The importance of the gelatinous matrix for the survival of eggs of Meloidogyne chitwoodi and M. fallax

Ghent University Faculty of Science Department of Biology Academic Year: 2013-2014 The importance of the gelatinous matrix for the survival of eggs of Meloidogyne chitwoodi and M. fallax Md. Rubel Mahmud Promoter Prof. Wim Wesemael Thesis submitted to obtain the degree of Master of Science in Nematology

The importance of the gelatinous matrix for the survival of eggs of Meloidogyne chitwoodi and M. fallax Md. Rubel Mahmud 1 Ghent University, Department of Biology, K.L. Ledeganckstraat 35, 9000 Gent, Belgium Summary- The survival of eggs of two quarantine temperate root-knot nematodes Meloidogyne chitwoodi and M. fallax were investigated in vitro by a series of experiments comparing the hatching of J2 from egg masses (EM) to that from loose eggs (LE). Both types of eggs were exposed to different temperature (20 C and 5 C) in water or soil (20% moist and dry soil) and also subjected to two different relative humidities (75.5% and 7%). After treatment percentage of hatching was determined. Results showed that hatching from both type of eggs was dependent on the temperature and the moisture status in soil over time. Hatching in water and soil was significantly higher at 20 C than at 5 C. In order to see the treatment effect, both types of treated eggs were kept in room temperature (20 C-25 C) for further hatching. Significantly higher hatching was observed from EM (> 90%) than LE (60-70%) for both species after a six week incubation in moist soil. Hatching gradually declined with the increase of exposure time. During incubation in soil at 5 C less than 5% hatching was observed. Upon return to favourable temperature, more than 70% hatching was observed from the EM compared to LE (< 15% from M. chitwoodi and < 6% from M. fallax) at six week incubation. Hatching was significantly higher from the treatments in moist soil than dry soil. There was also a clear difference between the hatching from intact egg masses compared to loose eggs, highest hatching was observed from EM. Survival of eggs from EM was found also significantly higher than LE after exposure to 75.5 and 7% RH. The percent unhatched eggs were higher in M. chitwoodi than M. fallax. As the major difference between EM and LE was the presence of the gelatinous matrix, we can conclude that this gelatinous matrix serves as a defensive barrier for the survival of eggs inside from abiotic stress in soil and water. Keywords- Root-knot nematodes, Meloidogyne chitwoodi, Meloidogyne fallax, gelatinous matrix, egg mass, loose eggs, survival, temperature, moisture, relative humidity. ------------------------------------------------------ 1 E-mail: mahmud_83plp@yahoo.com 1

Nematodes are the most numerous Metazoa on earth. They are either free living or parasitic in plants and animals. They can be found in every habitat in soil, freshwater, and marine environments (Cobb, 1915) and are essentially aquatic animals. Nematodes depend on moisture for their movement and active life; relative humidity and other important environmental factors directly and indirectly affect nematode survival. Plant-parasitic nematodes evolved some special structures viz. a hollow protrusible stylet and large esophageal glands for penetrating into the root cell and withdraw the nutrients from the cell (Davis et al., 2000). Plant-parasitic nematodes are an important constraint for global food security and damage caused by nematodes has been estimated $US 80 billion per year (Nicol, 2011). About 4100 species of plant-parasitic nematodes have already been identified and new species are continuously being described (Decraemer et al., 2013). Many plant-parasitic nematodes cause substantial losses of different crops throughout the world. Root-knot nematodes are included within the genus Meloidogyne and represent one of the most polyphagous and ubiquitous genera of plant-parasitic nematodes. These nematodes infect thousands of different herbaceous and woody monocotyledonous and dicotyledonous plants and cause serious losses to numerous agricultural crops worldwide (Eisenback & Triantaphyllou, 1991). About 2000 plants are susceptible to infection by root-knot nematodes and they cause approximately 5% of global crop loss (Sasser, 1985). The symptoms caused by root-knot nematodes are typical in below ground plant parts. Nematodes feed on the root and produce a visible gall or knot. The gall or knot may vary in size and shape if plants response differentially to Meloidogyne infection. A few above ground symptoms are noticed in root-knot infection. Highly affected plants suffer from wilting because galled roots limit the uptake of nutrients and water. Presently, 98 species were described in the genus Meloidogyne (Jones et al., 2013) and recently two new species were described in 2014 which makes 100 species (Wesemael, pers. comm., 24 august, 2014). Among these, 23 were found in Europe (Wesemael et al., 2011). Besides the direct cost root-knot nematodes cause indirect costs because of the quarantine status of some Meloidogyne species in several countries or regions. Some species like the temperate root-knot nematodes M. chitwoodi (Columbia root-knot nematode) and M. fallax (False Columbia rootknot nematode) have a broad host range and complete several generations per crop growing season (Santo et al., 1980; O'bannon et al., 1982; Ferris et al., 1993; Brinkman et al., 1996). Both species are prevalent in cooler climate (Wesemael et al., 2011). Meloidogyne chitwoodi has 2

been reported from Argentina, Belgium, France, Germany, The Netherlands, Portugal, Switzerland, Turkey, several states of the USA, Mexico and South Africa (EPPO, 2003). The species was first detected in the EPPO region in the 1980s but may have been present in the region since at least 1930s (EPPO, 2013). Meloidogyne fallax was identified in 1992 in a field north of Baexem, The Netherlands (Karssen, 1996). Later it was diagnosed in plastic tunnel house in France (Daher et al., 1996) and in Belgium (Waeyenberge & Moens, 2001), Germany and Switzerland. Recently it was also found in the UK (CABI, 2013). This species was also detected outside Europe in New Zealand, Australia and South Africa. They are able to parasitize many mono- and dicotyledonous plants, including some important economic crops like potato, carrot, tomato, wheat (Karssen et al., 2013) and black salsify. Meloidogyne chitwoodi readily reproduces on corn (Zea mays L.) and wheat (Triticum aestivum L.) but reproduces poorly on alfalfa (Medicago sativa L.) (O'bannon et al., 1982). The extent of damage on carrot is not only dependent on cultivar but also population density of M. chitwoodi and M. fallax, temperature, length of growing season and also soil texture (Wesemael & Moens, 2008). This means that these species are more difficult to control by means of crop rotations than other root-knot nematodes with a narrower host range. Management of root- knot nematodes is difficult due to their wide host range and their ability to persist between the main host in the field and also in weeds or other plants. Both have important phytosanitary status and pose similar economic risks to damage crops (Hockland et al., 2013). Since 1998, both species were classified as quarantine organisms by the European and Mediterranean Plant Protection Organization (EPPO, 2003) due to their economic impact that affects all major crops grown in the field and those are cultivated in glasshouses in North-Western Europe (Karssen, 1996). Root-knot nematodes are sedentary and endoparasitic in nature and complete most of their life cycle within the root tissue of the host plant. Meloidogyne females do not hold eggs internally but they deposit eggs into a gelatinous matrix that keeps them together on the host root surface or inside the root tissue and which is produced by six rectal glands. The female lay 30-40 eggs per day and in a suitable host more than hundred eggs are produced. First moult occurs inside the eggs to develop from J1 to J2 prior to hatching. Hatching of most of the species of Meloidogyne occurs in water, being driven mainly by the influence of temperature (Karssen et al., 2013). However, some species of Meloidogyne also alter the rate of hatching in response to host root diffusate, although host age also has an effect on the issue (Wesemael et al., 2006). Hatched pre- 3

parasitic J2 leave the egg masses and immediately search to find host tissue. They penetrate usually closely behind the root tip by using a protrusible stylet and secreting cell wall degrading enzymes (Abad et al., 2003). The parasitic J2 nematodes remain there and induce a feeding site. J2 try to form giant cells by karyokinesis with cytokinesis. Under favourable conditions, the J2 stage moults to the third-stage juvenile (J3) after that J4 and finally to the adult stage. The combined time for the J3 and J4 stages is much shorter than J2 or the adult. J2 can survive in the soil in a quiescent state for an extended period and using lipid reserve stored in the intestine (Perry et al., 2009). Adult females secrete a gelatinous matrix (GM) in voluminous amounts, sometimes more than the entire female body volume. The six rectal glands are arranged radially around the anal opening and reach the peak of their development and activity in the young female just before and during egg laying. These cells are large in size with large nuclei and nucleoli with dense cytoplasm; which are features of intensive metabolic activity. The GM is secreted through a duct into the rectum and pushed through the anus outside the female body where it surrounds the vulva into which the eggs are deposited (Bird & Rogers, 1965). The egg mass and components of the eggshell are important for the survival of the developing embryo and the fully formed juvenile stages within the egg (Perry et al., 2009). The gelatinous matrix is a specialized adaptation of Meloidogyne that provides some protection against desiccation, predators or microbial antagonists of soil and that consists of proteins, a muco-polysaccharide and various enzymes (Eisenback, 1985). Light and electron microscopic studies revealed that the GM is a complex material that consists of amorphous, fibrillar and spherical macromolecular structures that may have enzymatic or hormonal activity. The mesh structure in the GM may retain or bind water, thereby maintaining the developing eggs in a constant and moist environment (Orion et al., 1994). The alteration from a hydrated to a dehydrated state is accompanied by an overall shrinkage and hardening of the egg mass with a change in color from yellow to reddish-orange and brown. The GM shrinks and hardens when dried and gives mechanical pressure on the eggs to inhibit hatching during drought condition (Bird & Soeffky, 1972). The survival of plant-parasitic nematode eggs in soil, particularly within EM of root-knot and other nematodes, is a fascinating chapter in the ecological adaptation of organisms to a hostile environment. Survival strategies enable the nematode to persist either in soil or in plant tissue where activity may be limited for long periods of time by temperature extremes and desiccation 4

(Wharton, 1995). Root-knot nematode species exhibit a greatest eco diversity of their life cycles. Two distinct groups of Meloidogyne, thermophiles and cryophiles, can be distinguished. Meloidogyne chitwoodi, M. fallax and M. naasi are cryophiles and able to survive soil temperature below 10 C. J2 of M. chitwoodi and M. fallax at 25 C, could not survive desiccation when exposed to 33% and 59% relative humidity (RH). However, survival of individuals of both species was high at 98% RH. Survival of M. chitwoodi was better than M. fallax (Aslam, 2010). Desiccation is the state of extreme dryness, or the process of extreme drying, and the nematodes that survive this stress are in a dormant state called anhydrobiosis. The structural and biochemical adaptations are important to survive unfavourable conditions. In dry soil conditions, a dehydrating egg mass provides a little mechanical pressure on the eggs which inhibit hatch. This matrix act as an efficient barrier to water loss from the eggs inside (Orion, 1995). Meloidogyne species vary in temperature range for hatching. Meloidogyne chitwoodi and M. fallax survived longer period of time at 4 and 10 C than 20 C (Das et al., 2011). The optimum hatching temperature is generally indicative of the geographic region or seasonal preference of their host plants. A number of eggs in egg masses from Meloidogyne incognita ceased their development and went to a resting stage even when the environmental conditions were favourable (de Guiran, 1979). From the above findings, egg masses of the Meloidogyne spp can have better protection than loose eggs under adverse environmental conditions and may undergo a dormant stage to survive for long periods which makes it difficult to control them. Information about the survival of eggs in egg masses (EM) and loose eggs (LE) under different temperature and desiccated conditions of M. chitwoodi and M. fallax is limited. However, this knowledge is important for phytosanitary measures that are in place for these quarantine nematodes. So the purpose of the present study was: 1. To investigate the influence of temperature on the survival of eggs of M. chitwoodi and M. fallax. 2. To investigate the influence of desiccation on the survival of eggs of M. chitwoodi and M. fallax. 3. To determine the importance of the gelatinous matrix in the survival of eggs of M. chitwoodi and M. fallax. 5

MATERIALS AND METHODS Nematode Cultures A population of M. chitwoodi (Smakt, The Netherlands) and M. fallax (Baexem, The Netherlands) was maintained by the following methods- Culture on tomato plants Nematode cultures were maintained on tomato plants (Solanum lycopersicum cv. Marmande). Seedlings (3-4 leaf-stage) were transplanted into individual plastic pots (17 cm diam.) filled with sterilized (100 C, 16 h) soil (sand 87%, loam 9%, clay 4%) and kept under controlled glasshouse conditions (14 h day light, 24 ± 1 C). For each species approximately 3000 J2, collected from the ILVO stock cultures with the Baermann funnel technique (Baermann, 1917) were inoculated in separate pots. Three to four months after nematodes were added; the infected roots of the tomato plants were washed and egg masses were collected from the roots. Culture in closed containers Seed potatoes were washed and disinfected with 5% NaOCl (House hold bleach) for maximum 4 minutes, then rinsed with water to remove the NaOCl. The tubers were dried and stored at room temperature and exposed to daylight to allow them to sprout. 200 gm dried and sterilized (100 C, 16 h) white river sand was put in the closed container (plastic, transparent pot, 0.5 l, 9-10 cm diameter, closed with lid) and 30 ml sterile tap water was added. The sprouting potato tuber was placed in the container and then covered with a lid. After 10-12 days, 800-1000 second-stage juveniles (J2) were inoculated in each pot. The containers were then incubated at 20-22 C in the dark. 10-14 weeks after inoculation, newly egg masses were formed on the roots and these can be collected by gently rinsing the sand from the roots. Again the J2 were collected by Baermann funnel technique for further inoculation to maintain stock culture. Collection and Preparation of Egg Masses and Loose Eggs Collection of egg masses from infected tomato plants and potato tubers Egg masses were collected from 13- and 16-week-old tomato plants, 7 and 9 weeks after inoculation, respectively. The age of the tomato plant was chosen in relation to the life cycle of tomato under the glasshouse growing regime. Plants at the age of 13 weeks were vegetatively fully developed and had started flowering and fruit setting (in case of 16-week old plant). The tomato roots were washed to remove the soil and the infected root parts were separated from the uninfected root parts. In case of potato tubers in closed containers 12-14 weeks after inoculation 6

egg masses were formed. The sand was removed gently from the roots by soaking in water. To obtain intact egg masses from the roots, small root pieces (ca 5 mm in length) containing a female plus egg mass were collected for the hatching tests. Preparation of egg masses After cleaning the roots from soil, sand and debris, are excised in small pieces (2-3 cm). Pieces of roots with egg masses were placed in a plastic petridish. Female containing egg masses were picked one by one with the aid of fine forceps and scissors under binocular microscope. Finally the egg masses were transferred to shallow distilled water in a small petridish until to put into the sieve. Preparation of loose eggs After collecting the egg masses, 30-40 egg masses were picked randomly into a small flat plastic lid containing 3-4 drops of water. Each of the egg mass was gently crushed by a hand squasher to liberate the individual eggs but the eggs are not liberating completely because of highly sticky gelatinous material adhering with the eggs. Therefore the suspension was agitated by a vibriomixer (Vortex) for 1 minute to liberate more eggs from the remaining gelatinous matrix. Large debris and dusty materials were removed by using fine forceps. The individual eggs were transferred into 50 ml beaker until a certain volume is reached. The suspensions were homogenized by blowing air through the suspension. After homogenizing, 3 subsamples of 1 ml were taken with a micropipette and transferred to a counting dish. After 10-15 minutes waiting to allow the eggs to settle down the 3 subsamples were counted and the total number of eggs in the suspension was calculated. Preparation of sieve For keeping the egg mass and loose eggs, modified eppendorf tube (1.5 ml, diam. 1 cm) was used. Special cut of eppendorf tube lid was made to fix the sieve with lid to pass the juvenile from eggs. The mesh size of sieve was 50 µm. Two pieces (according to diameter of sieve lid) of sieve was used when egg masses kept in soil. Egg masses (randomly picked 3 egg masses) were kept in between two sieves. Single sieve was used when egg masses or loose eggs kept in water. 7

Hatching of Eggs in Water Inoculation of egg masses and loose eggs in water To keep the egg masses and loose eggs inside the water for hatching, small glass bottle (2 cm diam.) was used. The bottle was filled with 6 ml of tap water, and then the sieve with egg masses was put inside the glass bottle. The eggs in egg mass and loose eggs were exposed to 20 C and 5 C separately to examine the viability of eggs based on hatching behavior through different time points ( 3, 6, 9 & 12 weeks). Five (5) replications were maintained in each time (every 3 weeks) to harvest from water. Counting of juveniles and eggs from water From the water medium, hatched juveniles and unhatched eggs were counted every 3 weeks later from both types of eggs. After 3 weeks, remaining unhatched eggs inside the egg masses were liberated by exposing them to a 1% NaOCl solution for 5 minutes. Hatching of Eggs in Soil (moist and dry condition) Preparation of sterilized soil for inoculation The soil used for the inoculation of both types of eggs of M. chitwoodi and M. fallax were collected from ILVO. The soil was sterilized with 100 C for 12 hours. After sterilizing, the soil was stored in a polythene container and closed. Dry sterilized soil was soaked with water so that 20% moisture was maintained during setting of experiment. Inoculation of egg masses and loose eggs in soil Freshly 3 egg masses were picked and put gently in between the 2 sieves that fixed with modified eppendorf tube (1.5 ml). Then 16 ml prepared soil (soil with 20% moisture) was filled in a small plastic pot (2 cm diam.). The prepared sieve containing egg masses were put on top of 16 ml soil, and then again filled by rest 15 ml soil to make total volume of 31 ml so that the sieve was covered by the soil well as natural condition. For inoculation of loose eggs, certain volume of egg suspension (about 150 eggs) was sucked by micropipette and inoculated in the pot contained 31 ml soil. Watering was given in every week to maintain the 20% soil moisture. In a set of pots no water was added to observe the hatching behavior under drying out conditions. The both type of eggs were exposed to 20 C and 5 C separately to examine the viability of eggs by hatching behavior through different time points (3, 6, 9 & 12 weeks). Five (5) replications were maintained in each time (every 3 weeks) to harvest either from soil. Soil moisture was measured for dried out soil before every extraction. 8

Extraction and counting of juveniles and eggs from soil For the extraction of juveniles and eggs from the soil, the automated zonal centrifuge machine (Hendrickx, 1995) was used (Fig. 1). The machine takes a subsample of 500 ml from the soil suspension and transfers it together with MgSO 4 (separation liquid, density 1.20 kgm -3 ) into the rotor. In the rotor, the nematodes get separated from other components and are retained in the interface between water and MgSO 4. Fig. 1. Operational procedure of the automated zonal centrifuge machine Then, a kaolin suspension is added to the rotor to avoid soil particles and debris mixing with the nematode suspension at the moment the centrifugation is finished. As a final step, the nematodes solution is collected in a glass beaker (120 ml) through the hollow shaft of the rotor. The nematodes in the solutions were left to sink for at least 3 hours before the supernatant was removed by hand controlled vacuum pump (Vacuubrand BVC 21 NT VARIO) and nematodes were counted in the remaining 40 ml of the nematode suspension. Each harvesting time, the sieve containing egg masses from the soil was pulled out with a forceps and transferred into small pot (2 cm diam.) containing water. The egg masses in water were kept for 3 weeks at room temperature (25 C). Each week hatched juveniles were counted and after 3 weeks remaining unhatched eggs were counted under binocular microscope. In case of loose eggs every week newly hatched juveniles from the same egg suspensions were counted. 9

Desiccation Experiment Preparation of saturated NaCl and NaOH solution To obtain a constant relative humidity saturated NaCl and NaOH solutions were prepared (Table 1). In the atmosphere over any water solution of a non-volatile substance a definite water vapour pressure at a given temperature is reached when the vapour phase is in equilibrium with the liquid. The solutions were prepared by dissolving enough solid NaCl or NaOH to saturate at boiling point. After cooling a small amount of solid was added. After the mixture has cooled, considerably more solid was added and this was allowed to stand for several days to a week to ensure saturation. Then both solutions were ready for use. Table 1: Relative humidity values over saturated solutions at 25 C temperature- Compound Temperature ( C) Relative humidity (%) NaCl 25 75.5 NaOH 25 7 Exposure of egg masses and loose eggs to different relative humidity An artificial humidity chamber was made of an airtight 500 ml clear plastic container with a lid that tightly covered it. The saturated solution (20 ml) of NaCl or NaOH was poured into the container. Three intact egg masses or 150 loose eggs of M. chitwoodi and M. fallax were randomly picked from roots and placed into a embryonic glass. The embryonic glass was immediately transferred to the humidity chamber. Exposure was done for 1 hour inside an incubator at 25 C. After 1 hour exposure time the embryonic glasses were removed from the humidity chamber and egg masses and loose eggs were transferred into small glass bottles (2 cm diam.). The egg masses were put on a small sieve (mentioned above). The plastic tubes with the egg masses and loose eggs were kept at room temperature to check the viability of the eggs by counting the hatched juveniles on a weekly basis. Every week the water was refreshed for proper aeration to stimulate hatching. After 4 weeks unhatched eggs were counted by dissolving the egg masses in a 1% NaOCl solution. Finally the percentage of hatched juveniles was determined. Statistical Analysis Statistical analysis was done using SPSS 21.0. Multifactor ANOVA was used to examine significance of main and interactive effects. Tukey test was used to compare between means (P < 0.05) at 95% level of confidence. Whenever necessary, data were transformed to log to fulfil the conditions of parametric tests. 10

RESULTS Effect of temperature on hatching of eggs from EM and LE of M. chitwoodi and M. fallax in water At 5 and 20 C significant differences in hatching of M. chitwoodi and M. fallax from EM and LE were observed at the different observation times (Table 2). Temperature was a major factor for hatching of the two species (F = 6601) followed by egg type (F = 410). There was significant difference between EM and LE in terms of hatching at 20 C for M. chitwoodi. Hatching was almost double in EM than LE at 20 C (Table 3) and between two egg types, highest hatching was observed from EM of M. chitwoodi at 9 weeks (88.55%) and lowest from LE at 3 weeks (39.33%). Hatching of EM was significantly lower (74.17%) at 3 weeks compared to 12 weeks but no significant difference was observed between weeks 6, 9 and 12. In case of loose eggs there was no significance difference of hatching among weeks, but highest hatching was observed at 9 weeks (48.00%) (Table 3). Table 2. Significance of main and interaction effects on the hatching of M. chitwoodi and M. fallax from egg masses and loose eggs in water. Source of variation Degrees of freedom F value P value Species 1 61.636.000 Temperature 1 6601.213.000 Time 3 5.256.002 Egg type 1 410.176.000 Species Temperature 1 49.408.000 Species Time 3 2.678.050 Species Egg type 1 9.626.002 Temperature Time 3 4.879.003 Temperature Egg type 1 403.060.000 Time Egg type 3 1.092.355 Species Temperature Egg type 1 15.710.000 Species Temperature Time 3 3.000.033 Species Egg type Time 3 0.060.981 Temperature Time Egg type 3 1.409.243 Species Time Temperature Egg type 3 0.025.995 11

In contrast of 20 C, hatching was significantly lower at 5 C for both types of eggs of M. chitwoodi (Table 3). Few juveniles hatched and the highest percentage of hatching was observed after 3 weeks from EM (0.85%). From the LE no significant differences were observed between different weeks but highest hatching was observed at 12 weeks (1.33%). At 5 C, there was no significant difference between EM and LE. In case of M. fallax also at 20 C, comparable results were observed between EM and LE for different weeks. Highest hatching was observed at 6 weeks (95.41%) and lowest from LE at 12 weeks (57.87%). From EM, highest hatching was observed at 6 weeks (95.41%) and lowest at 3 weeks (82.54%). In case of loose eggs no significant differences of hatching were found up to 12 weeks. At 5 C, a drastic decline in hatching was observed from both types of eggs. Highest hatching was observed at 9 weeks with 2.56% and 1.20% from EM and LE, respectively. However there was no significant difference between EM and LE up to 12 weeks (Table 3). Table 3. Hatching behavior of eggs of M. chitwoodi and M. fallax after exposure to 20 C and 5 C temperature up to 12 weeks. Species Temperature ( C) Egg type Time (weeks after hatching) 3 6 9 12 M. chitwoodi 20 5 Egg mass Loose eggs Egg mass Loose eggs A b 74.17 A ab 82.18 A a 88.55 A a 87.99 B a 39.33 B a 40.13 B a 48.00 B a 45.60 C a 0.85 C b 0.30 C b 0.22 C b 0.26 C a 0.93 C a 0.40 C a 0.80 C a 1.33 M. fallax 20 5 Egg mass Loose eggs Egg mass Loose eggs A c 82.54 A a 95.41 A ab 91.05 A b 90.22 B a 60.00 B a 66.13 B a 63.07 B a 57.87 C a 1.46 C a 1.42 C a 2.56 C a 1.79 C a 0.53 C a 1.07 C a 1.20 C a 0.93 Means with same letter do not differ significantly at 0.05 level using Tukey test. The upper case letter represents the vertical comparison (EM & LE) of percent hatching over temperature of each species. The lower case superscripts represent the horizontal comparison (among time) of each egg type at certain temperature. 12

Cumulative hatching (%) Cumulative hatching (%) 100 90 80 70 60 Chit EM Fall EM Chit LE Fall LE 50 40 30 20 10 0 3 week 6 week 9 week 12 week Time (Weeks) Fig. 2. Bar graph showing the cumulative percentage hatch and ± SE from egg masses (EM) and loose eggs (LE) of M. chitwoodi (Chit) and M. fallax (Fall) for different weeks at 20 C in water. 3 2,5 2 1,5 1 0,5 Chit EM Fall EM Chit LE Fall LE 0 3 week 6 week 9 week 12 week Time (Weeks) Fig. 3. Bar graph showing the cumulative percentage hatch and ± SE from egg masses (EM) and loose eggs (LE) of M. chitwoodi (Chit) and M. fallax (Fall) for different weeks at 5 C in water. 13

The cumulative percentage of hatching differed significantly between M. chitwoodi and M. fallax. At 20 C, hatching from EM of M. fallax was 8.37%, 13.23%, 2.5% and 2.23% more than M. chitwoodi for 3, 6, 9 & 12 weeks respectively. In case of LE, M. fallax had 20.67%, 26%, 15.07% and 12.27% more hatching than M. chitwoodi (Fig. 2). At 5 o C temperature both species had lower percentage of hatching, but higher hatching was observed from eggs of M. fallax than M. chitwoodi (Fig. 3). Effect of temperature and moisture on hatching of eggs of M. chitwoodi for different time in soil Two temperature regimes (20 C and 5 C), moisture status of soil (moist and dry condition), egg type (egg mass-em and loose eggs-le) and different exposure times (3, 6, 9 &12 weeks) showed significant effects on the hatching and survival of eggs of M. chitwoodi (Table 4) and M. fallax (Table 5). Table 4. Significance of main and interaction effects of variables for the hatching of eggs of M. chitwoodi for different time in soil. Source of variation Degrees of freedom F value P value Temperature 1 108.992.000 Moisture status (MS) 1 512.551.000 Time 3 98.444.000 Egg type 1 485.666.000 Temperature MS 1 0.468.495 Temperature Time 3 11.691.000 Temperature Egg type 1 30.631.000 MS Time 3 24.695.000 MS Egg type 1 18.286.000 Time Egg type 3 4.951.003 Temperature MS Time 3 28.921.000 Temperature MS Egg type 1 13.907.000 Temperature Time Egg type 3 18.803.000 MS Time Egg type 3 8.238.000 Temperature MS Time Egg type 3 8.051.000 14

The study showed moisture status of soil to be a major factor on the hatching and survival of M. chitwoodi with an F value of 512.551 and egg type to be the most determinant on the hatching and survival of M. fallax with an F value of 985.806. The moisture status of the dry soil was shown in (Table 6) up to 12 weeks. Table 5. Significance of main and interaction effects of variables for the hatching of eggs of M. fallax for different time in soil. Source of variation Degrees of freedom F value P value Temperature 1 65.984.000 Moisture status (MS) 1 375.851.000 Time 3 73.859.000 Egg type 1 985.806.000 Temperature MS 1 189.903.495 Temperature Time 3 9.546.000 Temperature Egg type 1 194.442.000 MS Time 3 31.898.000 MS Egg type 1 3.467.065 Time Egg type 3 6.953.000 Temperature MS Time 3 3.312.022 Temperature MS Egg type 1 11.816.001 Temperature Time Egg type 3 2.999.033 MS Time Egg type 3 2.976.034 Temperature MS Time Egg type 3 0.825.482 Hatching of both species is mostly affected by moisture status of soil and egg type. When the EM and LE were exposed to 20 C in moist soil for 3 weeks, highest hatching of M. chitwoodi was observed from LE (59.33%) and lowest from EM (7.15%) (Table 7). Hatching from EM was higher in dry soil (15.31%) than moist soil (7.15%). On the other hand, significantly low hatching (11.73%) was observed from LE in dry soil compared to moist soil (Table 7). The treated EM and LE were kept in normal conditions (in water at room temperature) for 3 weeks after the treatment and further hatching was monitored to check the viability of eggs. It was clearly showed that at 3+3 weeks, highest cumulative percentage of hatching (hatching during 15

the treatment in soil + hatching in water at room temperature) was observed from EM (92.45%) and lowest from LE (66.40%). The treatment in dry soil significantly lowered hatching from EM (17.88%) and LE (12.53%) (Table 8). From the 6 weeks treatment onwards hatching during the treatment was below 26% for both type of eggs and no hatching differences were observed between EM and LE in moist soil. For dry soil hatching from EM was significantly higher than hatching from LE. After 9 weeks no significance difference of hatching was observed from EM between moist soil and dry soil. For LE significantly more hatching was observed in moist soil compared to dry soil at 9 and 12 weeks (Table 7). When the EM and LE were transferred to water and kept in room temperature, highest cumulative hatching was observed from EM of both soil conditions. In contrast, very low post-treatment hatching was observed from LE. Significantly lower hatching was observed from dry soil than moist soil ( Table 8). When both type of eggs were treated at 5 C very low hatching was observed compared to 20 C for both soil conditions. In general, highest hatching was observed from LE in moist soil. However, after 3 weeks highest hatching was observed from LE in dry soil (6.13%). From 6 weeks onwards below 5% hatching was recorded in both soils (Table 7). At 3+3 weeks, when both type of eggs were incubated at room temperature in water, highest cumulative hatching was observed from EM (77.20% and 72.45%) and lowest from LE (14.53% and 8.80%) for treatment in moist and dry soil respectively. For 6+3 weeks there was no significant difference in hatching between moist and dry conditions for both EM and LE. Hatching from EM was significantly higher than LE. At 9+3 and 12+3 weeks hatching from EM and LE in dry soil ceased. This was also seen for LE in moist soil (Table 8). In case of M. fallax, after 3 weeks treatment of eggs to 20 C, highest hatching was observed from LE (67.20% and 29.60%) than EM (40.13% and 9.94%) from moist and dry soil respectively and significant difference was observed in hatching between EM and LE (Table 9). Upon return to normal condition at 3+3 weeks, highest hatching was observed from EM (96.25%) and lowest from LE (71.87%). The treatment in dry soil significantly lowered hatching as observed from EM (46.21%) and LE (29.73%) (Table 10). From the 6 weeks onwards during treatment, highest hatching was observed from EM (68.56%, 55.47% and 49.30%) and LE (46%, 32.27%, and 27%) for 6, 9 & 12 weeks respectively. But in dry soil treatment the percentage of 16

hatching was significantly lower than moist soil. However significant differences were recorded in hatching between EM and LE up to 12 weeks. From the 6 weeks hatching from LE was found below 5% in dry soil up to 12 weeks (Table 9). When both eggs were kept in room temperature at 3+3 weeks, a substantial number of eggs were hatched from EM (96.25% and 46.21% ) than LE (71.87% and 29.73%) but hatching was very low from dry soil treatment of both types of eggs. About 15%, 30% and 23% further hatching was observed from EM treated in moist soil at 6+3, 9+3 and 12+3 weeks respectively but very few (3% for 6+3 weeks and later below 1%) eggs were hatched from LE up to 15 weeks. In contrast very low post-treatment hatching was observed from both type of eggs in from dry soil, but hatching was significantly higher from EM than LE (Table 10). When the both eggs were exposed to 5 C, very low (< 2%) hatching was found compared to 20 C from both soil conditions at 3, 6, 9 & 12 weeks (Table 9). However when the eggs were incubated at room temperature after treated with moist soil, a significant amount of cumulative hatch was observed from EM 70.81%, 76.17%, 68.79% and 51.77% and less than 5% hatch was recorded from LE at 3+3, 6+3, 9+3 & 12+3 weeks respectively. On the other hand from the EM of dry soil, cumulative hatching was 74.02% at 3+3 weeks and 61.26% at 6+3 weeks, then declined significantly up to 15 weeks. In case of loose eggs only 7.33% at 3+3 weeks and significantly declined (< 2%) from the 9 weeks. However significant differences were observed in hatching between EM and LE in different exposure time (Table 10). Table 6. The effect of temperature (20 C and 5 C) on moisture status in dry soil (initial moisture 20%) up to 12 weeks. Time ( week) 20 C 5 C % moisture % moisture 3 7 14 6 5 10 9 4 8 12 3.5-4.0 7 17

Table 7. Hatching of M. chitwoodi from egg masses (EM) and loose eggs (LE) when exposed to moist and dry soil at 20 C and 5 C temperature up to 12 weeks. Temp ( o C) 20 5 3 week treated 6 week treated 9 week treated 12 week treated Egg type Moist soil Dry soil Moist soil Dry soil Moist soil Dry soil Moist soil Dry soil EM 7.15 c 15.31 b 26.3 a 14.2 b 23.27 a 16.22 a 18.29 a 9.73 a LE 59.33 a 11.73 bc 26.13 a 6.13 c 18.40 a 4.53 b 12.26 a 2.40 b EM 0.15 b 0.16 b 0.28 b 0.29 b 0.17 b 0.43 b 0.17 b 0.39 b LE 3.47 a 6.13 a 2.13 a 1.60 ab 4.00 a 1.07 b 4.00 a 0.13 b Table 8. Cumulative hatching (hatching during the treatment in soil + hatching in water at room temperature) of M. chitwoodi when exposed to moist and dry soil at 20 C and 5 C temperature up to 15 weeks. Temp ( o C) 20 5 Egg type EM LE EM LE 3+3 week 6+3 week 9+3 week 12+3 week Moist soil Dry soil Moist soil Dry soil Moist soil Dry soil Moist soil Dry soil A a 92.45 B c 17.88 A a 76.36 BC c 16.85 A a 65.81 BC b 17.07 A a 63.63 C b 11.47 A b 66.40 BC c 12.53 AB b 26.93 CD d 6.67 BC b 18.40 D c 4.53 BC b 12.27 D c 2.40 A a 77.20 A b 72.45 A a 67.27 A a 51.29 A a 61.95 B c 0.85 A a 61.87 B c 0.46 A c 14.53 AB c 8.80 BC b 4.53 BC b 2.67 AB b 6.40 CD c 1.33 BC b 5.07 D c 0.13 Means with same letter do not differ significantly at 0.05 level using Tukey test. The upper case letter (s) represent the horizontal comparison of percent hatching from each egg type over time. The lower case superscripts represent the horizontal (Moist & dry condition) and vertical (EM &LE) comparison within the each week at certain temperature. At week number, number before plus indicate duration of treatment in soil and number after plus hatching was monitored up to 3 weeks in room temperature. 18

Table 9. Hatching of M. fallax from egg masses (EM) and loose eggs (LE) when exposed to moist and dry soil at 20 C and 5 C temperature up to 12 weeks. Temp ( o C) Egg type 3 week treated 6 week treated 9 week treated 12 week treated 20 5 Moist soil Dry soil Moist soil Dry soil Moist soil Dry soil Moist soil Dry soil EM 40.13 b 9.94 c 68.56 a 11.51 c 55.47 a 9.62 c 49.30 a 9.98 c LE 67.20 a 29.60 b 46.93 b 4.00 d 32.27 b 1.33 d 27.07 b 1.07 d EM 1.04 a 1.19 a 1.06 a 0.97 a 1.07 a 1.36 a 0.58 b 1.64 ab LE 1.33 a 1.87 a 1.87 a 0.80 a 3.20 a 1.33 a 2.13 a 0.80 b Table 10. Cumulative hatching (hatching occurred during treated in soil + hatching during room temperature from the same treated EM and LE) of eggs of M. Fallax when exposed to moist and dry soil at 20 C and 5 C temperature up to 15 weeks. Temp ( o C) Egg type 3+3 week 6+3 week 9+3 week 12+3 week 20 5 EM LE EM LE Moist soil Dry soil Moist soil Dry soil Moist soil Dry soil Moist soil Dry soil A 96.25 a B 46.21 c A 83.85 a C 15.60 c A 84.58 a D 10.18 c A 72.28 a D 10.89 c A 71.87 b B 29.73 d AB 50.13 b C 4.40 d B 33.07 b D 1.33 d B 27.07 b D 1.07 d A 70.81 a A 74.02 a A 76.17 a AB 61.26 a A 68.79 a BC 35.67 b AB 51.77 a C 25.24 b AB 5.33 b A 7.33 b AB 3.73 b A 6.67 b AB 4.80 c BC 1.33 d ABC 3.20 c C 0.80 d Means with same letter do not differ significantly at 0.05 level using Tukey test. The upper case letter (s) represent the horizontal comparison of percent hatching from each egg type over time. The lower case superscripts represent the horizontal (Moist & dry condition) and vertical (EM &LE) comparison within the each week at certain temperature. At week number, number before plus indicate duration of treatment in soil and number after plus hatching was monitored up to 3 weeks in room temperature. 19

Effect of desiccation on hatching of second-stage juveniles of M. chitwoodi and M. fallax Significant differences were observed between the two species in relation with hatching from egg masses and loose eggs of M. chitwoodi and M. fallax after 1 hour exposure at two different relative humidities (75.5% and 7%) at 25 C. Hatching of eggs was observed for 4 weeks after treatment. The study showed egg type to be a major determinant of the survival of the two temperate Meloidogyne species used with an F value of 600.816 followed by relative humidity (250.017) (Table 11). Table 11. Significance of main and interaction effects for the hatching of eggs of M. chitwoodi and M. fallax. Source of variation Degrees of freedom F value P value Species 1 42.170.000 Relative humidity (RH) 1 250.017.000 Egg type 1 600.816.000 Species RH 1 15.181.001 Species Egg type 1 22.678.000 RH Egg type 1 104.133.000 Species RH Egg type 1 11.570.002 The percentage hatching of M. chitwoodi and M. fallax in relation to different relative humidity is shown in Table 12. When the eggs of both species were subjected to different relative humidities, significant differences of hatching were observed between EM and LE. Significantly greater percentage of hatching was observed from egg masses than loose eggs for four weeks. The highest percentage of hatching was observed from egg masses of both species at 2 weeks at 75.5% relative humidity. In first week after treatment, more hatching of M. chitwoodi was observed from EM than LE in both RH (Table 12). For M. fallax this difference at week 1 was only seen at the lowest RH. From week 2 onwards hatching from EM was always higher than that from LE for both species. For M. chitwoodi hatching from EM was similar for both RH except for the hatching during week- 2. 20

The hatching from LE of M. chitwoodi was significantly higher at 75.5% RH during the first two weeks; afterwards there was no difference with 7% RH. Identical hatching was observed from EM between two RH except week-1 for M. fallax. However from the LE, significant differences were found between both RH at up to week-2 later found similar. But significant differences in hatching were observed between EM and LE of both species (Table 12). Table 12. Effect of relative humidity on the hatching of eggs from egg masses (EM) and loose eggs (LE) of M. chitwoodi and M. fallax after exposure to 1 hour at 25 C for 4 weeks. Species M. chitwoodi M. fallax Time (Weeks after hatching) RH (%) 1week 2 week 3 week 4 week EM LE EM LE EM LE EM LE 75.5 22.92 a 5.95 c 37.66 a 15.38 b 10.83 ab 2.23 c 2.42 bc 0.62 c 7 20.84 a 0.86 e 13.71 b 0.51 d 8.37 b 0.18 c 4.98 ab 0.02 c 75.5 19.08 a 15.99 ab 30.29 a 14.75 b 24.63 a 1.04 c 7.74 a 0.39 c 7 9.79 bc 2.42 d 22.88 ab 5.02 c 17.94 ab 0.48 c 5.64 ab 0.09 c Means with same letter superscripts do not differ significantly at 0.05 level using Tukey test. The superscript letter (s) represents the comparison of hatching per week from both species. Table 13. The effect of different relative humidity on hatching (%) of egg masses (EM) and loose eggs (LE) of M. chitwoodi and M. fallax after exposure to 1 hour at 25 C for 4 weeks. Species RH (%) EM LE M. chitwoodi 75.5 73.86 ab 24.20 d 7 47.92 bc 1.59 f M. fallax 75.5 81.85 a 32.20 c 7 56.27 b 8.04 e Means with same letter superscripts do not differ significantly at 0.05 level using Tukey test. The superscript letter (s) represents the comparison of cumulative hatching of both species. The cumulative percentage hatch after 4 weeks observation after the treatment is shown in Table 13. There were significant differences between hatching from EM and LE of both species. For both species the cumulative hatching was highest from EM at 75.5% RH and lowest from LE at 7% RH. Hatching from EM was higher than hatching from LE for both species and also hatching at 75.5% RH was higher than at 7%RH. At 75.5% RH, J2 hatching from LE was 50% less than EM of M. chitwoodi. On the other hand, 46% less hatching was 21

Cumulative hatched J2 (%) observed in LE from EM at 7% RH. In case of M. fallax, 50% and 48% less hatching was observed in LE from EM at 75.5% and 7% RH respectively. Hatching from EM and LE of M. fallax was higher than M. chitwoodi. EM from M. fallax had 8% and 9% and for LE had 8% and 7% more hatching than M. chitwoodi at 75.5% and 7% RH respectively (Table 13, Fig. 4). 90 80 70 60 50 40 30 75,5% RH 7% RH 20 10 0 EM Chitwoodi LE Chitwoodi EM fallax LE Fallax Egg type Fig. 4. The effect of different relative humidity (1 hr exposure to 75.5% RH or 7% RH at 25 C) on hatching of juveniles from egg masses (EM) and loose eggs (LE) of M. chitwoodi and M. fallax. Bars represent the cumulative % hatch after 4 weeks ± SE. DISCUSSION Three approaches were employed to investigate the putative role of the gelatinous matrix for survival of eggs of M. chitwoodi and M. fallax. 1. Hatching of eggs from EM and LE of both species in water at 20 C and 5 C. 2. Hatching of eggs from EM and LE of both species in soil at 20 C and 5 C during different incubation times (3, 6, 9 & 12 weeks) and further hatching at room temperature after incubation. 3. Hatching of eggs from EM and LE after exposure during 1 hour to higher (75.5%) and lower (7%) relative humidity. The development (Wallace, 1966) and hatching (Bird & Wallace, 1965) of nematodes in the genus Meloidogyne are dependent, in part, on external sources of heat and water. In this experiment, survival and hatching of both species was depending on egg type, temperature, 22

moisture and time. In an in vivo experiment, Towson & Apt (1983) found that the duration of the survival of Meloidogyne spp. is determined by temperature and moisture. In water the hatching was found to be mostly affected by temperature and egg type for different observation times. The hatching of eggs was significantly higher up to 12 weeks at 20 C and from 6 weeks onwards hatching was more than 80% from EM of M. chitwoodi and more than 90% from EM of M. fallax. Hatching from LE was significantly lower than hatching from EM. On the other hand when treated with very low temperature (5 C), hatching was almost negligible from both type of eggs (< 2%). It was reported that the optimum temperature for hatching of both species was 20 C, but J2 of M. chitwoodi hatched more between 20 and 25 C (Khan et al., 2014). Hatching of M. chitwoodi and M. hapla was reduced significantly at low temperature (7 C) (Inserra et al., 1983). At low temperature a number of unhatched eggs went to either a state of quiescence or diapause. Among species of Meloidogyne, the percentage of unhatched J2 that enter diapause varies from less than 10% for M. arenaria to 94% for M. naasi (Perry et al., 2013). In this study when both type of eggs of M. chitwoodi and M. fallax were exposed to 20 C in moist and dry soil, very few eggs were hatched during the treatment. Upon return to favourable condition, the highest hatching was observed from EM and very few eggs were hatched from LE. The longer that LE were exposed to the soil environment, the fewer the eggs remained viable (based on hatching). Better survival was observed for eggs in egg masses. LE were also infected by microorganisms which could have influenced the results. Orion et al. (2001) compared the infectivity of egg masses and separated eggs of M. javanica after infecting with microorganisms at different time points. Separated eggs were easily attacked by microorganisms, while eggs in egg masses were mostly uninfected. The gelatinous matrix protected the eggs and served as a barrier against the microorganisms. Sharon et al. (2001) also found that the saprophagous fungi, Trichoderma harzianum is not able to grow on gelatinous matrices but colonizes isolated eggs and J2 of M. incognita. In this study, after 3 weeks incubation in moist soil, a significant amount of hatching was observed in the soil from LE of both species. The relatively short time period allowed J2 to develop inside the eggs and sensing of moisture most likely stimulated immediate eclosion. From the dry soil treatment, hatching was significantly lower compared to moist soil for both type of eggs. Bird & Soeffky (1972) reported that in dry soil conditions, a dehydrating egg mass provides a little mechanical pressure on the eggs which inhibit hatch. Another study showed that an extra protective layer which appear as an extracuticular subcrystalline layer in 23

Meloidodera charis showed to slow the rate of water loss of unhatched J2 and help to protect against desiccation (Demeure & Freckman, 1981). However upon return to favourable conditions a significant number of hatch was observed from EM compared to LE even in more dry soil up to 15 weeks. Guiran & Demeure (1978) showed when EM are subjected to dry soil for 1 week J2 hatched quickly after subsequent exposure to optimum conditions for 20 days. On the other hand, survival of eggs was more pronounced when exposed to 5 C. During treatment very few hatching was observed from both types of eggs up to 12 weeks. When investigating the treatment effect subsequently at room temperature, more than 70% hatching occurred from EM at first 3-6 (3+3) weeks and more than 50% hatching was recorded after the treatment with moist soil up to 15 weeks for both species. During treatment in dry soil at 5 C very low (< 5%) hatching was observed from EM and LE of both species. Upon return to favourable conditions, more than 70% eggs were hatched from EM in water at 3-6 weeks and gradually declined. Significant amount of eggs were resumed from EM (> 50% up to 6-9 weeks) of both species when treated with 5 C compared to 20 C. Because at 20 C, water loss was faster than at 5 C. Lees (1953) showed that a slow rate of water loss enhances the chance of survival of Panagrellus silusiae. However, from the LE of both species, very low hatching was observed up to 15 weeks compared to EM. Higher hatching was observed from both types of soil at 3-6 weeks and gradually declined with the increase of exposure time. Vrain & Barker (1978) found more resistance in unhatched J2 of M. incognita to low temperatures and found that eggs are better adapted to lower temperature than hatched J2. They also investigated that a significant amount (20-30%) of eggs developed abnormally or died because of chilling injuries due to a lack of adaptation to lower temperature. The temperate species, M. hapla proved more robust and few eggs developed abnormally. Sayre (1964) showed that when egg masses were exposed to -30 C for 30 minutes, 60% of M. hapla survived to hatch but only 30% of M. incognita survived which may reflect the greater survival to chilling of M. hapla. A number of nematode species are able to survive anhydrobiotically (Evans & Perry, 2009). In this study we monitored the hatching after incubation in high (75.5%) and very low (7%) RH. Hatching was higher from both type of eggs at 75.5% and lower at 7% RH for both species. This result was similar to Aslam (2010), J2 of M. chitwoodi and M. fallax at 25 C could not survive desiccation when exposed to 33% and 59% RH. However, survival of individuals of both species was high at 98% RH. In my study highest hatching was observed 24