis terrestris
Material examined: BRAZIL.Maranhão: Isolate produced oogonia with eccentric spores (figure 16), and zoosporangium clavate (figure 17).The morphological data of this species agree with those presented in the original description.
Isolate produced typical oogonia with oospore (figure 19).The main distinguishing features of A. keratinophilus are the affinity for keratin substrates and abundant presence of antheridial branches involving oogonium profusely.Furthermore, the data agree with the descriptions by Rocha (2002), who presented the first description in Brazil, isolating soil samples from Parque Nacional de Sete Cidades, Piauí.This is the second record for Maranhão State.
The current anthropic pressure over ecosystems causes a strong environmental disturbance.Despite global commitments to reduce the impacts of human activities on ecosystems worldwide, loss of biodiversity and ecosystem exploitation continues rising.However, it is little known about the biodiversity of ecosystems in Brazil.Based upon this survey research, new records for Maranhão State and Brazilʼs Countrywide have been made.Most of the speciesʼ records presented herewith were commonly found worldwide (Marano et al. 2008, Steciow et al. 2012) and have been previously isolated in Brazil (Gomes et al. 2003, Gomes & Pires-Zottarelli 2008, Milanez et al. 2008, Nascimento & Pires-Zottarelli 2012).Moreover, many other studies were carried out in Piauí State (Negreiros 2008, Pereira 2001, Rocha et al. 2001, Sousa 2014, Trindade-Júnior 2013), but a single study in Maranhão State (Sales 2009).
These new records of Oomycetes occurrences enhanced the knowledge about such organisms in Brazil.
Study area -The municipality of Timon is located on the left bank of Parnaíba River, which borders Piauí State.Timon comprises an area of 1,743,246 km², with a population density of 89,18 individuals per km² and an estimated population of 161,721 inhabitants.The municipality has a warm tropical dry climate with little temperature variation, at times exceeding 40 °C, the minimum being rarely less than 22 °C.Centroid coordinates are 5º05'S latitude and 42º50'W longitude, with an elevation of at 69 m.a.s.l., situated 426 km far from the capital of Maranhão State, São Luís (IBGE 2013) (fi gure 1).The Parque Natural Municipal Lagoa do Sambico was created by Municipal Decree-Law 1754/2012 (Timon 2012).The park comprises an area of 80,502.25 m 2 .Plant communities are dominated by herbaceous vegetation.It has a natural lagoon called Sambico (Rima 2011).Five water and soil samples were obtained every two months from August 2014 to May 2015, at four different points of the lagoon, for a total of 40 samples.
Figure 10 Z o o s p o r a n g i a s p h e r i c a l , b u r s i f o r m , 18˗48 × 20˗60 μm diâm., terminal.Internal proliferation observed, with a straight or winding release tube forming with the supporting hyphae a right angle.Zoospores encysted 8˗11 μm diâm.Sexual reproductive structures not observed.Material examined: BRASIL.Maranhão: Timon, Parque Natural Municipal Lagoa do Sambico, soil and water samples at collection spots, 23-II-2015, J.B. Silva (A4/4-S2/4-S4/4).
in Piauí State.It is the second record for Maranhão State.