Comparative analysis of soil nematode biodiversity from five different fruit orchards in Osmaneli district, Bilecik, Türkiye

Turkey is in an advantageous position in fruit growing thanks to its location and ecological characteristics. It is known that there are many species and varieties richness in the country due to the fact that it is located in the Near East and Mediterranean gene centers (Erdem and Kos, 2022). The country is the homeland of many fruit species such as that of the apple (Malus domestica L.), pear (Pyrus communis L.), quince (Cydonia oblonga L.), hazelnut (Corylus avellana L.), pistachio (Pistacia vera L.), sour cherry (Prunus cerasus L.), cherry (Prunus avium L.), plum (Prunus domestica L.), walnut (Juglans regia L.), almond (Prunus dulcis L.), chestnut (Castanea sativa L.), fig (Ficus carica L.), grape (Vitis vinifera L.) and pomegranate (Malum granatum L.), which have an important place in fruit culture. In 2020, a total of 21,853,084 tons of production were realized with an increase of approximately 6% compared to the data of 2019 (Gerçekçioğlu et al., 2009).

The Bilecik Province is located in the southeast of the Marmara Region, between the intersection points of the Marmara, Aegean, Central Anatolia, and Black Sea regions. The region has many advantages in fruit and vegetable cultivation thanks to its location, changing altitude differences in its geography and the unique ecosystem that is surrounded by the Sakarya River. The cultivation of many fruits is well-known locally, such as peach in the Osmaneli district, pomegranate cultivation in the Inhisar district and walnut and cherry cultivation in the Gölpazarı district. Furthermore, Bilecik is at the midpoint of cities such as Istanbul, Bursa, Izmit and Eskisehir, and it also provides access to the Marmara, Central Anatolia and Mediterranean Regions by road and railway, thus providing easy access to airports and ports, and therefore to the market (Niyaz and Demirbaş, 2011) (Table 1).

Nematodes (Phylum Nematoda) are one of the most diverse groups of invertebrate animals, characterized by a simple body structure with wide variety of feeding habits, life strategies, and their important role in the soil food web. It is reported that nearly 25.000 nominal species have been identified (Zhang, 2012), and it is estimated that their diversity can reach up to 1.000.000 nematode species (Hugot et al., 2001).

Many species are free-living animals, which inhabit soils and (both freshwater and marine) sediments. Their feeding spectrum is diverse, including predatory, algivory, fungivory, omnivory, saprophagy, etc. (Yeates et al., 1993). Many species have become plant and animal, even human, parasites, causing important diseases and pests (Lee, 2002).

As with any other crop grown in Türkiye, damage to the tree and/or fruit by pests and diseases, including nematodes, reduces the grower’s profit. The effects of some plant parasitic nematodes on plant growth and yield are largely the result of the disruption that these organisms cause to the normal process of plant root growth and soil exploration for both water and nutrients. Nutrient deficiencies resulting from the failure of the plant root system to explore and exploit the soil adequately can also be a major consequence of a plant parasitic nematode attack.

The objectives of this study were, (i) to determine the soil nematode fauna of fruit orchards in Osmaneli District of Bilecik Province, (ii) to characterize of nematodes as soil bioindicators and (iii) to characterize the biodiversity of nematodes in regard to their host plant.

This study was established in Duzce University’s Faculty of Agriculture, Department of Agricultural Biotechnology, Nematology Laboratory, from April 2022 to March 2023.

Samples were collected at the Osmaneli district, Bilecik, Turkey, in April 2022 during a field survey. The sampling was done regarding fruit tree orchards soil habitats and along six different eco-habitats, namely: cherry, nectarine, olive, plum, walnut and peach trees. (Fig. 1; Table 2).

Figure 1:

Sampling sites, fruit tree orchards: cherry (a), nectarine (b), olive (c), plum (d), walnut (e), peach (f) trees.

Samples were collected from 60 sampling sites (10 samples from each fruit type). For each location, one soil sample was collected from a 15 × 15 cm plot. A total number of 60 soil samples were put into ziplock sampling bags, stored in portable cooler during transportation and brought to the nematology laboratory of Duzce University for the extraction process.

A modified Baermann’s (1917) funnel technique using 12 cm diameter petri dishes was used during the extraction of nematodes. After separating rocks, 100 g of fresh soil was evaluated from each sampling site. Plastic trays lined with paper towels were used for extraction and incubated for 48 hours in the nematology laboratory. Extracted nematodes were collected after 48 hours. Nematode suspensions were heated up to 60 °C for killing before fixation. A formalin solution of 4% was used for fixation and preservation of nematodes until preparing permanent glass slides. Extractions were labeled with the relevant sample number, transferred to plastic tubes, and stored at Düzce University Nematology Laboratory. The rest of the soil samples were also stored in the soil laboratory for having a backup requirement in case of future studies.

A 100 g soil sample from each sampling site was placed into a glass container each with three last instar larvae of the wax Moth Galleria mellonella (L.) and covered with a lid. (Bedding and Akhurst, 1975; Kaya and Stock, 1997). The samples were then stored at room temperature. After 10 days, dead larvae were collected and transferred to White traps to collect the emerging IJs (Kaya and Stock, 1997).

After picking up procedure, preserved nematodes were rinsed with purified water to remove the debris. A staining block of 1.25 cm deep which contained 96% ethanol with the extracted nematodes was placed in an incubator at 40 °C, and a few drops of glycerol: formalin (4 %) (1:99) were added and left at room temperature overnight. The next morning, a few drops of a solution of five parts glycerol and 95 parts of 96 % ethanol were added, and two-thirds of its cavity was covered with a glass square. A few drops of the glycerol: ethanol (5:95) solution were added every two hours for the gradual transition of the glycerin. At the end of the day, two drops of glycerol: ethanol (50:50) were added to the staining block. The next day, individual nematodes were covered with glycerol and permanent glass slides were prepared (Yoder et al., 2006).

Nematodes were identified manually by using an Olympus CH microscope (Olympus Optical, Tokyo, Japan). Classification of nematodes were determined by a taxonomical key (De Ley and Blaxter, 2005). and additional taxonomical data from Hodda et al. (2006) and Andrássy (2002; 2005; 2009) were included. Nematodes were identified mostly down to the genus level. Coloniser-persister classification of nematode life cycle properties (1–5) were obtained in agreement to Bongers (1990; 1999). The nematode feeding types classification was established according to Yeates et al. (1993) and Du Preez et al. (2022). The structure index and enrichment index were calculated according to Ferris et al. (2001) and Ferris and Bongers (2009) in order to obtain the maturity degree of the nematode community composition in the ecosystem. The Nematode Indicator Joint Analysis calculation system (Sieriebriennikov et al., 2014) was used to analyze food web structure, feeding type diagnostics and MI family indices.

The total number of identified nematodes reached up to 2418 individuals (number of female: 1036; male: 154; and juvenile: 1228) belonging to 54 species, 54 genera, 33 families and 11 orders (Table 3). Besides, the total nematode abundance showed variability among samples with an average number of nematodes per 100 gr of soil that were 2 to 145 individuals from the sampling sites (Table 2).

According to the maturity index analysis (Fig. 2; 3), mean values showed the highest maturity level at peach trees (MI value: 3,52), followed by; walnut trees (MI value: 2.49), cherry trees (MI value: 2.15), nectarine trees (MI value: 1.86), plum trees (MI value: 1.57) and olive trees (MI value: 1.42). According to the maturity index 2–5 analysis, mean values showed the highest maturity level at peach trees (MI value: 3,84), followed by; olive trees (MI value: 3.16), walnut trees (MI value: 2.96), plum trees (MI value: 2.19), cherry trees (MI value: 2.15) and nectarine trees (MI value: 2.03). According to the Sigma Maturity Index analysis, mean values showed slightly different values: highest maturity level was detected at peach trees (Sigma MI value: 3.35), followed by; cherry trees (Sigma MI value: 3.29), walnut trees (Sigma MI value: 2.62), nectarine trees (Sigma MI value: 2.08), plum trees (Sigma MI value: 1.84) and olive trees (Sigma MI value: 1.76). The plant parasitic nematode index analysis showed the highest PPI mean values at cherry trees (PPI value: 3.79), followed by; olive trees (PPI value: 3.02), plum trees (PPI value: 2.95), walnut trees (PPI value: 2.93), peach trees (PPI value: 2.71) and nectarine trees (PPI value: 2.43). The enrichment index analysis (EI), results showed the highest enrichment level at olive trees (EI value: 97.67), followed by; plum trees (EI value: 84.18), peach trees (EI value: 73.24), walnut trees (EI value: 71.97), nectarine trees (EI value: 53) and cherry trees (EI value: 24.24). According to the structure index analysis (SI), results showed the highest structure level at peach trees (SI value: 95.26), followed by; olive trees (SI value: 85.56), walnut trees (SI value: 78.78), plum trees (SI value: 31.99), cherry trees (SI value: 24.24) and nectarine trees (SI value: 6.74).

Figure 2:

Comparative maturity index analysis of nematode c-p classification from five fruit orchards in Osmaneli, Bilecik, Türkiye.

Figure 3:

Free-living nematode c-p classification from five fruit orchards in Osmaneli, Bilecik, Türkiye.

Food web analysis of soil properties provides a useful tool for predicting soil quality by enrichment and structure type parameters. Results showed that the two of the fruit orchards, nectarine and plum, nematode assemblage were yielded into a high enrichment class, which means disturbed, N. enriched, with low C:N value, high bacterial activity and conductive soil. Three of the fruit trees’ (Olive, walnut and peach) nematode assemblage were placed into maturing, N-enriched, with low C:N value, high bacterial activity, and regulated class. Nematode assemblage of the cherry tree orchard occurred at degraded, depleted, with high C:N value, more fungal activity and a conductive soil type (Fig. 4).

Figure 4:

Food web analysis (Enrichment/Structure indices) from five fruit orchards in Osmaneli, Bilecik, Türkiye.

According to results obtained by feeding types of cherry trees’ nematode assemblage, plant parasitic nematodes were found to be 69.1 %, followed by bacterivorous nematodes (19.4 %), fungivorous nematodes (9.1 %) and predator nematodes (2.3 %); nectarine trees: Bacterivorous nematodes (44.2 %), plant parasitic nematodes (38.6 %), fungivorous nematodes (16.7 %) and predator nematodes (0.5 %); olive trees: Bacterivorous nematodes (70.7 %), plant parasitic nematodes (20.9 %), predator nematodes (7.2 %) and fungivorous nematodes (7.2 %); peach trees: Predator nematodes (47.3 %), bacterivorous nematodes (24.7 %), plant parasitic nematodes (21.2 %) and omnivorous nematodes (6.8 %); plum trees: Bacterivorous nematodes (65.6 %), plant parasitic nematodes (19.8 %), fungivorous nematodes (12.1 %) and predator nematodes (2.6 %); walnut trees: Bacterivorous nematodes (41.4 %), plant parasitic nematodes (29.3 %), predator nematodes (15.6 %), fungivorous nematodes (11.8 %) and omnivorous nematodes (1.9 %) (Fig. 5).

Figure 5:

Feeding types and their relative abundance of nematodes at different fruit tree orchards.

According to results obtained by the classification of plant parasitic nematode feeding type differences, nematode assemblage in cherry tree orchards were: Ectoparasites (58.7 %), semi-endoparasites (28.1 %) and epidermal/root hair feeders (13.2 %); nectarine trees: Ectoparasites (70.7 %), epidermal/root hair feeders (19.1 %), semi-endoparasites (9.6 %) and migratory endoparasites (0.6 %); olive trees: Migratory endoparasites (79.5 %), semi-endoparasites (7.9 %), epidermal/root hair feeders (7.1 %), ectoparasites (5.5 %); peach trees: Ectoparasites (58.1 %), epidermal/root hair feeders (16.1 %), migratory endoparasites (12.9 %) and semi-endoparasites (12.9 %); plum trees: Semi-endoparasites (63.9 %), ectoparasites (27.1 %), epidermal/root hair feeders (5.6 %) and migratory endoparasites (3.5 %); walnut trees: Ectoparasites (49.5 %), migratory endoparasites (26.2 %), epidermal/root hair feeders (15 %), sedentary endoparasites (7.5 %) and semi-endoparasites (1.9 %) (Fig. 6).

Figure 6:

Distribution (%) of feeding types within the plant-parasitic nematodes from Osmaneli, Bilecik, Türkiye.

According to results obtained by the classification of plant parasitic nematode cp class differences, nematode assemblage in cherry tree orchards were PP5 (46.3 %), PP3 (40.5 %), and PP2 (13.2 %); nectarine trees: PP2 (68.2 %), PP3 (26.1 %) and PP5 (5.7 %); olive trees: PP3 (88.2 %), PP2 (7.1 %) and PP5 (4.7 %); peach trees: PP3 (51.6 %), PP2 (41.9 %) and PP5 (6.5 %); plum trees: PP3 (84.7 %), PP2 (11.8 %) and PP5 (3.5 %); walnut trees: PP3 (48.6 %), PP2 (36.4 %) and PP5 (15 %) (Fig. 7).

Figure 7:

Plant parasitic nematode c-p classification from five fruit orchards in Osmaneli, Bilecik, Türkiye.