Characterization of nematode community in apple orchard soil in the Northwestern Loess Plateau , China

This work was carried out in the Northwestern Loess Plateau (NLP), one of two main large-scale apple production areas in China. It is to investigate the role of nematodes in apple production areas, which cause apple replant disease (ARD). Soil samples from total eight sites in NLP were collected twice, in summer and autumn, respectively. The nematodes were extracted by washing-sifting-sucrose centrifugation and total nematodes in each sample were counted with the aid of an anatomical lens (40×). Furthermore, a sub-sample of one quarter of each nematode suspension was observed with a Motic microscope (400 and 1000×) and each nematode was identified to genus level using diagnostic keys. The results indicated that the characteristics of the nematode community did not vary significantly in four different types of soils. Especially, the diversity indices of nematode community did not show obvious differences. In addition, the analysis of nematodes with different feeding habits revealed that the overall number of Pratylenchus penetrans nematodes in the replanted orchard in NLP were far below the density threshold that could jeopardize apple trees. Thus, it appeared that the nematodes were not the leading causal agent for ARD in NLP.


INTRODUCTION
The Northwestern Loess Plateau (NLP) is a large-scale apple cultivation area, however, most orchards are old and while production is decreasing, they need to be replaced, which makes growers face apple replant disease (ARD).ARD is a soil-borne disease that often occurs when trees are replanted in soils which have history of apple growth.It has been reported from all major fruit-growing regions of the world (Mazzola and Manici, 2012), and ARD is often caused by a consortium of biological agents, mainly, including nematodes, bacteria, actinomycete, oomycetes and fungi species (Tewoldemedhin et al., 2011).Nematode is an important soil organism.Nematodes occur widely in various types of soil, with numerous species and great mass, and they play an important role in the soil ecological process by changing the food webs structure and decomposition approach, affecting the functions of soil ecosystems (Ferris et al., 2001;Yeates and Bongers, 1999).ARD is a soil-borne disease, and thus, a soil environment approach should be taken to explore the pathogenesis of the disease.A good soil environment is the premise for the healthy growth of fruit trees.Foreign ecological scholars have proposed that soil nematode communities have been widely used as bioindicators of ecosystem conditions (Yeates, 2003;Ritz et al., 2009;Sánchez-Moreno et al., 2010), due to their key positions in soil food webs (Neher, 2001).The utilization of nematode community analysis for indicating soil food web dynamics in agroecosystems has been reported by many researches (Ferris and Matute, 2003;Briar et al., 2007;Sánchez-Moreno et al., 2006;DuPont et al., 2009).
Ecological indices calculated from relative populations of different soil nematode groups have been widely applied to quantify the response of the nematode community to environmental changes in soil (van Eekeren et al., 2008;Neher et al., 2005;Todd et al., 2006).The ShannoneWeaver Index (H) is a useful measure of diversity of nematode community (Cheng et al., 2008;Zhang et al., 2009).Indices of free living nematode maturity (MI) and plant-parasitic nematode maturity (PPI) represent soil nematode life-history characteristics associated with r-selection (nematode with short generation time, high fecundity and large population fluctuation) and K-selection (nematodes with long generation time, few offspring and generally appearing later in succession), respectively (Neher, 2001).MI and PPI have often been used to estimate the functional responses of soil nematodes to environmental change (Chen et al., 2009;Yeates and Newton, 2009).
Given the above, we carried out the project in apple region; which will be discussed here.The objectives of our study were to determine the differences of four cropping histories on soil nematode abundance and diversity in NLP, and to explore the role of soil nematode in ARD.

Study site outline
The study was carried out in NLP area, which is the world's largest loess deposition area.The plateau is slightly north of central China (34-40°N, 103-114°E).The climate belongs to semi-humid region; the mean annual temperature here is about 8-14°C; annual precipitation varies from about 600-800 mm.

Soil sampling
Individual soil samples were collected in summer (June 3 to 9) and autumn (September 28 to October 6) of 2010 at eight sampling sites (each site being a replicate).The sites were located in Pinglu (S1) and Linyi (S2) counties of Shanxi province; Baishui (S3), Luochuan (S4) counties and Yintai (S5) district of Shaanxi province; Maiji (S6), Qinzhou (S7) districts and Jingning (S8) county of Gansu province, respectively (Figure 1 and Table 1).At each sampling site, soils with the following four cropping histories were sampled: 1) replanted apple orchards; 2) within rows of old apple orchards; 3) between rows of old apple orchards (=inter-row) and 4) fallow soil.The replanted apple orchards had been replanted five years ago and suffered from ARD.The old apple orchards had been planted twenty years ago.In both cases, soil was sampled within a radius of about 0.5 m around the taproot of the tree.The fallow soil was located close to the apple orchard, but had not previously been planted with apple trees (= soil bare).At each point, two replicate samples were taken to a depth of 20 cm.Thus, a total of 128 soil samples were collected (2 seasons × 8 sites × 4 cropping histories × 2 replicates).All soil samples were sifted through a 6 mm sieve to remove the plant roots or large stones, subsequently placed in black plastic bags and stored at 4°C.Each sample was extracted and counted separately and the values of the two replicate samples were averaged.

Nematode extraction and identification
Nematodes were extracted from 100 g wet soil by washing-siftingsucrose centrifugation, using sieves with 40 (350 µm) and 500 (25 µm) meshes.The nematodes were heat-killed at 60°C and preserved in triethanolamine formaldehyde (TAF) solution (Shepherd, 1970).Total number of nematodes in each sample were counted with the aid of an anatomical lens (40×); a sub-sample of one quarter of each nematode suspension was observed using an inverted compound microscope (Motic; 400 and 1000x).All nematodes in the samples were identified, to genus level if possible.The nematodes were assigned to the following trophic groups: (1) bacteriovores (BF); (2) fungivores (FF); (3) plant-parasites (PP); and (4) omnivores-predators (OP) (Yeates et al., 1993).C-p values were allocated according to Yeates and Bongers (1999) and ecological indices were calculated as shown in Table 2.The abundances of total nematodes and each taxonomic group were adjusted to the number of soil nematodes per 100 g dry soil.

Statistical analysis
The effect of cropping history on the ecological indices was analyzed separately for each season with one-way ANOVA using DPS V7.05, which was a statistical analysis software developed by Zhejiang university of China, function equated with SPSS.Differences among treatment means were analyzed with Duncan's multiple range test.

Richness and density of nematode
The effect of cropping history on nematode richness (number of genera) and density was significant difference both in summer and in autumn.In summer, nematode richness in fallow soil was the highest of the four cropping histories (Table 3).In contrast, the lowest nematode richness was found in the vicinity of apple trees in both replanted and old orchards.The richness in inter-row soil was intermediate and not significantly different from the other soils.Accordingly, nematode density in fallow soil was significantly higher than that in the other soils.
In autumn, nematode richness was significantly higher in replanted orchards and fallow soil than in the old orchards (Table 3).Nematode density was significantly higher in fallow soil than in the old orchards.Nematode density was intermediate in replanted orchards, but not significantly different from the other soils.

Nematode community structure
The effect of cropping history on Shannon-Wiener diversity (H'), Simpson diversity (D), Pielou evenness (J) and Margalef richness (SR) was significant both in summer and in autumn.In summer, the value of the indices did not significantly differ among the four cropping histories (Tables 4 and 5).In autumn, J and SR did not significantly differ among the four cropping histories.However, in autumn H' was significantly higher in replanted than in old orchards and D was significantly higher in replanted orchards than within rows in old orchards.

Functional group features
Among the soils, no significant differences were found in Structure Index (SI), Enrichment Index (EI), Maturity Index (MI), Plant-parasite Index (PPI) and PPI/MI (Tables 6 and 7).SI index and EI index in summer presented a similar variation.Namely, gradually decreased as follows: replanted soil, row soil, inter-row soil and fallow soil.Nevertheless, there were no significant differences among them.SI index and EI index in autumn did not vary in a consistent manner, but there were also no is the c-p value of the ith taxon, and f(i) is the frequency of the ith taxon Bongers,1990Yeates,1994 Plant-parasite index (PPI) v(i) is the c-p value of the ith taxon, and f'(i) is the frequency of the ith taxon Bongers,1990 obvious differences among themselves, respectively.Although MI index, PPI index and PPI/MI values for four cropping histories soils in summer slightly varied, the statistical significance was not obvious.Of course, PPI index was higher than MI index.The statistic results of values in autumn were analogous to those in summer.

Nematode feeding habits
The effect of cropping history on the number of BF, FF, PP, OP nematodes and Pratylenchus penetrans was significant both in summer and in autumn.In summer, no significant differences were found in the number of BF, PP and OP nematodes among the four cropping histories (Table 8).In summer, the number of FF nematodes and P. penetrans was higher in fallow soil than in replanted apple orchards and between rows in old apple orchards.However, in autumn the number of FF nematodes did not significantly differ among the four cropping histories.In contrast, in autumn the number of BF, PP, OP nematodes and P. penetrans did significantly differ among the cropping histories (Table 8).The number of BF was significantly higher in replanted orchards than in the other soils.The number of PP nematodes and P. penetrans was significantly higher in fallow soil than in the old orchards, whereas the number of OP was significantly higher in fallow soil than in all three other soils.

DISCUSSION
Soil nematodes have been widely used as bioindicators to assess soil condition as they respond to changes in the soil environment caused by land use, agricultural management, etc. (Bongers and Ferris, 1999;Dong et al., 2008;Liang et al., 2009;Neher et al., 2005).Ecological indices have been widely applied to explore  the response of nematode community to soil environmental changes (Todd et al., 2006;van Eekeren et al., 2008), the soil nematode community can be used as a biological marker of soil health (Wardle et al., 1995;Neher, 2001;Neher et al., 2012;Klass et al., 2012), and evaluate soil quality (Yeates and Wardle, 1996;Yeates and Bongers, 1999).The ecological indices of H', SR and MI are often used to assess the soil condition.H' and SR are linked to the diversity of soil nematodes, and MI can reflect changes in soil condition (Zhang et al., 2009).In addition, SI and EI parameters can also be an indicator for soil health (Ferris et al., 2001).They were the most frequently used indices in the field of soil nematode community providing ecology in recent years (Powell, 2007).The present results showed that ecological indices, e.g.H', D, J, SR, SI, EI, MI, PPI and PPI/MI, except H' and D varied in four cropping histories soils in summer, but no evident differences were found by statistical significance in summer and autumn.Based on the above fact and result, we concluded that there were no differences among four cropping histories soils in soil nematode community structure, and there is a stable soil ecosystem.However, with regards to nematode, it did not demonstrate that four cropping histories soils have the same soil condition and soil health only from the above ecological indices, the whole factors should be considered, not one by one.Many studies suggest that nematodes are useful  bioindicators in soil and aquatic ecosystems, especially for assessment of ecosystem health (Ferris and Bongers, 2006;Shao et al., 2008;Neher, 2010;Park et al., 2011).PP nematode is well-known pests affecting important agricultural crops such as apple, peach, cotton and soybeans, and is one of the causes of continuous cropping obstacles crop.On this account, some researchers thought that Meloidogyne incognita, P. penetrans and Heterodera glycines are the main pathogenic nematodes in continuous cropping obstacles crop (St Laurent et al., 2008;Sharma and Kashyap, 2004;Stetina et al., 2007;Koenning and Edmisten, 2008).Hoestra and Oostenbrink (1962) showed that high densities of PP nematodes played an important role in ARD, primarily Pratylenchus spp.P. penetrans Cobb is considered to be the primary nematode species involved in ARD (Van Schoor et al., 2009;Tewoldemedhin et al., 2011).Jaffee et al. (1982) described that P. penetrans was the most abundant para-sitic nematode of PP nematodes, once its counts was up to 140 P. penetrans per 100 cm 3 and it would result in significant stunting and root necrosis.Results of the number of different nematode feeding habits in NLP apple region showed that the largest number of PP nematodes appeared in fallow soil, which was 121 per 100 g dry soil, and the largest number of P. penetrans appeared in fallow soil which was 62 per 100g dry soil, and was below the density threshold that could jeopardize apple trees.Thus, it appeared that the nematode was not the leading causal agent for apple replant disease in NLP.

Conclusion
Nematode populations in soils subjected to different agricultural practices have shed additional light on the effects of tillage systems and environmental conditions, including temperature, moisture and soil compaction.This paper discussed the soil nematode community characteristic of replant soil, row soil and inter-row soil of old orchards as well as fallow soil in NLP apple region without taking into account the above factors and other microorganism effect, which need to be further studied.Thus, the variety in orchard soil nematode community performance could reflect the change in orchard soil environment quality.The understanding could provide a basis for the old orchard's update or alteration and prevention of apple replant disease.

Figure 1 .
Figure 1.Map of eight sampling locations (red ellipses) of apple orchards in the Northwestern Loess Plateau, China.
abundance of those guilds indicative of an increasingly structured food web b is the abundance of those bacterial and fungal feeding guilds indicative of the basal food web condition Ranges from 0-100 Increasing SI indicates increased environmental stability Ferris et al., 2001 Enrichment index (EI) abundance of those bacterial and fungal feeding guilds indicative of resource enrichment b is the abundance of those bacterial and fungal feeding guilds indicative of the basal food web condition Ranges from 0-100 Increasing EI indicates increased resource availability Ferris et al., 2001 Maturity index (MI)

Table 1 .
Soil characteristics and climate of eight sampling sites of the NLP.Data represent mean ± standard error of 8 replications.Within each column, values followed by different low case letters are significantly different according to one-way ANOVA and Duncan's new multiple range test (P≤0.05).

Table 2 .
Formulae used to calculate community indices for soil nematodes.Data represent mean ± standard error of 8 replications.Within each column, values followed by different low case letters are significantly different according to one-way ANOVA and Duncan's new multiple range test (P≤0.05).

Table 3 .
Richness (number of genera) and density (number per 100 g dry soil) in summer and autumn of soil with four different cropping histories: replanted apple orchards, old apple orchards (within rows and inter-rows) and fallow soil (n=8).Data represent mean ± standard error of 8 replications.Within each column, values followed by different low case letters are significantly different according to one-way ANOVA and Duncan's new multiple range test (P≤0.05).

Table 4 .
Shannon-Wiener diversity (H') and Simpson diversity (D) of nematodes extracted from soil with four different cropping histories in summer and autumn.Data represent mean ± standard error of 8 replications.Within each column, values followed by different low case letters are significantly different according to one-way ANOVA and Duncan's new multiple range test (P≤0.05).

Table 5 .
Pielou evenness (J) and Margalef richness (SR) of nematodes extracted from soil with four different cropping histories in summer and autumn.Data represent mean ± standard error of 8 replications.Within each column, values followed by different low case letters are significantly different according to one-way ANOVA and Duncan's new multiple range test (P≤0.05).

Table 6 .
Structural index (SI) and enrichment index (EI) of nematodes extracted from soil with four different cropping histories in summer and autumn.Data represent mean ± standard error of 8 replications.Within each column, values followed by different low case letters are significantly different according to one-way ANOVA and Duncan's new multiple range test (P≤0.05).

Table 7 .
Maturity index (MI), plant-parasite index (PPI) and PPI/MI of nematodes extracted from soil with four different cropping histories in summer and autumn.Data represent mean ± standard error of 8 replications.Within each column, values followed by different low case letters are significantly different according to one-way ANOVA and Duncan's new multiple range test (P≤0.05).

Table 8 .
Number of different feeding habits soil nematodes and P. penetrans extracted from soil with four different cropping histories in summer and autumn.Data represent mean ± standard error of 8 replications.Within each column, values followed by different low case letters are significantly different according to one-way ANOVA and Duncan's new multiple range test (P≤0.05).