Preface Nematology is an emerging science deals with nematodes infesting crop plants, free living and beneficial nematodes. Plant parasitic nematodes are biotrophic parasites, more than 4100 species associated with crop plants, which cause 12.3 per cent economic yield loss with the tune of $ 157 billion in worldwide. Many strategies have been developed for nematode management including the use of chemical nematicides, which are harmful to human health and environment. Biological control is now accepted as an alternative, economic and environmentally sound management strategy. The beneficial microbe’s viz., bacteria, fungi and actinomycetes play a main role in stimulating defence mechanism and promote the plant growth and also showed nematicidal activities by production of secondary metabolites and toxic compounds. On the other hand, a large number of beneficial nematodes especially entomopathogenic nematodes like Steinernema and Heterorhabditis having mutualistic bacteria Xenorhabdus and Photorhabdus, respectively, cause rapid death of insect host within short period. Entomopathogenic nematodes have been used as potential biocontrol agents against many insect pests viz., Lepidoptera, Coleoptera, Hemiptera and Dictyoptera in agri-horticultural crops. Based on the biocontrol potential of entomopathogenic nematodes it can be used in the pest combat strategies and bacteria, fungi and actinomycetes having biocontrol potential against plant parasitic nematodes. Keeping this view a book on “Beneficial Nematodes and Nematode Antagonistic Bioagents” has been formulated. This book comprises of two sections, the first section (chapter 1 to 16) deals with isolation, identification, molecular characterization, mass multiplication, formulations and shelf life of bacteria, fungi and actinomycetes. The second section (chapter 17 to 24) deals with isolation, identification, mass culturing of insect host and mass multiplication, formulations and field potential of entomopathogenic nematodes against economically important insect pests.

The basic principles of biological control of pests and diseases remain the same, whether applied to insect pests, nematodes or plant pathogens. In the recent years, there is a paradigm shift towards studying antibiosis and its application in biological suppression of pathogens, yet the classical bio-control will remain as relevant as ever. The following discussion is based on recapitulating the basic principles of classical biological control as applicable to plant parasitic nematodes. Biological control agents (BCAs) have been exploited for pest control in different ways. One way is to manipulate the naturally occurring BCAs by augmenting their populations to pest suppressive levels. In this approach no BCAs are introduced, but various means such as agronomic practices, introduction of organic matter etc. are adopted to favour the build-up of BCAs. The second way is the introduction of BCAs, either as inoculative or inundative release. In the former case, the BCAs are introduced in small quantities and allowed to build-up gradually in due course of time. However, in the latter approach, BCAs are introduced in large quantities so that quick pest suppression can be achieved. Each method has its own merits and demerits (Table 1).

Introduction The soil around plant roots that forms the rhizosphere is a dynamic, complex zone. All plant parasitic nematodes are obligate parasities and must enter this zone to reach their host and cause damage (Kerry, 2000). Nematodes in soil are subject to infections by bacteria and fungi. This creates the possibility of using soil microorganisms to control plant-parasitic nematodes. Bacteria are numerically the most abundant organisms in soil, and some of them, for example members of the genera Pasteuria, Pseudomonas and Bacillus have shown great potential for the biological control of nematodes. Nematophagous bacteria are distributed broadly, possess diverse modes of action, and have broad host ranges. A variety of nematophagous bacterial groups have been isolated from soil, host-plant tissues, and nematodes and their eggs and cysts (Siddiqui and Mahmood, 1999; Kerry, 2000; Meyer, 2003). They affect nematodes by a variety of modes: for example parasitizing; producing toxins, antibiotics, or enzymes; interfering with nematode–plant-host recognition; competing for nutrients; inducing systemic resistance of plants; and promoting plant health (Siddiqui and Mahmood, 1999). They form a network with complex interactions among bacteria, nematodes, plants and the environment to control populations of plant-parasitic nematodes in natural conditions (Kerry, 2000).

Introduction Plant parasitic nematodes are important pests, causing major damage to the world’s food and fibre crops. The management of nematodes is more difficult than that of other pests because nematodes mostly inhabit the soil and usually attack the underground parts of the plants. Nematodes in soil are subject to infections by bacteria and fungi. This creates the possibility of using soil microorganisms to control plant-parasitic nematodes. Biocontrol of nematodes was first studied by Duddington (1951). The development of biological control agents is also considered an effective alternative for nematode control on vegetables (Van Gundy, 1985; Kerry, 1987). There are more than 200 species of nematophagous fungi described. Based on the infection mechanism, they are commonly subdivided into three main groups: the nematode-trapping fungi that captures free living nematodes using specialized morphological structures (i.e., traps), the endoparasitic fungi that infects nematodes using adhesive spores, and the egg- and cyst-parasitic fungi that infect these stages with their hyphal tips (Barron, 1977). The nematophagous fungi that have received most attention for biological control of plant-parasitic nematodes include various species of the nematode-trapping fungi Arthrobotrys spp. (Stirling and Smith, 1998), and the egg-parasitic fungi Pochonia chlamydosporium (Kerry, 2001) and Paecilomyces lilacinus (Gaspard et al., 1990).

Introduction The need of the agricultural community was the force of motivation for the development of broad-spectrum pesticides towards the management of pests and diseases of cultivated crops. Advances in pesticide were measured exclusively by the assessment of its efficacy in management alone. But today food production has ceased to be a major concern, due to stable population levels and success of innovative agricultural technologies. At this juncture, the agrochemical industry that primarily recognized the farmers as their customers has failed to acknowledge the public as an important client. Perception of the public reflects that, the negative aspects of pesticides seem to outnumber their benefits. The increased reflection on environmental concern over pesticide use has been instrumental in a large upsurge of biological disease control. Development of fungicide resistance among the pathogens, ground water and foodstuff pollution and the development of oncogenic risks has further encouraged for the exploitation of antagonistic microflora in disease management

Introduction Advances in pesticide were measured exclusively by the assessment of its efficacy in management alone. But today food production has ceased to be a major concern, due to stable increase in population levels and success of innovative agricultural technologies. At this juncture, the agrochemical industry that primarily recognized the farmers as their customers has failed to acknowledge the public as an important client. Perception of the public reflects that, the negative aspects of pesticides seem to outnumber their benefits. The increased reflection on environmental concern over pesticide use has been instrumental in a large upsurge of biological disease control. Development of fungicide resistance among the pathogens, ground water and foodstuff pollution and the development of oncogenic risks has further encouraged for the exploitation of antagonistic microflora in disease management. Biological control of plant pathogens can be broadly defined as the reduction of inoculum density or disease producing activities of a pathogen or parasite in its active or dormant state by one or more organisms, accomplished naturally or through manipulation of environment, host or antagonist or by mass introduction of one or more antagonists. The decades of laboratory experiments have led to the explosion in the field of bio-control and introduced more than 50 commercial formulations comprising of fungi and bacteria. To have a successful management of plant diseases and to have a pollution free environment, the biocontrol agents has to be ensured for its availability for commercial use by the end users. To harness the potential of biopesticides by the stakeholders and end users the product has to be registered under Central Insecticide Board authorities concerned of the respective countries.

Introduction A sustainable approach to managing pests by combining biological, cultural, physical and chemical tools in a way that minimizes economic, health and environmental risks. The objective for IPM practitioners is to minimize risks to human health and the environment from the pest management actions implemented. IPM is a holistic approach that seeks to manage pests by using methods that are effective, economically sound, and ecologically compatible. IPM practitioners base decisions on information that is collected systematically as they integrate economic, environmental, and social goals. IPM promotes the use and integration of biological control agent as one of the strategies for pest and disease management. Because, biological control of plant pathogen is a potential alternative to the use of environment harming chemical pesticides in agriculture.

To study the characteristics of microorganisms, it is essential to culture and growth them. Growth is the increase in cell number which can be obtained by providing proper physical and chemical conditions. Medium is a substance that provides nutrients for growth and multiplication of microorganisms. Any medium for the cultivation of bacteria and fungi must provide certain basic nutritional requirements which include a carbon, nitrogen, phosphate and various mineral nutrients. The following is the medium composition for the isolation and culturing of different biocontrol agents

The isolation of microbes is commonly done using serial dilution technique. 1. Trichoderma Trichoderma is one of the important biocontrol agents. The colonies first produce mycelial growth and then turn yellow and finally produce a dark green color mat.

The discovery and use of synthetic chemicals has contributed greatly to the increase of food production industry by controlling pest and diseases. However, the use of synthetic chemicals has raised number of ecological problems apart from increasing the cost of cultivation. In recent years, scientists have directed their attention in exploring the potential of beneficial microbes for plant protection measures. Biocontrol agents exhibit antagonistic activity against plant pathogens and activate resistance mechanisms in the host despite improving plant growth. In agricultural soils the nutritional condition is depleted hence beneficial microbes decrease and increase the pathogenic microbes. To ensure the sustained availability of biocontrol agents mass production technique and formulation development protocols has to be standardized which also increases the shelf life of the formulation. Though these agents have a very good potential in the management of Pest and Diseases it can not be used as cell suspension under field conditions. The cell suspensions of these biocontrol agents should be immobilized in certain carriers and should be prepared as formulations. The organic carriers used for formulation development include peat, turf, talc, lignite, kaolinite, pyrophylite, zeolite, alginate, pressmud, sawdust, vermiculate, etc.

Bacteria are by far the common type of soil microorganisms, possibly because they can grow rapidly and have the ability to utilize a wide range of substances as either carbon or nitrogen sources. The bacteria that provide some benefit to plants are found near, on (or) even within the roots of plants (Kloepper et al., 1980) to protect the plants from pathogen attack called as antagonistic organism. Among the many potential bacterial antagonists associated with the plant roots the fluorescent pseudomonads have received prominent attention due to their abundance in plant rhizosphere and their ability to colonize roots of a wide range of crop plants. Pseudomonas fluorescens is a Gram negative, non spore forming, rod shaped bacteria producing pigments which are greenish, fluorescent and water soluble. It is found to be effective against the following fungal pathogens viz., Rhizoctonia solani, Macrophomina phaseolina, Fusarium udum, Pyricularia grisea, Fusarium oxysporum fsp. cubense. It is capable of surviving in the spermosphere, rhizosphere and phyllosphere of plants. The mechanisms of biological action exhibited inovles antibiosis, competition, lysis and induced resistance. Pseudomonas have an exceptional capacity to produce a wide variety of metabolites, including antibiotics, that are toxic to plant pathogens.

1. Raw Materials Culture of Bacillus subtilis Talc powder - 300 mesh

Shelf life for Pseudomonas fluorescens (TNAU Pf-1) Aim: To find out the shelf life of P. fluorescens (TNAU Pf-1) using different carrier materials Product: The talc based P. fluorescens (TNAU Pf-1) product is used for the study on shelf life. Selective medium (King’B medium) for Pseudomonads

Deoxyribonucleic acid (DNA) carries the genetic instruction for the biological development and functioning of all cellular forms of life. It is the blue print which contains the instructions to construct other cell components. Molecular biology experiments often require isolation of DNA for a variety of purposes like construction of DNA libraries, gene cloning, PCR and other marker technology, etc. The extraction of DNA from actively growing tissue is relatively easier yielding higher quantities of DNA. Principle The cell wall is ruptured and the proteins associated with DNA are removed by detergent like SDS or by interaction with phenol. Lipids are extracted with alcohol and DNA is precipitated by isopropanol. Materials Required Maize seed samples, Liquid nitrogen, CTAB (Cetyl trimethyl ammonium bromide), Chloroform, isoamyl alcohol, Sodium chloride, Glacial acetic acid, Isopropyl alcohol and 70% ice cold ethanol, TE buffer (5X) (24.2 g of Tris base, 5.71 ml of galcial acetic acid, 10 ml of 0.5M EDTA (pH 8.0) , dissolved in 1000 ml of distilled water). Prepare 1X TE as per the requirement.

Many Bacillus species are capable of producing a wide variety of secondary metabolites that are diverse in structure and function. The production of metabolites with antimicrobial activity is one determinant of their ability to control plant diseases. The activity is determined using PCR. PCR consists of an exponential amplification of a DNA fragment, and its principle is based on the mechanism of DNA replication in vivo. dsDNA is denatured to ssDNA, duplicated, and this process is repeated along the reaction according to the following formula

Agarose gel electrophoresis is used for the fractionation and characterization of nucleic acids. It is a simple and highly effective method for separating, identifying and purifying DNA fragments of various sizes. DNA can be checked for size, intactness, homogeneity and purity by this technique. Agarose is a purified form of agar and forms a gel by hydrogen bonding of the agarose monomers. Increasing the Agarose concentration of a gel reduces the migration speed and enables separation of smaller DNA molecules. Shorter molecules move faster and migrate further than longer ones. Aim: To study the size of the DNA extracted from the fungal culture. Principle: Ethidium Bromide added to the gel interacts with the bases of DNA and fluorescence orange when irradiated with UV light.

Introduction Studies on biocontrol agents have revealed the presence of a diverse range of antifungal metabolites derived from amino acid, polyketones and isoprenoid biosynthetic pathways, each of which has distinctive regulatory points and is subjected to different stimuli within organisms. In this experiment, the extraction of secondary metabolites from the culture of T. viride and Chaetomium globosum will be demonstrated.

Entomopathogenic nematode (EPN) viz., Heterorhabditis, Neosteinernema and Steinernema have been recovered from many regions of the world. They selectively infect many insects and a few other arthropods but do not adversely affect mammals or plants. They are actually vectors of pathogenic bacteria, cause relatively rapid death of the host (24 – 48 h) and show considerable potential in biological control of insect pests in the integrated pest management systems. EPNs are similar to insect parasitoids in that the immature form develops at the expense of one host individual; they are ecologically similar to both parasitoids and arthropod predators because the host individually killed. However, these nematodes differ from predators and parasitoids in two ways: (1) the nematode is mutualistically associated with a bacterial pathogen which actually kills the host, and (2) the pathogenicity of the bacterium has a great influence on the efficacy of the system (Ehler, 1990). The purpose of this chapter is to introduce EPN as biocontrol agents in this refresher course and to provide an overview of some current issues in biological control of insects with particular emphasis on their relevance to the use of EPNs. Biological control is defined as the action of natural enemies which maintains a host population at levels lower than would occur in the absence of the enemies. Natural enemies will include parasitoids, predators, nematodes, microbial pathogens and their gene products. Entomologists generally divide biological control into two categories: (1) natural biological control which is effected by native or coevolved natural enemies in the native home (origin) of a given insect and (2) applied biological control which due to human intervention. Applied biological control is further divided into classical biological control (introduction of exotic natural enemies) and augmentative biological control (enhancement of natural enemies already in place).

Introduction Corcyra cephalonica commonly called as rice meal moth or rice moth is a pest of stored foods, viz., cereals, cereal products, oilseeds, pulses, dried fruits, nuts and spices. Many of the natural enemies mass-bred in the laboratory for use in field against crop pests are dependent on either egg or larval stages of Corcyra due to the simple reason that it is easier and cheaper to produce natural enemies on different stages of Corcyra than on their original hosts. Morphology and Biology of Corcyra The eggs are oval and measure 0.5 x 0.3 mm. The white surface is sculptured and has a short nipple-like structure at one end. The larvae are generally creamish – white except for the head capsule and the prothoracic tergite, which are brown. There are well-developed prolegs on abdominal segments 3-6 and 10. A fully matured larva measures 15 mm. The last-instar larva spins a closely woven, very tough, double-layered cocoon in which it develops into a dark-brown pupa. The anterior portion of the cocoon has a line of weakness through which the adult emerges. The adults are small. The hind-wings are pale-buff, and the fore-wings are mid-brown or greyish-brown with thin vague lines of darker brown colour along the wing veins. The males are smaller than the females. Sexual activity usually begins shortly after adult emergence. There is a preoviposition period of about 2 days. Egg-laying mainly occurs during the night. The greatest numbers are laid on the second and third days after emergence, although oviposition may continue throughout life. Eggs take about 2-3 days to hatch. Optimum conditions for larval development of C. cephalonica are 30 – 32.5 °C and 70 per cent RH, at which, the period from egg hatch to adult emergence is only 26-27 days. There is considerable variation in the number of larval instars; however, males generally have 7 and females have 8. The last-instar larvae pupate within the food. The adults emerge through the anterior end of the cocoon, where there is a line of weakness. The sex ratio is 1:1. The adult moth is nocturnal and is most active at nightfall.

Although the Rhabditids have been studied from last one century, the field level use of Entomopathogenic nematodes the members of Rhabditids for the management of insect pest was followed only after Dutky who practically used EPNs in 1937 on Apple Codling moth. The taxonomy of Entomopathogenic nematodes of the order Rhabditidae is discussed in this lecture.

Basically we need to mount and prepare the slide for observation of various stages of EPN as detailed below in Table.1

I. In vivo culturing technique In vivo culturing means culturing of entomopathogenic nematodes (EPN) in the host. Generally rice meal moth, Corcyra cephalonica and greater wax moth, Galleria mellonella are used as insect hosts for rearing EPNs.Insect hosts such as Spodoptera, Helicoverpa and Bombyx mori are also used for culturing Steinernema and Heterorhabditis but these insects need plant host especially leaves for culturing. Corcyra can be grown in broken cumbu grains and Galleria in honey comb or artificial diet.

Introduction Entomopathogenic nematodes (EPNs) viz., Heterorhabditis, Neosteinernema and Steinernema have been recovered from many regions of the world and have been used commercially as biocontrol agents of insect pests. They are associated with mutualistic bacteria in the genus Xenorhabdus for Steinernematidae and Photorhabdus for Heterorhabditidae. The infective juvenile nematodes enter their host insect through natural openings (mouth, anus or spiracles), they release bacteria into the insect and cause rapid death of insect host (24-48 h). EPNs were used against many insect pests viz., rice leaf folder, Cnaphalocrocis medinalis (Srinivas and Prasad, 1991); tobacco cutworm, Spodoptera litura (Rajkumar et al., 2003); brinjal fruit borer, Leucinodes orbonalis (Hussaini et al., 2002) diamond back moth, Plutella xylostella (Singh and Shinde, 2002); Diaprepes abbreviates (Jenkins et al., 2007); Bemesia tabaci (QiuBaoli et al., 2008) and sugar beet beetle (Saleh et al. 2009).

Introduction Agricultural production in India is constrained by number of biotic and abiotic factors. India is a tropical country and has a climate which is conducive for insect pests which are, one of the major constraints in agricultural production, processing and storage. Annual crop losses due to insect pests and diseases in India are estimated to be 18 percent of the agricultural output. Insect pests are widespread in all the agroclimatic zones and large quantities of insecticides are used to control these pests. Indiscriminate use of insecticides, lack of adequate knowledge on proper application techniques and dosages, and frequent use of broad spectrum insecticides are common in India. There is a need to identify suitable alternative methods for the management of insect pests and among them, biological control using viruses, bacteria, fungi and entomopathogenic nematodes is gaining greater attention world over. Nematode families of Steinernematidae and Heterorhabditidae attracted most attention as they contain the entomopathogenic nematodes (EPN). Entomopathogenic nematodes are obligate parasites of insects that kill their hosts with the aid of bacteria (Xenorhabdus and Photorhabdus) carried in the nematode’s alimentary canal. The lethal obligate insect parasites are ubiquitously distributed without pathogenicity to mammals and beneficial non-target insects, and exempted from registration requirements. Currently there are about 98 valid EPN species belonging to Steinernema (79) and Heterorhabditis (19). Soil surveys conducted in divergent areas of Indian subcontinent have demonstrated high diversity of EPN across seasons, habitats, and geographic regions. There are several target pests in India that can be controlled with entomopathogenic nematodes and several workers have used EPN against cutworms, stem borer, white grubs, etc. under laboratory and field conditions and are compatible with existing IPM programmes.

Entomopathogenic nematodes (EPN) in the family Steinernematidae and Heterorhabditidae have enormous potential to control insect pests of economic importance. Since, chemical pesticides pose environmental hazards, these nematodes have received considerable attention as potential bio-insecticides because of their wide host spectrum, active host seeking, killing the host within 48 h, easy mass production, storage and application and are environmentally safe. One of the important criteria for a successful biocontrol agent is formulation of nematodes into a stable product, which has played a significant role in commercialization of these biological control agents. Apart from formulation field application and desired control of insect pests decides the success of EPN. Formulation of Entomopathogenic Nematodes EPNs have been known since 1929 but they became commercially available only during 1990s.The first attempts at formulating EPN were initiated in 1979 but the shelf life was very limited (one month). Infective juveniles carried on moist substrates such as sponge, vermiculite and peat require continuous refrigeration to maintain their viability. While formulating nematodes, two things are necessary, a long shelf-life and easy application method. The simplest method is impregnating a moist substrate (such as sponge, peat, vermiculite, etc.) with nematodes. The sponge needs to be squeezed in water before application, to release the nematodes, whereas nematodes from other carriers could be applied directly to the soil. However, these formulations are labour-intensive, require continuous refrigeration and lack economy of scale and therefore can be used on small scale only. Although the infective juveniles of entomopathogenic nematodes can be stored for several months in water in refrigerated bubbled tank, high cost and difficulties of maintaining quality preclude the deployment of this method. Therefore, nematodes are usually formulated into solid or semi-liquid substrates soon after they are produced.