' ’ V . Y) ,

’*1^.

Chapter 6

SUMMARY AND DISCUSSION SUMMARY AND DISCUSSION

The present study is an addition and significant contribution in 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 , the genus 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 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 the total impact on watermoulds. Numerical data was analysed statistically to correlate the findings. There was negligible difference in the values of temperature, pH, D.O. and free CO2 in ponds and lakes. However, the concentration values of total alkalinity, hardness, calcium, chlorides and phosphates were high in lakes as compared to ponds. In closed waterbodies the average range of temperature was 20°C - 30°C. At ‘moderate temperature range’ maximum species were isolated. When the temperature increased the number of species also gradually increased in the established sites. However, the correlation in them was insignificant. The members of Saprolegniales were common to all the sites especially in rainy and winter seasons, at low temperatures, whereas, at high temperatures, Monoblepharidales were dominant only at Lakaki Pond during summer. Pythium species occurred repeatedly throughout the year from almost all sampling sites, therefore showed their constant occurrence and temperature variations did not affect their occurrence. Achlya bisexiialis and Aphanomyces laevis tolerated a wide range of temperature (17.4°C and 32.1°C) therefore considered as temperature tolerant species. In all types of closed waterbodies pH was alkaline throughout the study and the average pH range was 7.9 to 8.9 therefore all the watermoulds are alkaline species. Maximum species of watermoulds were isolated at this range. However, only in rainwater ditches at slightly acidic conditions Allomyces javanicus, A. moniliformis, Achlya orion and Aphanomyces laevis were thrived. The members of Blastocladiales and Saprolegniales were found to occur at almost all pH ranges, however, the former order was not recorded at ‘neutrally alkaline’ conditions.

205 Monoblepharidales occurred at ‘alkaline’ to ‘highly alkaline’ whereas, Peronosporales at ‘neutrally alkaline’ to ‘highly alkaline’. Aphanomyces laevis showed adaptability towards different pH conditions. However, statistically pH seemed to be insignificant to the occurrence of watermoulds, but significant negative correlation was obtained with the occurrence of species at Empress Garden Pond. Concentrations of minimum and maximum ranges of D.O. were almost equal in all the closed waterbodies, consistently at 3.0 - 7.0 mg/lit. Values of D.O. fluctuated in rainy and summer seasons, whereas, in winter there was slight increase in value. Maximum species were isolated in summer at 2.8 mg/lit. At high D.O. concentration only A. laevis was isolated and showed its significance. The lowest D.O. was recorded at Lakaki Pond which mostly due to sewage and decaying, submerged Acacia nilotica pods which favoured only G. polymorpha and G. prolifera. Significant negative correlation was observed between D.O. and watermoulds only at Empress and University Garden Ponds, which were unpolluted, however, at other sites the correlation was insignificant.

At Katraj Lake free CO2 was absent in all seasons, whereas, in other lakes and Lakaki Pond it was absent in summer and rainy seasons. On the other hand it was present at all unpolluted ponds throughout the study period. Allomyces arbuscula, Achlya oblongata, A. orion, A. dijfusa, Aphanomyces laevis Dictyuchus sterile, Pythium proliferum, P. torulosum, P. catenulatum and

Saprolegnia anomalies thrived even in absence of free CO2. Aphanomyces laevis was the only species, which sustained in absence as well as at high concentration

of free CO2. Insignificant correlation was obtained between free CO2 and density of watermoulds. At all polluted sites total alkalinity, total hardness, calcium, chlorides and phosphates were in high concentrations. At minimum and maximum concentrations of total alkalinity A. laevis and P. torulosum were thrived. The members of tolerated minimum as well as maximum range, whereas, Chytridiomycetes were found to occur only at higher concentrations. Total alkalinity showed insignificant correlation with watermoulds.

206 Total hardness was very low at garden ponds. Watermoulds occurred at moderate concentrations. At minimum only D. carpophonis and at maximum A. laevis, S. anomalies and P. proliferum were recorded. Insignificant correlation was obtained between total hardness and watermoulds. At maximum concentration of calcium, not a single watermould was thrived, however, at minimum, Saprolegniales and Peronosporales were common. At University Garden Pond maximum number of species was isolated at the lowest concentration and only in this pond, significant negative correlation was obtained. Presence of magnesium did not affect the occurrence of watermoulds, which was proved statistically. High chloride values indicated more organic pollution at Katraj Lake and Lakaki Pond than Bhugaon and Pashan Lake. A. laevis, Blastocladia arborata, Gonapodya polymorpha and G. prolifera were present at high ranges, whereas, at low not a single watermould was present. Chloride and watermoulds showed insignificant correlation. At garden ponds low concentration of phosphate was recorded, but the decrease in level of phosphates in the rainy season, increase in winter and highest in summer was noted. At higher concentration of phosphate not a single watermould was recorded. Watermoulds occurred at any concentration of phosphate were recorded during study, thus, the occurrence of watermoulds was independent, which was proved statistically. Thus, from the results of two years presented in the previous chapters, it is very clear that at unpolluted sites D.O., pH and calcium governed the occurrence of watermoulds, whereas, at polluted sites combined effect of physicochemical parameters was observed and not a single factor was dominant. At almost all sites strong positive correlation was present between total hardness and calcium, magnesium, chlorides, total alkalinity. To assess their effect, multiple regression technique was followed and the high values of the coefficient of determination in this technique suggested that fit is good. Bhairavanath and Manoharachary (1985) claimed that temperature is the determining factor in the periodicity of Phycomycetes, however in the present

207 studies temperature was not only a determining factor but D.O., pH and calcium were the determining factors and negatively correlated with occurrence of watermoulds. Whereas, Misra (1982) noted that D.O. and calcium are positively correlated and temperature is negatively correlated with watermoulds. Dayal and Tandon (1963), Hughes (1962), Roberts (1963), Srivastava (1967), Dick (1976) and Madhusudan Rao and Manoharachary (1983) have claimed that slightly alkaline pH, more organic matter, oxygen, nitrogen and low water temperature are some of the factors having significant influence on fungal numbers, distribution and their phenology. However, watermoulds in the present studies favoured alkaline pH for their growth and high temperatures but did not show any influence of DO alone. Perrott (1960), Dayal and Tandon (1963), Robert (1963), Srivastava (1967), Hunter (1975) and Misra (1983) noted that low temperature favours the growth of watermoulds. Alabi (1971) reported the growth of watermoulds at high temperatures. Misra (1985) claimed that nutrient source and the nature of the available substrate appear to be the decisive factors for the active growth of fungi, whereas, Khulbe (1980) and Bhairavanath and Manoharachary (1983) considered pH as the decisive factor. In the first year of study, maximum number of species was isolated from the established sites as compared to that in the second year. However, during the study period, when all types of waterbodies including ditches were considered, the maximum species were recorded in the second year.The species diversity was more remarkable in rainy season followed by winter and least in summer. In the study period, the number of watermoulds were maximum at Lakaki Pond followed by University and Empress Garden Pond, whereas, minimum watermoulds were isolated at Bhugaon and Pashan Lake. The occurrence, distribution and frequency of watermoulds, mainly, Oomycetes was more as compared to Chytridiomycetes in closed waterbodies. The members of Oomycetes were present in polluted as well in non-polluted waterbodies, thus, Oomycetes did not show any specificity for their distribution, whereas, Chytridiomycetes were mainly restricted to polluted waterbodies. Rai and Misra (1977), Bhairavanath and Manoharachary (1983), Misra (1985) and Mishra and Dwivedi (1987) worked on alkaline ponds and lakes from different localities in India except Maharashtra. All these research workers except few found that Oomycetes are dominant over Chytridiomycetes, whereas, Mishra and Dwivedi reported the reverse results. Results in the present studies also revealed the dominance of Oomycetes in closed water bodies from Maharashtra. Thus in general it seems that Oomycetes rules over all other aquatic fungi. From the studies it is noted that most of the members of Chytridiomycetes require prominent organic substratum for their growth. Gonapodya and Blastocladia are the cases, which preferred only organic substratum especially angiosperm fruits (Thaxter, 1895; John and Das Gupta, 1958; Perrott, 1960; Manoharachary, 1981; Das Gupta and John, 1988; Usha Kiran and Dayal, 1992). In the present study decaying, submerged Acacia nilotica pods supported the growth of these fungi. The genera Achlya, Allomyces, Aphanomyces and Pythium were common in ditches and the established sites, whereas, Dictyuchus, Gonapodya, Blastocladia and Saprolegnia were restricted only to the established sites. The species, Achlya recurva, A. klebsiana and A. flagellata, Allomyces javanicus, A. moniliformis, Blastocladia incrassata, Brevilegnia longicaulis, Olpidiopsis achlyae, Rozella allomycis occurred only in rainwater ditches and showed their restrictions of habitat. The genus Achlya was the most dominant in all types of waterbodies with nine species and 31 % frequency occurrence. Saprolegnia, Pythium and Allomyces followed it with 11% occurrence. The occurrence and distribution of watermoulds was restricted to particular waterbodies. Gonapodya prolifera, G. polymorpha, Blastocladia arborata, B. incrassata, Saprolegnia anomalies were restricted only to most polluted water. Achlya bisexualis, Achlya sp. Blastocladia arborata, B. incrassata, Dictyuchus carpophorus, Gonapodya polymorpha, G. prolifera and Saprolegnia torulosa occurred only in ponds whereas species Achlya oblongata, A. orion, Saprolegnia anomalies and S. ferax were restricted to lakes. Seasonal periodicity at generic level was observed only in Gonapodya and Saprolegnia.

209 Periodicity at particular season and at particular site was observed in Achlya hisexnalis, Dictyuchus carpophorus, G. polymorpha and G. prolifera and Saprolegnia anomalies. The species which were isolated in summer season did not have specific seasonal periodicity as they occur either in rainy season or in winter. In rainy season maximum species were isolated in the second year of study, of these 11 species were restricted only to this season. During winter 14 species were isolated, of these, only three species, namely, Achlya sp., Saprolegnia ferax and S. torulosa were restricted only to winter. Out of the 28 species, only 10 species occurred in all seasons but most of them were sporadic. In temperate countries maximum abundance of watermoulds is in winter or in autumn and spring and decrease in summer (Forbes, 1935; Waterhouse, 1942; Perrott, 1960; Roberts, 1963). But in tropical countries like India, maximum numbers of watermoulds was recorded in rainy season followed by winter and least in summer in closed system (Dayal and Tandon, 1962, 1963; Srivastava, 1967; Manoharachary, 1979 a, b; Mer et al, 1980; Chowdhry and Agarwal, 1980; Manoharachary and Ramarao, 1981; Bhairavanath and Manoharachary, 1983; Gupta and Dubey, 1987). I agree with the above results. Whereas, Dayal and Tandon (1962 - 63) stated that winter is the ideal season for Phycomycetes. Alabi (1971), in tropical countries like Nigeria, noted that most of the fungi from Saprolegniaceae were recorded in dry season when temperatures were relatively high. Das Gupta (1982) and Manoharachary (1981) noted that period immediately after monsoon rains and winter were best for the collection of watermoulds in India. My observations are more or less similar. Eccentric forms were dominant in ponds and mostly in unpolluted ones whereas subcentric and centric forms were more in lakes and in polluted water. Eccentric species did not show seasonal periodicity, whereas, species with subcentric oospore had it in rainy and winter seasons; those with centric oospores had it only in winter. Hughes (1962), Alabi (1971), Manoharachary and Ramarao (1981) and Misra (1983) claimed that in tropical conditions the species with eccentric

210 oospores are more frequent than the species with centric or subcentric oospore. I also recorded similar results. In closed waterbodies, distinct periodicity is not observed in eccentric species but marked periodicity is observed in centric and subcentric species by Chaudhuri (1942), Zeigler (1958), Dick and Newby (1961), Hughes (1962), Srivastava (1967), Alabi (1971), Fox and Wolf (1977), Maestres and Nolan (1978) and Klich and Tiffany (1985). I also observed similar type of periodicity. However, Bhairavanath and Manoharachary (1983) and Manoharachary (1985) have reported that species with eccentric oospore are common in winter. Achlya, Allomyces, Aphanomyces, Blastocladia, Dictyuchus and Pythium are the conmion watermoulds in closed waterbodies from India. In tropical countries the genus Achlya is dominant with maximum species, whereas, in temperate countries Saprolegnia is dominant mostly in cool water. Harvey (1952 in Ho, 1975) stated that certain species of Saprolegnia did not survive in heavily polluted waters. On the contrary, in the present studies, S. ferax and S. anomalies were isolated from the polluted water. These seemed to be pollution indicator species. Khulbe (1983) also found the former species in the polluted water. These species are thus considered as indicators of water pollution. Aphanomyces is recovered from temperate as well as tropical countries throughout the temperature range in all seasons over the year. However, Thakur Ji and Dayal (1966) observed it more frequently in winter. A. laevis is the only species, which occurred in all concentrations of physicochemical parameters. Its occurrence is in polluted and non-polluted sites. Dick (1970) observed nonsexual reproductive structures on insect exuviae, whereas in present studies, abundant oogonia were seen on cockroach cadaver. A. laevis is present in closed as well as open waterbodies and soil, so considered as universal in occurrence. I also observed and confirmed larvicidal activity of A. laevis on mosquito larvae in several ditches and the laboratory. Manoharachary and Ramarao (1981), Madhusudan Rao and Manoharachary (1983) and Bhairavanath and Manoharachary (1985) reported that Achlya, Aphanomyces, Blastocladia, Olpidiopsis and Pythium are confined to

211 aquatic habitat and complete their life cycle in water and regarded as ‘indwellers’ or ‘active forms’ and ‘inhabitants’ of aquatic ecosystems as stated by Park (1972) and Dick (1976). They claimed that ‘indwellers’ form the major flora of aquatic environment. Allomyces might be ‘transient’ between aquatic and terrestrial ecosystem as stated by Madhusudan Rao and Manoharachary. In the present studies, species of the same genus may have different origin of potential inoculum. Based on observations different species in lakes and ponds are categorized differently. The ‘indwellers’ were restricted to ponds because of the submerged substratum and less wave action in water. The frequency of ‘aliens’ is more in lakes because of the addition of free flowing water from various sources. In lakes Allomyces arbiiscula, Achlya oblongata, A. orion, A. diffua and Saprolegnia ferax are considered as ‘aliens’; Aphanomyces laevis and Pythiiim torulosim are ‘inhabitants’ and D. sterile is the only ‘indweller’ species. In ponds A. proliferoides, S. torulosa and Allomyces arbiiscula are the ‘aliens’; Achlya bisexualis, A. diffusa, D. carpophorus, D. sterile, A. laevis, G. prolifera, G. polymorpha, Blastocladia arborata and B. incrassata are ‘indwellers’; A. proliferoides and P. torulosum are ‘immigrants’. Waterhouse (1942) reported Blastocladiaceae, Pythiaceae and Leptomitaceae from the Hogsmill river and commented that, most of the watermoulds have a seasonal periodicity. Hunter (1975) found maximum number of Saprolegniales followed by Peronosporales. Only one member of Chytridiomycetes was observed in her studies. Desale (1996) and Gandhe and Gandhe (2003) reported similar observations. In the present studies, though Saprolegniales was dominant, Chytridiomycetes were occurred in good number. Thus the lentic system proved to be favourable for the growth of Chytridiomycetes. D.O., pH and temperature controlled seasonal periodicity in river water. In the present studies, calcium was found as the third parameter governing the occurrence and periodicity of watermoulds instead of temperature. Aphanomyces laevis occur in polluted as well as unpolluted water in river as well as closed waterbodies. Achlya was the dominant genus in both watersystems. However, generic and species diversity was more in riverine system with

212 remarkable fluctuations in their occurrence, distribution and development of thallus. Strong water currents or wave actions often influence the open water systems and zoospores are migrated from one site to other. However, the closed water systems are with limited diversity, which remains unchanged for a longer period till fluctuations in physicochemical parameters.

213