' ’ V . Y) , ’*1^. Chapter 6 SUMMARY AND DISCUSSION SUMMARY AND DISCUSSION The present study is an addition and significant contribution in taxonomy of Indian watermoulds isolated from different closed water systems (Lentic water system) such as lakes, ponds and ditches from Pune City, Maharashtra State, India. The taxonomy of the isolated watermoulds mainly morphological characters reflected several interesting morphological variations. The exhaustive data of the occurrence, distribution and seasonal periodicity, major physicochemical parameters certainly add valuable information in ecology of the tropical watermoulds. Zoospore release mechanism from the zoosporangia of the different genera is a very diffent approach and probable criterion for classification of watermoulds. The results presented showed that distribution of watermoulds was different at all sites and often specific to the site. The studies on different water bodies exhibited more generic and species diversity of watermoulds in ponds than in lakes. In all, 11 genera and 28 species were isolated of which the order Saprolegniales, the genus Achlya and eccentric forms were dominant. While working on taxonomy of watermoulds several morphological variations were noted in some species. These variations were not only in vegetative thallii but also in reproductive structures. In Rozella allomycis the morphological variation was observed in resting spores, which were three times larger per segment than previously recorded spores by Sparrow (1960) and Kapadnis (1978). Blastocladia incrassata showed distinct variation in thallus branching which was mostly irregular, dichotomous or sometimes racemose, which totally differs than the branching system observed by Steciow (2001). She reported only thick terminal lobes in the thallus. In the species, Brevilegnia longicaulis variation was observed in sexual and asexual reproductive structures. In addition to the general characters some interesting findings like sympodial renewal of sporangia, presence of gemmae, pyriform, spherical, long stalked, hyaline papillae on hyphae and occasional occurrence of androgynous antheridia were also highlighted. The species of Brevilegnia showed remarkable 201 morphological diversity thus it might need taxonomical revision. In Saprolegnia torulosa the non-functional antheridial extension is at the base of the upper oogonium, which after disarticulation becomes the tip of the subsequent, catenulate oogonium, is an interesting feature in the genus Saprolegnia.ln the present studies, the functional oospheres were frequently observed in Achlya bisexualis, which after fertilization become functional oospores, however, many research workers recorded abortive oospheres very frequently. Though the genus Achlya is mainly characterized either by the presence of monoclinous, or diclinous and/or androgynous antheridial branches, A. reciirva predominantly showed androgynous antheridial branches, whereas in A. orion monoclinous antheridial branches were prominently noted in the present studies. In addition, A. orion showed sessile to subsessile oogonial stalks. Aphanomyces laevis and Pythium torulosum were recorded throughout the study period from all the closed waterbodies and showed their constant occurrence. Aphanomyces laevis was interesting as it showed larvicidal activity on mosquito larvae and can be a possible biological control. During the study period, two new species, namely Achlya species and Saprolegnia anomalies were proposed on the basis of different morphological characters. Of which in Achlya species distinctly large, balloon like projections from hyphal walls intermingling with true oogonia having brown oospores are the characteristic features, whereas, in Saprolegnia anomalies many proliferating tubes developed from the walls of sporangia and gemmae at the time of zoospore release which is a unique feature as compared to the other species of Saprolegnia except S. anisospora, where only gemmae develop proliferating tubes. Brevilegnia longicaulis and Saprolegnia torulosa are new to India. Blastocladia arborata, B. incrassata, Allomyces javanicus, Achlya bisexualis and A. diffusa are new to Maharashtra State. An Acacia nilotica L. pod is a new substratum record for the species of Gonapodya and Blastocladia. Laboratory experiments showed strict host specificity of Rozella allomycis and Olpidiopsis achlyae. When in cultures of Allomyces javanicus, A. moniliformis and A. arbuscula an endoparasite R. allomycis was inoculated, it 202 showed host specificity only for A. arbiisciila. Similarly for O. achlyae, cultures of Achlya orion, A. oblongata, A. bisexualis, A. flagellata were used and the latter was seen as a specific host. The taxonomical data on watermoulds exhibited a very wide range of morphological diversities. In this context, diversity in thallus organization, asexual and sexual reproductive structures and sexual reproduction in different genera and species was discussed. In addition, diversity in habitats, baits and colony growth of watermoulds was also described. Video recording of sequential events of isogametangial fusion and formation of zygote of Allomyces arbuscula was executed for the first time. Powell and Blackwell (1998) worked on the phenetic analysis of genera of the Saprolegniaceae and concluded that nonsexual characters dominantly provide intergeneric taxonomic separation and delineation of Saprolegniaceae, whereas, sexual (oogonial and antheridial) characters are generally more variable and perhaps of great utility when applied intragenerically. The video recording of zoospore (nonsexual structure) release from the zoosporangia of the different genera in the present studies gives support to the statement made by Powell and Blackwell. The extensive study of live sequential events like protoplasm differentiation leading to formation of zoospores, their movement within zoosporangium, actual mechanism of release of zoospores and their fate after release were recorded by using CCD camera with detailed specifications in different genera of zoosporic fungi. All these details and variations strongly support the above mentioned phenetic analysis put forth by Powell and Blackwell. Most striking feature of zoospore release was recorded in Rozella allomycis where zoospores get released from zoosporangia by three different methods: plug formation, pouch formation and pore formation, of these, video recording of the former two methods are innovative. In addition, video recording of mechanism of zoospore release in nine different genera was accomplished. The classificatory systems so far established are totally based on morphology and reproductive structures in the thallus but nobody has realized the importance of mechanism of zoospore release, which is probably an important 203 criterion to give a proper status to the genus in classification. According to Hohnk (1933) small vacuoles inside the zoospores get disappeared to create a strong pressure within the sporangium causing the passive escape of the zoospores. However, Dick (1973) stated that the pressure of hydration and differential viscosity causes the zoospore release, however he did not give convincing proof. But Dick and Johnson et al. (2002) agreed that there is no adequate hypothesis to explain spore release in watermoulds. Padgett and Johnson (2004) made video recording of zoospore release in Isoachlya and stated that it has greater significance in taxonomy for the confirmation of the species. In the present studies, the video recording of zoospore release in live cultures of watermoulds, the presence of gradually increasing movement of all zoospores within their boundary inside the zoosporangium at the time of release create a pressure on the thin discharge papilla which is a convincing evidence to explain zoospore release which could be a realistic proof to put forth the hypothesis of zoospore release. This may be significant and new criterion for the classification of the genera in watermoulds. Though the traditional criteria are useful in classification of watermoulds and accepted by mycologists, importance of phylogenetic studies based on molecular sequencing cannot be ingnored for accurracy. In the present studies, S. anomalies has been established as a new species on the basis of morphological features but its status has been confirmed by molecular sequencing and its position, by creating a phylogenetic tree. Though ponds and lakes are the types of closed water system, they were treated as separate entities in the present studies for the data analysis. The lakes under study have changing periphery as per the amount of rainfall during that year. However, the different water sources such as nearby rivers, streams and brooks also add some water in lakes. On the other hand, the ponds have stagnant water with limited periphery. Therefore, ‘aliens’, ‘immigrants’ and ‘transients’ were common in lakes whereas, ‘indwellers’ or ‘indigenous’ species were active in ponds. In polluted lakes one ‘indweller’, two ‘inhabitants’ and five ‘aliens’ were recorded. In unpolluted ponds two ‘immigrants’, six ‘indwellers’ and two 204 ‘aliens’ were noted. In polluted pond one ‘alien’ and due to availability of substratum five ‘indwellers’ were recorded. The impact of different physicochemical parameters and seasonal fluctuations influence the occurrence and periodicity of watermoulds in closed waterbodies. The correlation within different parameters and between occurrence of watermoulds and physicochemical parameters was studied for