Survival of most of the nematodes and their abundance will be noticed at 15

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1 DISCUSSION Identification of plant parasitic nematodes is the basic objective of this work has been made based on combined analysis of both classical and traditional taxonomy based on morphology and molecular taxonomy based on ITS of ribosomal DNA of test organism. Hirschmanniella oryzae, Xiphinema rivesi, Hoplolaimus indicus, Pratylenchus loosi, Heterodera oryzicola, Criconemoide sp, Helicotylenchus sp, Longidorus elongate, Trichodorus sp and Paratrichodorus minor were identified from soil samples of Rice fields. Meloidogyne incognita, Tylenchorhynchus sp, Trichodorus sp and Paratrichodorus minor were identified from soil samples collected from of Tomato fields. Rotylenchulus reniformis, Trichodorus sp and Paratrichodorus minor were identified from soil samples of Cotton fields. These phytonematode species belongs to three orders 1.Tylenchida, 2. Dorylaimida and 3.Triplonchida. Seven families from first order Tylenchida (Hoplolaimidae, Pratylenchidae, Criconematidae, Rotylenchulidae, Heterodoridae, Meloidogynidae and Dolichodoridae), two families (Longidoridae and Xiphinematidae) from second order Dorylaimida, and one family (Trichodoridae) from third order Triplonchida. Our results were verified with classical morphology based classification of Chitwood (1958) to understand the taxonomic position of nematodes identified from Rice, Tomato and Cotton crop fields. Thus our results are confirmed as correct and in tune with classical classification and taxonomic position of nematodes as there are no differences. These plant parasitic nematode infections were reported by earlier researchers also in various crops that are in our study (Prasad et al, 1985; 1992; 2009). In India, M. graminicola is the dominant species infecting rice. Meloidogyne sp infecting paddy, tomato and cotton was reported by Gaur et al., (1993). Ditylenchus angustus and Meloidogyne sp nematodes are major pests of deep water rice (Cox and Rahman, 1980). In irrigated rice, infections by Hirschmanniella sp. and Aphelenchoides besseyi are common. However, in upland agro ecosystem rice is infested by Meloidogyne and Pratylenchus species (Prasad and Somasekhar, 2009). This confirms that habitat variations effects nematode diversity (Chandel et al, 2002). Plant parasitic nematode was also reported on irrigated rice in Andhra Pradesh (Sharma and Prasad, 1995) and Karnataka (Prasad et al., 2001). Occurrence of M. triticoryzae was reported from Delhi, Uttar Pradesh and Haryana (Gaur et al., 1993). H. oryzicola is widely 81

2 distributed in Kerala, India (Raveendran et al, 1976; Rao and Jayaprakash, 1977; Venkitesan, 1979; Kuriyan, 1985; Charles and Venkitesan, 1990). Koshy et al. (1987) recorded Meloidogyne plant parasitic nematode on banana in Goa. Gupta et al. (1977) reported occurrence of a cyst nematode in paddy fields in Bankura and Burdwan in West Bengal. H. skohensis, was reported from rice and wheat fields of Kangra valley in Himachal Pradesh (Kaushal et al., 2000). Lesion nematodes Pratylecnhus species are reported on rice from many countries. About ten species of Pratylenchus were reported on rice and among them P. zeae and P. indicus are most common in rice. From India, Pratylenchus sp. particularly P. indicus and P. zeae have been recorded on rice in Andhra Pradesh, Assam, Gujarat, Kerala, Orissa, Madhya Pradesh, Rajasthan, Uttar Pradesh and West Bengal (Prasad et al., 1987). Several species of Hirschmanniella have been reported in association with irrigated rice all over the world. In India only two species i.e. H. oryzae and H. mucronata are resent and they are dominant species that infect rice crop. In several ectoparasitic nematodes, Aphelenchus avenae Bastian, 1965; Helicotylenchus sp. Steiner, 1945 ; Longidorus elongatus Thorne and Swanger,1936; Criconemoides; Tylenchorhynchus sp.; T. annulatus Golden,1971; Xiphenema elitum Khan et al., 1976; X. insigne Loos, 1949 and X. arbum Siddiqi,1964 were found to coexist along with the endoparasitic nematodes in the rice rhizosphere (Prasad et al., 1987). Meloidogyne incognita, M. javanica and the first report of M. floridensis were reported on tomato crop in Florida (Church, 2005). Since the host range is likely different from other Meloidogyne sp. (Taylor et al., 1982; Church, 2005). Meloidogyne sp, Rotylenchulus reniformis, Globodera rostochiensis and several ectoparasitic nematodes were known to attack tomato in many different parts of the world and it was reported earlier by Taylor (1967) and Dropkin (1980). Rotylenchulus reniformis is widely distributed in all cotton grown regions of India (Gulsar Banu, 2007). Rotylenchulus reniformis was first described from Hawaii, USA and is widespread in the tropics and subtropics (Stephen et al., 1991). About 19 Genera of plant parasitic nematodes are reported in cotton crop and in world until today (Nandini Gokte- Narkhedkar et al, 2002). Of these, the most important generic species in Indian context is Rotylenchulus reniformis (Reniform nematode), Meloidogyne incognita, Hoplolaimus sp. and Pratylenchus sp. The Rotylenchulus reniformis (Reniform nematode) has been recorded to be the key nematode species on 82

3 cotton in Central and Southern India while in Northern cotton-growing areas, the Meloidogyne incognita (Root knot nematode) is dominant (Nandini Gokte et al, 2002). The Changes in root growth of plants directly effect the root feeding nematodes and its diversity (Yeates, 1987; Verschoor et al., 2001). It also effects the plant parasitic nematode community structure. Eight species of plant parasitic nematodes were isolated from rice fields of red clay soil and roots. Hirschmaniella, Hoplolaimus and Pratylenchus sp were the most predominant nematodes showing highest population density from all soil and root samples collected from rice fields (Zin Thu Zar Maung et al 2010). Through our studies we conclude that the soil physicochemical and biological conditions of the rice field top soil layers, before flooding and during the draining period they have more abundance of plant parasitic nematodes. Other reports on rice field or wetland nematode community structures are scarce (Neher et al., 2005). The studies plant parasitic nematodes in rice, tomato and cotton field and their soil samples speaks about the sequential changes of nematodes, generally give a base for life and fundamental support the biodiversity of other animals and helps in the formation of food chains and food webs at rhizosphere, similar concepts were earlier proposed by various researchers (Beier et al., 2003; Ajah et al 2006; Muschiol et al., 2008). It also facilitates to maintain free living nematode populations for improving soil health conditions our studies are also important to understand and predict how seasonal changes affect the nematode population and density. Xiphinema rivesi, Trichodorus sp and Paratrichodorus minor are the most predominant nematodes showing highest population density from all soil and root samples collected from cotton and tomato crops. The largest numbers of plant parasitic nematodes found in the soil at a depth of 0-20cms only. Generally more than 70% of the rice root system is spread between 0-10 cms of upper soil layer and root length significantly decreased with depth. Hence our present finding is supported by root zone area and the plant parasitic nematodes abundance was more at depth 0-20 cm of soil is a correct observation. The largest numbers of plant parasitic nematodes were found in the soil depth of 0-20cms. In this, different types of genera of nematodes were found i.e. Pratylenchus, Tylenchus, Heterodera and Aphelenchoides and then were found at depth of 0-30cms. Similar findings were made earlier workers 83

4 Yeates et al; Some of the earlier workers reported that these nematodes numbers also decreased along with the increase of depth Verschoor et al; However, some families of Nematodes like Helicotylenchus sp, Trichodorus and Paratrichodorus sp were found at the deeper soil layers and Meloidogyne sps, Hoplolaimus sps and Criconemoides were also found in roots. Survival of most of the nematodes and their abundance will be noticed at 15 o C to 30 o C temperature (Ferris, 1985; Kimpinski, 1985). The nematodes become inactive when the temperatures range is below 0 o C to 15 o C. At high temperatures that range between 30 o C to 40 o C nematodes become inactive (de Leij et al, 1992). In our studies we noticed that less number of nematodes were recorded in summer due to high temperature and these observations are in tune with the statement given by de Leiz et al., (1992). In case of some nematodes, cold tolerance is noticed and it has mainly been divided into 1. Frigid tolerance and 2. Frigid avoidance (Grewal et al., 1994). The mechanism of frigid tolerance of nematodes is achieved by survival of nematodes under freezing conditions with water freezes only extra cellularly and which in turn finally results in frigid tolerance. This phenomenon is more widespread among nematodes than frigid avoidance (Shapiro-Ilan DI et al., 2006). Frigid avoidance means that nematodes can avoid the formation of ice in their body fluids by means of getting super cooling point down (Dai 2006). Nematodes cold tolerance varies with the environmental changes and thus they may adapt to the most useful and optimized strategies (Whartonet al, 2005). The geographical distribution, activity and life cycles of nematodes depend on the level of water in the soil (Ravi Chandra, 2008). Although some encysted larval stages of Hirschmaniella sps can survive for eight months under high moisture level, the viability of cyst decline rapidly. Excessive moisture leads to the decline of nematode populations (Hollis and Fielding, 1958), Hollis and Fielding also reported that, there is an inverse correlation with the amount of flooding and rainfall. Significant changes in population density are often associated with the periods of rainfall. Parris (1948) considered that root knot nematode activity increased with the rainfall as in the case of Ditylenchus dipsaci. The influence of soil moisture is evident in the relationship between rainfall and nematode population fluctuations (Minton et al, 1960). Minton el al., (1960) found this correlation in the case of the migratory plant parasite Hoplolaimus sp. 84

5 Many studies were made between phytonematodes diversity, its population and the soil texture that contain (silt, sand and clay) (Kohne et al, 2009). Soil texture was correlated with nematode community structure diversity by Porazinska et al., (1998). A study of the plant parasitic nematode diversity was carried out at the three field sites selected in the present study. Most of these phenomenons were common to both the paddy and tomato field soils with a few minor exceptions. But cotton fields shows some major difference when compare to paddy and tomato. The most common and abundant nematode is Pratylenchus sp, in all the three crops and their respective sites. This may be because of one common factor in that is the soil texture being basically clay with degrees of variation in the percentage of silt and sand. Based on our recordings we conclude that the number of genera in a soil habitat is having a direct relationship to its biodiversity. Similar opinion was expressed earlier by Ou et al., (2005). Changes in soil environmental conditions depend on soil type, its fertility, aeration, water availability etc. has a direct relation with diversity of nematode and its communities. It was noticed in our studies Nematode genus richness varies across soil ecosystems and an abundant food source at the rhizosphere accommodates great richness (Machia et al., 2003). At the same time, long-term anthropogenic disturbances such as tillage, especially in the fields, exerted great effects on soil nematode communities. Soil physical characteristics, different nutrients and elements influence the occurrence, distribution and population dynamics of nematodes (Mcsorley, 1998; Wang et al., 2004). In any organism the nucleotide base sequence is the primary source of biological variation (Powers et al., 1997). A wide range of molecular markers are now available for the analysis of this genetic variation and many of them can be used for taxonomic and diagnostic purposes in nematology (Powers and Fleming, 1998). Molecular methods for diversity assessment have been very helpful and already aided in understanding of other groups of organisms that are difficult or impossible to study and identify by any other means (Floyd et al., 2002). DNA barcoding method DNA sequences as markers for taxonomic identification and helps in biodiversity surveys (Hebert et al., 2003). The techniques what we used especially universal primers are fundamentally target microbial genes have significant advantages, e.g., convenience, high-throughput, and considerable savings in time (Puitika et al., 2007); It is a quicker and more efficient way of studying nematode diversity than traditional taxonomic 85

6 methods, which depend on morphological criteria (Floyd et al., 2002). Knowledge of genetic diversity in the case of plant-parasitic nematodes is essential in breeding plants with high resistance in host plants an ecologically very important method of pest nematode control (Hahn et al., 1994; Roberts, 2002). This molecular technology is an important forward step in enabling detection of taxa increased in all the beings and faster result time and wider adoption by technicians who are almost all skilled modern molecular biological techniques. In this, we were used a single universal forward primer and reverse primers set Nem_18S_F/rDNA1, Nem_26S_R /rdna2 to amplify all the target species of single nematode in a single tube, which was formed successful PCR bands on the agarose gel for the tested nematode samples (Fig 3.3). The use of multiplex reactions intensity bands in the PCR products helped to detect species of nematodes. Similarly, Floyd et al, (2005) also noticed and reported that the sensitivity of universal (Forward and Reverse) primer set gives it the ability to identify a mixed nematode population when only one nematode individual is taken for study. Hence, we have used above universal primers and got ITS gene sequences of individual plant parasitic nematodes. As many earlier researchers used them and got good results (Vrain et al.,1992). The ITS sequence that we got had considerable size variation in the ITS gene region i.e. 789bp size of ITS gene in Hirscmaniella oryzae, 896bp size in Xiphinema sp, 972bp size in Xiphinema sp, 802bp size in Trichodorus sp, 882bp size in Rotylenchulus reniformis, 899bp size of Trichodorus sp and 883bp size of Paratrichodorus sp. Powers et al., (1997) observed that the same nematodes genus and species that have similar ITS size length variation i.e. Heterodera, Globodera, Ditylenchus, Belonolaimus, Tricodorus and Hoplolaimus. Berrya et al., (2008) also took the help of ITS gene sequence in the detection of root-knot nematode, Meloidogyne sps, lesion nematode, Pratylenchus sps and dagger nematode, Xiphinema sps from parasites of sugarcane field crops. Thus our studies are in tune with other researchers who has employed both universal primers for isolation of ITS gene sequence are also used them for nematode identification (Vrain et al., 1992; Power et al., 1997). Earlier many researchers, Saeki et al (2003) observed that the detection of plant parasitic nematodes, Meloidogyne incognita and Pratylenchus coffeae from pure 86

7 culture of nematodes by using universal and specific primers. Zijlstra et al. (2000) and Wishart et al. (2002) observed that to identify different Meloidogyne sps from individual nematodes based on Internal Transcribed Spacer using different speciesspecific primers is highly useful. Subbotin et al. (2001) could identify Heterodera glycines in a mixture of nematodes that were found in case of soybean cyst nematode by using duplex PCR method. Oliveira et al. (2005) and Wang et al. (2004) distinguished four different species of Xiphinema and Trichodorida sps based on ribosomal DNA ITS gene sequences. However, considerable size variation in the ITS1 can occur. ITS1 can range from 870 bp to 1,354 bp in a nematode species i.e Xiphinema sp (Ye et al., 2004) and 791 and 2,572 bp in Coccinellidae beetles. It is due to the presence of repeated elements in the ITS1 sequences; these repeat elements showed variation in occurrence of frequency among species (Von der Schulenburg et al., 2001). Similar trends and companions were observed in our results with Xiphinema sp, 972bp size in Xiphinema sp, 802bp size in Trichodorus sp, 882bp amplifying better than X. elongatum and M. javanica, where we noticed variation in size of ITS gene sequence between different species of nematodes. In addition to the competition effect, it was also found that the Tm values of Trichodorus sp, X. elongatum and M. javanica were almost identical. Phylogenetic analyses: Several studies have shown that the ITS of rrna gene in plant parasitic nematode taxa might be too variable in length to allow construction of a plausible alignment assuring the reliability of subsequent phylogenetic analysis (Ferris et al. 1993; Subbotin et al.2001). By using our experimental genetic data we determined the molecular phylogeny of different nematode species based on sequence of ribosomal internal transcribed spacer (ITS) region (Strimmer and Haeseler, 1996; Swofford, 2001; Ronquist and Huelsenbeck 2003; Kumar et al., 2004). Phylogeny is the history of organism lineage as they change through time. It implies that different species arise from previous forms via decent and that all organisms from the smallest microbes to the largest plants and vertebrates were connected by the passage by genes along the branches of the phylogenetic tree that links all the life form (Ma et al., 2008). 87

8 In this study we determined how the families have been derived during evolution. The sequencing of nucleic acids provides a powerful approach in measuring evolutionary relationships. ITS region of rdna sequence is isolated from Hirschmanniella oryzae and the nematode is collected from Rice field. The quality of the sequence electropherogram was very high and on par with earlier works. It is approximately made up of 789 bp at the ITS region were sequenced using primer pair Nem_18S_F/rDNA1, Nem_26S_R /rdna2 (Vrain et al., 1992). Our analyses indicate a monophyletic grouping of the Tylenchina, including Steinernema and Hirschmanniella oryzae. The ITS region of Hirschmanniella oryzae sequence showed high similarity with the same family of Pratylenchus sp (GI ) of Italy and with Hirschmanniella sp (GI ) of USA. ITS region of rdna sequence Hirschmanniella oryzae and the nematode is isolated from Rice field. Phylogenetic analyses of Hirschmanniella oryzae ITS region of rdna sequence depicted similarity with three distinct nematode families Pratylenchidae, Dolichodoridae and Hoplolaimidae as all these families were belong to the same order of Tylenchida. Pratylenchus sp of Italy and with Hirschmanniella sp of USA with our sequence was formed a clade in different analyses of Neighbor Joining and Maximum parsimony with different bootstrap values (Figure.3.6a&b). similar observations were also made by many other researchers (Baldwin et al., 1995).The present phylogenetic hypothesis, based on expanded taxon sampling and more adequate phylogenetic analysis, agrees in general with the Tylenchina framework as proposed by Bert et al., (2008). NJ topology is based on phenetic method and this method measures the pair wise differences among sequences selected for the present study and the relationship is based on a distance matrix and its calculated values (Swofford et al., 2001). This matrix is later used to construct the phylogenetic tree, This algorithm construct a tree in stepwise manner i.e the two most similar sequences are grouped together and then the next most similar sequence is added to it and so on (Lindberg et al., 2011). Similar observations were noticed in our phylogenetic tree constructed based on our experimental results. Earlier workers, Adams et al., (1998), Pamjav et al., (1999) also reported similar opinion through their findings. 88

9 MP is a cladistic method and it is character based method. In this method, all possible topologies are evaluated and one that optimizes the evolution is chosen and made as the correct tree. Cladistic methods emphasize more on the evolutionary origin of species than the phenotypic relationships (Swofford et al., 2001). It assumes that a set of sequences descended from a common ancestor by mutation and selective processes without hybridization or other by horizontal gene transfers. These methods are best in comparing phylogenetic tree constructed for the study with other trees to determine different lines of evolution (Brinkman et al., 2011). This is the reason NJ and MP topologies were having different bootstrap replicate values and may shows variations. Hence, we have selected both types i.e. NJ and MP for the analysis of our data. The amplification of the ITS region using primer pair Nem_18S_F/rDNA1, Nem_26S_R /rdna2 yielded one distinct amplicon approximately having 896 bp in size for Xiphenema insigne. In our experiments Xiphenema insigne ITS sequence showed high similarity with Xiphenema sps (G ), Trichodorus sp (GI ) of UK and Longidorus sp (GI ) of Scotland and Xiphinema sp along with above mentioned species formed a clade. Xiphinema sp belong to family of Xiphinematidae and Longidorus sp is from family of Longidiridae. But these two families belong to the same order of Dorylaimida. Whereas Trichodorus sp are belongs to the family of Trichodoridae and from different order i.e. Triplonchida. Even though these species belong to different orders and families they are showing congruence because these species having similar function of common nature probably, i.e. they are transmitting virus through nematodes to the plants. From this we can conclude that even though there is a variation occurs at structural level of an organism, if the function is same, it may be expressed at ITS level In NJ and MP analyses it has showed high similarity with above species with different bootstrap values (Figure.3.9a&b). A comparative study of host plant and phytonematode was carried out to understand the relevance of ITS and its expression. ITS sequence of Hirschmanniella oryzae and Xiphinema insigne and Rice plant ITS gene sequences (collected from Genbank) were applied to the Cluster X2 to compare them with their similarities in the Genbank from NCBI. It showed a comparison and commonness between the 89

10 resulting sequence data of ITS region. Using Cluster X2 (Multiple sequence alignment), we have done phylogenetic analyses based on its results obtained earlier. In this, Maximum Parsimony and Neighbor joining topologies congruence between Hirschmanniella oryzae, Xiphinema insigne and Paratrichodorus sp of ITS sequence and the selected host plant Rice ITS gene sequences were comparative studies were made. Hirschmanniella oryzae ITS gene sequence and Host plant Oryza nipponbares (GI ) and Oryza jemont ( GI ) species of China country formed as one clade and showing similarity of 98 bootstrap replicate values and Paratrichodorus sp ITS sequence showed similarity with host Rice plant (Oryza sativa GI ) sequence of India, Oryza longistaminata (GI ) China and Oryza rufipogon (GI ) Australia they had different bootstrap replicate values in different analyses of NJ and MP. Xiphinema insigne ITS sequences did not resolved much congruence with host Rice plant sequences (Figure.3.11a&b). Trichodorus sp was isolated from tomato field and analyzed it s ITS sequence. In phylogenetic analyses studies, this sequence showed a high similarity with Trichodorus sp (GI ) of China. This sequence and our Trichodorus sp ITS gene sequence formed a single clade. In these two analyses of Neighbor Joining and Maximum parsimony our nematode ITS sequence formed as a single clade with different bootstrap replicate values. (Figure.3.14a&b). Trichodorus sp of ITS sequence and Tomato plant ITS gene sequences(collected from Genbank from NCBI) were applied to the Cluster X2 to compare them with their similarities. It showed a comparison and commonness between the resulting sequence data of ITS region. Using Cluster X2 (Multiple sequence alignment) we got results and we have done based on these results further phylogenetic analyses. In this Maximum Parsimony and Neighbor joining topologies, congruence the occurred between Trichodorus sp of ITS sequence and Tomato plant ITS gene sequences. Trichodorus sp ITS sequences showed similarity with host Tomato plant sequence of China, India (Lycopersicum esculentum GI ), UK (Lycopersicum esculentum GI ), Japan (Lycopersicum esculentum GI ) and Palestine (Solanum lycopersicum GI ) countries with different bootstrap replicates and still formed as one group in tree (Figure.3.16a & b) 90

11 Plant parasitic nematode Rotylenchulus reniformis was isolated from Cotton field and its ITS sequence was obtained. In phylogenetic analyses Rotylenchulus reniformis showed similarities with Rotylenchulus reniformis (GI ) of USA. In these analyses our sequence formed a clade with Rotylenchulus reniformis of USA. These species belongs to the same family Rotylenchulidae. It showed high similarity in these analyses of Neighbor Joining and Maximum parsimony and ITS sequence had formed a clade with different bootstrap replicate values. (Figure.3.19a&b). The phytonematode, Trichodorus sp was isolated from Cotton field and it s ITS sequence was obtained. In phylogenetic analyses Trichodorus sp showed similarities with Trichodorus sp of china. In these analyses our sequence formed the same clade with Trichodorus sp of China country. These species belongs to the same family of Trichodoridae and It showed high similarity with above species. There was no difference in Neighbor Joining and Maximum parsimony analyses and our sequence formed a clade with different bootstrap replicate values. ITS sequence of Paratrichodorus sp was isolated from Cotton field in our experiments. In phylogenetic analyses Paratrichodorus sp showed similarities with Paratrichodorus sp (GI ) of China and Rotylenchulus reniformis (GI ) of USA. In these Paratrichodorus minor from same Trichodoridae and Rotylenchulus reniformis from different family of Rotylenchulidae belongs to different order of Tylenchida. Even though these species were showed high similarity with above species, because Rotylenchulus reniformis species were host specific plant parasitic nematodes. There was no difference in these analyses NJ and MP of our sequence that was formed as a single clade with different bootstrap replicate values (Figure.3.22a&b). Rotylenchulus reniformis, Trichodorus sp and Paratrichodorus sp of ITS sequence and Cotton plant ITS gene sequences (collected from Genbank) were applied to the Cluster X2 to compare them with their similarities in the Genbank from NCBI. It showed a comparison between the resulting sequence data of ITS region. Using Cluster X2 (Multiple sequence alignment) obtained results, we have done phylogenetic analyses as per Thompson et al., (1997). In this Neighbor joining topologies, congruence between Trichodorus sp, Paratrichodorus sp Rotylenchulus 91

12 reniformis 1 and 2 of ITS gene sequence and Cotton plant ITS gene sequences. Trichodorus sp, Paratrichodorus sp Rotylenchulus reniformis 1 and 2 ITS gene sequences showed similarity with host Cotton plant different species (Gossypium herbaceum and Gossypium hirsutum) sequence of India and Gossypium raimondii (GI ) USA countries, these were formed as sister nodes and with Gossypium capitis GI of Brazil the above species were formed as one group with different bootstrap replicates values. (Figure. 3.24a&b) Blouin (2002) and Vilas et al. (2005) showed in the previous paper that mitochondrial genes and ITS gene of rdna are the best choices to search for potential cryptic species when using sequence data on small numbers of individuals in nematodes, trematodes and cestodes. Collins and Paskewitz (1996) identified cryptic species as those groups of closely related species that are difficult or impossible to distinguish by morphological traits. Anderson et al. (1998) reported that nematodes tend to be having much conserved morphologically but molecular techniques indicate that many presumed mono-specific species actually consist of several cryptic species. The concept of cryptic species has been proven in many organisms, such as the insect Astraptes fulgerator (Hebert et al., 2004), plant Grimmia laevigata (Fernandez et al., 2006), fungi Amanita muscaria (Geml et al., 2006) and Aspergillus fumigatus (Pringle et al., 2005), slugs, Arion sp (Pinceel et al., 2005), and in birds Phylloscopus sp (Olsson et al., 2005), and it is of common occurrence in nature. Adams et al. (1999) suggested and said that ITS1-rDNA provides useful phylogenetic characters to resolve phylogenetic relations among closely related sister taxa (Joyce et al., 1994, Adams et al., 1999). The present results shows and also proves again that Phylum Nematoda occur in a wide spectrum of ecological habitats and natural histories ranging from deep sea sediments to arid deserts, from interstitial bacterivores to obligate parasites with multiple intermediate hosts (Tchesunov et al., 1995; Schierenberg, 2005). In this classification basic clades are covered, but the class Enoplea has not covered the full ecological range the phylum. This explains that the evolution of ecological adaptations within each nematode taxon was depended on the rate of change in genes and ecophysiology (Paul De Ley. 2005). Phylum Nemata contains 2000 described species and it is divided into two classes, Adenophorea and Secernentea. Class Adenophorea consists of two subclasses 1. Enoplia, 2. Chromadoria and eleven 92

13 orders. Class Secernentea is divided into three subclasses 1. Rhabditia, 2. Spiruria and 3.Diplogasteria, and eight orders (Ley and Blaxter, 2002), between these 19 orders of phylum Nemata, seven orders include nematodes that are parasites or associated to invertebrate animals such as Annelida, Mollusca and Arthropoda (Cobb, 1914; Platt, 1994). Fig.No.3.25: Summarized SSU phylogeny of Nematoda with example taxa, ecological range and higher classification (adapted from De Ley & Blaxter, 2002). Compared with plant parasitic nematodes of ITS gene Sequence our Results. 93

14 Our results have been compared with modern phylogeny based classification of De Ley and Blaxter The plant parasitic nematodes are Hirschmanniella oryzae and Rotylenchulus reniformis belong to the Chromadorea class. Xiphinema rivesi belong to the Dorylaimia class. Paratrichodorus minor and Trichodorus sp species belongs to Enoplea class. In this classification it is noticed that in Chromadorea, a number of clades have arisen. These clades are classified as separate orders Chromadorida, Desmodorida, Monhystrida, araeolaimida, Plectida and Rhabditida. SSU (small sub unit) phylogenies place this taxon at the crown of Chromadoria and as sister group to the order Plectida. For this reason, De Ley and Blaxter (2002, 2004) classified it instead as order Rhabditida, thereby greatly expanding the contents of this taxon compared to all previous systems. Six clades have formed in Rhabditida and classified as Rhabditinia, Tylenchina, Myolaimina and Spirurina. Hirschmanniella oryzae and Rotylenchulus reniformis based on their rdna sequences which were coming under this sub order of Tylenchina, order of Rhabditida and class of Choromodorea (Figure. No.3.25). rdna gene sequence of Xiphinema sp belongs to the Dorylaimia class. In the Dorylaimia class five clades were formed and classified as Dorylaimida, Mermithida, Monochida, Dioctophymatida and Trichinellida. Dorylaimia has much greater past diversity and within it has more successful surviving clade in the order Dorylaimida. This includes many species of large predators, omnivores, as well as the plantparasitic family Longidoridae, of which some species transmit plant viruses. ITS sequences of Paratrichodorus minor and Trichodorus sp species belong to the Enoplea class. In class Enoplia organism especially live in diverse marine habitats, but multiple lineages are also found in freshwater sediments and/or moist soils (Ref). One of these lineages includes marine, freshwater and terrestrial taxa, suggesting that early Enoplia were characterized by much greater osmotic tolerance than early Dorylaimia (Huan et al., 2009)). Enoplia are interesting phylogenetically especially because of the occurrence of features that are presumably ancestral within nematodes, such as a highly indeterminate mode of development (Justine, 2002) and retention of the nuclear envelope in mature spermatozoa (Lee, 2002). One enoplian order that has clearly undergone extensive evolution in soils is order Triplonchida, which includes plant nematode parasites such as Trichodorus. These are convergent with dorylaims in a number of respects, e.g., they also have a protrusible tooth for feeding (called an 94

15 onchiostyle) and several species are known to act as virus vectors. Molecular marker data have shown that the triplonchid clade includes free living nematodes such as Tobrilus and Prismatolaimus, even though these are morphologically quite divergent from trichodorids. However, nobody could explain the reason. Order Chromadoria has undergone a wide range of evolutionary modifications in cuticular structure hence resulting in strikingly decorative ornamentations. Free living Chromadoria are on average noticeably smaller than Enoplia and Dorylaimia, correlated with a greater preponderance of rapidly reproducing bacterial feeders. Some of these species are among the smallest known predatory nematodes. The most distant relatives of C. elegans that can presently be efficiently cultured with C. elegans-like methods are certain bacterivorous species of the orders Monhysterida and Plectida (De Ley and Mundo-Ocampo, 2004). Sub order Tylenchina has not only given rise to zoo parasitic species, but also radiated into most diverse group of plant parasites and fungal feeders among the nematodes. These tylenchs are equipped with a protrusible stomatosylet that is convergent with, but clearly different from, the odontostyle of dorylaims and the onchiostyle of trichodorids (Paratrichodorus sp and Trichodorus sp). SSU sequences have confirmed the previously unpopular hypothesis that their closest relatives are morphologically very dissimilar Cephalobs (Siddiqi, 1980). Both groups are therefore now united in the suborder Tylenchina, which is another example of a more drastic change induced by the new phylogenies. Analogous to zoo parasitic Rhabditida order, the life cycle of some parasitic tylenchs includes an infective juvenile stage. However, most tylenchs and cephalobs do not have one single dispersive and enduring stage, but are instead capable of surviving harsh conditions throughout most of their life cycle. This has enabled them to compete very successfully with dorylaims in even the driest and coldest terrestrial environments. The major lineages within Tylenchina are the panagrolaims, a less clearly circumscribed amalgam of free living opportunists, fermentation specialists, insect pathogens and animal parasites. Parthenogenesis appears to be much more common in Tylenchina and Goldstein et al. (1998) speculated that this could in fact be linked to the mechanism of axis determination. 95

16 The evolution of ecological adaptations within each nematode taxa was forced by limitations on the rates of change in genes and ecophysiology. Reconstruction of the phylogeny of ITS - rdna gene sequences using information from several genetic loci to avoid problems of the non-correspondence between gene and species phylogeny, and using a larger number of representative isolates for each species. Adams et al. (1999) suggested that ITS-rDNA provides useful phylogenetic characters to resolve phylogenetic relations among closely related sister taxa (Joyce et al., 1994, Adams et al., 1999). It is also conclude that the results presented above describe the development of a simple and efficient method to identify the plant parasitic nematodes based upon the amplification of DNA through PCR. The results described have provided evidence that the primer sets Nem_18S_F/rDNA1, Nem_26S_R /rdna2 (Vrain et al., 1992) are good for detecting and identifying the tested nematodes and possibly also several other nematode species belonging to different orders and families. Molecular methods were applied to identify the plant parasitic nematodes. This molecular study used in dealing with nematodes DNA, which helps in future the studies of nematode biodiversity and in developing methods to control plant-parasitic nematodes without damaging beneficial organisms in the soil, depending on their genetic diversity. Our results were varified with classical morphology based classification of Chitwood (1958) and SSU DNA based classification of De Ley & Blaxter, (2002), to see the taxonomic position of nematodes identified and recorded from Rice, Tomato and Cotton crop fields. there were no differences or deviations from classical classification and taxonomic position of nematodes and they are totally matching De Ley & Blaxter, Overall, the ITS region becomes an important taxonomic feature in nematode identification. ITS versatility, specificity, effort of experimental manipulation, and growing ITS databases should accelerate its application in nematology. Its usefulness, however, will hinge on a careful evaluation of the relationship between ITS genetic variation and traditional taxonomic positions in the coming days. 96

17 Finally, we conclude that ITS based molecular phylogenic studies totally matched with classic taxonomy and traditional classification. Hence one can use ITS as a tool for both purposes. Moreover, even minor variation that one noticed at ITS gene sequence may help in detecting ecotypes or the future evolutionary predictions with reference to that particular nematode species. Most interesting finding in our studies with reference to plant parasitic nematode and its host plant suggested that ITS gene sequence helps in identifying nematode pest status and host plant susceptibility. Thus, these studies will help us to develop the Integrated Pest Management (IPM) package for a specific host plant and its variety. At the same time ITS supports Quarantine technology with reference to agro product export and import. 97