Preface Nematodes are a complex, diverse group of round worms that occur worldwide in essentially all the environments. Plant parasitic nematodes are recognized as serious pests of crop plants in recent years. The world-wide annual crop loss due to these obligate parasites have been estimated as $ 78 billion. The damage caused by nematodes are often overlooked. They are hidden enemies and the associated symptoms can also be attributed to nutritional and water related disorders. The epidemics of sugarbeet sickness due to cyst nematode, yellow disease of black pepper, molya disease on wheat and barley, the ear-cockle disease on wheat and others have led to the recognition of the science Plant Nematology as an important branch of Agricultural Sciences. The recognition of nematodes as serious deterrents to crop productivity was felt in India only in the 1961, when Dr. Jones from Germany reported the menace of potato cyst problems were identified in important crop plants. Compared to other disciplines of crop protection, Plant Nematology is the youngest deserving tremendous support to strengthen the discipline. The Department of Nematology at Tamil Nadu Agricultural University, Coimbatore has been in the forefront and has provided leadership in strengthening the discipline of Nematology in the country. Recently, Nematology has been included in the curriculum of few Agricultural Universities in India and as such, there is a paucity of text book in Nematology to cater the needs of students. In this direction, a sincere attempt has been made to bring out a comprehensive text book dealing with the relevant aspects of the subject, Agricultural Nematology. This book has been written as a text book primarily keeping in view of the requirements of undergraduate students of agriculture, horticulture and post-graduates specializing on Nematology and Entomology dealing with plant parasitic nematodes. This book provides a brief account of historical background including the developments in India and abroad, details of morphology, anatomy and taxonomy of plant parasitic nematodes, relevant nematological techniques and focus on nematode problems in important agricultural and horticultural crop and their management. The book also provides a brief account of Caenorhabditis elegans which is being used as a biological model in most of the biological research. The book will also be useful to those who are preparing for competitive examinations conducted by various central and state government agencies for recruitment. Every attempt has been made to provide necessary information from the view point of students dealing biological sciences.

Agricultural crops are being affected by a wide range of organisms such as insects, mites, fungi, bacteria etc. Another important and unseen enemy is known as plant parasitic nematodes. The study on nematodes is known as Nematology. Nematology is an important branch of biological science, which deals with a complex, diverse group of round worms known as Nematodes that occur worldwide in essentially all environments. Nematodes are also known as eelworms in Europe, nemas in the United States and round worms by zoologists. Many species are important parasites of plants and animals, whereas others are beneficial to agriculture and the environment. Nematodes that are parasites of man and animals are called helminths and the study is known as Helminthology. The name nematode was derived from Greek words nema (thread) and oides (resembling). This book focuses primarily on plant parasitic nematodes, their morphology, anatomy, nematological techniques, taxonomy, classification based on their feeding habits, symptom of damage to crops, interaction with other microorganisms, biology of important nematodes viz., Meloidogyne, Heterodera, Rotylenchulus, Tylenchulus and Radopholus and nematode pests of important crops and their management. Annual crop losses due to these obligate parasites have been estimated to be about $78 billion worldwide and $8 billion for U.S. growers. The estimated annual crop loss in Tamil Nadu is around Rs. 200 crores. The soil in a hectare of agroecosystem typically contains billions of plant parasitic as well as beneficial nematodes. The damage to plants caused by nematodes is often overlooked because the associated symptoms, including slow growth, stunting and yellowing can also be attributed to nutritional and water related disorders.

In light of the high population of nematodes, N.A. Cobb (1915) who is considered to be the father of American Nematology, provided a dramatic description of the abundance of nematodes. He stated, “if all the matter in the universe except the nematodes were swept away, our world still would be dimly recognisable. We would find its mountain tops, valleys, rivers, lakes and oceans represented by a film of nematodes.” The Papyrus Ebers suggested a knowledge of the human intestinal parasite, Ascaris lumbricoides and the tissue parasite, Dracunculus medinensis. In early Egyptian writings also these two human parasites have been described. In 1956, Borellus recorded his observations “Vinegar eels” the microbivorous or free living nematode occurring in most vinegar at that time. The statement “sowed cockle, reaped no corn” in Shakespeare’s “Love’s Labour’s Lost”, act 4, scene 3, as suggested by Thorne (1961) possibly the first record of plant parasitic nematodes in 1549. The nematode that Thorne suspected to be in that reference actually was described by Needham in 1743. Subsequently, discovery of microscope and developments in various disciplines of science led to the discovery of plant parasitic nematodes and the disease caused by them. Some of the important milestones on the history of plant nematology are listed below in chronological order.

Eventhough nematodes occupy nearly every habitat on earth, they are remarkably similar in morphology and life stages. Despite their structural complexity, certain basic principles are common to all nematodes. Nematodes are triploblastic, bilaterally symmetrical, unsegmented, pseudocoelomate, vermiform and colourless animals. The plant parasitic nematodes are slender, elongate, spindle shaped or fusiform, tapering towards both ends and circular in cross section. The length of the nematodes may very from 0.2 mm (Paratylenchus) to about 11.0 mm (Paralongidorus maximus). Their body width vary from 0.01 to 0.05 mm. In few genera, the females on maturity assume pear shape (Meloidogyne), globular shape (Globodera), reniform (Rotylenchulus reniformis) or saccate (Tylenchulus semipenetrans). The swelling increases the reproductive potential of the organism. Radially symmetric traits (triradiate, tetraradiate and hexaradiate) exist in the anterior region. The regions of intestine, excretory and reproductive systems show tendencies towards asymmetry. The nematodes have one or two tubular gonads which open separately in the female and into the rectum in the male which also have the copulatory spicules. The free living saprophytic nematodes are generally larger in size. The animal and human parasitic helminths may have length of few centimetres to even a meter or more. The helminth parasitising whale fish is about 27 feet long. The study on these animals and human parasites are known as Helminthology.

Life cycle of nematode has six stages. The egg stage, first stage juvenile (J1), second stage juvenile (J2), third stage juvenile (J3), fourth stage juvenile (J4) and the adult stage. The first four stages are the immature stages and are known as juvenile stages. The female lays eggs in soil or in plant tissues, singly or in groups as egg mass that hatch out into juveniles which are almost similar to adults appearance. The first moult occurs within the egg shell and the second stage juvenile comes out by rupturing the egg shells as J2. In case of Xiphinema index, the juveniles are reported to emerge from the egg before the first moult. The juvenile cuticle is shed after each moult. The egg The nematode eggs are oval in shape. The eggs are covered by three membranes, the external protein layer which is the secretion of uterus wall, the middle chitinous layer or the true shell secreted by egg itself and the inner lipid layer (vitelline layer). The chitin content in the egg shell vary in different species of nematodes.

Plant parasitic nematodes cause serious problems in sustained crop production. For a successful management of nematodes, their morphology, biology and host - parasitic relationship should be thoroughly studied. A thorough study is achieved only when its taxonomical position is known. Thus, in a simple way taxonomy can be defined as the theroretical study of classification of an organism. The systematic arrangement of organisms into groups on the basis of their relationship is termed as classification. Nematodes are placed in the group invertebrate, Kingdom Animalia. Hyman (1951) revised the phylum Aschelminthes and included six classes viz., Rotifera, Gastotricha, Echinodera, Priapulida, Nematoda and Nematomorpha.

Sampling for nematode population estimation Sampling for nematode communities has been the basis for the development of appropriate control strategy. The major purposes of sampling for nematodes include population estimation for general detection, advisory or predictive programme, disease diagnosis and also for research purposes. Plant parasitic nematodes are usually confined to top 20-25 cm soil and may vary depending upon the soil type, moisture content, host plant and climatic condition. Surface soil do not harbour any nematode. The roots and rhizosphere of severely infested plants do not harbour numerous nematodes as the nematodes tend to move to adjacent healthy plants. Hence soil and root samples need to be collected from plants showing partial symptoms. Samples should not be collected from dead or wilted plants.

The nematode feeding on plant can be divided into above ground feeders and below ground feeders. I. Above ground feeders a) Feeding on flower buds, leaves and bulbs Seed gall nematode : Anguina tritici Leaf and bud nematode : Aphelenchoides Stem and buld nematode : Ditylenchus

Most of the plant parasitic nematodes affect the root portion of plants except Anguina spp., Aphelenchus spp., Aphelenchoides spp., Ditylenchus spp., Bursaphelenchus cocophilus and B. xylophilus. Nematodes suck the sap of the plants with the help of stylet and causes leaf discolouration, stunted growth, reduced leaf size and fruits, lesions on roots, galls, reduced root system and finally wilting. Symptoms of nematode diseases can be classified as Symptoms produced by above ground feeding nematodes Symptoms produced by below ground feeding nematodes

Plant parasitic nematodes favour the establishment of secondary pathogens viz., fungi, bacteria, virus etc. The nematodes alter the host in such a way that the host tissue becomes suitable for colonization by the secondary pathogens. Even though the nematodes themselves are capable of causing considerable damage to the crops, their association with other organisms aggravate the disease. The nematodes cause mechanical wound which favours the entry of microorganisms. In some cases, the association of nematode and pathogen breaks the disease resistance in resistant cultivators of crop plants. Nematode - Fungus Interaction Nematode - fungus interaction was first observed by Atkinson (1892) in cotton. Fusarium wilt was more severe in the presence of Meloidogyne spp. Since then the nematode - fungus interations had received considerable attention on important crops like banana, cotton, cowpea, brinjal, tobacco and tomato. Some examples of nematode - fungus interaction are given in the following table.

Rice Plant parasitic nematodes cause serious damage to rice crop. Thirty two species belonging to 13 genera were observed in association with the crop. Among them few are considered to be important. They are the rice white - tip nematode (Aphelenchoides besseyi), rice stem nematode (Ditylenchus angustus), rice root nematode (Hirschmanniella oryzae), root-knot nematode (Meloidogyne graminicola) and the cyst nematode (Heterodera oryzicola). The white-tip nematode (Aphelenchoides besseyi) The yield loss due to the white-tip nematode is estimated to be as much as 17.4 to 54.1 per cent. The nematode is distributed in India, Bangladesh, Sri Lanka, Japan, Indonesia, Taiwan, Former USSR, Italy, Cuba and Madagascar.

Citrus (Citrus spp.) Citrus is an important fruit crop and ranks third in area which is followed by mango and banana. They are grown in 105,396 hectares in India. Among the oranges, mandarin oranges, limes and sweet oranges are very popular while lemons, grape fruits, pummelos, sour and bitter oranges are of minor importance. The Citrus spp. are grown in larger extent in the states of Maharashtra, Karnataka, Kerala, Tamil Nadu, Andhra Pradesh, Punjab, Haryana, Rajasthan, Assam, Orissa and Bihar. Nematode menace in citrus gardens are very much realised. In 1912 the citrus nematode, Tylenchulus semipenetrans was reported from California and found to cause ‘slow decline’ disease. More than 30 genera of plant parasitic nematodes are associated with the Citrus spp. Among them, Tylenchulus semipenetrans, Radopholus citrophilus, Meloidogyne spp. Pratylenchus spp. Xiphinema spp. and Hoplolaimus spp. are often associated with the crop.

Potato Potato is one of the most important vegetable crop grown in India. It thrives best in cool climate. Therefore it is a summer crop in the hills and a winter crop in plains. Twenty seven species of nematodes have been reported on potato among them the cyst nematodes (Globodera rostochiensis and G. pallida) and the root-knot nematodes (Meloidogyne incognita, M. javanica and M. hapla) are important in India. Cyst nematodes (G.rostochiensis and G. pallida) In India, the cyst nematode menace on potato was first reported by Dr. Jones during 1961 from Nilgiri Hills. Geographical distribution The potato cyst nematode is distributed in Central, South and North America, Europe, Iceland, Western USSR and South Africa. In India it is distributed in Nilgiri and Kodai Hills and in New Delhi.

Crossandra Crossandra (Crossandra undulaefolia) is one of the important commercially grown flower crop in Tamil Nadu. In recent years there is a marked decline in the cultivation of this crop in Madurai, Dindigul, Coimbatore, Salem, Thiruvannamalai and Tiruchirappalli districts. The common problem with the crop is death of plants in patches during the second and third year. This is due to the nematode-fungal complex disease in the crop. Investigations revealed the association of root-knot nematode, Meloidogyne spp. and the root-lesion nematode, Pratylenchus delattrei with the crop along with fungal patheogen, Fusarium solani. Root-knot nematode Both M. incognita and M. javanica are reported to cause damage and yield loss to the crop. The affected plants exhibit stunted growth, yellowing of leaves and also have chlorotic symptoms. The nematode in association with R. solani causes serious damage to the crop. The infested plants show root galls.

Black Pepper Black pepper (Piper nigrum) is indigenous to the tropical forests of Western Ghats of South India. The crop is grown in Kerala, Karnataka, Tamil Nadu, Andra Pradesh and Assam. The root-knot nematode, Meloidogyne incognita and the burrowing nematode, Radopholus similis are important pests of this crop. The root-knot nematode infested vines show yellowing of lower leaves and the leaves loose natural luster. The yellowing of leaves gradually progresses upwards. The affected vine leaves become flaccid and wither. The infested plant root show root galls of varying sizes. The burrowing nematode also causes serious damage to the vine. Var der Vecht (1950) first reported slow wilt in this crop due to R.similis infestation in Indonesia. The nematode in association with Fusarium and Rhizoctonia causes serious damage to the crop.

Nematode Control Plant parasitic nematodes can be controlled by several methods. The nematode control aims to improve growth, quality and yield by keeping the nematode population below the economical threshold level. The control measures to be adopted should be profitable and cost effective. It is essential to calculate the cost benefit ratio before adopting control measures. The nematode control methods are Regulatory (Legal) control Physical control Cultural control Biological control Chemical control

Nematode associated with insects are referred as entomophilic, entomogenous and entomophagous nematodes. Generally they belong to the superfamilies Tylenchoidea, Rhadbitoidea, Oxyuroidea and Mermithoidea of the phylum Nematoda. Nature of parasitism Christie (1914) divided the nematodes associated with invertebrates into three groups. Those nematodes which live in the alimentary tract of he invertebrates. Those nematodes which are more or less closely related to free living species and often have a combination of saprophagous and parasitic habits. Those nematodes which parasitizes the body cavity or tissue of their host. Entomophilic nematodes are group of parasites that cause debilitation, sterility or death of insects. Entomophilic nematodes vary greatly in size and shape having the insects as intermediate or as definite hosts. Many group of insects are parasitised by nematodes.

Model organisms are widely used in biological researches to describe basic biological processes. When selecting living organisms as models to work with, a wide range of characteristics are used which include: 1) rapid development with short life cycles, 2) small adult size, 3) ready availability and 4) tractability. Being small, growing rapidly and being readily available are crucial in terms of housing them, given the budget and space limitations of research and teaching laboratories. Tractability relates to the ease with which they can be manipulated. Many model organisms have been used including fruit fly, Drosophila melanogaster, the most obvious organism used for teaching Mendelian genetics and developmental biology; Escherichia coli, a favoured organism for molecular biological studies involving recombinant DNA technology. Research into the molecular and developmental biology of C. elegans was begun in 1974 by Sydney Brenner and it has since been used extensively as a model organism. C. elegans is a popular research organism as it possesses all the characteristics mentioned, yet shares many essential biological properties with humans. For instance, researchers who study apoptosis (programmed cell death) use C. elegans as an experimental organism in the hope of finding treatments for certain types of human cancers, such as leukaemia. C. elegans is easy and inexpensive to maintain in laboratory conditions with a diet of E. coli. Embryogenesis occurs in about 12 hours, development to the adult stage occurs in 2.5 days and the life span is 2-3 weeks. The short, hermaphroditic life cycle (~3 days) and large number (300+) of offspring of C. elegans allows large-scale production of animals within a short period of time. Since C. elegans has a small body size, in vivo assays can be conducted in a 96 well microplate. Laboratory stocks of C. elegans can even be stored in liquid nitrogen for 25 years without losing their viability. The transparent body allows clear observation of all cells in mature and developing animals. Furthermore, the intensively studied genome, complete cell lineage map, knockout (KO) mutant libraries and established genetic methodologies including mutagenesis, transgenesis and RNA interference (RNAi) provide a variety of options to manipulate and study C. elegans at the molecular level and for a more detailed presentation of genetic and genomic resources.

Glossary A A : A ratio calculated by dividing the length of the nematode by its maximum width. Abduce : To head away or way to Abduct : Non living Acanthiform : Look like thorn shape Acetylcholine : Chemical transmitter of nerve and nerve-muscle impulses in animals. Acute : Sharp pointed Active ingredient(a.i) : Chemicals in a product that are responsible for the pesticidal effect. Adanal bursa : Bursa enclose a part of the tail Aerolated : The transverse line present in the lateral fields Aestivation : Dormant during unfavourable dry condition Alae : Longitudinal thickening of cuticle forming wing like expansion (longitudinal alae, cervical alae, caudal alae and bursa) Alate : Possessing lateral field. Ambifenestrate : The occurrence of two openings in the vulval cone which are seperated by the vulval bridge as observed in some species of Heterodera. Amphid : Paired sensory organ located at the anterior.