Ecological Characterisation of Native Isolates of Heterorhabditis indica from Viti Levu, Fiji Islands

Culture of all the 35 isolates obtained during the survey of Viti Levu was maintained separately under laboratory conditions. Keeping isolates separately prevented their contamination with other isolates and preserved their genetic diversity (Kaya and Gaugler, 1993). All isolates were reared in last-instar of the host insect, G. mellonella at 25°C. IJ were harvested three to 5 days after their first emergence from the cadaver and were stored at 15°C as aqueous suspensions (in distilled water) in 750 ml tissue culture flasks for no longer than 6 days. Prior to testing, IJ were washed with distilled water and their viability was determined by microscopic examination (Agudelo-Silva et al., 1987). The viability was determined by counting the live nematodes under the microscope. If sample has more than 95% live nematode it was used in experiment. The concentration of IJ required in each bioassay was estimated from the mean of ten subsamples (1 ml) from each nematode suspension and final concentration was adjusted by volumetric dilutions. Since the extended laboratory culture of EPNs in the absence of outbreeding can be deleterious (Kaya and Gaugler, 1993), the strains used were kept in culture for less than five generations (Selvan et al., 1994; Shapiro et al., 1996). Also, to ensure production of IJ with good biological activity and virulence, the population was maintained by trapping the first emerging IJ and using them for initiating the next generation of IJ (Lindegren et al., 1993). The last instar of G. mellonella with average weight of 0.2 g to 0.3 g was used as test insect. G. mellonella is universally preferred experimental host insect for EPNs. Hence, its use allows comparisons between bioassays conducted in different laboratories around the world (Grewal et al., 1999). Except for single dose screening assays, use of soil was preferred over tissue paper in infectivity assays. Hence, soil with sandy loamy texture (73% sand, 10% silt, 17% clay) with 1.3% organic matter was used in bioassays.

A single-dose screening assays was conducted for all 35 nematode isolates to test their relative virulence against G. mellonella and to select the three top ranked isolates for further ecological characterisation. For single-dose screening assay, a 5.5-cm Petri dish was lined with a moistened filter paper. Approximately 50 IJ were transferred to each Petri dish to which five Galleria larvae were added. The host mortality and time duration to death were recorded. Based on mean time until death and mean proportional mortality, the three most virulent isolates were selected for further ecological characterisation.

Three experiments were conducted to determine the type of foraging and host finding strategy used by native isolates of H. indica. In the first experiment, a 100-mm Petri dish was lined with a moistened filter paper and sprinkled with 0.2 g of sand particles. Approximately, 100 IJ were transferred to each Petri dish, and the behaviour of the IJ was monitored for 10 min using a dissecting microscope at × 50 (Gungor et al., 2006). There were five replicates per isolate. A second experiment was conducted to evaluate the response of IJ to the host volatile cues. The experiment was conducted as described by Shapiro-Ilan et al. (2005). A 100-mm Petri dish was filled with 2% agar. Two 1000 µl pipette tips were inserted in the lid of Petri dish on the opposite side to each other. On a treatment plate, a G. mellonella larva was placed in one pipette tip and the other tip was left empty. In control plates, both tips were left empty. The Petri dish was sealed with paraffin film and was left for 1 hr to build the host cue gradient. After an hour, approximately 2000 IJ were placed on a small piece of filter paper positioned in the centre of the agar plate through a hole in the cover. After 2 hr, the number of IJ found in a 1 cm diameter circle under each pipette tip was recorded. Host-seeking ability was estimated as percentage of IJ found under the pipette tip with the host relative to the total number of IJ found under the host plus those found under the empty pipette tip. In control plates, none of the pipette tip had insect host and one side was randomly designated the host side for calculation purposes. There were five replicates for each isolate. In the third experiment, the ability of the IJ to find and infect a sedentary host at different soil depths was determined (Raja et al., 2011). The soil column bioassay was used to study the ability of the IJ to find hosts and calculating the invasion efficiency at different depths. A plastic tube of 11.5 cm height was filled with sandy loam soil up to 10.5 cm. A G. mellonella larva was constrained between the two layers of aluminium mesh that did not allow the larva to crawl. The aluminium mesh was placed at 0, 2, 5, or 10 cm soil depth, and 100 IJ in 1 ml of distilled water were added on the surface of each column. There were five replicates per isolate. After 2 days the columns were disassembled and the dead larvae were dissected and pepsin-digested to determine the number of nematodes that had penetrated into the body cavity of the host (Koppenhöfer and Fuzy, 2003). For pepsin digestion, the dissected cadaver was placed in 15-ml centrifuge tubes containing 4 ml of pepsin solution (2.3 g of NaCl and 0.8 g of pepsin in 100 ml of deionized H₂O, adjusted to pH 1.8 to 2.0 with conc. HCl). The tubes were incubated in water bath at 37°C for 1½ hr. Following incubation, 10 ml of 0.1% Tween80 solution was added to each tube. The tubes were vortexed for 20 sec and contents were poured into a 10-cm Petri dish. All nematode stages present in the suspension were counted under × 50 magnification.

LC50 of three isolates of H. indica were investigated against G. mellonella larvae. A 50 µl drop containing 1, 3, 5, 10, and 25 IJ were applied to 24-well tissue culture plate. To control plate only 50 µl of distilled water was added. Then two grams of sandy loamy soil, the moisture of which was adjusted to 12% (w/w), was added to each well. One last-instar of G. mellonella was added to each well. The plates were covered and incubated at 25°C. Mortality was assessed after 48 hr by probing. In the absence of a response to the probe, the larva was considered dead. Twenty G. mellonella larvae were tested per concentration.

The experiment was conducted to evaluate pathogenicity of the three selected native isolates of H. indica at different temperatures using host G. mellonella and to establish the range of temperatures at which the native isolates can infect insect larvae. Initially, the experiment was conducted as described by Koppenhöfer and Kaya (1999) but in the second attempt slight changes were made in methodology. In the first failed attempt, air dried soil was used, whereas in second attempt a moisture level of the soil was adjusted to 12% (w/w). Three 24-well tissue culture plates were used. 50 IJ in 20 µl of distilled water were applied to each well. The plates were placed in incubators at 10^°C, 15^°C, 20^°C, 25^°C, 30^°C, 35^°C, and 40^°C. After 1 hr for acclimatisation, one last instar larva of Galleria was added to each well. The plates were covered in polythene bags to avoid evaporation and desiccation of IJ. The first plate was used to record larval mortality, time of death, and number of nematodes that penetrated per larva at various temperatures. To record time of death, the wells were examined at 12 hr interval for 3 weeks and each larva was probed. In the absence of response to the probe, the larva was considered dead. Dead larvae were removed from the wells and thoroughly washed in distilled water. The cadavers were dissected and digested in a pepsin solution (Mauleon et al., 1993). Dissection and digestion were done 1 day after death at 25°C and 35°C, 2 days after death at 20°C, and 3 days after death at 15°C. The second plate was used to record the time of the first emergence of progeny at various temperatures. The plates were checked daily and cadavers were removed from the plate and placed in a Petri dish and further incubated at respective temperatures until the emergence of the IJ. The White traps were monitored daily and the first day of IJ emergence was recorded. Cadavers from which no IJ had emerged were dissected to determine the fate of the infection. The third plate served as a control to which only 20 µl of distilled water was added.

Experiments were conducted to evaluate the reproductive capacity of three selected native isolates of H. indica in host G. mellonella at different temperature and to establish reproduction thermal niche breadth of these isolates. Each well of 24-well tissue culture plates was filled with 0.5 g of sterile, air dried sandy loamy soil whose moisture level was adjusted to 12% (w/w). To each well 50 IJ in 20 µl of distilled water were applied. One last instar larva of Galleria was added to each well. The plates were kept at 25°C for 1 day to standardise the number of IJ penetrating into the larvae. The plates were placed in incubators at 10°C, 15°C, 20°C, 25°C, 30°C, 35°C, and 40°C and were covered in polythene bags to avoid evaporation and desiccation of IJ. The cadavers were removed from the tissue culture plate, thoroughly washed to remove soil or any nematode attached on the surface and placed in White traps which were further incubated at respective temperatures until the emergence of the IJ. Small White traps were used for easy collection of all the emerging IJ. The Small White traps were made using 5.5-cm Petri dishes and small plastic lids. The plastic lid was inverted and placed in Petri dish. It was lined with filter paper with edges touching the water in the outer Petri dish. There were 24 cadavers per isolate per temperature. The White traps were monitored daily. The IJ were harvested till they stop emerging and pooled together and stored at 15^°C. Ten White traps were randomly selected for counting the total number of IJ emerging from the cadaver and progeny was estimated by counting five subsamples under ×50 magnification.

Before the experiment, the IJ stored at 15°C were allowed to adapt to room temperature (25°C) for 2 hr. Thermal tolerance was evaluated as described by Ma et al. (2013) with a little modification. Approximately 2000 IJ were suspended in 1 ml of Ringer solution (9 g NaCl, 0.4 g KCl, 0.4 g CaCl₂ and 0.4 g Na₂CO₃ dissolved in 1000 ml of distilled water and autoclaved) (Lacey, 1997) and incubated at 40°C for 1, 2, 3, and 4 hr. After each interval period, 9 ml distilled water was added to the tube and tubes were kept for an additional 24 hr at room temperature (25°C) to allow the nematodes to recover from the heat shock. A sample of 50 µl was taken for recording the live and dead nematodes. Live nematodes were counted using a stereomicroscope. Nematodes were considered live if they were naturally moving or responded to probing with a fine needle. For each isolate, IJ maintained at 25°C were used as control. There were five replicates for each isolate.

An experiment was conducted to evaluate the effect of soil moisture on the capability of seeking out and infecting host G. mellonella and to establish the range of moisture levels at which they can infect host larvae. 100 IJ in 20 μl of water were placed on the bottom of each well of 24-well tissue culture plates. The wells were filled with 2 g of sterile, air dried sandy loamy soil whose moisture level had been adjusted to 5%, 8%, 10%, 15%, 20%, and 25% (w/w). One last instar larva of Galleria was added to each well and wells were completely filled with soil. In control plate only 20 µl of distilled water was added. The plates were incubated at 25°C, covered in polythene bags to avoid evaporation and desiccation of IJ. The dead larvae were recovered after 3 days and washed thoroughly to remove soil or any nematode attached on the surface. The cadavers were dissected and enzymatically digested to record the number of nematodes that had penetrated into it. Since it was not possible to dissect all cadavers at the same time, the cadavers were stored in the freezer to be dissected later. There were twenty-four replicates per soil moisture level.

Desiccation tolerance of native isolates was evaluated based on the procedure described by Shapiro-Ilan et al. (2005). Approximately 2000 IJ of each isolate were applied to 5-cm diameter filter paper disks (Whatmann No. 1). The excess water was removed from the filter paper disc by vacuum filtration. The filter paper containing IJ was placed on a lid of a 5-cm Petri dish and left open at room temperature (25°C) for 15 min for excess moisture to dry. Later, they were transferred to desiccator and exposed to relative humidity (RH) levels of 85%. RH level of 85% was established by using 60 ml of saturated salt solutions of KCl (Solomon et al., 1999). After 24 and 48 hr, the IJ were rehydrated by immersion in 10 ml of sterile water for another period of 24 hr. The number of dead and live IJ was counted in 50 µl of sample using a stereomicroscope. The IJ were considered dead if they did not show any mobility and response on probing with a needle. All treatments were incubated at 25°C. There were five replicates for each isolate.

Hypoxia tolerance was evaluated as described by Zadji et al. (2014) with slight modification. Approximately 10,000 IJ were suspended in 0.5 ml distilled water and transferred to a 0.5 ml Eppendorf tube. At this concentration, the dissolved oxygen level can reach zero within 10 min (Grewal et al., 2002). The tubes were closed and kept in the dark at 25°C for 24 hr and 72 hr. After treatment, the nematodes were suspended into 15 ml water in a Petri dish and kept at 25°C in the dark for an additional 24 hr. Open Eppendorf tubes constantly kept at 25°C in the dark were used as controls. 50 µl of sample was taken for recording the live and dead nematodes in the sample and counting was done using a stereomicroscope. There were five replicates for each isolate.

To determine the optimal storage conditions for H. indica, IJ were collected and diluted to 1000 IJ/ml with sterilized deionized water. A concentration of 1000 IJ/ml is the best to store Heterorhabditidae (Acevedo et al., 2006). 50 ml of this suspension was collected in BD falcon 750-ml tissue culture flask and stored at 5°C, 10°C, 15°C, 20°C, and 25°C. There were two replicate flasks for each temperature and flasks were sampled after 1, 2, 4, 8, and 16 weeks. Survival was determined in subsamples of approximately 100 IJ per flask under a dissecting microscope. Nematodes were considered live if they were naturally moving or responded to probing with a fine needle.

To facilitate comparison among isolates, the performance of each isolate for each ecological trait was scored as described by Salame et al. (2010). The isolate was scored as 1 if performance was not significantly different from the highest level for that trait, -1 if performance was not significantly different from the lowest level for that trait, and 0 if performance was between the highest and lowest, or not significantly different from either. The scores among traits were then added for each isolate and comparison was made.

Statistical analyses were performed on the data collected from each set of experiments. Excluding the single-dose screening assays and survival capacity in storage, all the experiments were conducted twice. To investigate the difference between the two trials independent samples t-test was used. In all cases, the results of both trials were similar and were combined for analysis. For screening test, the time until death and proportional mortality were calculated, and isolates were ranked. Isolates having low rank for time until death and proportional death were selected. For rest of the experiments, before conducting statistical analysis all percentage data was transformed using the arcsine square root transformation. For reproduction assay, the data was normalized by log transformation (Campbell and Wraight, 2007). Non-transformed means are presented in figures. For host cue experiment, Independent samples t-test was conducted between each isolate and corresponding control to study the response of individual isolate toward the host volatile cues. When required, insect mortality data was corrected according to Abbott (1925). For vertical soil experiment data was assessed using analysis of variance (oneway ANOVA) at the 5% level of significance (Freund and Littell, 1981) and Tukey’s test was used to compare means. LC50 values of EPN isolates were calculated using probit analysis (Finney, 1971). Overlap of the 95% fiducial limits was used to determine significance (Taylor et al., 1998) and values were not considered to be significant whenever the ranges overlapped (Grewal et al., 1993). The effect of storage temperature on survival was analysed using repeated measures analysis of variance (Koppenhöfer and Kaya, 1999). Statistical analysis was performed using SPSS 21.0 software for Windows XP.

The time taken by different isolates to kill the insect host ranged from 36.48 ± 1.07 hr to 44.8 ± 3.7 hr. Isolates GALA, VDTA, RASUTA and BATTA took least time (36.48 ± 1.07 hr) to cause 100% mortality, whereas, isolate STS took maximum time (44.8 ± 3.7 hr) to cause 64% of host mortality. The 35 isolates were ranked according to the time until death and proportional mortality (Figs. 1, 2).

Figure 1:

Figure 2:

Two isolates, GALA and VDTA were selected as both showed 100% host mortality in least time (36.48 ± 1.07 hr) and they both belong to different climatic regions of Viti Levu. DUNT also recorded 100% mortality but took more time to cause mortality (39.3 ± 2.7 hr). DUNT was selected for further ecological characterisation because it was suspected to harbour a different species of symbiotic bacteria. Incidentally, all the three isolates were recovered from different locations as well as different habitat type. Isolate GALA was recovered from Galoa (Windward side of the island) along riverside among wild ginger plants. Isolate VDTA was recovered from grass land in Vuda region (Leeward side of the island) and Isolate DUNT was recovered from sand dune along coastline in Sigatoka.

All the IJ of native isolates of H. indica were seen moving on the surface and did not show any standing, body waving or jumping behaviour. However, in storage, in aqueous suspension, dense aggregation of IJ was observed (Fig. 3).

Figure 3:

All the three isolates were highly attracted towards the volatile cues given by host insect and showed positive movement toward the host (Fig. 4). There were significant differences in percentage movement of IJ between treatment and control for three isolates for isolate GALA, treatment plates (Mean percentage ‘M’ = 82.8, Standard Deviation ‘SD’ = 6.2) and control (M = 46, SD = 10.9); t = -6.4, df = 8, p = 0.002; for isolate VDTA treatment plates (M = 81.8, SD = 1.7) and control (M = 44.7, SD = 12.9); t = -6.3, df = 8, p < 0.001 and for isolate DUNT treatment plates (M = 80.7, SD = 5.02) and control (M = 46.9, SD = 8.06); t = -7.8, df = 8, p < 0.001. However, there was no difference in percentage movement of IJ among three isolates of H. indica (F = 0.28; df = 2; P = 0.75).

Figure 4:

In the vertical soil columns, all the isolates were able to reach to the depth of 10 cm and caused 100% mortality of host. The number of nematodes that penetrated into host at different soil depth was highest at 2 cm depth and lowest for 10 cm depth. At 2 cm depth, 30 ± 5.3 IJ/larvae, 28.4 ± 4.7 IJ/larvae and 27 ± 2.7 IJ/larvae were recorded for isolate DUNT, VDTA and GALA, respectively (Fig. 5), whereas, at 10 cm depth, 6.8 ± 1.7 IJ/larvae, 11.6 ± 1.7 IJ/larvae and 8.6 ± 3.2 IJ/larvae were recorded for isolates DUNT, VDTA and GALA, respectively (Fig. 5). There was no significant difference in penetration rate at 0 cm, 2 cm or 5 cm (P > 0.05) for all the tested isolates. However, at 10 cm all the three isolates showed significantly less penetration rate than the other depths (F = 70.5; df = 3; P < 0.001). There were no significant differences in penetration rates among three tested isolates of H. indica (F = 0.9; df = 2; P = 0.38) at different soil depth.

Figure 5:

The LC50 values of three EPN isolates DUNT, VDTA and GALA against G. mellonella ranged from 2.8 IJ/larvae to 3.8 IJ/larva in bioassays at 48 hr after application (Table 1). The dose-response test (LC50) revealed no significant differences among three tested isolates of H. indica χ2 (11, N = 3) = 2.06, P = 0.99.

All the tested isolates of H. indica were able to infect and cause mortality of Galleria larvae at temperature range of 15°C to 35°C (Fig. 6A). No host mortality was observed at 10°C. At 40°C, larval mortality in treatment and control were equal and no penetrated nematodes were observed in treated larva. The highest mortality occurred between 25°C and 35°C (100% for all the three isolates), somewhat less at 20°C (62% for isolate DUNT and GALA; 75% for isolate VDTA); and low mortality at 15°C (45% for isolate DUNT; 58% for isolate VDTA; 41% for isolate GALA) (Fig. 6A). Temperature had a significant effect on the time of death (F = 3603.6; df = 4; P < 0.001). Time to death of larvae was shortest at temperature ranging from 25°C to 35°C (2-2.2 days) and longest at 15°C (17.4-17.7 days) (Fig. 6B). There was no significant difference in time to death at temperature 25°C, 30°C, and 35°C. Time taken by three isolates to kill the host at different temperatures was also not significantly different (F =1.9; df = 2; P =0.14). Number of nematodes penetrating into host larvae was highest at 25°C (31 ± 2.9 IJ/larva for isolate DUNT; 32.8 ± 3 isolate VDTA: 33.1 ± 3.4 for isolate GALA) and the lowest was observed at 15°C (5.8 ± 1.9 IJ/larva for isolate DUNT; 6.9 ± 2.3 isolate VDTA; 6.5 ± 2.4 for isolate GALA) (Fig. 6C). There were significant differences in number of nematodes that penetrated host at different temperatures (F = 378.6; df = 4; P = < 0.001). However, there was no significant difference in penetration rate among the isolates (F = 0.4; df = 2; P = 0.9) at different temperatures.

Figure 6:

Progeny emergence from cadavers was observed between 20°C and 30°C with no IJ emerging at 15°C and 35°C (Fig. 7A). Although insect larval death occurred and the nematodes established in the cadavers, no IJ emerged at 15°C and 35°C. At 20°C, only 37%, 50%, and 45% of the cadavers of isolate DUNT, isolate VDTA and isolate GALA respectively, produced progeny. At 25°C to 30°C, 100% cadavers for all the three isolates produced progeny. The shortest emergence time of IJ was observed at 30°C (7.8 ± 1.51 days) for isolate VDTA and the longest was at 20°C (19.7 ± 1.87 days) for isolate DUNT (Fig. 7A).

Figure 7:

There were significant differences in day of emergence at different temperatures (F = 261; df = 2; P < 0.001). However, no differences in day of emergence was detected among three tested isolates of H. indica (F = 1.9; df = 2; P = 0.14) at different temperatures. The highest number of emerged IJ was observed at 25°C (average 127,000 for isolate VDTA, 112,800 for isolate GALA, and 88,200 for isolate DUNT) and the lowest number was observed at 20°C (average 61,600 for isolate VDTA, 68,800 for isolate GALA, and 48,800 for isolate DUNT) (Fig. 7B). The number of emerged IJ at 25°C was significantly higher (F = 168.8; df = 2; P < 0.001) than at 20°C and 30°C. Isolate DUNT progeny production was significantly lower than isolate VDTA and isolate GALA (F = 38.01 df = 2; P < 0.001). There was no significant difference in progeny production between isolate VDTA and isolate GALA of H. indica (P > 0.05).

Survival of the tested nematode isolates decreased with time. Moderate survival was observed until 2 hr of exposure with 69%, 78%, and 64% of IJ of isolates DUNT, VDTA, and GALA surviving, respectively. After 4 hr, survival of isolate VDTA was highest (21.6%) compared to isolate DUNT (16.4%) and VDTA (13.2%) (Fig. 8). The survival of IJ after exposure at 40°C was significantly different for the tested isolates (F = 5.9; df = 2; P = 0.005). Exposure time to heat also had significant effect on IJ’s survival (F = 643.7; df = 3; P < 0.001). No mortality was observed in the control treatments. Isolate VDTA was significantly more tolerant than isolate GALA (P = 0.03). However, there was no significant difference in heat tolerance between isolate VDTA and isolate DUNT (P = 0.112).

Figure 8:

All three native isolates of H. indica caused 100% mortality of G. mellonella larvae at all moisture levels except for 5% (Fig. 9). At 5%, all the tested isolates failed to cause any mortality of G. mellonella.

Figure 9:

Their penetration rate increased until 10% of moisture level, and decreased with further increase in moisture level. Significant difference in number of nematodes penetrating into host at different moisture levels was detected for all the tested isolates (F = 827; df = 5; P < 0.001). However, there was no difference in penetration rate for moisture levels 8% and 25%. Similarly, there was no difference in penetration rate for moisture level 10% and 15% also. No significant differences in penetration rates were detected among the three tested isolates (F = 1.1; df = 2; P = 0.3) at different moisture levels.

Survival of IJ under 85% RH was affected by exposure time (F = 7.4, df = 1; P = 0.01), with 5.0 to 7.1% and 0.8 to 1.5% of IJ surviving on day 1 and 2 of the experiment, respectively (Fig. 10). The survival of IJ after desiccation did not differ significantly among the isolates (F = 0.2; df = 2; P = 0.7). Survival recorded in all control treatments during the 48 hr of the experiment was more than 95%.

Figure 10:

A gradual reduction of hypoxia tolerance, as indicated by IJ survival was observed during 72 hr exposure. The survival of IJ after hypoxia treatment differed significantly among isolates (F = 10.9; df = 2; P < 0.001) and exposure times (F = 225.7; df = 1; P < 0.001). Survival recorded in all control treatments during the 72 hr of the experiment was higher than 95%. After exposure to hypoxic conditions for 24 hr and 72 hr, survival of IJ varied between 83.9% and 89.6%, and 56.08% and 68.3%, respectively (Fig. 11). Above 80% the population of all tested isolates survived 24 hr of exposure to hypoxic conditions. After 72 hr, the highest survival was observed with isolate GALA (68.3%) thus showing the highest tolerance, which was significantly higher (Tukey test, p < 0.01) than in the other isolates tested.

Figure 11:

Storage capacity of the three isolates of H. indica was determined at temperatures ranging between 5°C and 25°C. There was a steady decline in the numbers of IJ alive in water and by week 16 of storage almost all IJ of H. indica were dead (Fig. 12). The optimal storage temperature for H indica in this study was observed around 15°C, followed by 10°C. The least survival of H. indica was observed at 5°C.

Figure 12:

Survival of all the isolates was affected by storage temperature with significant differences among temperatures after 1, 2, 4, 8, 12, and 16 weeks of storage (F = 2546.1; df = 4; P < 0.001). There was a significant effect of duration of storage on survival (F = 2764.3; df = 5; P < 0.001). Significant differences in % survival was detected among three tested isolates (F = 14.2; df = 2; P < 0.001) with isolate GALA surviving at a higher rate than the other isolates (Tukey test, p < 0.05).

The ecological traits selected to identify the best candidate were reproductive capacity, heat tolerance, hypoxia tolerance and storage stability. Other traits were not considered as there was no significant difference among these isolates with respect to them. A maximum score of two points could be reached. However, all of the examined populations had moderate to low scores. The highest score was that of the isolate GALA (2 points) (Table 2), mainly due to its reproduction capacity and good score in environmental stress assays. The lowest score –3 was recorded for the isolate DUNT.