The embryo in each nematode egg develops through embryogenesis to a first-stage juvenile (J1), which, in some longidorids and trichodorids, hatches. However, in most species of plant-parasitic nematodes, the J1 moults within the egg to the second-stage juvenile (J2). It is this invasive J2 that hatches and then feeds on a host plant. There is little variation in the average size of nematode eggs, irrespective of the size of the adult, and the eggshell of plant-parasitic nematodes typically consists of three layers, an outer vitelline layer, a middle chitinous layer and an inner lipid layer. The lipid layer is the main permeability barrier of the eggshell and makes the egg resistant to chemicals, including non-fumigant nematicides. Physiological adaptations, such as different states of dormancy, are an essential component of the survival of nematodes in the absence of a host and are frequently associated with the unhatched juveniles (Perry 1989, 2002). In the majority of species of plant-parasitic nematodes, the juvenile hatches provided environmental conditions, including temperature and moisture content of the soil, are favourable. However, in some species, co-evolution of host and parasite has resulted in a sophisticated relationship whereby the nematode does not hatch unless stimulated by chemicals emanating from the host roots. These emanations have been termed root diffusates, root leach-ates or root exudates. Root diffusates is the preferred term of the present authors because 'diffusate' conveys the idea of volatile and non-volatile components diffusing through the soil and establishing a concentration gradient; thus, it is an especially apposite term in relation to hatching and attraction of nematodes.

The hatching process can be divided into three phases: changes in the eggshell permeability, metabolic activation of the juvenile, and eclosion (or hatch from the egg). The chronological order of the first two phases differs between genera. For example, in Meloidogyne spp., activation of the juvenile appears to occur first and causes eggshell changes; in others, such as Globodera spp., alteration of eggshell permeability characteristics appears a necessary pre-requisite for activation of the juvenile (Perry 2002). The agents for initiation of these responses vary between species and genera of nematodes but have been studied most extensively in species of root-knot and cyst nematodes. Hatching and survival attributes of these species are associated with the 'packaging' of eggs into ecological units (Perry and Moens

Fig. 1.3 Egg masses of Meloidogyne chitwoodi stained with Phloxine B; the posterior end of the adult female (arrowed) is visible outside the root. (Courtesy Wim ML Wesemael, Institute for Agricultural and Fisheries Research, Belgium)

2011). Females of root-knot nematodes lay eggs into a gelatinous matrix, which comprises an irregular meshwork of glycoprotein material (Sharon and Spiegel 1993). The gelatinous matrix surrounds the eggs and retains them in a package termed an egg mass (Fig. 1.3). With cyst nematodes, the death of the mature females is followed by polyphenol oxidase tanning of the cuticle resulting in a hard, brown cyst. Egg masses and cysts can each contain several hundred eggs. Egg packaging units similar to cysts and egg masses are not found in animal-parasitic or free-living nematodes.

Hatching of Meloidogyne spp. is, in general, temperature dependent and hatching occurs when temperatures are favourable without the need for stimulus from root diffusates. However, there are exceptions and a proportion of the unhatched juveniles of M. hapla, M. triticoryzae and M. chitwoodi, for example, have been shown to be dependent on root diffusates for hatch, especially in later generations during a host growing season (Gaur et al. 2000; Perry and Wesemael 2008). Although a few other species from other groups (e.g. Rotylenchulus reniformis and Hypsoperine ottersoni) hatch in response to host root diffusates, this phenomenon is most common among the cyst nematodes but even in this group reliance on host stimulation for hatch varies. Globodera rostochiensis and G. pallida, have a very restricted host range and are almost completely dependent on host diffusates for hatch, whereas H. schachtii, for example, has a very wide host range (some 218 plant species, including many weeds) and hatches well in water (Perry 2002). Heterodera avenae has a large hatch in water but a relatively narrow host range; however, the hosts are very common (Turner and Rowe 2006). The dependence of G. rostochiensis and G. pallida on a plant-derived hatching stimulus is an obvious control target, with the aim of inducing hatch in the absence of a host plant and thus causing the nematodes to die of starvation. However, although much research effort has been expended in elucidating the chemicals, termed hatching factors, in root diffusates, there has been no successful control strategy using analogues of the hatching factors to induce hatch in the field.

Host root diffusates induce a cascade of inter-related changes leading to eclosion, and the sequence of events has been discussed in detail by Jones et al. (1998) and Perry (2002). Unhatched J2 of Globodera and Heterodera spp. are surrounded by perivitelline fluid, which contains trehalose. Trehalose generates an osmotic pressure that reduces the water content of the J2 and inhibits movement because the turgor pressure is insufficient to antagonise the longitudinal musculature. For

Fig. 1.3 Egg masses of Meloidogyne chitwoodi stained with Phloxine B; the posterior end of the adult female (arrowed) is visible outside the root. (Courtesy Wim ML Wesemael, Institute for Agricultural and Fisheries Research, Belgium)

hatching to occur, the pressure needs to be removed. In G. rostochiensis and some other species, this is achieved by a change in permeability of the inner lipoprotein membranes of the eggshell via HF binding or displacing internal Ca2+ (Clarke et al. 1978). In both G. rostochiensis and G. pallida, a 5 min exposure to host diffusate is sufficient to stimulate hatch (Perry and Beane 1982), suggesting the involvement of a receptor-ligand interaction between the HF and the eggshell lipoprotein membrane. The change in eggshell permeability enables trehalose to leave the egg, with a concomitant influx of water and subsequent rehydration of the J2 to a water content commensurate with movement. The eggshell of G. rostochiensis remains rigid during the hatching process and there is no evidence of enzyme involvement. Devine et al. (1996) demonstrated that the potato steroidal glycoalkaloids, a-solanine and a-chaconine, induce hatch of G. rostochiensis; glycoalkaloids are known to destabilise lipid membranes during which leakage of trehalose is possible. However, enzymes have been implicated in softening of the eggshell prior to eclosion in other species, including Xiphinema diversicaudatum, Aphelenchus avenae and M. incognita; in M. incognita lipase activity has been positively correlated with hatch (Perry et al. 1992). Rehydration of the J2 of G. rostochiensis is accompanied by increased metabolic activity due in part to removal of osmotic pressure and hydration and in part to direct stimulation of the J2 by root diffusate. Changes in gene expression of G. rostochiensis J2 appear to occur during or immediately after the hatching process (Jones et al. 1997), but more work is needed on the molecular aspects of the hatching response.

The J2 of Globodera spp. uses its stylet to cut a regular series of perforations in the subpolar region of the eggshell, and the J2 hatches through the resulting slit. J2 of D. dipsaci use a similar approach, except that the stylet thrusts are more random and the J2 uses its head to force open the slit in the eggshell. In Pratylenchus penetrans and H. avenae, a single stylet thrust penetrates the eggshell and the head extends this into a tear.

Once hatched, nematodes are vulnerable to environmental extremes and have to locate a host to start feeding. For example, under optimal conditions for movement, J2 of G. rostochiensis must locate a host root and set up a feeding site within 6-11 days of hatching otherwise it will exhaust its energy reserves and die (Robinson et al. 1987). Hatching in response to host root diffusates has the advantage of ensuring that the nematodes hatch and leave the protection of the egg and cyst when host roots are close by; thus, synchrony of host availability and nematode hatch is advantageous for nematode survival.

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