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Rice is regarded as one of the most staple food for a major part of the world's population, especially in Asia, Latin America, the Middle East, and the West Indies. Around 90 percent of total rice is produced in Asia alone that makes it as an Asian crop (Mohanty, 2009). Other then Asia, Rice is also becoming an important cereal food and supporting lives of many people in Central America, Europe and Africa. About 50 million tons of rough rice is to be increased per year by 2015 to meet world's demand where the projected demand for Asia is an additional 38 million tons (Pandey, 2008; Mohanty, 2009). Rice is also the most important food grain with respect to human nutrition and energy intake, which provides more than one fifth of the calories consumed worldwide. Interestingly, India is the world's second largest producer of white rice accounting for about 80% of all world rice production. Rice in India is the most dominant crop, and a staple food for the people of the eastern and southern parts of India. The earliest remains of cultivated rice was found in the north and west parts of India and date from around 2000 BC. In India, Rice production is an important part of the national economy. According to United Nations report, Global production of rough rice will rise to a record high of 710 million metric tons in 2010. Rice production in India which is the world's second-largest grower and consumer is expected to increase 15 percent to 151 million tons according to the UN Food and Agriculture Organization report.
With the increasing population, demands for food have been increasing. Increasing the productivity of food by decreasing the severity of biotic and abiotic factor will be a challenging task. Plant parasitic nematodes are the hidden enemy of many crops and are one of the many groups of harmful organism which depend on plants for their survival and reproduction. Plants parasitic nematode can cause damage to almost all kinds of crops but due to their subterranean habit and microscopic size they remain invisible to the naked eye. The estimated annual yield losses due to plant parasitic nematodes in the world's major crops is recorded about 12.3% and 14% in the developing countries (Sasser, 1987) and Worldwide losses to rice only from nematodes are estimated at around 10%. In India, recent studies shows that plant parasitic nematode is responsible for both the quantitatively and qualitatively yield losses amounting around Rs.240 billion every year (Khan, 2008). Beside this direct damage, these nematodes also serve as predisposing agents in development of disease complexes in association with agents like fungi, bacteria and virus.
Root know nematodes, Meloidogyne graminicola are known to cause substantial decrease in rice yield. M. graminicola is also reported from several places as having significant adverse effects on yields in most types of rice. The important plant parasitic nematode species varies with respect to geographical region and agro-ecological conditions. In India, Among the Meloidogyne species that occurs widely on rice, Meloidogyne graminicola is reported as the main species present. Meloidogyne graminicola is considered to be a major constraint in upland rice production in India with a range of biotypes that seriously poses problems for cultivars. M. graminicola has increasing importance on lowland rice farmimg systems where water conservation results in intermittent flooding of fields. Yield losses in rice caused by Meloidogyne graminicola ranges from 20-80% and about 11-73% in upland and in intermittently flooded conditions, respectively (De Waele & Elsen, 2007). Tolerance to plant parasitic nematodes is also dependent on water management practices. The changes in climatic condition, especially water scarcity helps researchers think to develop production technologies for cultivation of rice with less water condition which may increase activity of M. graminicola (De Waele & Elsen, 2007). Rice varieties showing resistance to nematodes may represent the practical and effective means of managing these nematodes in small scale rice farming systems. The absence of classical plant breeding solutions and the limitations of chemical treatment also represent an excellent opportunity for biotechnological applications. Because of the lacking in knowledge, the nematode problems are not recognized properly. More knowledge on the interaction between rice plant and nematodes is important and necessary to get the nematode problem under control.
The most evolutionary advanced adaptations for plant parasitism by plant parasitic nematodes are the products called parasitism genes (Gao et al., 2002). The Parasitism proteins are secreted by the nematode and play a direct role in plant parasitism. These Parasitism proteins secretions mostly originate from the pharyngeal gland cells, but secretions from the chemosensory amphids might also be important (Davis et al., 2004). Many Parasitism genes has already been reported from plant parasitic nematodes including Globodera rostochiensis, Meloidogyne incognita, Heterodera schachtii etc. However, surprisingly none of this parasitism gene from Meloidogyne graminicola has not been reported so far. With the above background, the aims of the present study are
To Clone the putative plant cell wall degrading enzymes from the rice nematode Meloidogyne graminicola
To analysed the cloned putative plant cell wall degrading enzymes from the rice nematode Meloidogyne graminicola by application of various molecular tools and techniques.
The experimental work will involves the following step, Firstly, finding out whether these genes are really present in our nematode, Meloidogyne graminicola DNA/RNA , Secondly, functional analyses studies by in situ hybridization to check their expression in the gland cell of the nematode, and Thirdly, to check the expression of the genes in different life stages of Meloidogyne graminicola. In vitro rice plants will also be used in the experiment for culturing Meloidogyne graminicola.
2. LITERATURE REVIEW
2.1.THE HOST PLANT: RICE
Rice (Oryza sativa) is cultivated in about 150 million ha of land worldwide (Bouman et al., 2006) and the major staple food in south-east Asia and also commonly grown in West Africa and South America. Rice feeds about one half of the world's population. The average yield of rice is about 3-4 tons ha-1 (Padgham et al., 2004). Rice is the grain with the second-highest production in the world next to maize and the most important grain with respect to calorie intake and human nutrition thereby providing more than one fifth of the calories consumed worldwide. The cultivation of Rice is well-suited to countries and regions having low labor costs accompanied with high rainfall. Although the parent species of rice are native to regions of South Asia and some parts of Africa, trade and exportation of rice over centuries have made it commonplace in many cultures worldwide. Nearly two thousand varieties of rice are grown throughout the world. The International Rice Research Institute (IRRI), Philippines has more than 83,000 varieties in their gene bank. The differences in these varieties are related to morphology of the plants and grains, ramification, productivity, resistance to falling, as well as resistance and tolerance to biotic factors and non-biotic factors.
2.2. ORIGIN AND HISTORY
Rice cultivation was first noticed in China (Hemu Du region) around 5000 B.C. and in Thailand around 4500 B.C. Later they appeared in Cambodia, Vietnam and southern India. Oryza sativa which is a common Asian rice and known to be originated from the Far East at the foot of the Himalayas. O. sativa japonica grew on the Chinese side and an irrigated rice of temperate zone with medium or short grains, also called round grain and O. sativa indica on the Indian side and irrigated rice of warm tropical zones, with long, thin and flat grains. The majority of the varieties belongs to this species and characterized by its plasticity and taste qualities. Another common species of Africa, Oryza glaberrima, is an annual species and believed to originate from West Africa, covering a vast region that extends from the central Delta of the Niger River to Senegal. The Asian rice, Oryza sativa was adapted to farming system in the Middle East and Mediterranean Europe in an around 800 B.C.
2.3. RICE FARMING SYSTEMS
2.3.1. IRRIGATED RICE
Irrigated rice farming systems accounts for about 55% of the world area under rice cultivation and accounts for 75% of the world total production. Generally, Land for rice cultivation is prepared while wet and water kept held in reservoirs. In Asia, transplanted rice is most common but in some parts, direct seeding is also used. In this system, seeds are pre germinated and grown in wet seed-beds for a period ranging about 2 weeks. Whereas in direct seeding, seeds are frequently pre germinated and can be broadcasted by hand or machine-drilled in puddled soil or even spread over the water by airplane as practice in developed country. In this farming system, Rice production ranges from 5 tons per ha in the rainy season and more than 10 tons per ha in dry season, when adapting modern technologies.
2.3.2. THE RAINFED LOWLAND RICE
The rainfed lowland rice occupies 25% of the total world area under rice cultivation and accounts for 17% of world production just next to irrigated rice. It grows over a compacted soil and in bunded fields that able to retain water between 0-25 centimeters of low level and 25-50 cm of medium level. Rainwater or a local reception tank feeds this non-irrigated rice. The major concern in this type of production system is the risk of drought and unexpected floods.
2.3.3. UPLAND OR DRYLAND RICE (IN MOUNTAINS OR PLATEAUS)
The Upland or dryland rice represents approximately around 13% of the total world area under rice cultivation and accounts for 4% percent of global rice production. In this production system, Land is prepared for planting rice and the rice is dry-seeded. Lack of humidity and normally poor soil is the major problem and thus affecting the crops and yields which are usually very low. This type of ecosystem is common in Brazil, Madagascar, India and other parts of Southeast Asia. In Asia, this production system is mainly observed near the river banks when the level of water goes down at the end of the rainy season.
2.3.4. DEEPWATER OR FLOOD-PRONE RICE
Deepwater or flood-prone rice is common in regions of Southeast Asia including Bangladesh, Thailand, Cambodia, and in West Africa and South America also. In this production system, Water level is 1 to 5 meters deep and supplied by rivers, lakes, tides etc. Usually, seeds are broadcasted in ploughed fields, and unbunded, in regions where the level of water rises quickly after the onset of the monsoon. Rice production in this system is usually low due to droughts and flood and the low production potential of cultivars grown with few inputs. Interestingly, this production system supports the needs of more than 100 million people, most of them living on small family farms and in rural areas.
2.4. PESTS AND DISEASES OF RICE
Rice diseases are mainly caused by fungi, bacteria, viruses, nematodes and mycoplasma-like organisms. Insects, mammals, birds, rodents and weed are also considered as major pests of rice (Jahn et al., 2007; Khiev et al., 2000). Rice sheath blight disease (Rhizoctonia oryzae) occurs throughout the rice-growing areas in tropical, subtropical and temperate countries. This disease causes 20-25 percent yield reduction but can cause 50% yield loss if susceptible cultivars are planted. In tropical Asia, it causes a yield loss upto 6%. Lowland rice in subtropical Asia and temperate is affected by blast, Magnaporthe grisea whereas upland rice in tropical Asia, Latin America, and Africa is affected. Brown spot, Bipolaris oryzae, is distributed in all rice-growing countries worldwide including Asia, Africa and America. In 1945, Sir John Woodhead reported that brown spot was the main cause of the 1942 Bengal famine (cited in Ou 1985). Vidhyasekaran and Ramadoss (1973) also reported that severe infections can reduce yield by 20 - 40%. Yield losses of 14-41% were observed in epidemic area of India whereas 16 - 40% losses estimated in Florida, USA under favorable conditions. Bacterial blight of rice caused by Xanthomonas oryzae pv. oryzae can cause 6-60% yield losses upon infection. Bacterial leaf streak (X. oryzae pv. oryzicola) is also an important rice disease and can reduce grain weight. Rice Tungro disease (RTD) is mainly caused by Rice Tungro Spherical Virus (RTSV) and a Rice Tungro Bacilliform Virus (RTBV) and is also one of the most destructive diseases in South and Southeast Asia. The Disease symptoms are caused mainly by infection by the rice tungro bacilliform virus. Among the pests, Rice stem borer (Scirpophaga incertulas) is one of the major pests of rice which is particularly destructive in Asia, the Middle East and the Mediterranean regions. Plant parasitic nematodes such as M. graminicola, Ditylenchus angustus and H. oryzae are responsible for rice yield losses.
2.5. DEFENSE RESPONSE OF RICE
Pathogens including nematodes, fungi, viruses, bacteria and oomycetes attack plants and deliver virulence factors or effector molecules into the plant cells for causing disease by increasing their virulence. On the other side, plants are capable to protect themselves against this invasion by inducing complex defense mechanisms, such as changes in biochemical, morphological and molecular characteristics including production of antipathogenic compounds, expression of defense related genes and apoptosis (programmed cell death) (Van Loon et al., 2006). Dangl & Jones (2001) described the first defense system of plants, a passive defense, as the formation of physical or chemical barriers against pathogenic attacks which is sufficient to stop most of the microbial entries into the plants. The plant has corresponding resistance genes (R-genes) and pathogen has virulence genes (Avr-genes) in a host-specific defense system. The plant's R-protein encoded by R-gene binds to the corresponding pathogen's Avr-protein and triggers a chain of signal transductions resulting in the activation of inducible defense response and disease resistance. Odjakova & Hadjiivanova (2001) also described that R- and Avr-gene mediated recognition as gene for gene recognition. According to Kiraly et al., 2007; Odjakova & Hadjiivanova (2001); Wojtaszek (1997), in a non host-specific recognition of pathogen of an incompatible interaction, the signal transduction and defense response are activated by the recognition of pathogen or cell wall derived-signal molecules which are called exogenous or endogenous elicitors respectively and are produced during infection. The Mi-gene was first found in Lycopersicon peruvianum, a wild tomato species, is the best known R-gene that gives resistance against infection of Meloidogyne arenaria, M. javanica and M. incognita and has been successfully introduced into modern tomato cultivars. Hussey & Janssen (2002) mentioned resistance genes in wild potato species against several Meloidogyne spp. and many of them had been successfully incorporated into modern potato cultivars and other economically important crops. Bari et al. (2009); Jones & Dangl (2006) reported a 'zigzag' model of plant immune system. In this system, pathogen associated molecular patterns (PAMPs) by host encodes PRRs (pattern recognition receptors) which results in PTI (PAMP triggered immunity). Successful pathogens secrete effectors that suppress PTI. Thus, disease is induced by pathogen resulted in effector triggered susceptibility (ETS). Plants recognize a given effector and activate effector-triggered immunity (ETI) results in disease resistance. Plant disease resistance is enhanced and pathogen growth is restricted by the activation of PAMP triggered immunity (PTI) or effector-triggered immunity (ETI).
Fig.Â A zigzag model illustrates the quantitative output of the plant immune system.
Source- The plant immune system, Jonathan D. G. Jones & Jeffery L. Dangl. Nature 444, 323-329(16 November 2006) doi:10.1038/nature05286
According to Odjakova & Hadjiivanova (2001), a hypersensitive response (HR) is induced and different defense genes are expressed by the action of those compounds as secondary messengers. Kiraly et al. (2007) reported that induction of HR was not led by every types of recognition. Zinov'eva et al. (2004) mentioned that after the reaction against plant parasitic nematodes, two types of cell deaths was occurred of which first one was cell necrosis and second type was genetically programmed cell death or apoptosis. Systemic resistance against new infections to other plant parts could be acquired by plant after this primary infection and this phenomenon was described by Durrant & Dong (2004) as SAR (systemic acquired resistance). The defense response of rice to M. graminicola infection has not yet been observed and there is limited information of defense response of rice to other nematodes also.
2.6. CONSTRAINTS FOR RICE PRODUCTION
Rice production is highly affected by various factor including both biotic and abiotic stresses. The biotic factors affecting rice yields are diseases and pests. Economically important diseases of rice include Sheath Blight, Rice ragged stunt and tungro. The fungus Magnaporthe grisea, causes Rice blast which is regarded as the most significant disease affecting rice yield. There is also another fungus, Cochliobolus miyabeanus, which causes brown spot disease in rice. Rice pests are the organisms having the potential to reduce the yield or value of the rice or rice seeds, Jahn et al., (2007) and include weeds, pathogens, insects, rodents, and birds. Among the abiotic stress, Drought is the main factor affecting rice in upland rice and deficiency of essential nutrients also affects rice yields, especially the micronutrient deficiency in upland or aerobic rice while sometimes nutrient toxicity affects plant growth. Soil acidity can also be another problem in upland rice. Nematodes have also been reported to cause serious growth depressions and severe yield losses in rice.
2.7. NEMATODE PROBLEMS IN RICE
More than 35 genera and 200 species (Prot et al., 1994) of nematodes have been reported in rice.These nematodes can be a major constraint to the high yields of rice. In upland rice, Meloidogyne spp. and Pratylenchus spp. are known to cause more damage while In deep water rice, very few nematodes have been reported including M. graminicola, Ditylenchus dipsaci, causing the ufra disease (Prot et al., 1990). Aphelenchoides besseyi, cause white tip disease in rice and found in most ecosystems (Bridge et al., 1990), while other nematodes are not distributed homogeneously across the ecosystems (Prot et al., 1994). The foliar nematode parasites, Aphelenchoides besseyi and Ditylenchus dipsaci, cause visible symptoms in the foliage and hence are detectable easily. In addition to Aphelenchoides besseyi, Hirshmaniella spp., cause major problems in irrigated rice. However, there have been reports of M . graminicola or other Meloidogyne species infesting deep water rice apart from the observations that a species of Meloidogyne, referred to as M. ezigua, occurs in the deep water rice farming region in Thailand (Hashioka, 1963 ; Kanjanasoon, 1962 ; Ou, 1972)
2.8. THE ROOT-KNOT NEMATODES: MELOIDOGYNE GOLDI, 1892
The root-knot nematodes are known to be responsible for billions of dollars of economic loss worldwide commercial crop production each year on over 5,000 host species (Sasser and Freckman, 1987). The most significant factors that determines severity of the disease caused by Root knot nematodes is the ratio of males and females. Females are known to contribute more to root-knot nematode disease than males. Females feed vigorously and for longer period as compared to males, due to the increased energetic demands of producing and laying eggs. Root-knot nematodes are the plant-parasitic nematodes belonging to the genus Meloidogyne. Meloidogyne spp. is considered as one among the most economically damaging genera of plant-parasitic nematodes on horticultural and field crops. It is distributed worldwide, obligate parasites of the roots of thousands of plant species, including monocotyledon and dicotyledon herbaceous and woody plants. Meloidogyne means apple-shaped female. Root-knot nematodes an obligate sedentary endoparasite distributed both in tropics and temperate regions (Dhandaydham et al., 2008). According to description of Eisenback & Triantaphyllou (1991), root knot nematodes, Meloidogyne spp., are sexually dimorphic; females are globose and 0.3-0.7 mm in diameter with a slender neck which embedded in root tissue; their vulva is present near anus and is subterminal; body cuticle is thin, annulated and whitish. Male nematodes are vermiform, 1-2 mm long and free-living in soil; they have robust spicules but bursa is absent. As these nematodes are endoparasites, their stylet is short with moderately sclerotized. According to Hunt et al. (2005), excretory pore of these nematodes is often located near to stylet base; their eggs are deposited in a gelatinous matrix which is present outside the body; the juveniles (J2) of these nematodes are vermiform, slender and about 450 Âµm long having weakly sclerotized stylet. According to Shurtleff & Averre (2002), average length of the stylet of the root knot nematodes is 10-20 Âµm but may have a little variation among J2, male and female. The life cycle of root knot nematodes comprise of four juvenile stages and adult. Ferraz & Brown (2002); Karssen & Moens, (2006) mentioned that second stage juveniles (J2) of this nematodes hatched from eggs; invaded the host root and induced feeding site called giant cells. According to Abad et al. (2008); Bird et al. (2009), completion of two root-knot nematode genome sequencing reveals that M. hapla encodes approximately 14,200 genes in a compact 54 Mbp genome whereas 86 Mbp of M incognita genome encodes approximately 19,200 genes. Both of these root-knot nematode genomes compact gene families which is comparable with the free-living nematode, Caenorbabditis elegans. Both the nematodes encode large suites of enzymes that uniquely target the host plant. These genes are thought to acquired through horizontal gene transfer
The systematic position of root-knot nematodes as mentioned by De Ley & Blaxter (2002) is: Phylum: Nematoda; Class: Chromadorea; Order: Rhabditida; Suborder: Tylenchina; Infraorder: Tylenchomorpha; Superfamily: Tylenchoidae; Family: Meloidogynidae; Genus: Meloidogyne. De Waele & Elsen (2007) reviewed that 92 nominal Meloidogyne species had been described by 2006. According to Jung & Wyss (1999), they are economically the most important nematode species having wide host range and can reproduce in more than 2000 plant host species. Major species of Meloidogyne include M. arenaria, M. exigua, M. graminicola, M. hapla, M. incognita, M. javanica, M. mayaguensis. M. chitwoodi andM. falax. M. graminicola is considered as the most common and important nematodes for rice root-knot according to Hunt et al. (2005); De Waele & Elsen (2007); Jepson (1987).
Fig. Male and female root-knot nematode morphology
From Eisenback, J.D. 1985. (Not to scale) The male (left) is vermiform, 1400um long, motile, with one gonad, spicules and longitudinal muscles, whereas the female (right) is globose, 800um long, sedentary, with two
Gonads and no spicules or longitudinal muscles
2.9. THE ROOT KNOT NEMATODE: MELOIDOGYNE GRAMINICOLA
2.9.1. MELOIDOGYNE GRAMINICOLA
M. graminicola is a common species of the tropics and subtropics where it infects rice and it is facultative, meiotic parthenogen, with a haploid chromosome number of 18. M. graminicola is a sedentary endoparasitic nematode and first reported by Golden and Birchfield in 1965 in barnyard grasses in the Louisiana State, United States. The rice root knot nematode M. graminicola is known to be established in India, China, Nepal, Bangladesh, Laos, Thailand, United States and Vietnam (Yik et al., 1977, 1979; Kihn, 1982; Poudyal, 2005). De Waele & Elsen (2007) described that Meloidogyne graminicola as the most damaging Meloidogyne species on rice in shallow intermittently flooded land and upland conditions and also reported that it was not only distributed in main rice producing area, specially the South and Southeast Asia, but also found to be distributed in Brazil, Colombia, USA and South Africa and is prevalent in rainfed (upland), irrigated (lowland) and deepwater rice ecosystem. Rao and Israel (1973) reported that this nematode completed life cycle in 26-51 days in India on the other hand, Bridge and Page (1982) reported that this nematode species completed life cycle in less than 3 weeks at 22-29Â°C in Bangladesh resulted in building up of population densities during a single crop cycle. Meloidogyne graminicola has been reported from rice growing regions of India (Isreal, Rao & Rao, 1963; Roy, 1973), Laos (Manser, 1968), Thailand (Buangsuwon et al., 1971), U.S.A. (Golden & Birchfield, 1968 ; Yik & Birchfield, 1979), and Bangladesh (Hoque & Talukdar, 1971 ; Page et al., 1979). It has been reported mainly from rice growing in upland conditions and nurseries (Buangsuwon et al., 1971 ; Israel, Rao & Rao, 1963 ; Manser, 1968 ; Rao & Israel, 1971, 1972), and also reported to be absent from the rice crop grown in flooded fields (Buangsuwon et al., 1971 ; Manser, 1968).
2.9.2. SYMPTOMS OF MELOIDOGYNE GRAMINICOLA ON RICE
The common symptoms due to Meloidogyne graminicola infection on rice includes characteristic hooked-like galls on roots, newly emerged leaves appear distorted and crinkled along the margins, stunting, chlorosis of young plants and heavily infected plants flower and mature early. Deep water rice varieties can elongate to come above the water surface; however, upon severe infection with M. graminicola they are unable to grow and drown, leaving patches of open water in flooded fields (Bridge & Page, 1982). Small galls appear on the roots as beaded, clubbed or spindle-shaped, which coalesce upon heavy infection. Characteristic hooked and swollen root tips are apparent which prevent root elongation (De Waele & Elsen, 2007).
Fig. Meloidogyne graminicola root galls on rice seedlings. Source-http://www.warda.org/publications/Warda_Nemaotde.pdf. 6thDec2010.
Fig.Characterisics hooked tips caused by Meloidogyne graminicola. Source-http://www.warda.org/publications/Warda_Nemaotde.pdf. 6thDec2010.
2.9.3. LIFE STAGES OF MELOIDOGYNE GRAMINICOLA
The life stages of M. graminicola consists the Second stage juveniles or J2 that penetrate the root behind root tip. This leads to giant cell formation due to feeding by juveniles and hypertrophy of cells which further leads to the development of galls. The gall then continues to swell, with females of J4 and males of J4 inside the galls. As the life cycle goes on, the male leaves the root while female stays inside the root and start laying eggs in a gelatinous matrix outside the root. J2 hatch from the eggs and attracted towards the root and the life cycle carries on. The life cycle is completed in one to two months depending on environmental conditions. However, there are reports that the life cycle of the rice Root Knot Nnematodes under favourable conditions at 25-30Â°C takes 19 days (Bridge & Page, 1982). This short life cycle allows buildup of M. graminicola populations during a single crop cycle at a faster rate. M. graminicola normally feeds on the cells and interferes with nutrient uptake, water uptake, and translocation due to root damage (Caillaud et al., 2008). The feeding cells are rapidly turned into multinucleate giant cells which act as metabolic sinks and compete with rice (Singh et al., 2006).
Fig. Life cycle of Root knot nematode. Source-
2.9.4. RICE YIELD IN RESPONSE TO MELOIDOGYNE GRAMINICOLA INFECTION
The rice root-knot nematode found to attacks the rice plant at all the growth stages. It is regarded as one of the limiting factors in rice production in all rice farming systems. There is an estimated reduction of 2.6 percent in the yield of rice for every 1000 nematodes present in young seedlings as recorded in upland rice farming system and in irrigated rice, damage is mainly caused in the nurseries before transplanting or before flooding in the case of direct seeding. Greatest yield decline is reported under non flooded conditions (Plowright & Bridge, 1990; Prot & Matias, 1995; Tandingan et al., 1996; Soriano et al., 2000; Soriano & Reversat, 2003), however, great yield losses can also occur in drought-prone rain fed lowland systems (Padgham et al., 2004). Soriano et al. (2000) recorded yield losses due to M. graminicola which was ranged from 11% to 73% in simulations of intermittently flooded
2.9.5. MANAGEMENT PRINCIPLES OF OF MELOIDOGYNE GRAMINICOLA
There are various control measures that are available for managing rice root knot nematodes. This includes biological, physical, chemical, cultural, mechanical, resistant cultivars and mechanical control. The common cultural controls that are put into practices include continuous flooding of rice field, raising the rice seedlings in flooded soils and also crop rotation. M. graminicola control can be achieved by continuous flooding of rice plants in deep water (Bridge et al., 2005). Rao and Israel (1971) and Soriano et al. (2000) proposed that early-season flooding can reduce M. graminicola damage on rice as the Meloidogyne juveniles cannot invade the roots in flooded conditions. Rotation of crop with marigold (Tagetes sp.) is also found to be effective in lowering the root knot nematode populations due to its nematicidal properties
There are several nematicidal compounds which can be used as chemical control. Rice seeds can be treated with Entomopathogenic nematode along with carbofuran. Other control methods can be dipping in systemic chemicals such as oxamyl or fensulfothion, phorate, carbofuran etc. Soriano et al. (2003) reported that carbofuran controlled the rice RKN and improved yield of the first rice crop but did not affect the second rice crop.
2.10. NEMATODE (ROOTKNOT AND CYST NEMATODE) PARASITISM GENE
Root-knot nematodes which are highly successful parasites evolved a very specialized feeding relationship with the host plant or crops to cause the destructive root-knot disease. It initiate their parasitic relationship with host by releasing their secretions into root cells which in turn stimulate the root cells of the host to become specialized feeding cells which are the sole source of nutrients essential for the nematode's survival. A deeper understanding of the basic principles and mechanisms of root knot nematode parasitism is critical for discovering new targets in root-knot nematodes to develop novel crop resistance using biotechnology tools.
Fig. Model of nematode parasitism of a plant cell.
The parasitism genes expressed in the Root knot nematode's esophageal gland cells and encode secretory proteins that are released through its stylet to direct the interactions of the nematode with its host plants. The products which are collectively called as parasitome, of parasitism genes secreted into susceptible host tissue modulate the complex changes in function, morphology, and gene expression in host root cells to form feeding cells. Gheysen & Jones (2006); Ferraz & Brown (2002) described that juveniles of Meloidogyne entered the root tip and migrated intercellularly towards the root tip. After reaching the root apex, they turned back and migrated again intercellularly until they found a suitable place near vascular cylinder for feeding site induction. Plant-parasitism is believed to be evolved at least three times independently, but morphological adaptations for plant parasitism are surprisingly similar among all plant-parasitic nematodes. Plant-parasitic nematodes are well equipped with stylet to tear the cell walls and allow exchange of solute between plant and parasite and also have well-developed secretory gland cells associated with esophagus that produce secretions released through the stylet into host. In order to be parasitism, the nematode must be able to penetrate the roots of host plants and migrate through root tissues. Considering the small size of root-knot and cyst nematode infective J2 stage, plant cell walls pose formidable obstacles so; nematodes release a mixture of cell-wall-digesting enzymes to break structural plant cell-walls. Above this, the most interesting things appear to be the nematode-directed formation of the feeding cells by both the root-knot nematodes as giant-cells and cyst nematodes as syncytia. Gheysen & Jones (2006) also suggested that both induction and maintenance of giant cells were controlled by stylet secretions. These secretions were originated from dorsal and subventral pharyngeal glands of feeding nematodes. Karsen & Moens (2006) reported that like other Root knot nematodes, M. graminicola started feeding on a group of giant cells established in the phloem or adjacent parenchyma. These giant cells (2-12, usually about 6) are multinucleated and produced by repeated endomitosis without cytokinesis. The cells of the neighboring pericycle of the nematode undergo hypertrophy and hyperplasia and produce a typical gall or root-knot, usually after one or two days after the penetration of J2.
Gheysen & Jones 2006 also reported that the nematode genes expressed solely in subventral gland cells were most similar to the genes those produced cell wall degradation enzymes from bacteria and were not present in symbiotic bacteria. Zenov'eva et al. (2004) summarized several gene products isolated from subventral glands of nematodes which included lipoprotein, cellulose-binding protein, chitinase, pectinase, proteinase, and endoglucanase. PARASITISM GENE IDENTIFICATION
A number of arrays have been approaches to identify the nematode parasitism genes and proteins have been devised and tried. Many of these approaches mainly focus on esophageal-glands due to their involvement in parasitism. The identification of parasitism genes has proven to be difficult due to the microscopic size of the plant-parasitic nematodes, which makes it hard to collect enough material for analysis (Vanholme et al., 2004). Immunoaffinity purification was used to enrich secreted proteins, which results in the finding of a secreted protein (endo-1,4- Î²-glucanase) from the subventral glands of the cyst nematode Globodera rostochiensis (Smant et al., 1998). Another method to be mentioned is the analysis of collected nematode secretions by 2D gel electrophoresis and microsequencing. This has also proved successful for the beet cyst nematode Heterodera schachtii (De Meutter et al., 2001) and the root-knot nematode Meloidogyne incognita (Jaubert et al., 2002b). Recently, mass spectometry is also used for direct identification of proteins secreted by M. incognita, revealing proteins with host cell reprogramming potential (Bellafiore et al., 2008). The peptide sequence from an antigen purified with an esophageal-gland-specific monoclonal antibody was previously used to isolate the first parasitism gene from a plant parasitic nematode (Smant et al., 1998), encoding a Î²-1,4-endoglucanase enzymes (cellulase). Further studies also reveal the expression of cyst nematode endoglucanase genes and their associated products specifically from subventral gland cells of the nematode, cellulolytic activity of the enzymes, and secretion of cellulases from the stylet in plant during migration of infective J2 stage inside the host roots (Wang et al., 1999). These studies also confirmed and refined the previous reports of secreted cell-wall-modifying enzymes from plant parasitic nematodes (Deubert et al. 1971). An important discovery of the nematode endoglucanases enzymes was their strong resemblance to prokaryote (glycosyl hydrolase family 5) endoglucanases and little similarity to endoglucanases of eukaryotes and no similarity to any gene of Caenorhabditis elegans (Smant et al., 1998).