The genus Allomyces ( Blastocladiomycota ) in the State of Piauí , Brazil

(The genus Allomyces (Blastocladiomycota) in the State of Piauí, Brazil). Brazilian ecosystems have been intensively exploited for agricultural expansion, however, the diversity of zoosporic organisms in such biomes remains little known. Therefore, further research is required to better understand their role within these ecosystems. Studies with zoosporic fungi were carried out and 22 Allomyces isolates were obtained from soil samples collected at six municipalities from Piauí State. After identification procedures, the taxa were grouped into four species: A. anomalus R. Emers., A. arbusculus E.J. Butler, A. moniliformis Coker & Braxton, and A. neomoniliformis Indoh. A. arbusculus had the highest rate of resistant sporangia viability (10%) and the largest geographical distribution in Piauí, occurring in seven out of ten sites studied. Countrywide, they occur within 14 municipalities from three states. Greater knowledge about the geographical distribution of Allomyces in Brazil is being pioneered in the State of Piauí. Novel information regarding the diversity and occurrence, as well as taxonomic characteristics of the isolates is presented herein.


Introduction
Allomyces is a genus characterized by filamentous mycelium and dichotomous branching.It presents planogametes and zoospores with a single posterior flagellum.The genus was established by Butler (1911) based upon the species A. arbusculus, in India.It was reclassified from Chytridiomycota to a new phylum Blastocladiomycota (Fungi) (James et al. 2006).
The genus Allomyces (Blastocladiales) comprises nine species and has very wide geographical distribution, occurring in five continents (Kirk et al. 2008).Such genus is represented by several species in the United States, Mexico, Costa Rica, Brazil and Argentina, which indicates a wide geographic distribution in both temperate and tropical regions of the Western Hemisphere.So reasonably one would assume that future research on this taxon could also find a similar distribution in the hottest regions of the Eastern Hemisphere (Hawsksworth et al. 1996, Steciow & Eliades 2001).
Biodiversity studies of zoosporic organisms, including Blastocladiomycota, in all Brazilian biomes are still very scarce, and the current limited knowledge about these organisms is also due to the very few research specialists in Brazil (Jerônimo et al. 2015).As a matter of fact, these organisms play a major role in the balance of ecosystems and soil fertilization process, since they are found in different ecosystems where they act as organic matter decomposers, by reducing the various types of substrates to simple elements, then ready to be mineralized by subsequent decomposers (Powell 1993, Barr 2001, Shearer et al. 2007).
It has been already known that most Brazilian ecosystems are being over-exploited for agricultural expansion.Conversely, the diversity of this group of organisms within the biomes remains little known, and therefore further studies are needed in order to enhance the knowledge about their diversity.Although Allomyces is the first genus within the Blastocladiomycota phylum to be reported in Brazil (Wolf 1941), there is lack of information about the diversity and geographic distribution of such taxon (Pires-Zottarelli & Rocha 2010).
However, recent studies have contributed to better understanding of the biodiversity and distribution of zoosporic organisms in the State of Piauí (Rocha et al. 2001, Rocha 2006, Pereira & Rocha 2008, Rocha et al. 2010a, 2010b, 2014, Trindade-Junior & Rocha 2013, Rocha & Macêdo 2015).The present study brings new information regarding the genus Allomyces in Piauí.The characteristics of the isolates are described herewith.
Samples were collected from surface water samplings (100 ml) in sterilized glass jars; and topsoil (200 g) was collected at 10 cm depth in the soil profile, with the aid of a sterile spatula, in polyethylene bag labeled with information on the collection site.The samples were transported to the zoosporic fungi Laboratory at the Universidade Federal do Piauí and prepared as per multiple-baiting (adapted) method (Emerson 1941, Sparrow 1960, Barr 1987, Steciow & Eliades 2001, Marano et al. 2008).The water samples were placed in Petri dishes (100 mm diameter), and then cellulosic organic substrates (corn straw, sorghum seeds, onion cataphylls, cellophane and filter paper) were added to the media; the substrates were previously prepared for colonization by zoosporic organisms.From soil samples, aliquots of 20 g were placed in Petri dishes (100 mm diameter) filled with sterile distilled water (30 ml), and after a brief agitation of soil, it was decanted for 20 minutes and then the same substrates were added.The Petri dishes were incubated at room temperature (30-32 o C) under light and dark photoperiod for seven days.And then after that, the substrates were mounted on glass slides and examined under a microscope (Olympus BX 41, Tokyo, Japan).The colonized substrates were then transferred to Petri dishes filled with sorghum seeds and sterilized distilled water for colony replication.
In order to study the life cycle and description of Allomyces species, the isolates were dried on filter paper at room temperature (30-32 o C) for eight weeks.Also, to reestablish Allomyces life cycle, the filter paper containing the resistant structures was placed into a Petri dish with sterile distilled water and sorghum seeds.After the incubation at room temperature (30-32 o C) for 24 hours, the resistant structures germinated and released the zoospores, reestablishing the life cycle.
The viability rate is calculated in order to establish the germination percentage of resistant structures (Emerson & Wolfson 1954).Furthermore, we mounted three Petri dishes with filter paper and filled them with dehydrated resistant zoosporangia as well as sterile distilled water, and then after incubation for 24 h, a 100 resistance zoosporangia were accounted for, and from these, we quantified the zoospores released.After germination and colony growth, 20 measurements were taken to assess the vegetative and reproductive structures as well as the upper and lower values of sizevariation interval.The species have been identified within the literature, such as Emerson (1941), Sparrow (1960) and Wolf (1941).After identification, the species were then described.These species were incorporated into the Culture Collection, Laboratory of Zoosporic Fungi, Universidade Federal do Piauí (Zoosporic Fungi of Brazil, ZFBR).

Results and Discussion
The species of the genus Allomyces found in the State of Piauí were the following: A. arbusculus E. J. Butler 1).
In South America, besides Brazil, Allomyces was also recorded for Argentina, where A. arbusculus, A. macrogynus and A. neomoniliformis occur (Steciow et al. 2012).The life cycles of Allomyces, according to Emerson (1941), are represented by three subgenera.The subgenus Euallomyces develops gametophytes, which produce mobile gametes fusing and forming planozygotes.The latter develop into sporophytes producing thin-walled zoosporangia and resistance zoosporangia.The zoospores formed in the thinwalled zoosporangia are haploid and develop into sporophytes, whereas the zoospores formed in the resistance zoosporangia are diploid and develop into gametophytes.
In the subgenus Cystogenes, resistance zoosporangia produce diploid zoospores, which encyst, undergo meiosis and produce gametes that fuse and develop into sporophyte.Unlike the subgenus Brachyallomyces which produces diploid zoospores, but they not develop into gametophytes.

Allomyces neomoniliformis
Resistance zoosporangium viability: 8%.The pores within the thick-walled resistance zoosporangium are more spaced out than other species (figure 27).They were also deciduous while leaving the capsule at maturity (figures 26 and 27).We observed the growing of thin-walled zoosporangia as well as resistance zoosporangia in a cymose arrangement (figure 26).As a matter of fact, there were no significant differences in comparison to the original description of the species.
The resistance zoosporangia are remarkably resistant structures to environmental conditions and may remain viable for long periods.Emerson (1941) observed that, when completely dried, the resistance zoosporangia can remain viable for several hours at 100 o C or a number of years at ordinary temperatures.There have been reports on their conservation viability of nearly ten years.The viability rate, observed in this study, was generally lower for all species, which reached at most 10%. A. arbusculus (resistance zoosporangium) showed the highest viability rate (10%) and the largest geographical distribution in the State of Piauí, occurring within seven out of ten studied sites.Its occurrence was also found within 14 municipalities in three Brazilian States.
All isolates of the species reported for Piauí were obtained from soil samples.

Figure 1 .
Figure 1.Geographic distribution of the genus Allomyces in Brazil and in the State of Piauí.

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A. arbusculus belongs to the subgenus Euallomyces; A. anomalus belongs to the subgenus Brachyallomyces, while A. moniliformis and A. neo-moniliformis belong to the subgenus Cystogenes.