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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

1. Regulatory (Legal) control
2. Physical control
3. Cultural control
4. Biological control
5. 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.

Objectives

• To study working principles of simple microscope
• Study different parts of simple microscope

Introduction

This is the first ever microscope. It was founded by Leeuwenhoek in 17^th century. He combined a convex lens with a specimen holder, for seeing the magnified images of small objects. It looks like a magnifying glass because it has the capacity to magnify between 200 and 300 times. A simple microscope is a small focal lens used to view the magnified images of small objects. It is just like a hand lens and glass with relatively lower magnification power.

Objectives

• To study working principles of compound microscope
• Study different parts of compound microscope

Introduction

A compound microscope is an optical instrument consisting of two short focal-length convex lenses that are used to observe the highly magnified images of small objects by naked eyes. The compound microscope is capable of magnifying up to 1000 the image of a small object by complex system of lens arrangement. The working distance varies from 0.14 to 4 mm. Compound microscopes generally require the sample to be seen to be sufficiently thin, or translucent, to allow the light to pass. Such microscopes provide high magnification, but the view is two-dimensional.

Objectives

• Differentiate between fixatives and stains.
• Describe methods of staining.
• List the commonly used fixatives and stains and their composition.
• Describe the uses of above mentioned solutions and stains.

Introduction

Every time a part of an organism is removed, the action of bacteria will soon disintegrate, dry or putrefy it. They must be kept as close to normal as possible or in other words preserved and fixed in order to study these. Chemicals are required in many practical of Biology. Some of these chemicals preserve and specifically colour certain cell parts while others are used as solutions. You will learn about certain fixatives (preservatives) and stains in this chapter.

Objectives

• Better understand the process and different stages of mitosis.
• Prepare your own specimens of onion root in which you can visualize all of the stages of mitosis.

Principle

The genetic information of plants, animals and other eukaryotic organisms resides in several (or many) individual DNA molecules, or chromosomes. For example, each human cell possesses 46 chromosomes, while each cell of an onion possesses 8 chromosomes. All cells must replicate their DNA when dividing.

DNA replication in eukaryotes is followed by the process called mitosis which assures that each daughter cell receives one copy of each of the replicated chromosomes. During the process of mitosis, the chromosomes pass through several stages known as prophase, metaphase, anaphase and telophase. The actual division of the cytoplasm is called cytokinesis and occurs during telophase. During each of the preceding stages, particular events occur that contribute to the orderly distribution of the replicated chromosomes prior to cytokinesis.

An onion root tip is a rapidly growing part of the onion and thus many cells will be in different stages of mitosis. The onion root tips can be prepared and squashed in a way that allows them to be flattened on a microscopic slide, so that the chromosomes of individual cells can be observed easily. The super coiled chromosomes during different stages of mitosis present in the onion root tip cells can be visualized by treating with DNA specific stains, like Feulgen stain and Acetocarmine stain.

Objectives

• Better understand the process and different stages of meiosis.
• Prepare your own specimens of onion root in which you can visualize all of the stages of meiosis.

Principle

Meiosis is a type of cell division in which the number of chromosomes is halved (from diploid to haploid) in the daughter cells, i.e., the gametes. It occurs in cell that produce gametes in sexually reproducing organisms. The division is completed in two phases, meiosis I and meiosis II. Meiosis I is a reductional division in which the chromosomes of homologous pairs separate from each other. Meiosis II maintain same number of chromosomes, hence, is called as equational division. Stages of meiosis can be observed in a cytological preparation of the pollen mother cells of the anthers of f lower buds.

Objectives

• Find out the number and types of gametes and gametic recombination in monohybrid, dihybrid and trihybrid crosses.

Principle

Gametes are reproductive cells that join together to form a new cell called a zygote during sexual reproduction. Pollen in seed-bearing plants, is the gametophyte producing male gamete. Female gametes (ovules) are included in the ovary of plant. At maturity a sexually reproducing individuals forms the gametes. Homozygote generate only one form of gamete. Homozygous referring to a diploid cell or organism possessing two identical alleles of a specific gene (e.g. AA or aa). Heterozygote generate different types of gametes. Heterozygous referring to a diploid cell or organism possessing two different alleles of a specific gene (e.g. Aa).

Objectives

• Better understand the monohybrid cross.
• Calculation of monohybrid phenotypic, genotypic and testcross ratios.

Principle

Monohybrid cross deals with the study of inheritance of one pair of contrasting characters. The alleles of contrasting character selected in the monohybrid cross will show exact dominance and recessive relationship. It is also defined as a cross is made between two true-breeding parents differing for a single trait, producing an F₁ generation. These plants are intercrossed to produce an F₂ generation. Monohybrid crosses study led to the Mendel’s law of segregation. Monohybrid cross was first produced in pea by Mendel to explain the “law of segregation”. When parents with distinct phenotypes are crossed, in F₁ generation, only one type of plants appears phenotypically. However, segregation can be observed in F₂ progeny and the population can be divided into two phenotypes classes. After counting the individuals. It is observed that 75% of them express the dominant phenotype and 25% of them express recessive phenotype.

Objectives

• Better understand the dihybrid cross.
• Calculation of dihybrid phenotypic, genotypic and testcross ratios.

Principle

A cross is made between two true-breeding parents differing in two pairs of genes, producing an F₁ generation. These plants are inter-crossed to produce an F₂ generation. Mendel second law i.e., Law of independent assortment can be explained by studying the inheritance of two characters at a time, simultaneously. When two phenotypically distinct parents differing with respect to two characters governed by two pair of genes are crossed together. Each pair of genes assorts independently into all possible combination forming four kinds of progeny, two of these are parental type and two are recombinant types.

Objectives

• Better understand the law of independent assortment.

History

The law was proposed by Gregor Johann Mendel with the other laws together called as ‘Mendelian laws of inheritance’, in 1866. He used pea plant as a model organism, and studied 7 characters.

Principle

The Law of Independent Assortment, also known as “Inheritance Law”, states that alleles of different genes assort independently of one another during gamete formation. Independent assortment for two genes can be explained by assuming that the two genes are located in two different chromosomes. The two alleles of a gene will be located in the two homologues of the concerned chromosome. Independent separation of these two pairs of chromosomes at anaphase I of meiosis will lead to the independent segregation of the genes located in them. Thus any allele of one gene is equally likely to combine with any allele of the other gene and pass into the same gamete. Independent segregation of two genes produces four different types of gametes in equal proportion. A random union among these gametes gives rise to sixteen possible zygotes. These zygotes yield a 9:3:3:1 phenotypic ratio, which is known as the typical dihybrid ratio. When two pairs of independent alleles enter into F₁ combination, both of them have their independent dominant effect. These alleles segregate when gametes are formed but the assortment occurs independently at random and quite freely.

Objectives

• Chi-square test for goodness of fit of genetic ratios.

Introduction

Karl Pearson developed the chi square test of statistical significance which is commonly used in Mendelian and population genetics. Chi square (χ²) test is a test of statistical significance which is used to test the significance difference between observed and expected frequencies or ratios. The purpose of χ² test is to decide if a set of observed data is according to an expected ratio or if it agrees well with an expected or theoretical distribution.

The chi square test of independence allow us to evaluate whether the segregation of alleles at one locus is independent of the segregation of alleles at another locus. To distinguish between inheritance patterns that obey Mendel’s laws versus those that do not, a conventional strategy is to make crosses and then quantitatively analyze the offspring’s. Based on the observed outcome, an experimenter may make a tentative hypothesis. For example, it may seem that the data are obeying Mendel’s laws. Hypothesis testing provides an objective, statistical method to evaluate whether the observed data really agree with the hypothesis.

The underlying principles behind a statistical approach is to evaluate the goodness of fit between the observed data and the data that are predicted from a hypothesis. This is sometimes called a null hypothesis because it assumes there is no real difference between the observed and expected values. A high deviation would be found between the observed and expected values. If this occurs, the hypothesis is rejected. Hopefully, the experimenter can subsequently propose an alternative hypothesis that has a better fit with the data.

The null hypothesis is a particular claim concerning how the data is distributed. More will be said about the construction of the null hypothesis later. The null and alternative hypotheses for each chi square test can be stated as