Literature Review and Research Proposal

on

Adaptation of Meloidogyne spp. to drought stress

Academic Year 2014-2015

Submitted by

Md. Iqbal Hossain

Postgraduate International Nematology Course (PINC)

Faculty of Science, Department of Biology, Ghent University,

Ghent 9000, Belgium

Promoter

Prof. dr. Wim Wesemael

Co-promoter

Prof. dr. Roland N. Perry

Contents

Part A: Literature Review ........................................................................................................................ 1

1. Introduction ................................................................................................................................. 1

2. Importance of Meloidogyne .......................................................................................................... 1

3. Life cycle biology of Meloidogyne sp. .......................................................................................... 2

4. Adaptation strategies in Plant parasitic nematodes ........................................................................ 3

5. Effect of temperature on different life stages of nematodes ........................................................... 5

6. Adaptation of nematodes in cold stress ......................................................................................... 7

7. Adaptation of nematodes in osmotic stress ................................................................................... 8

8. Biochemical changes at different adaptation mechanism .............................................................. 9

Part B: Proposal..................................................................................................................................... 11

1. Background Information ............................................................................................................ 11

2. Hypothesis ................................................................................................................................. 12

3. Experimental procedure ............................................................................................................ 12

3.1. Culturing of nematodes ..................................................................................................... 12

3.2. Collection of egg masses .................................................................................................... 12

4. Adaptibility testing .................................................................................................................... 13

4.1. Experiment: Determine the survival capability of Meloidogyne spp. at different generations13

Part C: References ................................................................................................................................. 14

Part D: Addendum ................................................................................................................................. 27

Table 1. Time schedule and expected deliverables ............................................................................. 27

1

Part A: Literature Review

1. Introduction

Nematodes are bilaterally symmetrical multicellular worm like animals, the most abundant metazoa on

earth. They are found in almost all ecology (Ferris et al., 2001); even, they are discovered as an

anhydrobiotic (Moens & Perry, 2009). Nevertheless, a layer of liquid is essential for their activity and

motility. Environmental factors such as temperature, relative humidity etc. have also an effect on the

survival mechanism of nematodes.

Nematodes are living as free-living organism, plant-parasitic and animal-parasitic. Among the 25000

described nematode species (Hodda, 2011 ), about 4100 species are plant-parasitic in nature (Decraemer

& Hunt, 2013). Most of the plant-parasitic nematodes (PPN) attack the roots of plants but very few are

capable to cause disease on above ground plant parts. They cause several diseases of plants that results in

heavy losses in crop production all over the world regarding quality and quantity. About 12.3% of annual

agricultural crop production are destroyed by them which cost $157 billion (Hassan et al., 2013).

Distribution and severity of PPN species is diverse. For example, Nacobbus spp. are found only in North

and South America; species like the carrot cyst nematode Heterodera carotae is host specific whereas

Meloidogyne spp. are globally distributed with a large host range (Nicol et al., 2011). Furthermore, root-

knot nematodes (Meloidogyne spp.) are obligate plant parasites and one of the most destructive PPN

genus (Hunt & Handoo, 2009).

2. Importance of Meloidogyne

Root-knot nematode (Meloidogyne spp.) is a polyphagus group of obligate plant-parasitic nematodes

which has highly adaptive capability and has been found worldwide (Karssen et al., 2013). It causes

disease in thousands of monocotyledonous and dicotyledonous plants (Eisenback & Hirschmann, 1991) .

2

Nearly100 species are discovered in this genus (Onkendi et al., 2014) but only six of them are most

widely distributed and responsible for considerable damage (Adam et al., 2007). Among them,

Meloidogyne incognita, M. javanica and M. arenaria are highly abundant in tropical climates but also in

green houses of temperate regions; M. chitwoodi, M. fallax and M. hapla are major species in temperate

climate. Furthermore, in respect of changing global trade pattern and crop production system, M. minor

and M. enterolobii species are becoming emerging threats (Wesemael et al., 2011) for the temperate and

tropical region respectively. As a result, M. chitwoodi, M. fallax and M. enterolobii have been reported as

quarantine pest by European and Mediterranean Plant Protection Organization (EPPO) (EPPO, 2011).

Two closely related cryophilic species M. chitwoodi and M. fallax were first identified at Pacific

Northwest of USA in 1980 (Golden et al., 1980) and at Baexem, the Netherlands in 1992 (Karssen,

1996), respectively. M. chitwoodi was also recorded in Argentina, Belgium, Germany, Netherlands,

Portugal, Switzerland, Russia, Australia, Mexico and South Africa (EPPO, 2004; Karssen et al., 2013),

M. fallax was observed in France, Belgium, Switzerland, UK and Germany (Dahler et al., 1996; Schmitz

et al., 1998; Fourie et al., 2001; Marshall et al., 2001; Nobbs et al., 2001; Waeyenberge & Moens, 2001;

Karssen et al., 2013). In Belgium, both species are causing severe quality damage to vegetables and

turning into a threat to food canning industries (Wesemael & Moens, 2008). Besides, M. hapla is mostly

associated with different dicot plant where as M. minor is observed only two times in potato field but very

common in several sport grounds in UK, Netherlands and Belgium golf courses along with M. naasi

(Karssen et al., 2004; Viaene et al., 2007).

3. Life cycle biology of Meloidogyne sp.

Meloidogyne spp. is a group of obligatory plant pathogenic nematodes, endoparasitic in nature. Females

are sedentary and remain inside the root tissue. They lay eggs in a confined gelatinous egg sacs (Karssen

et al., 2013). They complete their first moulting inside the eggs. Second-stage juveniles (J2) are hatched

from eggs and start searching for a host plant. Generally, they enter into the host tissue near the root tip by

3

physical means (stylet) as well as cell wall degrading enzymes (Wieczorek et al., 2014). They move

intercellularly in the cell wall compartment towards root tip and turn around when they reach the apical

meristem cells and move further to reach the differentiating vascular zone (Wyss et al., 1992; Goto et al.,

2013). There, they look for cells to induce them as a multinucleate giant cells (Bartlem et al., 2014) that

also act as transfer cells (Jones & Northcot, 1972). For their growth and development, they take nutrients

from these cells and follow 3 successive moults to be adults. Parthenogenetic reproduction is common for

root-knot nematodes (Castagnone-Sereno, 2006). Adult females start to lay eggs, while vermiform

malesleave the host tissue.

4. Adaptation strategies in plant-parasitic nematodes

According to Wharton (2004), ‘survival strategies’ refer to a specific behavior of organisms at their

biological and physical challenges. During their life cycle, different plant-parasitic nematodes also follow

several survival strategies against environmental extremes and host response (Perry, 2011). Two types of

dormancy, ‘quiescence’ and ‘diapause’ often lengthen survivability of unhatched larvae in soil living

nematodes including RKN (Perry, 1989). Cryobiosis, thermobiosis, anoxybiosis, osmobiosis and

desiccation are different survival mechanisms that are also observed in nematodes (Wright & Perry,

2006). First discovered plant-parasitic nematode, Anguina tritici has also special adaptation aptitude

against desiccation (Needham, 1743). Commonly, life cycle of a PPN is divided into different distinct

stages such as egg, four juvenile stages (J1, J2, J3 and J4) and adults and different major PPN also

exhibits different adaptation mechanism at their different life stages.

Eggs of RKN are laid in mucoid protein mass that provides safety to eggs water loss and predators

(Eisenback, 1985). Furthermore, extreme temperature makes this glycoprotein shriveled and hard that

promotes mechanical pressure and hinders the hatching of J2 larva that is prone to drought environment

(Wright & Perry, 2006). Additionally, gelatinous matrix defends the eggs from the attack of some soil

microorganism as was observed in M. javanica (Orion & Kritzman, 2001). During dry conditions, a dried

4

eggshell can also decrease water loss rate by changing its permeability (Ellenby, 1968). Additional

research on eggshell permeability was done by Wharton (1980) and he observed that the lipid layer of the

eggshell played a major role for lowering the water loss during desiccation. Not all PPN have survival

capability. Mostly, the species with limited hosts such as potato cyst nematodes (Globodera rostochiensis

and G. pallida) gained well adaption features for undesirable circumstances (Perry, 2002). The unhatched

larvae of this nematode can survive many years in cyst without host (Turner & Rowe, 2006). Ellenby

(1946) observed that the permeability of cyst wall exterior and eggshell of G. rostochiensis changed at

desiccated conditions which helped cyst wall and eggshell to restrict their water losing rate compared to

favourable conditions. This helps the unhatched susceptible J2 to survive in the egg and hatching factors

(host root exudates) are obligatory for this nematode to hatch their J2 (Wright & Perry, 2006). J2 of

soybean cyst nematode Heterodera glycines hatched in water at optimum conditions but at the end of the

plant growing season when environment is becoming complex, most of the J2 remains in the cyst and

hatching is solely dependent on hatching factors, irrespective to the origin of hatching factors; i.e. natural

(Ishibashi et al., 1973) or artificial (Thompson & Tylka, 1997). Other cyst nematodes, such as H. carotae

(Greco, 1981), H. goettingiana (Greco et al., 1986), H. sacchari (Ibrahim et al., 1993) contain the same

characteristics. Even, RKN, M. chitwoodi and M. triticoryzae (Gaur et al., 2000; Wesemael et al., 2006)

also showed the same features for hatching of J2.

Under drying condition, species of Anguina and Ditylenchus showed coiling and clumping like features in

their life cycle that helps to avoid water loss by reducing their surface (Crowe & Madin, 1975).

Ditylenchus myceliophagus also lessen their water loss becoming coiled (Womersley, 1978).

Furthermore, at the last stage of the crop growing season, D. dipsaci, stops their development at J4 stage

due to shortage of food and make large aggregations and start coiling. This attribute known as ‘eelworm

wool’ that helps them to remain alive for many years at desiccated condition by the sacrifice of peripheral

J4s of this aggregation (Ellenby, 1969). Rice grain nematodes, Aphelenchoides besseyi also stops their

multiplication at ripening stage of rice and adults become coiled and clumped which helps them surviving

5

for 2-3 years with dry grains (Perry & Moens, 2011). On the other hand, J2 of 2 gall producing nematode

Anguina tritici and A. pacificae become anhydrobiotic but remain uncoiled and survived many years

(McClure et al., 2008). So, coiling is not obligatory for survival during desiccation. Nevertheless, D.

dipsaci and A. tritici, can overcome desiccated environment and survive many years in above ground

plant parts (Moens & Perry, 2009). Anguina tritici, D. dipsaci and Tylenchus have been showed

potentiality of being enliven from desiccation after 20 year (Perry & Moens, 2011), 28 years (Fielding,

1951) and 39 years (Steiner & Albin, 1946) respectively. Cuticle lipid content of desiccated J4 of D.

dipsaci and J2 of A. tritici have been increased compared to hydrated juveniles of those nematodes (Bird

& Buttrose, 1974; Preston & Bird, 1987). Sheath retaining is also a survival mechanism against

desiccated condition. Young adults of Rotylenchulus reniformis remains confined with all 3 moulted

cuticle of their larval stage which facilitates them of being dormant in adverse condition until situation

becomes amiable for their survival (Gaur & Perry, 1991). Entomopathogenic nematode Heterorhabditis

megidis also retains cuticle sheath that provides them lowering their drying in unfavorable environment

(Menti et al., 1997).

Dauer phenomenon is a special characteristic of nematodes that helps them to survive at stress condition.

Dauer is very common in C. elegans where dauer larvae restrict its biological activity and survive periods

at desiccated situation (Kenyon, 1997). J2, J3, or J4 stages are performed as dauer in A. tritici, C. elegans

and D. dipsaci respectively (Bird & Bird, 1991). Pine wood nematode Bursaphelenchus xylophilus, also

contains a dauer stage that facilitates the transportation to pine trees with the help of insect vector

Monochamus (Mota & Vieira, 2008).

5. Effect of temperature on different life stages of nematodes

Temperature is known as influential on life cycle and biology of nematodes. Different activities such as

growth and development, mobility, infection capability and hatching are affected by its surrounding

temperature (Davide & Triantaphyllou, 1968; Wallace, 1971; Tzortzakakis & Trudgill, 2005).

6

Meloidogyne arenaria requires at least 12.2°C with short photoperiod for their egg mass production

(Yeon et al., 2003).

For certain range of temperature; development of nematodes increase with enhancing of temperature

which is termed as temperature niche breadth (Anderson & Coleman, 1982). For example, growth

development rate of C. elegans (wild type) increases 2.1 times and 1.3 times when they have been kept at

25°C and 20°C respectively instead of 16°C (Stiernagle, 2006). Additionally, temperature plays a key role

on host-nematode association in soil ecology. Nematode resistant hosts, for example: tomato, cotton and

other crops became vulnerable at increasing thermopiriodism (Ferris et al., 2013). Meloidogyne resistance

gene, Mi becomes ineffective when a host has been kept at higher temperature continuously (Holtzman,

1965; Laterrot & Pecaut, 1965; Dropkin, 1969). Furthermore, same result was observed at in vitro studies,

where host resistence against M. incognita has been lost due to heat stress (Haroon et al., 1993). Several

experiments were done to reveal the effect of temperature on nematodes. Ploeg and Maris (1999)

examined the effect of five soil temperatures on fecundity rate and period of M. incognita on tomato host

plant. They can complete their life within an average of 16.2°C and 30°C. Furthermore, optimum

temperature, moisture and aeration are obligatory for hatching out of Meloidogyne juveniles (Bergeson,

1959). Meloidogyne javanica and Heterodera glycines were tested on susceptible and resistant hosts at 5

different temperatures (20°C, 24°C, 26°C, 28°C and 32°C). Parameter on biological activity such as

quantity of egg-masses, total eggs, females and successful infestation (%) of J2 for M. javanica were

significantly higher at 28°C over other temperatures on both type of hosts. Same temperature also

significantly increases J2 infective capability (%), amount of female’s total population (Female no. + no.

of cyst) for H. glycines on a susceptible host. However, temperature did not show any effect on the total

population of cyst nematodes on resistant plants. Females of M. javanica produced a higher number of

eggs in a susceptible host at 4 different soil temperatures (20°C, 26°C, 28°C and 32°C) but a significant

increase was only observed at 32°C (Campos et al., 2011). An observation was done on survival character

of eggs of two RKN under different soil temperatures in laboratory along with field observation. Eggs of

7

M. hapla were better in viability than that of M. javanica but became destroyed when kept for a longer

period at -2°C in soil. Opposite results were seen when eggs of both species were kept at 36°C and 40°C.

This proves overwintering capability of M. hapla and M. javanica in field at temperate and tropical region

respectively (Daulton & Nusbaum, 1961).

6. Adaptation of nematodes in cold stress

Nematodes that can be recognized as a cold survivor are capable to carry on their life after facing freezing

temperatures for longer periods; nematodes of temperate regions which can survive at freezing

temperatures are included in this group (MacGuidwin & Forge, 1991; Dimander et al., 1998). Nematodes

that are living in Arctic (Coulson & Birkemoe, 2000) and Antarctic region (Wharton, 2003) along with

sea ice (Gradinger, 2001), alpine sites (Hoschitz & Kaufmann, 2004) and glaciers (Christner et al., 2003;

Hodson et al., 2008) definitely have special adaptation behaviour to cold in their life cycle (Wharton,

2011).

The main constraints to survive in freezing conditions are destruction of body cells especially intracellular

freezing due to osmotic stress (Mazur, 1984). There are nematodes that can tolerate intracellular freezing

(Wharton & Ferns, 1995; Salinas-Flores et al., 2008). Panagrolaimus davidi is one of the nematodes

collected from the Antarctic (Wharton, 2011) that shows this aptitudes (Wharton & Ferns, 1995; Wharton

et al., 2003; Wharton et al., 2005). Feeding behavior has an effect on the tolerance capability of P. davidi

(Raymond & Wharton, 2013). Panagrolaimus davidi can also survive against extracellular ice

development (Wharton et al., 2005). Generally, nematodes survive in cold as being anhydroboitic but

desiccation should be done before exposure to freezing (Wharton, 2002). Nevertheless, P. davidi

performs an external dehydration tactic which is known as ‘cryoprotective dehydration’ (Wharton &

Barclay, 1993). Cell membranes of this nematode act as a little barrier to ice formation (Wharton & Ferns,

1995). The embryo or J1 remains in supercool condition inside eggs when ice is around the eggs.

Eggshell plays key role to protect eggs from adverse freezing (Wharton, 1994).

8

This ‘supercool’ characteristic is also observed in unhatched J2 of cyst nematodes, G. rostochiensis and

G. pallida when cyst were enclosed in ice, placed at −38°C. Cyst wall and eggshell provide support to

keep alive J2 against freezing (Wharton et al., 1993; Wharton & Ramløv, 1995; Devine, 2010).

Acclimatization to low temperatures can increase the adaptation capability of nematodes to cold stress

(Ehlers et al., 2005). It was observed in entomopathogenic nematodes, such as in Steinernema feltiae, S.

anomalae and H. bacteriophora (Brown & Gaugler, 1996). Furthermore, placing J2 of M. hapla at 4°C

for 12 h also increases their cold tolerance capability to sustain in 5% polyethylene glycol (at −4°C)

(Forge & MacGuidwin, 1990, 1992).

7. Adaptation of nematodes in osmotic stress

Maintaining osmotic pressure between the body fluid (pseudocoelomic fluid) and the surrounding water is

challenging for nematodes. They keep themselves isometric to hyperosmotic pressure in different

habitats. Fresh water nematodes are hyperosmotic in nature but their body fluid concentration remains

relatively constant (Wright, 2004) whereas marine nematodes retain themselves isometric that helps them

surviving in sea water (Wright, 2004). However, habitat of estuarine nematodes is affected by marine and

fresh water current where ionic composition and osmotic pressure are always changing at diurnal period

(Forster, 1998).

Nematodes living in sea do not face any problem in saline water. About 3.5% salinity is found in sea

water which is equivalent to 0.06 N NaCl or 1000 mmol/kg. Some nematodes are observed in terrestrial

springs that are rich in different minerals (Hodda et al., 2006). Even, nematodes such as Microlaimus,

Theristus and Bathylaimus are reported to live in > 10% salinity habitat; for example intertidal habitats of

Zanzibar western coast (Olafsson, 1995). Furthermore, nematodes are highly adapted in saline lakes of

Australia where NaCl concentration is at least 9.3% (Bayly & Williams, 1966; Deckker & Geddes, 1980).

Nematodes are likely to avoid osmotic stress. They like to live in less stressful places. The free living

nematode, C. elegans moves toward less concentrated areas (Choe & Strange, 2007). Moreover, J2 of M.

9

hapla (Prot, 1978) and M. incognita (Le Saux & Queneherve, 2002) perform the same characteristics and

show avoiding actions to higher concentrated habitats. Furthermore, Steinernema carpocapsae (Pye &

Burman, 1981) and Rotylenchulus reniformis (Riddle & Bird, 1985) also show the same survival habit.

According to Willmer et al. (2005), nematodes can overcome osmotic stress by two mechanisms, either

changing their own body fluid concentration like to outer fluids concentration or being tolerant to external

concentration; and they are termed as osmoconformers and osmoregulators respectively. Ascarissuum

(Hobson et al., 1952) and J3 of P. decipiens (Fuse et al., 1993) are the best examples of osmoconformers

and osmoregulators respectively. However, some organism perform osmobiotic features to avoid external

high concentration (Kellin, 1959).

Cuticle and eggshells (Wharton, 1980) also provide physical barrier to restrict water flux that also help

nematodes to survive against osmotic stress. Less permeable cuticle reduces water loss rate that protects

C. briggsae in stress environment but the resistance of cuticle lessen when nematodes become old (Searcy

et al., 1976). Additionally, this physical tolerance aptitude (Wharton et al., 1988) is also observed in D.

dipsaci when they have been kept in 1.6 M NaCl for 1 day (Viglierchio et al., 1969). On the other hand,

high permeable cuticle of isosmotic marine nematodes, offer them to endure well in saline sea water

(Wharton, 2011).

8. Biochemical changes at different adaptation mechanism

Trehalose is a disaccharide that provides nematodes tolerance against high temperature (Jagdale &

Grewal, 2003), desiccation, cold or different global stresses by accumulating this natural chemical into

the nematode body (Grewal et al., 2006). Panagrolaimus davidi (Wharton et al., 2000), N. battus eggs

(Ash & Atkinson, 1983), S. kushidai (Ogura & Nakashima, 1997), and S. carpocapsae (Qiu & Bedding,

1999) accumulated higher trehalose when they are exposed to cold. Enhancing desiccated stress (Perry et

al., 2012) as well as higher temperature stress (Jagdale & Grewal, 2003) causes more metabolism of

10

trehalose in EPN genera Steinernema. Moreover, trehalose synthesis increases in another EPN genera

Heterorhabditis exposed to both high or low temperature (Jagdale et al., 2005).

Synthesis of heat shock protein also increase stress tolerance capability in nematodes. Accumulation of

heat shock protein increases at exposure to heat or cold stress (Devaney, 2011). Transgenic

Heterorhabditis bacteriophora that contains the heat shock protein gene hsp70A enhances its

thermotolerance capability (Gaugler et al., 1997). Increasing formation of Hsp90 has been reported in

eggs at 5°C but the amount of that protein does not increase in J2 at the same temperature (De Luca et al.,

2009).

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Part B: Proposal

1. Background Information

Plant-parasitic nematodes cause diseases of plants at roots and other parts of plants. They are responsible

to damage about 12.3% of annual crop production in the world that costs $157 billion (Abad et al., 2008).

Root-knot nematode (RKN), the most widely distributed obligatory plant-parasitic nematodes, are

polyphagous and the most economically damaging group of nematodes to agricultural production. The

average threshold limit to crops is generally from 0.5-2 juveniles/g of soil (Greco & Di Vito, 2009). Till

2013, 98 species (Jones et al., 2013) of this obligate nematode have been reported that can cause disease

to monocotyledons, dicotyledons, herbaceous and woody plants (Eisenback & Hirschmann, 1991).

Among them, Meloidogyne incognita, M. javanicaand M. arenariaare the most damaging nematodes in

tropics, M. chitwoodi, M. fallax, and M. hapla causes considerable crop loss in cooler regions (Adam et

al., 2007). Furthermore, M. chitwoodi and M. fallax are very important species due to their quarantine

status along with their damaging capability to economic crops like potato. These species are polycyclic at

optimum temperature in crop growing seasons (Brinkman et al., 1996). Due to climate change, southern

Europe and the Mediterranean countries become drier. Moreover, life cycle of temperate nematodes in

this region becomes more diversified that affects their survival mechanism as well as their infectivity rate

to plants. Proper managing and restricting distribution of these quarantine nematodes and knowledge on

survival mechanisms and infectivity of these nematodes is very much essential. To gain that knowledge it

is necessary to cope up the upcoming RKN problems in Europe at challenging warming climate. The

experiments in this thesis will be set up with the following objectives:

1. To investigate the adaptability and infectivity of Meloidogyne spp. at different temperatures

2. To examine the adaptability of Meloidogyne spp. to drought stress

12

2. Hypothesis

Main survival stage of Meloidogyne is egg mass which provides protection for eggs, J1 and J2

(unhatched) with the help of its gelatinous matrix at different harsh ecology. Egg mass will be kept at

different drought conditions (on the basis of temperature and moisture levels). Hatching capability,

survivability and infectivity of following generations will be key factors to assess their adaptive

capability. It is assumed that successive generations will become more adapted and as such more

aggressive.

3. Experimental layout

3.1. Culturing of nematodes

Root-knot nematodes, Meloidogyne spp. will be cultured on susceptible tomato plants (Solanum

lycopersicum cv. Moneymaker) in glasshouse at 20°C-26°C with 14 h light period at ILVO, Merelbeke,

Belgium. Tomato seedlings will be transplanted individually in plastic pots containing sterilized soil.

Freshly hatched J2 will be used to infect the susceptible plants for getting the egg masses.

3.2. Collection of egg masses

Egg masses will be collected from 12-16 weeks old tomato plants, after 8-12 weeks after inoculation.

Adhering soil should be discarded from plant roots and egg masses should be collected from roots using

sharp blade.

13

4. Adaptability testing

4.1. Experiment: Determine the survival capability of Meloidogyne spp. at different

generations

Collected egg masses will be put under different temperatures and relative humidities for certain time

where as control treatment will be the egg mass without any temperature treatment and at 100% RH. 1st

generation J2 will be calculated during a hatching experiment and they will be used to inoculate

susceptible tomato host plants. 2nd

generation egg masses will be collected and calculated and kept again

at different temperatures and relative humidities. This cycle will be continued during the time frame of

thesis work. In the glasshouse tomato plants with RKN will be stressed (drought stress) and egg masses

will be collected from these plants (if they are formed). J2 hatching from this egg masses will be

examined for adaptation to drought stress as described above.

14

Part C: References

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L., Anthouard, V., Artiguenave, F., Blok, V.C., Caillaud, M.-C., Coutinho, P.M., Dasilva, C., De

Luca, F., Deau, F., Esquibet, M., Flutre, T., Goldstone, J.V., Hamamouch, N., Hewezi, T., Jaillon,

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Poulain, J., Robinson-Rechavi, M., Sallet, E., Segurens, B., Steinbach, D., Tytgat, T., Ugarte, E.,

Van Ghelder, C., Veronico, P., Baum, T.J., Blaxter, M., Bleve-Zacheo, T., Davis, E.L., Ewbank,

J.J., Favery, B., Grenier, E., Henrissat, B., Jones, J.T., Laudet, V., Maule, A.G., Quesneville, H.,

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the metazoan plant-parasitic nematode Meloidogyne incognita. Nature Biotechnology 26, 909-

915.

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Anderson, R.V. & Coleman, D.C. (1982). Nematode temperature responses - A niche dimension in

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Bird, A.F. & Bird, J. (1991). The structure of nematodes. Academic Press, pp 94.

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27

Part D: Addendum

Table 1. Time schedule and expected deliverables

Tasks Sept Oct Nov Dec Jan Feb Mar Apr May Jun Jul Aug Sep

Collection,

reading and writing of

relevant

literatures on

research topics

Submission

of literatures

Lab works: Nematode

culture

Egg mass

collection

Adaptability testing

Data

analysis

Thesis

writing

Thesis defence