Paper No. : 08 Biology of Parasitism

Module : 33 Plant Parasitic

Development Team

Principal Investigator: Prof. Neeta Sehgal Head, Department of Zoology, University of Delhi

Paper Coordinator: Dr. Pawan Malhotra ICGEB, New Delhi

Content Writers: Dr. Vartika Mathur, Sri Venkateswara College, University of Delhi Dr. Tom O. G. Tytgat, Department of Molecular Plant Physiology, Institute for Water and Wetland Research, Radboud University Nijmege, The Netherlands

Content Reviewer: Prof. Virender Kumar Bhasin Department of Zoology, University of Delhi

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Biology of Parasitism ZOOLOGY Plant Parasitic Nematodes

Description of Module

Subject Name ZOOLOGY

Paper Name Biology of Parasitism

Module Name/Title Morphology, biology, life cycle and infection of crop plants by parasitic nematodes

Module ID 33; Plant Parasitic Nematodes

Keywords PPN, Triplonchida, , Tylenchida, root parasitic nematodes, ecto-endoparasites

Contents

1. Learning Outcomes 2. Introduction 3. Phylogeny 4. Biodiversity 4.1. Triplonchida 4.2. Dorylaimida 4.3. Tylenchida 4.3.1 Above Ground Feeders 4.3.2 Root Parasitic Nematodes 5. Summary

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Biology of Parasitism ZOOLOGY Plant Parasitic Nematodes

1. Learning Outcomes

After completing this module, you will be able to:

 understand the economic and ecological importance of plant parasitic nematodes.  learn the phylogeny of the plant parasitic nematodes.  understand their biodiversity.  identify and classify them based on their morphological features and mode of parasitism.  distinguish them based on their trophic niches.

2. Introduction

Plant parasitic nematodes (PPN) nematodes are usually small, soil-borne pathogens and the symptoms they cause are often nonspecific. They are extremely diverse and abundant in all types of soil, and plants regularly encounter them during their lifetime (Sohlenius, 1980). There are more than 4100 described species of PPN (Decraemer & Hunt, 2006). With only a few exceptions, PPN are root feeders. Their presence is not only the key driving force of plant succession in natural environments (De Deyn et al., 2003), but they also collectively represent a significant constraint to the global food security with annual crop damages estimated to be more than $US80 billion per year (Nicol et al., 2011).

Nematode infection affects the plant at different levels. Infested fields show a reduced number of plants, and infected plants have a lower biomass due to a disturbed physiology (Figure1). Crop damage is not only limited to the direct -plant interaction itself, but very often nematode infected plants are more susceptible to fungal or bacterial infection (Abawi & Chen, 1998). Certain nematode species can serve as vectors for a number of important plant viruses (Brown et al., 1995), or may enhance crop losses associated with insect as well as weed pests (Alston et al., 1991).

Figure 1: A healthy sugar beet versus a beet infected with the cyst nematode Heteroderas chachtii. 3

Biology of Parasitism ZOOLOGY Plant Parasitic Nematodes

3. Phylogeny

Most of the nematode species are microscopic in size and lack obvious distinguishing characteristics. Therefore, classification within the phylum has been very difficult. Based on small-subunit ribosomal sequences of 53 species, Blaxter et al. (1998) proposed a molecular evolutionary framework for the Nematoda comprising five major clades, all of which including parasitic species (Figure 2). It suggests that parasitism arose independently at least four times, and plant parasitism three times.

4. Biodiversity

Evolutionary adaptation to plant parasitism led to some striking morphological changes (Hussey, 1989). Almost all plant-parasitic nematodes possess a hollow, protrusible, hypodermic needle-like axial spear, which serves as the interface between the plant and the nematode.

Figure 2: A phylogenetic hypothesis for the phylum Nematoda based on small subunit sequences (Blaxter et al., 1998). C+S indicates clade Chromadorida plus Secernentea; S+P indicates Secernentea plus Plectidae. Plant parasitism occurs in three widely separated orders.

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Biology of Parasitism ZOOLOGY Plant Parasitic Nematodes

It can be used to penetrate the plant cell wall, to withdraw nutrients from the cytoplasm, or to release secretions. These secretions are synthesised in the pharyngeal glands, which have enlarged considerably when nematodes adapted to parasitism. Two different types of pharyngeal glands are recognised: subventral and dorsal glands (Figure 3). Each gland is a single cell, with a long cytoplasmic extension terminating in an ampulla, which is connected to the pharyngeal lumen via a valve-like structure (Robertson & Wyss, 1979; Endo, 1984; Endo & Wergin, 1988). Secretory proteins are processed in the well-developed rough endoplasmic reticulum and Golgi-apparatus, and collected in secretory granules (Hussey & Mims, 1990) (Figure4). These granules are transported via microtubuli towards the ampulla, where the secretions are released into the pharyngeal lumen.

Figure 3 : Diagram of the anterior region of a second-stage juvenile of a tylenchid plant-parasitic nematode (Hussey R.S., 1989). Two types of pharyngeal glands are recognized : one dorsal and two subventral. The subventral gland extensions end of the metacorpal pump chamber. The dorsal gland extension runs anteriorly until the ampulla, which is located closely behind the base of the stylet.Via a valve structure, the ampullae of both types of pharyngeal glands are connected to the pharyngeal (esophageal) lumen. The protrusible stylet has a narrow lumen throughout its length. Close to the tip, the lumen has a ventrally located orifice. The stylet isused to pierce cell walls, to release pharyngeal gland secretions, and to withdraw nutrients from the cytoplasm of a plant cell.

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Biology of Parasitism ZOOLOGY Plant Parasitic Nematodes

Plant-parasitic nematodes occur in three widely separated orders: Triplonchida, Dorylaimida and Tylenchida (Blaxter et al., 1998), with the majority being in the latter. Although there are some similarities between these taxa, it is evident that evolution to plant parasitism occurred convergently in the three orders.

Figure 4: Diagram of a pharyngeal gland cell dissected from a cyst nematode (Smant G., 1998). mRNA, coding for secreted proteins, is translated on the rough endoplasmic reticulum. The precursor proteins are modified in the lumen of the endoplasmic reticulum, and transported to the golgi-bodies for further processing. Small vesicles, containing the mature secretory proteins, bud from the golgi–bodies and fuse thereafter to form large secretory granules. The secretory granules are transported via microtubuli through the cell extension towards the ampulla. Probably by the process of exocytosis, the contents of the secetory granules are released via the valve into the pharyngeal (oesophageal) lumen.

4.1. Triplonchida

All triplonchid plant-parasitic nematodes are migratory ectoparasites on roots (Wyss, 1997).The species of the family have five secretory gland cells, one dorsal and two anterior and two posterior subventral glands, which end in the basal bulb of the pharynx. Their stylet (onchiostyle) is a modified mural tooth. Trichodoridae (Figure 5.1) explore the surface of the root, and feed on epidermis cells. During feeding, which rarely exceeds a few minutes, the ventrally curved stylet is continuously thrust into the perforated cells (Wyss, 1981).Injection of secretions results in the formation of a feeding tube (Wyss et al., 1979). Due to the relatively large width of the feeding tube, they are the only root parasitic nematodes capable of removing large organelles from the cytoplasm. If

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Biology of Parasitism ZOOLOGY Plant Parasitic Nematodes

the epidermis cells survive the feeding, Tobraviruses can be successfully transmitted by the nematodes. Trichodorids are considered to be little advanced in their mode of parasitism as the cytoplasm of several hundred cells has to be withdrawn to produce a few eggs within a week (Wyss, 1981).

4.2. Dorylaimida

The axial spear of the Longidoridae is composed of an odontostyle and an odontophore; the former is thought to have evolved from a mural tooth and has a dorsal aperture.It is formed in the anterior part of the pharynx and moves forward during moulting to replace the previous odontostyle, at the same time the odontophore is reformed in situ (Coomans & De Coninck, 1963).The length of the stylet enables the Longidoridae to feed deeper inside the root tissue.They have three pharyngeal glands, one dorsal and two subventral. Most of the time, root tips are selected as feeding sites, and root tip galls develop (Wyss, 1997). Xiphinema index (Figure 5.3) feeds on a column of cells: it starts feeding for a few minutes on one cell, and afterwards progresses to deeper laying cells.Dorsal gland secretions are injected into the cells and seem to liquefy the cytoplasm (Robertson & Wyss, 1979).After ingestion of the solutes by the nematode, the cells die of necrosis.Probably also in response to the secretions, the neighbouring cells become hypertrophied and binucleate (Wyss et al., 1988). In case of Longidorus (Figure 5.2), stylet secretions seem to induce a gall-like structure containing uninucleate hypertrophied cells surrounding the necrotic feeding cells (Griffiths & Robertson, 1984).

Figure 5: Schematic representation of feeding sites of some selected migratory root-parasitic nematodes.1: Triplonchid nematodeTrichodorus sp.; 2: Dorylaimid nematode Longidorus elongatus; 3 : Dorylaimid nematode Xiphinema index; 4-7 : Tylenchid nematodes ; 4 : migratory ectoparasite Tylenchorhynchus dubius; 5 : sedentary ectoparasite Criconemella xenoplax; 6 : migratory ecto-endoparasite Helicotylenchus sp.; 7 : migratory endoparasite Pratylenchus sp. (Reproduced from Wyss, 2002)

4.3. Tylenchida

Tylenchida are thought to have evolved from rhabditid forms, the hollow spear being a fusion of sclerotisedrhabdions (Jones, 1965). The tylenchid spear has a narrow lumen with ventral aperture 7

Biology of Parasitism ZOOLOGY Plant Parasitic Nematodes

close to its tip, and because it is formed in situ from the wall of the stoma it is called stomatostylet. All tylenchids have three pharyngeal glands: one dorsal and two subventral. Plant parasitic nematodes are often classified according to their trophic niche (Wyss, 1997; Ferris & Ferris, 1998), and the tylenchids will be reviewed as such.

4.3.1. Above Ground Feeders

Above ground feeders can be divided into three groups: bulb, stem and foliar nematodes. Although these nematodes are sometimes found in the soil, they migrate up the stems of plants and enter bulb, stem, foliar or floral tissue. The most common of the stem nematodes are species from the genus Ditylenchus. D. dipsaci is a known migratory endoparasite, which feeds on parenchymatous tissue in stems and bulbs of onion, alfalfa, clover, etc. Feeding often causes swelling and distortion as a result of the breakdown of the middle lamellae of cell walls. Certain juvenile stages exhibit the phenomenon called anabiosis and can survive in a desiccated form for many years.This often causes them to be spread from one place to another in plant material.The pine wood nematode, Bursaphelenchus xylophilus, is vectored by a cerambycid beetle from diseased to healthy pine trees (Linit, 1988). It causes pine wilt disease, which has become a major problem in native pine forests in Japan, southern China and the USA. Depending on the age and physiological conditions, heavily infected trees may die within a few weeks to a year after infection (Abawi & Chen, 1998). Anguinatritici is a parasite of cereals, causing leaf, stem or seed galls. Dry seed galls contain quiescent juveniles. The nematodes revive with moisture, and infect developing shoots or move up the plant. When flower primordia form, they enter this tissue, and stimulate development of galls instead of normal seeds (Ferris & Ferris, 1998).

4.3.2. Root Parasitic Nematodes

According to their parasitic strategy, they can be classified into different categories (Wyss, 1997; Tytgat et al., 2000):

 Migratory ectoparasites stay vermiform throughout their life cycle and feed for a short time at several places along the root. Several genera from the Tylenchida belong to this group. Tylenchorhynchus (Figure 5.4) possesses a short stylet, and feeds on epidermis cells, causing little damage to the plant roots. No galls are induced. Belonolaimus feeds on deeper root tissue.  Sedentary ectoparasites like Paratylenchus and Criconemella (Figure 5.5) have been observed feeding on the same cell for many days (Rhoades & Linford, 1961; Hussey et al., 1992). Depending on the species, the food cell can be an epidermal or cortical cell.  Migratory ecto-endoparasites, also called semi-endoparasites, can feed ecto-parasitically on roots, but very often they partially invade the roots to feed on cortical or outer stellar cells. They remain vermiform throughout development. Examples are Hoplolaimus and Helicotylenchus, often parasites of grasses. Helicotylenchus spp. may invade roots completely, where they feed from a single cell for several days with alternating periods of salivation and ingestion (Jones, 1978a). The feeding cell (Figure 5.6) is surrounded by a few metabolically active cells with dense cytoplasm but without nuclear enlargement. Feeding tubes were detected in the feeding cell (Jones, 1978b).  Migratory endoparasites are found in the subfamily of the Pratylenchinae (Figure 5.7), with Pratylenchus and Radopholus being the most economically important genera. Every life stage of 8

Biology of Parasitism ZOOLOGY Plant Parasitic Nematodes

Pratylenchus spp. can enter or leave the root. They mainly feed on cortical cells. Many cells are killed during their intracellular migration (Zunke, 1990), and this makes the plants susceptible to invasion by other organisms.  Sedentary endoparasites have evolved the most advanced and specialised strategies of root parasitism. They induce specific permanent feeding cells or feeding structures from which they derive their food throughout development and reproduction. The invasive developmental stages are vermiform, but depending on the genera, it can be freshly hatched second-stage juveniles (J2) (e.g. Globodera, Heterodera, Meloidogyne) or adult females (e.g. Nacobbus, Rotylenchulus, Tylenchulus). Soon after feeding cell induction, the nematodes become sedentary due to a degeneration of somatic musculature. The female is always sedentary. Male juveniles stay or become vermiform again before maturity and regain their mobility.

Only few sedentary endoparasites derive their food from modified cells in the cortex of their host roots (Figure 6). Trophotylenchulus obscurus invades the roots at the J2 stage (Vovlas, 1987), and the sedentary female feeds from a metabolicaly active single uninucleate cell. Female juveniles of Tylenchulus semipenetrans feed as migratory ectoparasites, and it is only the young adult female that enters the root. Six to ten cortical cells around an empty cell, in which the head of the nematode remains protruded, are transformed into metabolically highly active cells with enlarged nuclei (Himmelhoch et al., 1979; B’Chir, 1988). Although Verutus volvingentis belongs to the subfamily Heteroderinae, which all induce syncytia or giant cells in the vascular cylinder of the root, it induces a syncytium within the cortex of the root (Cohn et al., 1984).

Figure 6: Schematic representation of feeding sites of some selected sedentary endoparasites. Reproduced from Wyss (2002).

Most sedentary endoparasites migrate to the vascular cylinder of their host roots, where they select one or a few cells that become transformed into permanent feeding sites (Figure 6). These sites can be assigned to three groups: uninucleate giant cells, multinucleate giant cells and syncytia. Single uninucleate giant cells are generally found in woody host plants of noncyst forming heteroderids such as Cryphoder autahensis. 9

Biology of Parasitism ZOOLOGY Plant Parasitic Nematodes

The giant cells are primarily induced in the pericycle, from where they expand into the stele. The nucleus of these cells is extremely enlarged and deeply invaginated to ensure an increased rate of nuclear- cytoplasmic exchange. The wall of the giant cell is considerably thickened at the head end of the nematode; elsewhere it contains numerous pit fields with many plasmodesmata for transport of solutes from adjacent cells. Feeding tubes have not been recorded (Mundo-Ocampo & Baldwin, 1984).The life cycle of Rotylenchulus reniformis is unique. Freshly hatched second stage juveniles undergo three successive moults in the soil without feeding. Young adult females invade the roots and induce a syncytium in the pericycle (Rebois et al., 1975). Cyst nematodes of the genera Globodera and Heterodera are important parasites of crops mainly in temperate regions of the world and their host range is usually restricted to one or few related families. They induce and maintain in the vascular cylinder of their host plants multinucleate syncytia that arise from expanding cambial cells whose protoplasts fuse after partial cell wall dissolution. The juvenile stages of Nacobbus behave like Pratylenchus spp. in that they damage the plant roots by intracellular migration (Maggenti, 1981). This is at least the case for the J2, while the subsequent juvenile stages (J3 and J4) apparently do not feed. A switch to sedentary endoparasitism occurs after the final moult, when the young females induce their typically spindle-shaped syncytia in the vascular cylinder (Souza & Baldwin, 1998). Hyperplastic responses in cortical cells are initiated concurrently, leading to the formation of galls in which the female becomes embedded. Root galls are also formed in roots infected by root-knot nematodes (Meloidogyne spp.). These sedentary endoparasites are the most serious soil pathogens globally. In contrast to the cyst nematodes, root-knot nematodes are more frequent in areas with warm and hot climates. Meloidogyne spp. invariably induce and maintain multinucleate giant cells. These cells develop by the expansion of about half a dozen cambial cells within the differentiating vascular cylinder of host plants. Each becomes multinuclear by repeated synchronous mitoses in the absence of cytokinesis.

5. Summary

 Plants regularly encounter plant parasitic nematodes (PPN) because of their high abundance and diversity.  More than 4100 species of PPN have been described.  PPNs are largely root feeders.  PPNs collectively cause annual crop damages of more than $US80 billion per year.  Molecular evolutionary framework for the Nematoda reveals five major clades, all of which include parasitic species. It demonstrates that animal parasitism arose independently at least four times, and plant parasitism three times.  Almost all plant-parasitic nematodes possess a hollow, protrusible, hypodermic needle-like axial spear, which serves as the interface between the plant and the nematode. It can be used to penetrate the plant cell wall, to withdraw nutrients from the cytoplasm, or to release secretions.  Plant-parasitic nematodes occur in three widely separated orders: Triplonchida, Dorylaimida and Tylenchida, with the majority being in the latter. Despite some similarities between these taxa, it is evident that evolution to plant parasitism occurred convergently in the three orders.  Plant parasitic nematodes can be functionally classified according to their mode of action into above ground feeders and root feeders. Within the root feeders, migratory ectoparasites, sedentary ectoparasites, migratory ecto-endoparasites, migratory endoparasites and sedentary endoparasites can be distinguished. 10

Biology of Parasitism ZOOLOGY Plant Parasitic Nematodes