|Archived at http://orgprints.org/3881|
Seasonal population dynamics of inoculated and indigenous steinernematid nematodes in an organic cropping system.
Otto Nielsen (e-mail: on[a]kvl.dk)
Holger Philipsen (e-mail: email@example.com) (corresponding author)
Zoology Section, Department of Ecology.
The Royal Veterinary and Agricultural University
Thorvaldsensvej 40, 3, DK-1871 Frederiksberg C, Denmark.
This study was based on naturally occurring and inoculated populations of steinernematid nematodes. The nematode populations were monitored spring and autumn in two following years in an organic cropping system and changes in population size were related to the presence of potential insect hosts. Nematode population were estimated in terms of nematode incidence (percentage of positive soil samples) by using Tenebrio molitor larvae as bait. The population of naturally occurring nematodes (Steinernema affine (Bovien) and S. feltiae (Filipjev)) was generally at a low level (0-17 % incidence for S. affine and 0-18 % incidence for S. feltiae). Inoculated S. feltiae established well in half of the plots where inoculation had been performed and reached incidences of 87 %. Establishment of inoculated nematodes, and population dynamics in general, was clearly influenced by the presence of insect hosts. In crops with high densities of potential hosts (Sitona lineatus in pea and partly Delia radicum in cabbage), nematode incidence increased from spring to autumn whereas nematode incidence remained unchanged or decreased when few hosts were present (in barley, carrots, alfalfa and leek).
Key words: Delia radicum, entomopathogenic nematodes, population dynamics, Psila rosea, Sitona lineatus, Steinernema affine, S. feltiae.
Steinernematid nematodes (Rhabditida: Steinernematidae) are obligate pathogens of insects as their life cycle under natural conditions only can be completed within an insect host (entomopathogenic nematodes). The nematodes are present in soil as infective juveniles and these may infect and kill insects occurring in the soil. The nematodes live in a close symbiotic relationship with bacteria of the genus Xenorhabdus (nematode-bacterium complex) and are together highly pathogenic to many insects species (Burnell & Stock, 2000; Boemare et al., 1997).
Surveys have been conducted in several habitats throughout the world to document the presence of steinernematid nematodes. In agricultural soils in Northern Europe, which is the focus of this study, the most common species appeared to be Steinernema affine (Bovien) and S. feltiae (Filipjev) (Nielsen & Philipsen, 2004a; Sturhan, 1996; Hominick et al., 1995; Boag et al., 1992; Ehlers et al., 1991; Griffin et al., 1991; Blackshaw, 1988; Husberg et al., 1988;).
To detect the nematodes, a technique based on bait insects has often been used. In this method, nematode-susceptible larvae are placed in soil samples and the larvae are observed over time to reveal infections by insect parasitic nematodes. The advantage of the method is its selectivity for insect pathogens and the fact that it includes the ability of the nematodes to successfully infect an insect.
So far, there have been relatively few studies that have followed entomopathogenic nematode populations over time (Hummel et al., 2002; Millar & Barbercheck, 2002; Campbell et al., 1998; 1995; Sturhan, 1996; Rovesti et al., 1991; Hominick & Briscoe, 1990a) and little is known about factors influencing the survival and population dynamics of these nematodes under field conditions.
An important factor for entomopathogenic nematode populations is most likely the availability of susceptible hosts in their environment. Unfortunately, only few studies of entomopathogenic nematode occurrence have included studies of insect populations in the same area (Mrá… ek & Be… váÍ , 2000; Campbell et al., 1995; Bednarek & Mrá… ek, 1986).
The aim of the present work was to study naturally and inoculated populations of entomopathogenic nematodes in an organic cropping system and relate the observations to the availability of insects host. The obtained knowledge can be used to better understand the population dynamics of entomopathogenic nematodes and in turn give help to design cropping systems where populations of entomopathogenic nematodes can be favoured as a buffering factor against populations of insects pests. This strategy is often referred to as inoculative and/or conservation biological control (Eilenberg et al., 2001).
Materials and methods
The organic cropping system studied was situated at Research Center Årslev (Danish Institute of Agricultural Sciences, Department of Fruit, Vegetables and Food Science).The cropping system consisted of six 1-hectare fields (Field N106-N111) that for several years had been grown organically without application of fertiliser or manure. The crops were grown in a fixed rotation of barley, nitrogen-catch crop (alfalfa and clover mixture), carrots or onions, cabbage or leek and pea (see Thorup-Kristensen (1999) for details). The soil was an Agrudalf soil which in the upper 25 cm layer consisted of approximately 15 % clay, 27 % silt and 55 % sand (Thorup-Kristensen, 2001).
Occurrence of entomopathogenic nematodes was estimated as the number of positive samples (nematode incidence). Each estimate of nematode incidence within a field was based on 125 soil samples (4.5 cm diameter x 15 cm length) from 5 plots (25 samples per plot). Each plot was circular with a diameter of 3 m (7 m2) and the plot centres were distanced by 10 m on a transect in the field. The transects followed the direction of soil amendments and plant rows.
Soil samples were taken spring (May-June) and autumn (September-October) in 2001 and 2002, respectively, in four of the six fields (N106, N108, N109, N111). For convenience, these four fields will be denoted Field I, II, III and IV, respectively, throughout the text. In addition, another set of plots was sampled in each of the fields. These plots were inoculated with S. feltiae nematodes and were situated parallel (15 m away) to the transect without nematode inoculation (only four plots were established in Field III and IV). The plots were inoculated on 21 May, 2001 (after sampling of soil) with S. feltiae nematodes obtained from E-nema, Raisdorf, Germany. In addition, Field III and IV were inoculated with S. feltiae (DK1 (isolated in a cabbage field at Hegnstrup, Denmark, 1997)). The plots in Field III were inoculated on 27April, 2001 (before spring soil samples were taken) and the plots in Field IV were inoculated on 8 May, 2001 (after spring soil samples were taken).
Both nematode strains were inoculated into holes in the soil (1-2 cm diameter x 5-10 cm deep) to simulate the patchy distribution of entomopathogenic nematodes which has been observed by other authors (Bohan, 2000; Spiridinov & Voronov, 1995). The holes were distanced by 20-25 cm in a grid (120-140 holes per plot). Nematodes from E-nema were applied as infective juveniles (approximately 20,000 infective juveniles per hole (~ 350,000 nematodes per m2)) and DK1 was applied by placing two nematode infected Tenebrio molitor larvae per hole. The Tenebrio larvae had been infected in the laboratory using approximately 75 nematodes per larvae. 36 T. molitor larvae from the batches of inoculated larvae were kept in the laboratory and checked for infections. 44 and 17 % of the larvae inoculated to Field III and IV, respectively, were infected. Because of the relatively poor infections, inoculation with DK1 was given up and nematodes from E-nema were inoculated instead.
Soil samples were stored at 5° C until baiting was performed (up to two months after sampling of soil). To reduce the possible effect of soil moisture on nematode isolation, soil sampling was avoided when the soil was very dry or very wet. The water content of the soil was estimated for each field and sampling date by drying subsamples of the soil at 80° C. The water content varied from 11.3 – 18.2 % w/w (mean = 14.1 %).
Each soil sample was mixed and divided into two subsamples. The subsamples were baited in two different periods (0-7 weeks apart). Baiting was performed in plastic jars that were placed in the dark at 18-24° C. One T. molitor larva was added per jar. The larvae were checked after one week. Dead larvae were replaced by a new larva and jars were checked again the following week where baiting was ended. Dead larvae (0-4 per sample) were placed individually on water traps to collect nematodes emerging from the larvae. Water traps were constructed in 5-cm Petri dishes by placing a piece of filter paper (approximately 2 x 2 cm) on top of a plastic lid and surrounded by shallow water. If one or more of the larvae from a sample were infected, the sample was denoted positive. The bait method has been evaluated by Nielsen et al. (manuscript).
Identification of nematodes was based on morphology of heat fixed specimens of infective juveniles from the water traps. Two species (S. affine and S. feltiae) were present, and these could easily be distinguished by their body size, body curvature and shape of tail. Generally, large quantities of nematodes were available allowing identification to be based on several specimens.
Soil living insects were quantified in the above mentioned plots once during each growing season by taking 8 soil samples per plot. The samples included the root of the crop plant and the surrounding soil (1-4 litres of soil per sample depending on root size). Special attention was given to plant specific insects like cabbage root fly, Delia radicum (Diptera: Anthomyiidae) in cabbage, carrot root fly, Psila rosae (Diptera: Psilidae) in carrots and the weevil, Sitona lineatus (Coleoptera: Curculionidae) in pea and alfalfa/clover. Cabbage root flies and carrot root flies were quantified when they were in the pupal stage in September whereas S. lineatus was quantified in July when the major part was in the larval stage. All insects were extracted by suspending the soil sample in water and followed by flotation and sieving as described by Nielsen & Philipsen (2003).
Nematode bait data were analysed separately for each nematode species and results are presented as mean percentages of positive samples (incidence) with standard error for each transect (bait results from each plot were pooled to give one value). For statistical tests, the data were treated as binary data (sample either positive or negative) and transformed (logit) using statistical modelling in the GENMOD Procedure provided by SAS (Statistical Analysis System 6.12). The given probabilities are based on c 2 -analyses. Insect data are presented as mean numbers with standard errors. Each estimate is based on the 4-5 plots in a transect. Analysis of variance was performed using the GLM procedure provided by SAS (insect numbers within a plot was pooled to give one value).
Incidence of naturally occurring entomopathogenic nematodes in the cropping system was generally at a low level (Figure 1). The maximum incidence was 18.4 % for S. feltiae (Field II, spring 2001) and 17.3 % for S. affine (Field III, autumn 2001). In plots of Field IV without inoculation of S. feltiae, naturally occurring S. feltiae were not observed (n=575) and S. affine was only present in four samples in Field I (n=875) and nine samples in Field II (n=875).
Inoculation of E-nema nematodes was performed in all fields on 21 May, 2001 just after the sampling in spring. The nematodes had established well in Field I with an incidence of 56.2 % in the autumn of 2001. In Field II, the nematodes had a poor establishment with an incidence of 10.4 % in the autumn of 2001. This was not statistically higher (P=0.18) than in the plots without inoculation (5.7 %). The plots in Field III and IV were also inoculated with S. feltiae DK1. In Field III, the nematodes had been inoculated 18 days before the sampling in spring, and this resulted in an incidence of 32.9 % compared to the absence of S. feltiae in the neighbouring transect. The results for Field III and IV in the autumn of 2001 revealed that inoculated nematodes had established in both fields.
Plots with nematode inoculation were for practical reasons not sampled in spring 2002. The results from autumn 2002, however, clearly document that inoculated nematodes were still present in Field I (62.1 % incidence) and Field IV (87.0 % incidence). In Field II and Field III, nematode incidence in inoculated plots was less than 10 %.
Inoculation of S. feltiae seemed to suppress S. affine as the incidence of S. affine generally was lower in S. feltiae inoculated plots. This is most obvious in Field IV where the increase in S. feltiae from autumn 2001 to autumn 2002 in inoculated plots was accompanied by a decrease in S. affine incidence. An analysis including all S affine data from the autumn of 2001 and 2002 showed that the incidence was significantly lower in plots where S. feltiae had been inoculated compared to plots without inoculation (P=0.02). However, if the fields and periods were analysed separately, only the difference in Field IV in autumn 2002 was significantly different (P=0.003).
The incidence of nematodes in plots within a transect was generally relatively homogeneous (low standard errors, Figure 1), but some of the transects deserve a closer study (Figure 2). In Field I, S. feltiae had much higher incidences in the middle of the transect and in Field II in inoculated plots, the highest incidences were seen towards one end of the transect (plots 4 and 5). Inoculation of S. feltiae to the plots did not change this pattern, although identical incidence was observed in three of the plots in autumn 2001. Field III also gave some remarkable results as the two nematode species tended to be negatively correlated. Further, S. affine persisted longer than S. feltiae. Due to the patchy distribution, the maximum level of the two nematode species on a plot basis far exceeded the maximum levels per transect (Figure 1). Thus the maximum incidences of S. feltiae and S. affine in a plot were 44 and 38 %, respectively (Figure 2).
Plant specific soil living pests insects were quantified in both years (Table 1). The highest number of insects were observed in pea (10.9-16.0 S. lineatus larvae per plant) followed by D. radicum in cabbage (7.1-7.3 pupae per plant). In alfalfa/clover and carrots, plant specific insects were found in low numbers only (0.1-1.0 insects per sample). Barley and leek fields were not surveyed as these fields normally host no or few plant specific soil living pests in Denmark No statistical difference (P>0.05) was found between the number of insects occurring in plots with or without S. feltiae inoculation. In addition to the insects mentioned in Table 1, a range of other insects was observed. These were, however, not plant specific and present in low numbers only, and were thus regarded to play a minor role as nematode hosts.
The presence of potential insect hosts had a positive effect on nematode populations (Table 1, Figure 1). This was most pronounced in pea and with inoculated nematodes, but an effect was also observed in cabbage. Here S. affine increased from 3.2 % in the spring 2001 to 10.0 % in the autumn in plots with S. feltiae inoculation (P=0.04), and from 5.7 % to 17.3 % in plots without inoculation (P=0.04). S. feltiae increased from zero to 8,9 % in plots without inoculation, and inoculated populations were maintained at a relatively high level (33 % incidence).
The occurrence of entomopathogenic nematodes has been studied in many countries (Hominick, 2002) and the presence of the species S. feltiae and S. affine in the present cropping system is in accordance with observations made in agricultural soils in other countries in northern Europe (Nielsen & Philipsen, 2004a; Sturhan, 1996; Hominick et al., 1995; Boag et al., 1992; Ehlers et al., 1991; Griffin et al., 1991; Blackshaw, 1988; Husberg et al., 1988). The level of naturally occurring nematodes was generally low (<20 %) and could be a result of the soil type which resulted in very hard and compact soil under dry conditions as well as a tendency to plasticity under wet conditions. However, incidences below 20 % have been observed in many and diverse agricultural soils (Nielsen & Philipsen, 2004a; Chandler et al., 1997; Miduturi et al., 1996; Boag et al., 1992; Griffin et al., 1991; Blackshaw, 1988; Husberg et al., 1988). We used T. molitor as bait in our study whereas most of the above mentioned studies used Galleria mellonella. Due to this and the lack of experimental standards for bait analyses, different studies should be compared with caution. The method applied in our study, has recently been evaluated by Nielsen et al., (manuscript) and here the aspects of measuring incidence or density is also further discussed.
The present study was based on a large number of samples per field and the populations were followed over time. This enabled a presentation of a detailed picture of the spatial and temporal distribution of the nematodes. Another detailed study was performed by Sturhan (1996) who followed a German cropping system (with sugar beets, barley and wheat) for 2½ years. He found, that S. affine was the dominating species and that nematodes appeared in all plots at least once during the observation period. He also found that the level of nematodes fluctuated over time, but there was no correlation with cropping practise. Sturhan (1996) did not include observations on the host population. This is necessary in order to understand the population changes and more studies of agricultural systems are required before general conclusions can be given on entomopathogenic nematode population dynamics in such systems.
A number of studies have concluded that entomopathogenic nematodes have a patchy distribution (Bohan, 2000; Campbell et al., 1998; Cabanillas & Raulston, 1994; Stuart & Gaugler, 1994; Boag et al., 1992) and that the distribution pattern probably reflects the spatial availability of hosts and their movement during the early stages of nematode infection (Bohan, 2000). In the present study, the number of insects per plot was relatively even (low standard errors, Table 1) and this can probably explain, why also the nematodes were relatively evenly distributed (low standard errors, Figure 1). Another reason is that nematodes had been inoculated uniformly to half of the plots and there is a tendency that standard errors increase with increasing incidence in plots where only the natural population was present (Figure 1).
It is evident from the present study that availability of potential hosts is a key factor in the determination of entomopathogenic nematode occurrence. Pea was the crop with the highest number of insects (S. lineatus) and this resulted in both years in high incidences of nematodes in autumn. It is known from other studies that S. lineatus is highly susceptible to entomopathogenic nematodes (Jaworska & Ropek, 1994) and one weevil larvae of the size of S. lineatus can produce 1,000-2,000 infective juveniles (Nielsen & Philipsen, 2004b). Also cabbage root flies are potential hosts of entomopathogenic nematodes (Nielsen & Philipsen, 2004b; 2003), but the level of these insects was relatively low in the study and the growing of cabbage had only a minor positive effect on the nematodes.
A close link between entomopathogenic nematode populations dynamics and the presence of hosts was also observed by Mrá… ek & Be… váÍ (2000) and Bednarek & Mrá… ek (1986). The findings are not surprising as host availability is an essential factor for increasing nematode numbers. Thus, the low level of hosts in the majority of fields could – in addition to the influence of soil characteristics – be another explanation for the generally low incidence of naturally occurring steinernematids.
In Field I naturally occurring S. feltiae incidence increased during winter from 6.4 % to 15.5 % (P=0.026). The explanation for this is probably that the nematodes in autumn were still clumped in or around host cadavers, but had dispersed in the following spring. This could either have been active movement or passive movement caused by field work.
Establishment of inoculated nematodes was successful in two of the fields (I, IV) and the basis for this success seemed to be availability of hosts. It is relevant to distinguish between nematode persistence and nematode recycling. In Field I and II inoculated populations did not increase during the observation period and it is possible that the isolated nematodes were remains of inoculum. In Field IV a large increase was seen from autumn 2001 to autumn 2002 and this could be a consequence of nematode propagation in the field. Alternatively, most inoculum must have survived and dispersed, but this seems more unlikely and the observation of S. feltiae infected S. lineatus larvae in the summer of 2002 (data not shown) directly proves that this insect served as host under field conditions. Also, the incidence of nematodes in inoculated plots in the autumn in pea fields (Field I in 2001 and Field IV in 2002) was much higher than in the other fields, which strongly indicates that the isolated nematodes were not only inoculum. However, distinguishing between persistence and recycling requires that the nematodes are studied for much longer periods or that labelled inoculum is used.
Introduced nematodes have also been followed in other studies and the general picture is that nematodes levels only can be raised for a short period of few days or weeks (Millar & Barbercheck, 2001; Campbell et al., 1998; Duncan & McCoy, 1996; Smits, 1996; Ferguson et al., 1995; Rovesti et al., 1991). Short term survival was not measured in the present study as the focus was on establishment through recycling rather than on nematode persistence. The lack of effect of the inoculated nematodes on the insect populations, however, indicates that persistence from inoculation in May and until the major occurrence of insects in July was low.
The possible competition between nematode species is another interesting observation. The topic has been studied by several authors although field based observations are rare (e.g. Millar & Barbercheck, 2001). In their study the exotic nematode S. riobrave was introduced to an area with the two endemic species S. carpocapsae and H. bacteriophora and the results indicated that H. bacteriophora was negatively affected while S. carpocapsae was unaffected by S. riobrave. An important factor is the level of the two competing species. Low incidences could allow space for both species. For instance, it was possible for S. affine to increase its incidence in S. feltiae inoculated plots and negative effect on S. affine was most pronounced in Field IV where S. feltiae establishment was very successful. As the inoculated and the natural occurring S. feltiae could not be distinguished, it is not possible to give a conclusion about intraspecific competition. Further, it is not possible to conclude whether competition and replacement actually happened in the field or whether it was caused by competition for bait larvae only.
The inoculated nematodes did not significantly reduce the level of plant specific insects (Table 1). The only population which was relatively abundant in spring was the inoculated population in Field I in 2002. It was, however, not tested whether these nematodes had an effect on the insects occurring in the barley field.
The conclusion of the present study is that entomopathogenic nematodes occurred at a low level but inoculation could be a way to increase their incidence. The availability of host insects was found to be very important for successful establishment of inoculated nematode populations and survival of entomopathogenic nematode population. This is in agreement with the general expectations and the strength of our study is that the nematode-insect relationship was quantified and that the quantification was based on an extensive sampling design of nematodes and insects.
We wish to thank DARCOF (Danish Agricultural Research Center of Organic Farming) for financial support and the company E-nema, Kiel, Germany for providing nematodes. The technical assistance of Hanna Hansen and Anne Anttila is highly appreciated.
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Figure 1. The incidence of Steinernema feltiae or S. affine in the four fields in spring and autumn 2001 and 2002, respectively. Light grey bars illustrate plots with naturally occurring nematodes only and dark grey bars illustrate plots where S. feltiae was inoculated during the observation period (inoculation indicated by an arrow (dotted line = DK1)). Each bar is the mean and standard error of 4-5 plots (25 soil samples per plot) (x = no sampling).
Figure 2. The occurrence of Steinernema felt and S. affine within selected transects. Each bar is the mean of one plot (25 soil samples). Inoculation of S. feltiae is indicated by an arrow.