Introduction

Any life in soil or water is represented by various organisms such as bacteria, fungi, protists, microarthropods, nematodes, and earthworms (Cifuentes-Croquevielle et al. 2020). These microbiotas, mesofauna, macrofauna, and microfauna are usually used as potential biological indicators. Nematodes are multicellular organisms that are found in soil and water films surrounding soil particles and are mostly used as a bioindicator in soil ecosystems (Ridall and Ingels 2021). Due to the ubiquitous distribution and occupation of a wide range of habitats, nematodes are helpful in measuring changes in the function and status of soils (Ridall and Ingels 2021). Nematodes are valuable indicators of soil ecosystem health and pollution and eutrophication in aquatic habitats (Sánchez-Moreno and Ferris 2018).

Diversity of living organisms, including nematodes, changes according to the geographical locations due to changing ecological and edaphic factors. In soil ecosystems, nematodes occupy the most niches; however, little is known about the ecological factors and their relationship with the soil parameters (Háněl 1993). Soil edaphic and climatic factors such as moisture, temperature, and pH are essential for nematode community structure (Griffiths et al. 2003). Bakonyi et al. (2007) found that the structure and diversity of the nematode communities are more sensitive to little fluctuations in soil moisture and temperature. Temperature changes affect the distribution of nematodes and result in short generation times (McSorley 2003; Treonis and Wall 2005; Shokoohi et al. 2019). Some reports have mentioned the impact of soil moisture and temperature on nematode populations like Helicotylenchus in alfalfa (Shokoohi et al. 2019) and Ditylenchus in maize filed (Franco-Navarro and Godinez-Vidal 2017). Natural forests and cultivated land have been shown to have a different population of nematodes in Mexico (Franco-Navarro and Godinez-Vidal 2017). Besides, land-use type affects the nematode community in which the forest bears more beneficial nematodes (Li et al. 2022). Different land-use types experience different disturbances and management practices resulting in changes in soil quality or soil health. The effect of land-use changes on the biodiversity of soil organisms has not been investigated in South Africa.

Magoebaskloof is a mountainous area in the Limpopo Province of South Africa between the towns of Haenertsburg and Tzaneen, which is covered by a natural evergreen subtropical forest. However, many farmlands for cultivating kiwi and avocados have been established along the rivers that pass through the mountains, resulting in a decline in the protected areas (Mkwalo 2011).

The purposes of our study were (1) to assess the composition of soil nematode communities and (2) to evaluate the relationship between nematodes with physicochemical properties in the soil.

Materials and methods

Soil sampling

Soil samples were collected from different locations in Magoebaskloof designated K1- forest tree (not disturbed for 50 years), K2- wetland (close to a river), K3- prepared land for kiwi farm, K4- oak tree (close to a river), K5- old established kiwi (37-years-old), and K6- 8-year-old kiwifruits (Table 1; Fig. 1).

Table 1 Localities of the soil samples of different sites in Magoebaskloof, in Limpopo Province, South Africa
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Fig. 1
figure 1

Sampling location and six sites in Magoebaskloof, Limpopo Province, South Africa. Upper panels: Limpopo map (stars indicating the sampling areas). Lower panels: K1 = forest tree (no disturbed for 50 years), K2 = grassland (close to a river), K3 = prepared land for kiwi farm, K4 = oak tree (close to a river), K5 = 37-year-old kiwi, and K6- 8-years-old kiwifruits

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A total of 60 samples were collected from the locations (Table 1). Ten soil samples from each site were collected separately from 10 to 30 cm below the soil surface after removing the aboveground plant debris. The subsamples cover each location for the root zone and roots to recover the various group of nematodes. Samples stored at 4 °C in cooler boxes were transferred to the Nematology laboratory of the Aquaculture Research Unit (the University of Limpopo, South Africa) for processing and identification of the nematodes.

Nematode identification

Nematodes were extracted from 200 g of soil from each location on the same day of collection using a modified tray technique (Whitehead and Hemming 1965; Shokoohi 2022). The nematodes were counted with a stereomicroscope (Zeiss; Discovery V8; Germany), and their genera identification was finalized using a light microscope (Zeiss, Lab.A1; AX10; Germany). Nematodes were then fixed with a hot 4% formaldehyde solution and transferred to anhydrous glycerin (De Grisse 1969) for species identification. The nematode genera were identified according to the classification provided by Andrássy (2005), Geraert (2008), and Shokoohi and Abolafia (2019).

Soil properties analysis

Chemical properties of soil, including ammonia, nitrate, phosphate, and hardens, were evaluated at the Aquaculture Research unit laboratory using a Hach spectrophotometric device (USA) based on the protocol instructed by the company. The pH was measured using Thermo Scientific Orion 3 Star pH Benchtop (USA).

Statistical analysis

The relationships between nematode population density (MPD) and frequency of occurrence (FO) of each nematode genus identified were expressed as prominence value (PV) for each locality to determine which genera were predominant in Limpopo Province in Magoebaskloof soil. The PV was calculated using the equation (Norton and Schmitt 1978).

$$\text{P}\text{V}=\text{P}\text{o}\text{p}\text{u}\text{l}\text{a}\text{t}\text{i}\text{o}\text{n}\,\text{d}\text{e}\text{n}\text{s}\text{i}\text{t}\text{y} \times \sqrt{\text{f}\text{r}\text{e}\text{q}\text{u}\text{e}\text{n}\text{c}\text{y}\, \text{o}\text{f}\, \text{o}\text{c}\text{c}\text{u}\text{r}\text{r}\text{e}\text{n}\text{c}\text{e}}$$

Additionally, frequency of occurrence (FO%) = (Number of samples containing a genus/number of total samples) × 100; relative abundance (RA) = total number of individuals of a particular genus per g soil and root sample in all samples/number of samples, including those with zero counts for that genus; population density (PD) = mean number of individuals of a particular genus/number of positive samples; absolute frequency (AF) = number of samples containing a genus/ total number of samples collected × 100; absolute density (AD%) = density of the genus/ total no: of samples collected × 100; relative frequency (RF%) = frequency of the species/ total frequency of all the species × 100; were also calculated.

Nematode biodiversity indices, including Shannon Index (H’) (Colwell 2009) was calculated. Nematode community analysis was assessed using NINJA (Nematode Indicator Joint Analysis) online software (Sieriebriennikov et al. 2014). Finally, to evaluate the soil factors’ relationship, including pH, ammonia, nitrate, phosphate, and hardens of the soil, locality, and nematode, a principal component analysis (PCA) was conducted based on Renčo et al. (2019). A principal component analysis (PCA) utilizing XLSTAT (Addinsoft 2007) was used to ordinate the sites by the abundance of nematode genera. Soil properties were used as supplementary variables to identify relationships with the abundances of the main nematode genera. The scores values were determined for each variable based on each of the principal components, and the scores for the first two components were used to form a two-dimensional plot (PC1 and PC2) based on eigenvalues given by the software XLSTAT. The data were normalised using log transform.

Data visualization

The network of nematode genera compositions in different land-use types was analysed using Gephi 0.10.1 software (Bastian et al. 2009). First, each one of the soil sample places had their latitude and longitude inserted in the table as a site node and, using Geo Layout plugin, were distributed in space accordingly and marked to be settled. After that, the remaining nodes representing the nematodes found in this study were inserted, and their placements were calculated by the Yifan Hu layout, which placed nodes based on the strength of connections each one had to the locked nodes of the site. In this way, stronger connections led to the node representing the nematode being closer to the site node. In addition, the thickness of the connecting lines and arrows also represented the strength of the connections. By its inherent characteristics, Yifan Hu layout also made the visualization of single connected nodes easier to identify.

Results

Analysis of the nematode communities

Thirty-one species belonging to 30 genera were identified (Table 2). Seventeen species were collected in natural forest, 15 species in association with grassland, 10 species in prepared kiwi land, 17 species in association with oak tree, 12 were associated with 37-year-old kiwi orchard, and 14 species in 8-year-old kiwi orchard (Table 2). From the overall genera collected, Acrobeles and Mesorhabditis were detected only in natural forest, Eucephalobus and Diphtherophora only in grassland, Pristionchus only in prepared kiwi land, Trypilina only in oak tree, Cervidellus and Pratylenchus only in 8-year-old old kiwi orchard (Table 2).

Table 2 Mean abundance of nematode species associated with different plants in Limpopo Province
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Eight plant-parasitic species (PPN), including Psilenchus sp., Tylenchorhynchus sp., Meloidogyne sp., Rotylenchulus parvus, Rotylenchus brevicaudatus, Pratylenchus sp., Nanidorus minor, and Xiphinema sp., were identified from the soil samples of kiwifruits (Table 2). Variations were observed for MPD in all nematode species over the localities/orchards (Table 3). Out of the 60 soil samples from six locations, the most prevalent nematode encountered were Ditylenchus, Acrobeloides, and Nanidorus with 100% of FO% and 8.1, 7.8, and 4.0 of prominence value (PV), respectively (Table 3). For 37-year-old kiwi, 23 J2 of M. hapla were detected per 200 g of soil.

Table 3 Community analysis of nematodes associated with different sites in Magoebaskloof, Limpopo Province, South Africa
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The occurrence of some species was not identified at some of the localities. For instance, R. brevicaudatus was not found in Nooyenskopie. Besides, S. brachyurus was not found in natural forest, and grasslands. The PV of Tylencholaimus mirabilis (55.9) indicates its high density in natural forest, where only this species was detected (Tables 2 and 3). In contrast, the least PV of 1.1 was observed for Pristionchus and Xiphinema (Table 3).

Indices of the nematode communities

Community analysis with NINJA indicated that there was a significant (p < 0.001) difference in the maturity, plant-parasitic, enrichment, and structural indexes and the herbivore, fungivore, bacterivore, and omnivore footprints within the assessed crops (Table 4). Changes in the sigma maturity index and plant-parasitic index status of the natural forest, and kiwi orchards samples were both significant (Table 4). The indices of the nematode communities were generally inconsistent between sites due to their ability to identify ecosystem disturbance (Table 4). Shannon Index (H´) and Maturity Index (MI) were the indices that identified ecosystem disturbance of the 37-year-old kiwifruit plants. As indicated in Table 4, the Shannon Index is the lowest in 37-year-old (H´ = 2.0), which was lower than in natural forest. MI was the highest in natural forest (2.8) compared to the other sites. Total nematode biomass (p < 0.001) was the highest (2.4 ± 0.1) in the rhizosphere of the okra. The metabolic activities (metabolic footprints) of various nematode guilds (herbivores, bacterivores, fungivores, predators, and omnivores) identified ecosystem disturbance well throughout the study, in which the activities were lower in the 37-year-old kiwi and 8-year-old kiwi plants. Additionally, Basal Index (BI), Enrichment Index (EI), Structure Index (SI), and composite and structure footprints distinguish between the natural forest and other sites throughout the study.

Table 4 Nematode community indices, biomass, and metabolic footprints associated with six soil types at Magoebaskloof, Limpopo Province, South Africa. Each data is an average of the individual (mean ± S.D).
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Correlation of selected soil parameters with nematodes

The result indicated a positive correlation (p < 0.05) between clay with Pseudacrobeles (r = 0.684), Acrobeloides (r = 0.658) and Meloidogyne (r = 0.668) species. In contrast, clay percentage has a strong negative correlation (r = -0.900) with Filenchus. Sand showed a strong negative correlation (p < 0.05) with Alaimus (r = -0.823) and Trypilina (r = -0.830). Copper showed a positive correlation with Psilenchus (r = 0.705). In contrast, no correlation between copper and other nematodes was detected. pH of the soil showed a positive correlation only with Acrobeloides (r = 0.813) and Mesodorylaimus (r = 0.779). Phosphorus showed a positive correlation with Butlerius (r = 0.641), Clarkus (r = 0.738), Acrobeles (r = 0.642), Mesorhabditis (r = 0.646), and Tylencholaimus (r = 0.646). Nitrate showed a positive correlation with Prismatolaimus (r = 0.684) and Aphelenchoides (r = 0.761) and a negative correlation with Acrobeloides (r = -0.688) and Mesodorylaimus (r = -0.676). Ammonia showed a positive correlation with Mesodorylaimus (r = 0.742) (Fig. 2).

Fig. 2
figure 2

Correlation of the selected soil parameters with the nematodes associated with different crops in six sites of Magoebaskloof in Limpopo Province, South Africa

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Distribution of nematodes in study sites

The results of the analysis of the soil samples collected from each site (K1-K5) were subjected to the PCA to study the correlation of the nematode genera (Fig. 3). An accumulated variability of 63.86% was observed in the analysis of nematodes and sites, where 35.97% for F1 and 27.89% for F2 were detected. The contribution of nematode genera and sites of the study to the PCA, indicated that Tylenchorhynchus, Tylencholiamus, Butlerius, Clarkus, Mesorhabditis, and Acrobeles were dominant in natural forest (K1). In contrast, Rotylenchulus and Pratylenchus were dominant in the sites with kiwifruit plantations. Whereas in grassland, Aphelenchus, Diphtherophora, and Ditylenchus were dominant (Fig. 3).

Fig. 3
figure 3

Biplot of principal component analysis (PCA) of the nematode species in different sites of Magoebaskloof, Limpopo Province, South Africa. K1 = forest tree (no disturbed for 50 years), K2 = grassland (close to a river), K3 = prepared land for kiwi farm, K4 = oak tree (close to a river), K5 = 37-year-old kiwi, and K6 = 8-years-old kiwifruits

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Relationship of soil parameters and PPN through the kiwi orchards

PCA (Fig. 3) explains 63.86% of the variation, 35.97% explained by PC1 and 27.89% explained by PC2, in which the soil chemical had a significant effect on the distribution of the nematode species. The contribution of nematodes, soil variables, and study sites to the analysis indicated that Rotylenchulus and Mesodorylaimus were dominant in substrates characterized by clay. In contrast, Filenchus was dominant in substrates characterized by phosphate. Aphelenchus, Tobrilus, Diptherophora, Ditylenchus, Eucephalobus, and Panagrolaimus were dominant in the site with more sand percentage. The result indicated that pH was not directly correlated with the nematodes in any site. Besides, the result indicated that nitrate had a positive correlation to Plectus and Prismatolaimus.

Data visualization

A network projection was done using Gephi to show the distribution of nematodes found in the different soil samples considering the geolocation of the sites (Fig. 4). The proximity of the 8-year kiwi, and old kiwi farm was apparent with an opposite group of the other four sites (oak tree, prepared kiwi land, grass, and forest tree). Each site had nematodes that were exclusively found in them except for the old kiwi farm. Emphasis should be made on Tylencholaimus mirabilis, one of the two omnivorous nematodes with a very strong connection to the forest tree site. Tobrilus was shared among prepared kiwi land and grass, while Filenchus had shared connections with both and forest tree. Plectus and Xiphinema were connected to oak tree, and Forest tree and Tylenchorhynchus had connections to both, with stronger connections to forest tree but also connected to grass. The remaining nematodes were more distributed and found between the two separate geographical clusters, with Mesodorylaimus, Pseudocrobeles, and Rotylenchulus closer to the old kiwi group. There was also of note that Aphelenchus and Ditylenchus, two out of the tree fungivorous nematodes found, were strongly connected to the grass site. And in opposite groups, Clarkus, the only predator nematode, was connected just to the forest tree and old kiwi farm.

Fig. 4
figure 4

Network analysis of the relationship of nematodes and study sites in Magoebaskloof, Limpopo Province, South Africa

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Discussion

Natural ecosystem changes over time due to human being activities. Elimination of the forest canopy considerably modifies the site microclimate, mainly light and water conditions (Swanson et al. 2011), which leads to changes in the composition, diversity, and habitat structure of understory plant species, which have potential implications for the soil biology (Neher et al. 2017). This phenomenon explains the dynamics of the microclimate of the Magoebaskloof mountain in Limpopo Province, South Africa. Any disturbance of the soil will lead to a progression of bacteria and fungi and the associated food web, with an initial decrease and then an increase in biodiversity (van Bruggen and Semenov 2000). The same result was obtained by Gömöryová et al. (2009), who indicated that the characteristics of microbial activity, including basal respiration, microbial biomass, N mineralization, and catalase activity were significantly lower in the acidic soil (Renčo et al. 2019). The same result was obtained in the present study, where the bacterivores nematodes were less dominant in the kiwi farms. The network analysis showed only Cervidellus was connected with 8-years kiwi farm. On the other hand, old kiwi farm was only connected with Mesodorylaimus (omnivores), and no bacterivores such as Pseudacrobeles, Acrobeloides, and Panagrolaimus were connected with a strong affiliation. The data visualization showed that besides some punctual exceptions, there were abundant connections towards the opposite group of the old and 8-years kiwi farms. Several studies showed that agricultural management practices, including tillage, chemicals, and human activities, distract the soil ecosystem and affect soil nematode diversity (Zhao and Neher 2012; Renčo et al. 2019). The result indicated that the total number of genera and species from the present study was similar to those in temperate regions (Thornton and Matlack 2002). The same result was obtained in the present study as the Magoebaskloof is the temperate region in Limpopo Province. The greatest Shannon index (H’) (2.4) was measured in the nematodes associated with the oak tree. In contrast, the lowest Shannon Index (1.8) was in grassland, which was located very close to the bank of the river. The low biodiversity was due to soil saturation with water and, therefore, the low oxygen accessible to the nematodes. Additionally, soil biodiversity was lower in cultivated soil than in natural lands. This agrees with the result of Franco-Navarro and Godinez-Vidal (2017), as they have detected less nematode diversity in maize fields than in natural forests in Brazil. The greatest dominance of genera was found in natural forests, where T. mirabilis, Clarkus, and Butlerius, omnivores/predator/bacterial feeders, were the main genera. This was a sign of the undisturbance of soil with high microbial potential. The presence of omnivorous nematodes indicates the soil food web was diverse and relatively stable, as observed in natural forest soil of the present study. Since predators and omnivores are susceptible to disturbances, their absence was an indication that the community in our sites disturbed structure due to agricultural practices (Ntalli et al. 2019), the result obtained in the present study. However, omnivores nematodes (Mesodorylaimus) were observed only in kiwifruit sites, implying that due to utilizing compost and animal manure (personal communication with the farmer), soil microbial activities balanced and resulting in a higher population of bacterivores. Hence, due to sufficient food sources, omnivores appear more than in other study sites. Omnivores nematodes are the main may have a direct or indirect effect on the microbivores biomass by controlling their population (Laakso and Setälä 1999). Nematode trophic structure in sites of the study was dominated by plant-parasitic and bacterial feeders (represented mainly by the order Rhabditida), a similar situation to that observed in studies of temperate regions (Pen-Mouratov and Steinberger 2005). The most abundant trophic groups in natural forests and grasslands were predator, bacterial, and fungal Feeders nematodes. Bacterial and fungal feeders’ nematodes are more abundant in disturbed soils, suggesting that disturbance fosters the growth of bacterial and fungal prey populations (Thornton and Matlack 2002). The natural forest studied was very close to the bank of the river and exposed to human activities for kiwifruit production. Therefore, soil disturbance affects the natural forest tree and associated soil biodiversity. The correlation matrix showed that A. complexus was positively correlated with phosphate. In contrast, it had not associated with ammonia and copper. Butlerius butleri had a moderate positive correlation with phosphate. This was because nematodes have a great diversity of specific ecological requirements. Most nematodes are considered to have a high density in freshwater ecosystems if a suitable environment exists (Traunspurger et al. 1997). Several studies also found that some nematodes are tolerant of anoxic conditions for a short period, giving them a competitive advantage in anaerobic situations related to intense eutrophication (Morrissy et al. 2021). The result also indicates that M. hapla was observed in most sites, except for the prepared kiwi land (K3). This species is among the most dangerous plant-parasitic nematodes for various crops, and possibly, it was distributed through human activities to all sites. Therefore, due to its aggressive nature for the plant roots, it may change the biodiversity of nematodes because of the high offspring and reproductive rate.

Conclusion

Magoebaskloof is a high rainfall and temperate region with an afrogreen tree forest in the Limpopo Province of South Africa. In this study, the biodiversity of nematodes showed a high biodiversity observed in association with natural forest tree. Despite human activities, natural forests showed stable soil conditions. Additionally, the sites with kiwifruits showed less biodiversity and low stability of the soil condition. Therefore, the result implies that the natural environment bears a higher soil quality with the proper number of beneficial nematodes. Also, it can be concluded that aquatic freshwater nematodes such as Tylencholaimus mirabilis and Acrobeles complexus are the primary component of freshwater meiofaunal communities that can be used as an indicator of pollution and soil conditions as these species were only found in natural forest tree of Magoebaskloof, in Limpopo Province, South Africa. Tylencholaimus mirabilis was found in high numbers associated with forest tree, with a positive correlation with nitrate (r = 0.646), and a negative correlation with copper (r = -0.378). Despite the low correlation of T. mirabilis with copper, analysing other metals may reveal the pollution of the soil through natural and anthropogenic influences.