Influence of soil conditions , spore densities and nematode age on Pasteuria penetrans attachment to Meloidogyne incognita

Several soil variables as soil texture, moisture and temperature, Pasteuria penetrans spore densities and nematode age were tested in the laboratory for their effects on spore attachment to Meloidogyne incognita juveniles. Percentage of juveniles with endospores attached and number of spores per juvenile increased with concentration of spores applied to soil. In a clay-loam soil, hundred-percent attachments were reached at densities of 5 × 10 spores per g of soil and above. Attachment was greater in loamy-sand than in sandy-loam and clay-loam soils, but lower when soil moisture was under 10% than when it was over 25%. Numbers of juveniles with spores attached were greater when soil temperatures were 25oC and 35oC than at 15oC and lower in 7-30 days old juveniles than in 0-6 days old juveniles. All factors that favored nematode mobility in soil increased Pasteuria spore attachment to M. incognita juveniles.

Endospores of P. penetrans are non-motile and thus, contact with nematodes will depend on nematode movement in soil (Stirling et al., 1990).Therefore, any factor that increased nematode mobility should also increase spore attachment.
Although the dynamics of acquisition of Pasteuria spores by Meloidogyne juveniles in soil is not yet fully understood, several biotic and abiotic factors have been suggested to affect it in field and greenhouse experiments, as Pasteuria spore densities and time of exposure (Stirling et al., 1990;Adiko and Gowen, 1994), soil texture (Mateille et al., 1995), soil moisture (Dutky and Sayre, 1978) and soil temperature (Hatz and Dickson, 1992;Freitas et al., 1997).However, most of these experiments have been carried out in field or greenhouse trials on diverse pathosystems, involving mainly M. javanica or M. arenaria, and the influence of nematode age on spore attachment has never been tested.Therefore, experiments in laboratory under controlled conditions are necessary to confirm or refuse these hypotheses.
The objective of this work was to test and determine, under controlled conditions, the influence of several soil and environmental factors on P. penetrans spore attachment to Meloidogyne incognita (Kofoid and White) Chitwood aiming at a better understanding of the process of acquisition of Pasteuria spores by Meloidogyne juveniles in soil.

Soil source
A clay-loam soil (40% sand: 30% silt: 30% clay) was collected from a fallow field at the National Agricultural Research Center (NARC), Tsukuba, Ibaraki, Japan.Sandy-loam (66% sand: 17% silt: 17% clay) and loamy-sand (76% sand: 12% silt: 12% clay) soils were prepared adding and mixing commercial sand (1-2 mm grade) to the clay-loam.Soils were sterilized twice at 120ºC for 20 min, air dried in an oven at 40ºC for two days, moistened up again to 25% soil moisture and stored for two weeks at 25ºC, before using them for experimental purposes.

M. incognita source
A M. incognita line maintained on susceptible tomato cv.Kyoryoku-beiju (Takii Seeds, Inc. Japan) at NARC, Tsukuba, Ibaraki, Japan was used as nematode inoculum.Juveniles of M. incognita were collected from egg masses 24-72 h after hatching.A suspension of 200 J2s in 1 ml of water was pipetted to each cup.Soil cups were then sealed and incubated again at 25ºC for four days, before nematode recovery.

Estimation of spore attachment
Different treatments were applied to soil cups and eventually, nematodes were recovered from 20 g of soil by the Baermann method (72 h).Pasteuria attachment was estimated in samples of 35 J2s by percentage of J2s with spores attached and spore intensity (total number of spores in a population of 35 J2s divided by the number of J2s with spores attached).All treatments were replicated six times.

Influence of soil texture on spore attachment
Plastic cups containing 20 g of clay-loam, sandyloam or loamy-sand soils were inoculated with Pas-teuria spores (10 4 spores per g of soil) and nematodes.Nematodes were recovered four days later and spore attachment estimated.

Influence of soil moisture on spore attachment
Plastic cups containing 20 g of clay-loam soil with 10 4 spores per g were air-dried at 30ºC and then divided in three groups that received 0, 3 or 5 ml of distilled water per cup, respectively.Cups were inoculated with M. incognita and four days later nematodes were recovered and spore attachment estimated.Soil moisture was determined by measuring the weight loss of four soil samples of 20 g from each treatment after drying for 24 h at 105ºC, resulting in three treatments (soil moisture: 9.5%; 25.2% and 41.6%).

Influence of soil temperature on spore attachment
Plastic cups with 20 g of clay-loam soil were inoculated with Pasteuria (10 5 spores g -1 ) and nematodes and then incubated at 15ºC, 25ºC and 35ºC for four days, before recovery of nematodes and Pasteuria attachment estimation.

Influence of juvenile age on spore attachment
Plastic cups were filled with 20 g of clay-loam soil and inoculated with Pasteuria (10 5 spores per g of soil).M. incognita J2s were recovered continuously during 30 days from egg masses and stored in a refrigerator at 10ºC.Juveniles were divided in ten age groups: [25-27] and [28-30] days old.Cups were then inoculated with 200 J2s of a specific age group, incubated for four days at 25ºC and nematodes recovered by the Baermann-funnel method.Number of J2s with spores attached and number of spores per juvenile were counted.

Statistical analysis
Data were analyzed by ANOVA using the statistical package SPSS v.8.0.When F values were significant, means were compared by the HSD Tukey test (P < 0.05).
Kolmogorov-Smirnov and Levene's tests were performed to check for normality and homocedasticity, if significant, numerical data were log 10 (x + 1) transformed and percentages arcsine square root transformed before being analyzed by ANOVA.Regression analyses were performed to determine the influence of endospore densities and M. incognita J2 age on Pasteuria attachment to M. incognita J2s.Curves with the highest R 2 were chosen as best fit.

Influence of Pasteuria spore concentration in soil
In the clay-loam soil, hundred-percent attachments were reached at concentrations of 5 × 10 5 spores per g of soil and above.There were no significant differences in spore intensities from 10 3 to 10 4 spores per g of soil, but numbers increased over 10 spores per J2 at spore densities of 10 5 and at 10 6 were more than 100 per J2 (Table 1).
Percentage of attachment and spore intensity were positively correlated to Pasteuria spore density in soil (P < 0.01).Linear and exponential regression models were fit when percentage of J2S with spores attached «a» and spore intensity «b» were dependent variables, and log 10 [Pasteuria spore densities] «x» was the independent variable (Figs. 1 and 2).100.0 ± 0.0 f 100.0 ± 3.4 d Values are expressed as average of six replicates ± standard deviation.Numerical data were log 10 (x + 1) transformed and percentages arcsine square root transformed, analyzed by ANOVA and means compared by the HSD Tukey test.Values followed by the same letter within a column are not significantly different (P < 0.05).

Influence of soil texture
In soils with 10 4 spores per g, percentage of attachment was significantly greater as proportion of sand in soil increased (Table 2).Attachment was greater in loamy-sand (62.2%) than in sandy-loam (40.0%) and in clay-loam (26.7%) soils.Spore intensity was also greater as proportion of sand increased, clay-loam (1.7) sandy-loam (2.6) and in loamy-sand (4.5).

Influence of soil moisture
Percentage of attachment increased with soil moisture but there were no significant differences in spore intensity (Table 2).At Pasteuria spore densities of 10 4 spores per g of soil, when soil moisture was low (9.5%)only 8.3% of J2s had spores attached, which was significantly lower than when soil moisture was 25.2% or 41.6% (23.3% and 32.2% of J2s with spores attached, respectively).

Influence of M. incognita juveniles age
Percentage of attachment was significantly lower when 7-30 days old juveniles were used (72.6%)than for 0-6 days old juveniles (94.8%) (P < 0.05).Spore intensity was greater on 1-3 days old juveniles (9.5) than in 4-6 days old (6.2) and in 7-30 days old (3.3).Percentage of attachment and spores per juvenile were negatively correlated to the age of juveniles.Third order polynomic curves were fit for percentage of J2s with spores attached «a» and spore intensity «b», respectively when juvenile age «z» was the dependant variable (Figs. 3 and 4).

Discussion
Percentage of M. incognita juveniles with spores attached and number of spores per juvenile were positively correlated with Pasteuria spore densities in soil.Stirling et al. (1990) reported increases in attachment to a different species, M. arenaria, with greater spores concentrations in soil and suggested that endospore densities of at least 2.5 × 10 5 per g of soil were needed to ensure an average of 20-50 spores per nematode and reduce damage caused by Meloidogyne populations.M. arenaria J2s inoculated in soils with 10 3 , 10 4 , 10 5 and 10 6 Pasteuria endospores per g of soil gave an average of 36, 58, 92 and 100% attachment and 1.2, 3.4, 9.6 and 51.0 endospores per juvenile respectively, after three days in the soil (Hewlett and Serracin, 1996).Our results are similar to these previous reports and show that in our M. incognita population, 100% attachments and more than 50 spores per J2 are reached after four days in clay-loam soils, at spore concentrations of 5 × 10 5 per g of soil and when Pasteuria spores are uniformly distributed in soil.
In our experiments, both attachment parameters, percentage of attachment and number of spores per juvenile, were positively correlated with proportion of sand in soil.Mateille et al. (1995) found that Pasteuria occurrence was greater in sandy soils in a survey in Senegal and suggested that sandy soil may favor endospore attachment to Meloidogyne spp.A significant reduction in nematode reproduction has also been reported in sandy-loam soils with P. penetrans when compared to loam or clay-loam soils (Singh and Dhawan, 1992).Our results show a direct relationship between proportion of sand in soil and spore attachment to M. incognita juveniles.This phenomenon could be caused either by a favored mobility of nematodes in sandy soils, or related to the movement and distribution of spores in the soil pores by a more uniform distribution of spores in sandy soils or a retention of spores within clay aggregates, which would make spores unavailable for attachment to nematodes (Mateille et al., 1996).
Pasteuria attachment to Meloidogyne J2s has also been reported to increase with temperature from 15ºC to 30ºC in M. javanica (Stirling et al., 1990) and M. arenaria (Hatz and Dickson, 1992;Freitas et al., 1997).Spore attachment to M. javanica was double at 27ºC than at 18ºC (Stirling et al., 1990).In P. penetrans infested soils, highest attachment to M. arenaria J2s occurred when soil was maintained at 20-30ºC for 4 days and greater temperatures reduced endospore attachment (Freitas et al., 1997).In our experiment Pasteuria spores attached to the cuticle of M. incognita at 15, 25 and 35ºC but maximum attachment was obtained at 25ºC.Temperatures below 25ºC decreased percentage of attachment and number of spores per juveniles and temperatures above 25ºC reduced number of spores per juvenile.Two mechanisms have been suggested to explain this phenomenon, the increased mobility of nematodes at greater temperatures (Bird and Wallace, 1965) and activation of temperature-dependent proteins and carbohydrates involved in the attachment process (Davies and Danks, 1993).
Studies on soil moisture effects have produced variable results.Dutky and Sayre (1978) did not find any correlation between attachment and soil moisture.On the contrary, moistening dry soil for three days before adding M. incognita J2s increased endospore attachment (Brown and Smart, 1984).In our study soil moisture of 9.5% showed lower attachment rates than soil moistures of 25.2% or 41.6%, which can be caused by the reduced mobility of nematodes when soils are dry.Percentage of juveniles with spores attached and number of spores per juvenile were negatively correlated to nematode age.Increasing the storage time of nematodes in water suspensions can lead to a quiescent-anoxybiotic stage (Antoniou, 1989), in which nematodes stop movement and chances of contact with Pasteuria spores are reduced.We observed that approximately 14.5 % of the 1-3 days old J2s were non-mobile and that 19.8% were inactive after 28-30 days storage at 10ºC.These differences were not significant at the P < 0.05 level.Nevertheless, it has been stated that the longer the state of quiescence the slower the return to normal conditions (De Guiran, 1977).Therefore, older juveniles would require longer time to become active and the time moving through the Pasteuria infested soil would be reduced.A reduction in endospore attachment caused by longterm storage of nematode suspensions in water was also reported by Freitas et al. (1997).These authors suggested that the surface coat of J2s, needed for endospore was lost during the water storage.According to Davies et al. (1991), the length of time taken for juveniles to be encumbered by spores in a water suspension was dependent on nematode age.De Silva et al. (1996) also showed an effect of nematode age on detachment of spores, detachment occurred only from one-day-old juveniles and not from five old days juveniles.This indicates that there may be changes in the surface coat of juveniles with age that affect attachment of spores.Changes in the cuticle with age have been observed in Caenorhabditis briggsae (Himmelhoch et al., 1977).
These results show that Pasteuria attachment to M. incognita juveniles is highly dependant on soil and nematode factors and that very favorable conditions are necessary to obtain high percentages of attachment, which would result in reductions in nematode damage.In less favorable conditions, high concentrations of spores or additional measures to increase infection rates would be necessary to be applied for a profitable application of P. penetrans in nematode control.

Table 1 .
Influence of P. penetrans spore densities in soil on percentage of M. incognita J2s encumbered with endospores and spore intensity

Table 2 .
Influence of soil conditions on percentage of M. incognita J2s encumbered with endospores and spore intensity Values are expressed as average of six replicates ± standard deviation.Numerical data were log 10 (x + 1) transformed and percentages arcsine square root transformed, analyzed by ANOVA and means compared by the HSD Tukey test.Values followed by the same letter within a factor and column are not significantly different (P < 0.05).
Regression curve for percentage of M. incognita J2s with Pasteuria spores attached vs juvenile age.Regression curve for spore intensity vs juvenile age.