International Research Journal of Biological Sciences ___________________________________ ISSN 2278-3202 Vol. 2(6), 31-34, June (2013) Int. Res. J. Biological Sci. Isolation and Characterization of Some Indian Hyphochytriomycetes Dubey Manish Kumar and Upadhyay R.S. Laboratory of Mycopathology and Microbial Technology, Centre of Advanced Study in Botany, Banaras Hindu University, Varanasi- 221 005, Uttar Pradesh, INDIA Available online at: www.isca.in Received 19th March 2013, revised 8th April 2013, accepted 5th May 2013 Abstract In the present study, three species of Hyphochytriomycetes were isolated, identified and described. They are Rhizidiomyces hirsutus Karling, Rhizidiomyces apophysatus Zopf and Rhizidiomyces bulbosus Karling. Among them, Rhizidiomyces bulbosus Karling species is being reported for the first time in India. Keywords: Hyphochytriomycetes, Rhizidiomyces and Rhizidiomyces bulbosus Karling. Introduction Material and Methods Hyphochytriomycetes (Hyphochytriales) is a small group of chytrid-like organisms that contain about 23 known species1. They are strikingly similar in morphology and development to many of the true chytrids, but are distinguishable from them by the presence of single anterior tinsel-type flagellum on their included them in the class zoospores. Fuller2 Hyphochytriomycetes of phylum Hyphochytriomycota. According to Berbee and Taylor3 the Hyphochytriomycota, Labyrinthulomycota and Oomycota belong to the Kingdom Stramenopila. Hyphochytriomycota, consisting of a single order Hyphochytriales, has been classified into three families, namely Anisolpidiaceae, Rhizidiomycetaceae and Hyphochytriaceae on The the basis of their thallus structure4,5,2,6. Hyphochytriomycota are a small group of little known fungi with almost no economic importance; but molecular and ultrastructural evidences place them together with the biflagellate heterokont organisms such as Oomycota and Chromistan algae7. Isolation: Baiting technique11,12 was used for the recovery of Hyphochytriomycetes. Samples of water and soil were collected at random and taken to the laboratory. Each sample was divided into triplicates, which were introduced in separate Petri dishes and flooded with 40 mL of sterile deionized water. Each triplicate was baited with chitin (purified shrimp exoskeleton) and keratin (purified snake skin). All triplicates were incubated at ambient room temperature for two weeks. The baits were periodically examined under a microscope for about two weeks and when the isolates became visible on the baits, they were transferred to 1/4YpSs (yeast extract peptone soluble starch) agar medium containing 300 ppm penicillin G and 300 ppm streptomycin sulfate. All isolates were obtained in to pure form by a series of regular subculture carried out on 1/4YpSs agar. Stock culture of all the isolates was maintained on 1/4YpSs agar slants. The cultures were stored at 10°C and subcultured after every three months onto fresh media. All specimens were deposited at the Laboratory of Mycopathology and Microbial Technology, Centre of Advanced Study in Botany, Banaras Hindu University, Varanasi, India. In India the study of Hyphochytriomycetes started as early as 1935, when Chaudhuri and Kochhar8 reported Rhizidiomyces apophysatus from the oogonia of Achlya klebsiana. Few years later, Mundkur9 reported the same species on A. klebsiana. However, the real start of research on Hyphochytriomycetes in India should be credited to J.S. Karling, a mycologist of USA who visited India in 1963 and described 6 species of Hyphochytriomycetes10. The purpose of this study was to throw light on description and information pertaining to habitats, substrates, and geographical locations of the following Hyphochytriomycetes isolated from fresh water and soil sources. Three species of Rhizidiomyces were isolated and illustrated in this paper. The detail of their life history, with particular emphasis on their delepmental stages on soild media is described. Rhizidiomyces bulbosus Karling species is newly recorded in India. International Science Congress Association Observation and identification: Thallus morphological features and developmental pattern of the isolates were examined using a light microscope on 1/4YpSs agar, PYG broth and on baits. The isolates were examined by light microscopy to assess range and variation in thallus structural features, including sporangial shape and size, discharge apparatus, number of discharge pores/ tubes, type of zoospores discharge, flagellation of the zoospore, possession of apophysis and morphology of rhizoidal system. The type of habitat and substrata from which each culture was originally isolated were analyzed. Identification and characterization of isolates were made with the help of Sparrow's `Aquatic Phycomycetes'11 and Karling's `Chytridiomycetarum Iconographia'13 and other relevant taxonomic literatures. 31 International Research Journal of Biologicall SSciences _______________________________________ ___________ ISSN 2278-3202 Vol. 2(6), 31-34, June (2013) Int. Res. J. Biological Sci. Results and Discussion yces hirsutus14: On Taxonomic account of species: Rhizidiomyc 1/4YpSs agar: Thallus monocentric, euca carpic; sporangium usually spherical, variable in size, 40-1300 m in diameter, multiple, short, tubular rhizoidal main axis ari arising from the base or from several peripheral points of sporangiu ium (figure 1. a and c.). On surface of mature sporangium walll w with 2-18 or more hair-like appendages of up to some distance,, w which develop into an extrametrical well-developed, profusely bbranched rhizoidal system. Terminal branches were finely div ivided and sharply pointed at the tips (figure 1. a). Apophysis w when present intraor extramatrical and variable in size and sha shape. Zoosporangia with long exit tubes, usually 1-3 in numbbers, elongate and tapering; the tubes contain numerous refrac active globules and bodies of various sizes (figure 1. b andd c). As the tubes elongated, they remain simple or branched on once to several times and the tips of the broad branches often enlarg rge to form ovoid to subspherical swellings. The contents of zoosp sporangium emerge as an undifferentiated naked protoplasmic maass which develops a vesicular membrane and underg rgoes cleavage into zoospore rudiments (figure 1. d). The zoo oospores get librated by the rupture of vesicular membrane and a get encysted to enlarge directly into the sporangium. Zoospores Zo (4×8 m) highly variable in shape; elliptical, oblo long, oval, or spherical with several minute refractive globules and a a stout anterior flagellum. Resting spores unknown. Color off colony c white. Specimens examined: Pond mud from f Alfred Park, Allahabad, U.P., 23 June 2012. Isolated on shri rimp chitin. Distribution: India, Argentina, USA, U Taiwan, Poland, New Zealand and Brazil Mature sporangial wall with multiple m elongated hair-like appendages is the main characte cter of this species15. Many variations of thallus structure occu cur in axenic culture, which is 16 the similar as described by Karling’s K . This species is saprophytic in nature and occur mo ostly in ponds. It is one of the most widely distributed species off Rhizidiomyces and observed in most parts of the world. Figure-1 Rhizidiomyces hirsutus Karling. a. Matur uring sporangium (S) with rhizoidal main axis (R) arisin sing from the base or from several peripheral points of sporangium. b. Empty sporangia with 3 discharge tubes (arrows). c.. Young Y sporangium (S) with long tapering discharge tube (T). d. Maature sporangium (S) with mass of quiescent spores (Z) (Z liberated through the disruption of vesicu icular membrane. Bars = 20 µm for a, c and d; 50 µm for fo b International Science Congress Association n 32 International Research Journal of Biologicall SSciences _______________________________________ ___________ ISSN 2278-3202 Vol. 2(6), 31-34, June (2013) Int. Res. J. Biological Sci. Rhizidiomyces apophysatus17 On 1/4YpSs agar: Zoosporangia monoc ocentric, eucarpic, gregarious, sessile, spherical when young bu but become oval or broadly pyriform, 20-60 µm, with a long ta tapering cylindrical discharge exit tube (figure 2. a); delimited from rom the intramatrical portion of the thallus by a cross wall at matu aturity. Intramatrical apophysis (3.5-5 µm) spherical to ovoidal, py pyriform or broadly fusiform with extensively branched rhizoid idal system arising from its base. The first indication of zoospo pore discharge from a sporangium was the appearance of clear ar papilla from the sporangium wall. When the tube reaches 5--15 µm in length, some of the protoplasm moves in to clear ar area and shows the movement of the protoplast from the spo porangium into the vesicle, which results in the formation off w wide single apical discharge pore at the apex of discharge tubee (Fig. 2. a). On the completion of sporangium discharge, pr protoplast contents through the tube and the uncleaved protopplasm is all in the vesicle, similarly as described by Fuller18. This coenocytic, protoplasmic mass immediately cleaves to form swarming zoospore initials and discharged as a mass. A After the discharge, the zoospores begin to move extrasporangiall vvery slowly with a rocking motion. These movements of the zzoospores increase and they swim away in all directions. Zoo oospores (3×6 µm) broadly elliptical and oblong with numerou ous small refractive globules. Resting spores unknown. Color of co colony white. Specimens examined: Pond mud from Alfre red Park, Allahabad, U.P., 23 June 2012. Isolated on shrimp chitin. Distribution: India, New Zealand, Argentina, a, Poland, USA and Brazil Weekly parasitic on oogonia of Sapro rolegnia ferax, S. asterophora, and Achyla polyandra in Germ many17; A. mixta in 19 Switzerland ; A. conspicua in North Caro rolina20,21,22,23,24 and New York25, U.S.A.; A. flagellata in Japan26 and U.S.A.4; and A. klebsiana in India9. Apparently, itt is a facultative parasite which can be cultured readily on synthetic tic medium22. It also occurs on pollen grains when they are used to bait soil samples27. It is one of the most studied species of Rhizid zidiomyces. Rhizidiomyces bulbosus16 On 1/4YpSs agar: Thallus epibio biotic, monocentric, eucarpic, nonapophysate, spherical, 25–70 µm in diameter, with a hyaline smooth wall; usually developing 1 to t 3 exit papillae at maturity. Rhizoids often irregular, ending bluntly, b usually reduced and limited in extent, arising genera rally from the base of the sporangium, rarely from 2 to 3 places pl on the periphery. Exit papillae grow out and enlarge to become be bulbous, subspherical, or broadly clavate, 8-20 µm in dia iam (figure 2. b).The bulbous exit papillae emerge from proto toplasm which subsequently cleave into zoospores. The zoospore ore discharge as an initial burst and remains quiescent for 1-2 minu inutes. The released zoospores encysted at the apex of the exit pap apillae, where they developed in to sporangia forming heaped-up p groups of sporangia on solid agar as described by Karling16. Zoospores Zo small, hyaline with refractive globules, ovoid, 3-4.5 µm µ in length. Resting spores unknown. Color of colony white. Specimens examined: Soil samples sa from Alfred Park, Allahabad, U.P., 25 July 2012. Isola olated on snake skin. Distribution: USA Fairly smaller zoospores produced d and development of 1 to 3, relatively short, bulbous, subspher erical or broadly clavate exit papillae instead of long, tapering g necks for the discharge of zoospores is the main characte ter of this species16. Many variations of thallus structure occ ccur in axenic culture. This species is saprophytic in nature and d occurs mostly in soil. Figure-2 a. Rhizidiomyces apophysatus Zopf develop loping a long tapering cylindrical exit tube (T). b. Rhizid idiomyces bulbosus Karling with h ttwo subspherical bulbous (B) Bars = 40 µm International Science Congress Association n 33 International Research Journal of Biological Sciences ________________________________________________ ISSN 2278-3202 Vol. 2(6), 31-34, June (2013) Int. Res. J. Biological Sci. Hyphochytriomycetes has been mentioned for the first time to India by Chaudhuri and Kochhar8 and subsequently by Mundkur9 and Karling10, but despite being cosmopolitan and ubiquitous in its distribution, it never generated fascination among Indian scientists and researchers. Thus, the record of Hyphochytriomycetes of India is rather scanty as only six species have so far reported from India. 5. Karling J.S., Some zoosporic fungi of New Zealand. IX. Hyphochytriales or Anisochytridiales, Sydowia, 20, 137–143 (1967) 6. Alexopoulos C.J., Mims C.W. and Blackwell M., Introductory Mycology, 4th ed., John Wiley and Sons, Inc. (1996) 7. Fuller M.S., Hyphochytriomycota, In “The Mycota,” Vol. VII, “Systematics and Evolution” (D.J. McLaughlin, E.G. McLaughlin, and P.A. Lemke, eds.), Part A, Springer-Verlag, Berlin, 73–80 (2001) 8. Chaudhuri H. and Kochhar P.L., Indian water moulds. I., Proc. Indian Acad. Sci. Sect., B, 2, 137-154 (1935) 9. Mundkur B.B., Fungi of India, Suppl. I., Sci. Monogr. Counc. Agr. Res., India (1938) The results of the present study indicate that Hyphochytriomycetes are of fairly common occurrence in Indian aquatic and terrestrial habitats. R. hirsutus Karling, R. apophysatus Zopf and R. bulbosus Karling are isolated, described and illustrated for the first time on solid media in India. All of them are saprotrophic on broad range of substrate. The bulbous subspherical exit papillae with irregular rhizoids, possession of fairly extensive rhizoidal system oriented on its base or sides and unique pattern of discharge tubes formation are key characteristic features of R. bulbosus Karling, R. hirsutus Karling and R. apophysatus Zopf respectively. 12. Fuller M.S. and Jaworski A., Zoosporic fungi in teaching and research, Southeastern Pub. Co., Athens, Georgia, 303 (1987) Conclusion 13. Karling J.S., Chytridiomycetarum Iconorgraphia, Luberecht and Cramer, Monticello, New York, 383-392 (1977) The results of present study showed a taxonomic description and ecological/distributional data of three species of Rhizidiomyces. This study also provides a taxonomic key with figures for their identification using solely morphological and developmental characters on solid media. R. bulbosus Karling is mentioned for the first time in our country, being also the first record of it outside U.S.A. Thus, this investigation would certainly lead to better understanding of role, diversity and ecology of Hyphochytriomycetes of India. Acknowledgements The authors gratefully acknowledge Serena Rasconi, Oslo University, Norway along with Shu-Fen Chen, Tainan, Taiwan for invaluable assistance in providing taxonomic literatures. The authors express their appreciation to Frank H. Gleason, University of Sydney, Australia and Ram Dayal, Varanasi for their helpful suggestions and encouragement. 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