Nematodes as Plant Pathogens PDF

CSB 324 Introduction to phytonematodes  Introduction to nematodes  Nematodes roundworms belonging to the Phylum Nematoda  the second most abundant and diverse multicellular animals on earth after insects.  Nematode species are very difficult to distinguish be...

CSB 324 Introduction to phytonematodes  Introduction to nematodes  Nematodes roundworms belonging to the Phylum Nematoda  the second most abundant and diverse multicellular animals on earth after insects.  Nematode species are very difficult to distinguish because of their subtle differences in their morphology.  Number of cells that constitute the nematode’s body, except the gonads are predetermined  Size increase after that number is reached proceeds by cell division as in other animals  Introduction to nematodes  over 80,000 genera have been described, of which over 15,000 are parasitic.  Because of their abundance, nematodes are an important component of every ecosystem.  They occupy nearly all ecological habitats from marine to fresh water, free living in the soil or as parasites of animals, insects and plants.  Of all the escribed genera, 50 % are marine dwellers; 25 % free- living or fresh-water 15 % animal parasites and 10 % plant parasites.  Some are beneficial as entomopathogens  Nematodes from different habitats Freshwater Plant parasitic Human parasiticInsect parasitic Free-living Marine nematode nematode Nematode/ nematode nematode nematode entomopathogenic  Nematode morphology  Nematodes are roundworm with unsegmented bodies.  They are bilaterally symmetrical and triploblastic i.e. possessing three layers: ectoderm, mesoderm, and endoderm.  Nematodes are thin and round in cross section  They have no circulatory system and transport nutrients through the body via fluid in the pseudocoelom.  They have a complete digestive system with separate orifices for food intake and waste excretion, a pattern followed by more complex animals.  The body cavity is a pseudocoelom, which lacks the muscles of coelomate animals that protects the body from drying out, from digestive juices, or from other harsh environments. . General morphology of a nematode  There are different morphologies  Mostly nematodes are eel- shaped  Some exhibit sexual diamorphism  Female root knot nematodes are pear-shaped  Reniform and Citrus nematodes are kidney-shaped (Rotylenchulus spp. and Tylenchulus spp. respectively)  General morphology of nematodes  Although this cuticle allows movement and shape changes via a hydrostatic skeletal system, it is very inelastic and so does not allow the volume of the worm to increase.  Therefore, as the worm grows it has to molt and form new cuticles.  The cuticles do not allow volume to increase so as to keep hydrostatic pressure inside the organs very high.  For this reason, the roundworms do not possess circular muscles (just longitudinal ones) as they are not required.  This hydrostatic pressure is the reason the roundworms are round Ecdysis in nematodes  General morphology of nematodes: The nervous system  Nematodes have a simple nervous system, with a main ventral nerve cord and a simple dorsal nerve cord.  Sensory structures at the anterior end are called amphids, while sensory structures at the posterior end are called phasmids.  There are no circular muscles, so the body can only undulate from side to side.  Contact with solid objects is necessary for locomotion;  its thrashing motion varies from mostly to completely ineffective at swimming. Amphids vs phasmids  General morphology  Nematodes generally eat bacteria, fungi and protozoans, although some are filter feeders.  Excretion happens through a separate excretory pore.  Nematodes also contract bacterial infections within excretion pores.  The type of cephalic framework can give an idea as to what the nematode feeds on.  Most fungal and algal feeders have a mural tooth that is used to pierce the cells while plant parasitic nematodes have a ´spear´ used to pierce plant cells. Cephalic frameworks and feeding types Morphology of the head  Reproduction  Reproduction is usually sexual.  Males are usually smaller than females (often much smaller) and often have a characteristically bent tail for holding the female for copulation.  During copulation, one or more chitinized spicules move out of the cloaca and are inserted into genital pore of the female.  Amoeboid sperm crawl along the spicule into the female worm.  Nematode sperm is thought to be the only eukaryotic cell without the globular protein G- actin.  Eggs may be embryonated or unembryonated when passed by the female, meaning that their fertilized eggs may not yet be developed  Reproduction  In free-living roundworms, the eggs hatch into larva, which eventually grow into adults;  in parasitic roundworms, the life cycle is often much more complicated.  Nematodes possess a wide range of modes of reproduction  some are hermaphroditic – they keep their self-fertilized eggs inside their uteruses until they are ready to hatch  the juvenile nematode will then ingest the parent nematode through a process termed Indotokia matricida.  Reproduction  Meloidogyne spp exhibit a range of reproductive modes including:  sexuality (amphimixis),  facultative sexuality  meiostic parthenogenesis (automixis)  mitotic parthenogenesis (apomixes). Nematode taxonomy and classification  Traditional classification  Mostly dependent on morphological characteristics of nematodes especially the presence or absence of phasmids.  Two classes;  Adenophoria; without phasmids;  Secernentea; with phasmids  Modern Classification  Based on small subunit ribosomal DNA (SSU rDNA).  The small subunit is the 16s component of the rDNA.  Current (2001, 2002, 2004 and 2006) integration by Paul de Ley (UC Riverside) and Mark Blaxter (University of Edinburgh).  Two Classes Enoplea and Chromadorea  Order Dorylamia in Enoplea has members that are of Agricultural importance  Most plant parasites under Chromadorea (Tylenchids)  Tylenchids constitute 94% of plant parasitic nematodes belonging to several families, and genera, e.g. root knot nematodes, lesion nematodes, rice nematodes etc Table 1: Morphological characters that delineate Chromadorea from Enoplea Character Chromadorea Enoplea Amphid Pore like/ Slit like Pocket like Post labial pore/ spirals Cuticle Annulated Smooth or finely striated Oesophagus 3 parts with 3 to 5 glands Cylindrical or bottle shaped Morphology of nematode oesophagus Shapes of amphids  PLANT PARASITIC NEMATODES  They possess a stylet, which distinguish them from saprophytic nematodes (see diagrams below).  They are generally smaller than saprophytic nematodes.  Piercing and thrusting muscles move stylet and allow the nematode to puncture host cell and suck cell fluids to the digestive system.  Digestive enzymes move through the stylet and it is through this process that viruses are acquired and transmitted.  There are two types of stylets possessed by nematodes that are used to separate them into classes:  1) Stomatostylet ( Chromadorea)  2) Odontostylet (Class Enoplea  Types of stylets Types of stylets A Stomatostylet and B Oontostylet  LIFE CYCLE OF A TYPICAL PHYTONEMATODE  Duration of life cycle of most phytonematodes is 3-4 weeks from commencement of egg-laying to the appearance of eggs of the next generation.  Stages involved in the cycle:  1. Egg – in the soil, in or on plant roots  2. Juvenile (Larva)  1st stage juvenile develops in the egg; 1st molt occurs within the egg  2nd juvenile: hatches from the egg; 2nd molt  3rd stage juvenile: in the soil, in or on plant; 3rd molt  4th stage juvenile; 4th molt Lifecycle of a typical phytonematode  Growth and development  Growth of the nematodes requires Molting  During molting, the nematode becomes quiescent and halts its feeding  Enzymes from the hypodermis digest part of the old cuticle  New cuticle is secreted by the hypodermis under the remains of the old one  Nematode resumes movement and crawls out of the old cuticle .  Lifecycle of phytonematodes  Pratylenchus spp. from 2nd stage juvenile on, moves freely into and out of roots.  Then the life cycle progresses as outlined below.  Lifecycle of Meloidogyne spp  Develop into 2nd stage juveniles within the eggs.  The 2nd stage juveniles then emerge to move about in soil in search of susceptible roots.  Once inside the root, Meloidogyne juveniles become immobile and develop into sausage-shaped individuals.  The enlarged sausage-shaped juveniles (after 8-9 days in a favorable host and under favorable temperatures) stop feeding and undergo three molts in succession in a period of 2-3 days.  The resulting individuals are differentiated into adult females and males.  The female resumes feeding and enlarges and becomes pear-shaped. From the time that the 2nd stage juvenile found a favorable position in the roots it lost its ability to move.  The male regains the ability to move while the female remains in one position.  Female starts egg-laying by depositing several eggs in a gelatinous matrix that usually protrudes from the root surface.  Lifecycle of Meloidogyne spp.  Feeding types of phytonematodes 1. Surface feeders:  Feed on cells of the root epidermis and on root hairs  Generally do not penetrate root cortex  e. g. Trichodorus spp.  Paratylenchus spp commonly known as Pin nematode Paratylenchus spp ffect of stubby root Trichodorus spp. Nematode (Trichodorus spp  Phytonematode feeding types 2. Ectoparasitic feeders  Feed on cells in the cortex  They penetrate with their stylets and sometimes with a small portion of the anterior body.  e. g. Belonolaimus spp., Xiphenema spp., Longidorus spp. and Rotylenchus spp.  Feeding types of phytonematodes 3. Migratory endoparasites  Habitually enter plant tissues and move about actively.  E.g.Pratylenchus spp ( lesion nematode)., Ditylenchus spp ( stem and bulb nematode)., Radopholus spp ( burrowing nematode) and Aphelonchoides spp (leaf/foliar nematode). Nema wool Pratylenchus spp Effect of Ditylenchus spp Effect of Rodopholus spp Effect of Aphelonchoides spp  Phytonematode feeding types 4. Sessile endoparasites  Are adapted to a sessile life and do not move  Adult female are swollen and remain in one position while males are elongate and active  They induce the formation of specialized cells in the host  e. g. Meloidogyne spp (root knot nematode)., Heterodera spp. (lemon –shaped Cyst nematode). Globodera spp. (globe-shaped Cyst nematode) and Naccobus spp. ( Fakse root knot nematode) emale root knot nematode False root knot nematode Heterodera spp Globodera spp DIAGNOSIS OF DISEASES CAUSED BY NEMATODES Observation of symptoms and signs 1.  It may be possible to diagnose a few nematode-induced plant diseases by careful examination of infected plants  to look for specific signs and symptoms (galls, cysts, stubby roots, “nema wool” (for stem and bulb nematodes), and areas of internal leaf necrosis filled with nematodes (for foliar nematodes). 2. Soil and plant tissue assay and Microscopy  In the absence of obvious signs, soil-borne nematodes must be diagnosed by assaying soil and root samples  Nematodes tend to congregate in “hot spots”, so a composite set of samples will most accurately reflect nematode populations  The time of year and where nematodes are expected in the soil profile must also be considered when samples are taken.  Once the nematodes have been isolated from soil or plant tissue, microscopy is used to look at morphological characters  After extraction, nematodes must be identified and counted under a microscope.Other parts of the plant must also be incubated to let nematodes move out  Extraction of nematodes from soil There is a number of extraction methods. These include: Washing and Sieving: a number of nestled sieves are used with the highest perture size at the top and the smallest aperture at the bottom Baermann funnel Modified Baermann Funnel/ tray method Decanting and sieving Modified Baermann tray Root incubation Foliar puncturing technique method Baermann funnel and incubation  Extracting cyst nematodes Washing and drying method Oostenbrink Elutriator Fenwick can  Centrifugation-floatation technique  Thismethod is used to extract all life stages including eggs,sessile, moving and dead nematodes.  Uses gravitational force to float out nematodes  Traditionally termed ‘The sucrose method’  Other ‘heavy’ chemicals such as iodixanol solution, and Ludox solution  Diagnosis of diseases caused by nematodes  Host specificity test in specific-host nematodes  Molecular identification  Culturing isolation DNA extraction Identification Alignment Sequencin With existing data g Nematode dispersal and dissemination