Introduction

The center of origin of olive trees is in the Eastern Mediterranean regions (Besnard et al., 2011). Even now, olive cultivation constitutes a key element of the Mediterranean agricultural sector and Mediterranean countries dominate world olive oil and table olive production and consumption (Blondel et al., 2010). Tunisia is the largest southern Mediterranean country producing olive and olive oil (Gharbi et al., 2014) and it is the fourth largest producer worldwide after Spain, Italy and Greece (Jackson et al., 2015). During 2017–2018, the Tunisian olive oil production reached 280,000 tons (ONAGRI, 2018). The competitiveness of Tunisian olive oil production could be related to a significant expansion of area planted. Olive trees intended for oil production extend over 1.8 million hectares which constitute 79% of the surface dedicated to fruit farming and 34% of crop lands (Gharbi et al., 2014). The olive growing areas spread from the northern to the southern regions, where a wide range of edaphoclimatic conditions prevail. In the North and in some regions of the Center, olives are cultivated in association with other annual cultures or fruit trees, whereas in the South they are exclusively cultivated in monoculture (COI, 2017). These plantations are mainly extensive due to the weather conditions of Tunisia, known by the weakness and irregularity of rainfall (Gharbi et al., 2014). Nevertheless with irrigation two other systems of plantation are available, intensive and super-intensive (Allalout & Zarrouk, 2013).

The intensification of olive crops may disturb the agro-ecosystem (Loumou & Giourga, 2003) and make olive trees more susceptible to some diseases (Trapero et al., 2009) such as plant-parasitic nematode diseases (Castillo et al., 2010; Nico et al., 2002). Research has revealed that olive trees serve as a suitable host plant for highly diversified plant-parasitic nematode communities, including endoparasitic and ectoparasitic species. Over 250 species have been detected in association with the olive rhizosphere in many countries of the Mediterranean Sea and worldwide (Ali et al., 2014; Ali et al., 2017; Archidona-Yuste et al., 2019; Archidona-Yuste, Cantalapiedra-Navarrete, et al., 2020a; Palomares-Rius et al., 2015). The root-knot nematodes (Meloidogyne spp.) and root-lesion nematodes (Pratylenchus spp.) are widely distributed through olive soils, especially in nurseries and intensive cropping systems where irrigation conditions are favorable to their multiplication (Aït Hamza et al., 2018; Ali et al., 2014; Ali et al., 2016; Ali et al., 2017; Archidona-Yuste et al., 2018; Archidona-Yuste, Cantalapiedra-Navarrete, et al., 2020a; Castillo et al., 2010; Nico et al., 2002; Palomares-Rius et al., 2015). These two nematodes are considered the most damaging plant-parasitic nematodes of cultivated olive (Ali et al., 2014; Castillo et al., 2010; Nico et al., 2002; Nico et al., 2003). Conversely, other harmful species of nematode to olive such as Heterodera mediterranea, Tylenchulus semipenetrans, Rotylenchulus macrosoma and R. macrodoratus are less distributed in olive orchards (Ali et al., 2014; Ali et al., 2017; Archidona-Yuste, Cantalapiedra-Navarrete, et al., 2020a; Castillo et al., 2010; Palomares-Rius et al., 2015).

The diversity and structure of plant-parasitic nematode communities infesting olive trees could be influenced by different environmental (soil physicochemical properties and climate, and substrate origin in nurseries) and agronomic factors (orchard and soil management systems, irrigation regime, olive cultivar and age of plantation) (Aït Hamza et al., 2018; Ali et al., 2017; Archidona-Yuste, Wiegand, et al., 2020b; Aїt Hamza et al., 2015; Benkovic-Lacic et al., 2016; Palomares-Rius et al., 2015). In fact, in olive orchards, nematode abundance and diversity of plant-parasitic nematode communities were influenced by olive cultivar (Chafaa et al., 2014; Palomares-Rius et al., 2015), season (Chafaa et al., 2014) and orchard and soil management practices (Ali et al., 2017; Aїt Hamza et al., 2015; Benkovic-Lacic et al., 2016; Palomares-Rius et al., 2015). However, soil physicochemical properties were the main factors driving the structure of these communities (Aїt Hamza et al., 2015; Palomares-Rius et al., 2015). In nurseries, the diversity and structure of plant-parasitic nematode communities were affected mainly by climate (rainfall and minimum temperature) (Aït Hamza et al., 2018). Otherwise, recent studies exploring spatial structure in the diversity of plant-parasitic nematode communities revealed that soil was the most influential factor driving these communities while agronomic management practices showed less influence then expected (Archidona-Yuste, Wiegand, et al., 2020b).

In Tunisia, the investigation of plant-parasitic nematode communities associated with cultivated olive has still not been undertaken except for some taxonomic studies (Guesmi et al., 2016; Guesmi-Mzoughi et al., 2016; Guesmi-Mzoughi et al., 2017; Palomares-Rius et al., 2014). The works done until now have described a new species Pratylenchus oleae which was detected on cultivated olives suffering from tree decline (Palomares-Rius et al., 2014). Some other species were detected for the first time on cultivated olive such as Rotylenchus incultus and R. eximius (Guesmi-Mzoughi et al., 2016) and Longidorus euonymus, Longidorus glycines, Xiphinema conurum, Xiphinema meridianum and Xiphinema robbinsi (Guesmi-Mzoughi et al., 2017). Symptoms of nematode attacks have mainly appeared in intensified orchards, and it is still difficult to convince farmers about nematode parasitism. Therefore, this current study aims to: (i) identify and determine the population densities of plant-parasitic nematodes infesting olive trees in the main producing area in Tunisia; (ii) evaluate the effect of agronomic characteristics (including degree of intensification, irrigation regimen and olive cultivar) and soil type of the studied olive orchards on the diversity of plant-parasitic nematode communities.

Material and methods

Olive orchards description and sampling

This study was conducted in 123 commercial olive orchards located in the main olive production regions in Tunisia: Beja, Zaghouan and Nabeul in the North, Sousse, Monastir, Mahdia (Sahel regions), Kairouan, Kasserine, Sfax and Sidi bouzid in the Center. For each department, at least two olive orchards were sampled: about 20 in each of Nabeul, Beja and Zaghouan, 18 in Sahel regions and 44 in the other departments (Fig. 1). The different agronomic characteristics (including degree of intensification, irrigation regimen and olive cultivar) and soil type of the studied olive orchards are presented in Table 1. The degree of intensification in olive orchards were differentiated into three categories including: (i) traditional olive orchards which are mostly rainfed and sometimes irrigated manually when intercrops particularly barley and bean were present, with low plant density (less than 100 olive trees/ha), local varieties, low inputs in fertilizer and pesticides, and manual harvesting, (ii) intensive orchards with drip irrigation, high plant density (around 200 olive trees/ha), local varieties, sometimes intercrops such as legumes (potato, tomato, cucumber and pepper) or fruit crops (pomegranate, almond and peach trees) were present, high inputs in fertilizer and pesticides, and manual or mechanical harvesting, and (iii) super-intensive orchards with drip irrigation, very high plant density (between 1250 and 1666 olive trees/ha), imported varieties, no intercropping, high inputs in fertilizer and pesticides, and mechanical harvesting. Olive orchards were also classified according to soil intensification with irrigation and the presence of intercrops. Additionally, the soil type was analyzed by a private company “Food Quality” in Tunisia.

Fig. 1
figure 1

Map of olive sampling sites in Tunisia (Color should be used in print). (Green circles corresponds to sampling sites in each region)

Full size image
Table 1 soil and agronomic characteristic of olive sampling sites
Full size table

Samples were collected in the spring and autumnal periods of 2013, 2014 and 2015. Depending on the size, soil and agronomic characteristics of each olive orchard, 3 to 30 trees were randomly selected for sampling. Active roots were collected with a shovel under the canopy of each olive tree from 5 to 40 cm depth, discarding the upper 5 cm of the soil profile. This ensured that roots from weeds or other herbaceous plants were unlikely to be sampled. Four sub-samples were collected from each tree and were thoroughly mixed to obtain a single representative sample per tree. In total, 717 root samples were collected from 123 olive orchards. The number of sampled olive orchards per region, soil and agronomic characteristics is described in Table 1.

All studies carried out until now on olive nematode communities were interested in soil nematodes as bio-indicators of the disturbance of the soil environment (Ali et al., 2017; Aїt Hamza et al., 2015; Palomares-Rius et al., 2015). However, to our knowledge, there is no study that has used root nematodes in such subjects, although plant roots can have net effects on the soil microbial community structure and functioning (Bonkowski et al., 2009). Therefore, this study will use, for the first time, plant roots to describe plant-parasitic nematode communities infesting olive trees.

Nematode extraction, identification and quantification

Nematodes were extracted from a 10 g sample of each representative root sample using a sugar centrifugal-flotation method (Coolen, 1979). Before extraction, roots were washed free of soil and cut into small pieces. Plant-parasitic nematodes were identified at the genus level and enumerated under a stereomicroscope at 80x magnification using a hand-tally counter. The nematode identification was based on morphological traits of each genus (Mai & Mullin, 1996; Nickle, 1991; Siddiqi, 2000). When necessary, observations of specimens under a microscope were carried out to distinguish between closely related genera.

Identified plant-parasitic nematodes were separated according to their feeding strategies into three groups: 1) ectoparasites, including migratory ectoparasites of roots that can feed in the cortical tissues (eg., Helicotylenchus, Rotylenchus, Criconemoides, Paratylenchus), root surface tissue feeders (eg., Tylenchorhynchus) and algal, lichen or moss feeders that feed by piercing (eg., Tylenchus); 2) migratory endoparasites (eg., Pratylenchus, Zygotylenchus) and 3) sedentary endoparasites (eg. Meloidogyne, Heterodera) also including sedentary semi-ectoparasites (eg. Rotylenchulus) (Siddiqi, 2000; Yeates et al., 1993).

The number of specimens of each genus was determined from 10 g of root sample. This includes the number of juveniles, adults and sedentary stages for endoparasites. The prevalence of each genus was calculated as the percentage of samples where this genus was detected.

Finally, the density of plant-parasitic nematodes was calculated per olive orchard as the average of the nematode density determined from all samples in the same site, and expressed as the nematode abundance.

Nematode diversity indexes

Several indexes were considered to assess the abundance and diversity of the nematode communities: the total number of plant-parasitic nematodes per 10 g of roots (N) (Korenko & Schmidt, 2006); genera richness (G) which is the total number of genera per 10 g of roots (Korenko & Schmidt, 2006); relative abundance (Ar) (Ar = n *100/N) where (n) is the number of specimens per group of plant-parasitic nematode (ectoparasites, migratory endoparasites and sedentary endoparasites) (Dajoz, 1971) and the Shannon-Wiener diversity index (H′) (H′ = − \( \sum \limits_{\boldsymbol{i}}^{\boldsymbol{G}}\left(\frac{\boldsymbol{ni}}{\boldsymbol{N}}\right)\ast \boldsymbol{\ln}\left(\frac{\boldsymbol{ni}}{\boldsymbol{N}}\right) \)) (Shannon & Weaver, 1949).

These indexes were calculated using PRIMER6 software (Clarke & Warwick, 2001) except for relative abundance which was calculated in an excel file.

Data analyses

In order to explore the effects of soil and agronomic factors (irrigation regimen, intensification, intercrops and variety) on the diversity of plant-parasitic nematode communities, redundancy analysis (RDA) (Borcard et al., 2018), non-parametric tests and ANOVA analysis were performed using the vegan library with R version 4.1.2 software and SPSS® 16.0. software, respectively. In these analyses, each olive orchard was considered as statistical unit.

Before analysis, data were tested for normality and homogeneity of variances by Kolmogorov-Smirnov and Levene test at 5%, respectively.

Two redundancy analyses (RDA) were performed. Categorical variables (irrigation regimen, intensification, intercrops and variety) were converted to dummy variables before analysis. For the first RDA, nematode abundances were used as the response variables, and agronomic factors and soil texture were used as explanatory variables. Two matrixes were used. The abundance matrix was composed of sampled olive orchards in rows and nematode genera in columns, while the matrix of explanatory variables was composed of sampled olive orchards in rows and agronomic factors and soil texture in columns. For the second RDA, ecological indexes were used as response variables with the same explanatory variables. Then the ecological diversity matrix was composed of sampled olive orchards in rows and ecological indexes in columns. (Borcard et al., 2018; Renčo et al., 2020).

Furthermore, a two-way ANOVA was performed to analyze the effect of intensification with soil type, intensification with intercrops and soil type with intercrops on nematode abundance and ecological indexes. When a significant interaction for independent variables was observed, one-way ANOVA was performed, with a post-hoc Duncan test considering dummy variables. Conversely, when there was no interaction between factors, two one-way ANOVA of each factor was followed by the same posthoc test at probability 5% were performed. In addition, data corresponding to nematode densities and ecological indexes were compared according to the degree of intensification of olive orchards, intercrops and soil type using the non-parametric tests Kruskal-Wallis and Mann-Whitney at 5% probability.

For RDA and two-way ANOVA analyses, data were converted to log10 (X + 1) before analysis to improve normality.

Results

Diversity of plant-parasitic nematodes associated with olive roots

Identification studies based on morphological traits identified 11 genera of plant-parasitic nematodes associated with olive roots in the 123 olive orchards visited. Most of them were ectoparasites: Criconemoides spp., Helicotylenchus spp., Paratylenchus spp., Rotylenchus spp., Tylenchorhynchus spp. and Tylenchus spp.. The group of sedentary endoparasitic nematodes was represented by three genera that are Heterodera spp., Meloidogyne spp. and Rotylenchulus spp.. In addition, Pratylenchus spp. and Zygotylenchus spp. were identified as migratory endoparasites (Table 2).

Table 2 Importance of plant-parasitic nematodes identified on olive roots
Full size table

The most prevalent genera of plant-parasitic nematodes identified on root samples were, in decreasing order, Helicotylenchus spp., Pratylenchus spp. and Meloidogyne spp.. However, Meloidogyne spp. and Pratylenchus spp. were the most abundant nematodes (Table 2).

Effect of soil and agronomic characteristics on the diversity of plant-parasitic nematode communities

The first RDA was performed to evaluate the effect of soil and agronomic factors on the composition of the plant-parasitic nematode communities (Fig. 2). The corresponding RDA model was highly significant (p = 0.001) as were the canonical axes RDA1 and RDA2 which explained 16.3% and 9.6% of the variance, respectively (Supplementary Table 1). As illustrated in Fig. 2, the composition of plant-parasitic nematode communities was discriminated among soil and agronomic factors. The abundances of Pratylenchus spp., Paratylenchus spp. and Meloidogyne spp. were positively correlated with the irrigation regimen and intercrops. However, the abundance of Pratylenchus spp. was negatively correlated with the level of intensification and olive variety. Helicotylenchus spp. was positively correlated with the soil type (Fig. 2).

Fig. 2
figure 2

RDA biplots of the relationship between nematode abundances and soil and agronomic factors (Color should be used in print). (Cric:Criconemoides; Helic:Helicotylenchus; Hete:Heterodera; Meloi:Meloidogyne; Para:Paratylenchus; Prat:Pratylenchus; Rotu:Rotylenchulus; Roty:Rotylenchus; Tyle:Tylenchorhynchus; Tyl:Tylenchus; Zyg:Zygotylenchus)

Full size image

In order to explore the diversity of plant-parasitic nematode communities in relation to soil and agronomic factors, a second RDA was performed (Fig. 3). Its model was highly significant (p = 0.001) as were the canonical axes RDA1 and RDA2 which explained 18.8% and 6% of the variance, respectively (Supplementary Table 2). The RDA biplot showed that soil and agronomic factors have an effect on the diversity of plant-parasitic nematode communities. In fact, total nematode number (N), genera richness (G), local diversity (H′), the relative abundance of sedentary endoparasite nematodes, and the relative abundance of migratory endoparasites were positively correlated with the presence of intercrops in irrigated soils. However, the relative abundance of migratory endoparasites was negatively correlated with the level of intensification. However, the relative abundance of ectoparasites was positively correlated with soil type and variety (Fig. 3).

Fig. 3
figure 3

RDA biplots of the relationship between nematode ecological indexes and soil and agronomic factors (Color should be used in print). (N:total number of plant-parasitic nematodes; G:genera richness; Loge_H_prime: Shannon-wiener diversity index; Ar-ecto:relative abundance of ectoparasites; Ar-endo-mig:relative abundance of migratory endoparasites; Ar-end-sed:relative abundance of sedentary endoparasites)

Full size image

For more detailed information, one-way non-parametric ANOVA (Kruskal-Wallis test, Mann-Withney test) was performed (Tables 3 and 4). Results showed that the intensification of olive farming induced the increase in the total number of plant-parasitic nematodes (N) on olive roots, linked especially to the multiplication of Pratylenchus spp. and Meloidogyne spp. in intensive cultivations (Table 3). However the abundance of Meloidogyne spp. seems to be related to the intensive orchards in the presence of intercrops, while Pratylenchus spp. is more related to the super-intensive orchards without intercrops (Tables 3 and 4).

Table 3 Impact of intensification of olive orchards on the plant-parasitic nematode densities and ecological indexes
Full size table
Table 4 Impact of the presence of intercrops and type of soil on the plant-parasitic nematode densities and ecological indexes
Full size table

In fact, the intensification of soil in association with irrigated intercrops such as legumes (potato, tomato, cucumber and pepper) or fruit crops (pomegranate, almond and peach trees) which are suitable hosts for Pratylenchus spp. and Meloidogyne spp., enhances the multiplication of plant-parasitic nematodes (N), genera richness (G) and the relative abundance of endoparasite nematodes (Ar endo-sed, Ar endo-mig,) (Figs. 2, 3 and Tables 3 and 4). However the abundance of ectoparasites, mainly Helicotylenchus spp., was more important in traditional olive orchards (Tables 3 and 4).

In addition, the one-way ANOVA analysis demonstrated that sandy soils are more suitable for multiplication of Rotylenchulus spp., Meloidogyne spp., Heterodera spp. and Rotylenchus spp.. In contrast, Pratylenchus spp. are more abundant in clay soils (Table 4).

Two-way ANOVAs revealed that there was a statistically significant interaction between the effects of intensification and soil type on the abundance of Rotylenchulus spp. (F(3) = 4.319, p = 0.006), Pratylenchus spp., (F(3) = 59.174, p = 0.000), Zygotylenchus spp. (F(3) = 3.331, p = 0.022), the total number of plant-parasitic nematodes (N) (F(3) = 29.202, p = 0.000), the relative abundance of ectoparasites (F(3) = 6.426, p = 0.000) and the relative abundance of migratory endoparasites (F(3) = 10.453, p = 0.000). Comparisons with the Duncan test showed that Rotylenchulus spp. were more abundant in sandy soils in traditional olive orchards, Pratylenchus spp. were more abundant in clay soils in super-intensive systems, while Zygotylenchus spp. were more abundant in sandy-clay soils in intensive olive orchards. The total number of plant-parasitic nematodes (N) was higher in clay soils in super-intensive orchards, and sandy and sandy-clay soils in intensive orchards. The relative abundance of migratory endoparasites was higher in clay and sandy soils in super-intensive olive orchards, whereas ectoparasites were less abundant in clay soils under super-intensive system.

The two-way ANOVA also showed a significant interaction between the effects of intensification and intercrops on the abundance of Meloidogyne spp. (F(1) = 3.920, p = 0.050), Pratylenchus spp. (F(1) = 29.354, p = 0.000), the Shannon diversity index (H) (F(1) = 6.144, p = 0.015) and the relative abundance of migratory endoparasites (F(1) = 7.624, p = 0.007). It was revealed that the abundance of Meloidogyne spp. and Pratylenchus spp. were higher in intensive olive orchards in the presence of intercrops. The Shannon diversity index was higher in traditional olive orchards in the presence of irrigated intercrops, and intensive orchards with or without intercrops. Migratory endoparasites were more abundant in traditional olive orchards with intercrops.

However, the interaction between soil type and intercrops showed significant effects only on the abundance of Pratylenchus spp. (F(2) = 3.300, p = 0.040) and the relative abundance of migratory endoparasites (F(2) = 4.495, p = 0.013). In fact, Pratylenchus spp. were more abundant in clay soils without intercrops and sandy soils with intercrops. Migratory endoparasites were less abundant in sandy-clay soils without intercrops.

Discussion

Diversity of plant-parasitic nematode communities

Symptoms of damage caused by plant-parasitic nematodes has appeared in Tunisian olive orchards mainly on those that are conducted under intensive and super-intensive systems.This study was therefore carried out to decipher the nematofauna associated with olive trees in our country, which has been the subject of our recent research (Guesmi et al., 2016; Guesmi-Mzoughi et al., 2016; Guesmi-Mzoughi et al., 2017; Palomares-Rius et al., 2014). Surveys were conducted in those regions most known for olive orchards in Tunisia, and identified 11 genera of plant-parasitic nematodes infecting olive roots which are Criconemoides spp., Helicotylenchus spp., Heterodera spp., Meloidogyne spp., Paratylenchus spp., Pratylenchus spp., Rotylenchulus spp., Rotylenchus spp., Tylenchorhynchus spp., Tylenchus spp. and Zygotylenchus spp. This nematofauna seems to be not very diverse when compared to those identified on olive trees in Spain with 38 genera (Archidona-Yuste et al., 2019; Palomares-Rius et al., 2015) and in Morocco with 47 genera (Ali et al., 2017). This could be explained by three reasons; first, our study was interested only in plant-parasitic nematodes infecting olive roots, so nematodes which spend most of their life cycle in soil could be not detected. Secondly, roots were washed free of soil prior to extraction, which would make some ectoparasites underrepresented. The third reason is that the studies carried out in Spain and Morocco were interested in parasitic nematofauna not only of cultivated olive, but also of wild olive, which support greater nematode diversity (Ali et al., 2017; Archidona-Yuste et al., 2019; Palomares-Rius et al., 2015).

All nematode genera identified in this study have been detected on olive trees in several countries of the Mediterranean Basin (Ali et al., 2014; Ali et al., 2017; Archidona-Yuste et al., 2019; Archidona-Yuste, Cantalapiedra-Navarrete, et al., 2020a; Aїt Hamza et al., 2015; Castillo et al., 2010; Chafaa et al., 2014; Palomares-Rius et al., 2015). Among potentially damaging plant-parasitic nematodes, Meloidogyne spp., Pratylenchus spp., and Heterodera spp. were infecting olive roots in Tunisian orchards. The root-knot nematodes are among the most dominant and frequent taxa infecting olive trees in wild and cultivated orchards (Ali et al., 2016; Archidona-Yuste et al., 2018) as well as in nurseries (Aït Hamza et al., 2018). Several species were detected in olive trees such as Meloidogyne arenaria, Meloidogyne hapla, Megalaima incognita, M. javanica, Megachile baetica and Malthonica lusitanica (Aït Hamza et al., 2018; Ali et al., 2014; Ali et al., 2016; Archidona-Yuste et al., 2018). Recently, two new species were identified. M. spartelensis infecting wild olive trees in Northern Morocco (Ali et al., 2015), and Musca oleae infecting wild and cultivated olives in Southern Spain (Archidona-Yuste et al., 2018). So olive trees are good hosts for Meloidogyne spp. and infection by these pests could cause serious damage to this crop, mainly in olive nurseries (Aït Hamza et al., 2018). Feeding by root-knot nematodes (Meloidogyne spp.) induces distortion of secondary roots, heavy root galling and the formation of permanent feeding cells, the giant cells, and stellar destruction in olive roots (Abrantes et al., 1992; Castillo, Vovlas, Subbotin, & Troccoli, 2003a; Nico et al., 2002). Additionally the root-lesion nematodes (Pratylenchus spp.) were frequently detected in the olive rhizosphere. More than twelve species of Pratylenchus spp. have been recognized on cultivated and wild olive orchards but not all of them are dangerous for olive trees (Ali et al., 2014; Ali et al., 2017; Archidona-Yuste et al., 2019; Archidona-Yuste, Cantalapiedra-Navarrete, et al., 2020a; Castillo et al., 2010; Palomares-Rius et al., 2015). The presence of non-pathogenic species of root lesion nematodes in olive rhizosphere could be explained by the association of suitable host plants as intercrops in olive orchards (Archidona-Yuste et al., 2019). However, Pratylenchus penetrans and P. vulnus have been demonstrated to be pathogenic to olive trees (Lamberti & Baines, 1969; Nico et al., 2003) and the damage caused by P. vulnus can lead to olive tree decline (Lamberti, 1969). Recently, Platygaster oleae was identified infecting wild and cultivated olives suffering tree decline in southern Spain and Tunisia (Palomares-Rius et al., 2014). Considering the cyst nematodes (Heterodera spp.), a few species have been detected in association with olive trees. They are Heterodera avenae, H. mediterranea and H. riparia and other species which remain unidentified (Aït Hamza et al., 2018; Ali et al., 2014; Ali et al., 2017; Archidona-Yuste et al., 2019; Archidona-Yuste, Cantalapiedra-Navarrete, et al., 2020a; Palomares-Rius et al., 2015). Among Heterodera species, H. mediterranea is specialized to both cultivated and wild olives. The infection by this species causes cellular alterations in the cortex, endodermis, pericycle, and vascular parenchyma tissues of olive roots that could lead to olive tree decline in young plantations and nurseries (Castillo et al., 1999). The reniform nematode, Rotylenchulus spp., was detected in some root samples. To our knowledge, this is the fifth report of this nematode in the olive rhizosphere from countries of the Mediterranean Basin after Italy, Spain, Greece (Palomares-Rius et al., 2018) and Morocco (Ali et al., 2017). Two species have been described on cultivated olives which are Rossia macrosoma and R. macrodoratus (Inserra & Vovlas, 1979; Castillo, Vovlas, & Troccoli, 2003b; Palomares-Rius et al., 2018). These species have been demonstrated to be pathogenic to olive (Castillo, Vovlas, & Troccoli, 2003b; Inserra & Vovlas, 1979). Feeding by R. macrosoma on olive roots induce the formation of syncytia (Castillo, Vovlas, & Troccoli, 2003b), while infection by R. macrodoratus causes the establishment of uninucleate giant cells which serve as feeding cells (Inserra & Vovlas, 1979). Although R. reniformis has not been found infecting olive in natural conditions, olive can be a potential host of this nematode as reported in several studies under controlled conditions (Al-Sayed & Abdel-Hameed, 1991; Badra & Khattab, 1980).

Influence of soil and agronomic factors on plant-parasitic nematode communities

This study showed that farming system (traditional, intensive and super-intensive), soil intensification by irrigation with the association of intercrops, and soil type are the main agronomic factors driving the diversity of plant-parasitic nematode communities. Our results are partially in agreement with those obtained by Palomares-Rius et al. (2015) and Ali et al. (2017). Indeed, our study demonstrated that the total number of plant-parasitic nematodes was significantly higher in irrigated than in non-irrigated olive orchards, and in the presence of intercrops. Similarly, research achieved in Morocco demonstrated that irrigated olive orchards are more infested with plant-parasitic nematodes than non-irrigated olive orchards (Ali et al., 2017). In fact, irrigation enhances the development of roots which increases the multiplication of nematodes (Castillo et al., 2010; Nico et al., 2002). Additionally, it has reported that irrigation waters are a source of contamination by plant-parasitic nematodes (Faulkner & Bolander, 1970; Hugo & Malan, 2010). However, Palomares-Rius et al. (2015) showed that the irrigation regimen did not influence any of the tested diversity indexes except for the Shannon index, which was significantly higher in irrigated olive orchards compared to that under a rainfed regime. Concerning the association with intercrops, the same researchers demonstrated that plant-parasitic nematodes are significantly more abundant in organic olive orchards with the presence of intercrops than in orchards under conventional management with the absence of intercrops (Palomares-Rius et al., 2015). The association of irrigated intercrops with olive trees in intensive cropping system increased the multiplication of endoparasites, especially Meloidogyne spp. and Pratylenchus spp.. This could be linked to the nature of intercrops such as tomato, pepper, almond and peach trees, which are suitable host plants for the multiplication of root-knot nematodes (Perry et al., 2009) and root-lesion nematodes (Castillo & Vovlas, 2007).

Our study revealed that the diversity of plant-parasitic nematode communities is different between olive orchards under super-intensive regimes, and orchards under intensive regimes with the presence of intercrops. In fact, plant-parasitic nematode communities were significantly less rich in genera, less diversified and more homogenously distributed in communities in super-intensive systems than in traditional systems. However, it is the opposite for olive orchards with intercrops. These results suggest that the intensification practices affect the structure of the plant-parasitic nematode communities in favor of the most pathogenic nematodes for olive trees that could increase their population densities feeding on olive roots. This hypothesis has been verified for diverse forest and annual crops (Kimenju et al., 2009; Yeates & Bongers, 1999; Zhang et al., 2017). The intensification of olive orchards increased the multiplication of endoparasite nematodes on olive roots, mainly Pratylenchus spp. and Meloidogyne spp.. But the root-knot nematodes were more dominant in olive orchards under an intensive regime in the presence of intercrops, while lesion nematodes were more dominant in olive orchards under super-intensive regime without intercrops. The abundance of Meloidogyne spp. in irrigated olive orchards was also cited by Aїt Hamza et al. (2015) and Ali et al. (2017). Because of this, our study focused on the impact of the presence of intercrops in olive orchards, which enhances the multiplication of the most dangerous nematode Meloidogyne spp. This concurs with studies carried out by Archidona-Yuste et al. (2018) which revealed the importance of the irrigation regimen and the presence of intercrops, in driving the distribution of root-knot nematodes in cultivated olive in Southern Spain. However, each Meloidogyne species responds differently to these factors. In fact, M. javanica was the main species correlated to irrigated soils with intercrops in contrast to M. arenaria (Archidona-Yuste et al., 2018). This species was highly abundant in high-density cultivated olive orchards in the center of Morocco where cultivated areas are generally distributed by agricultural practices and human activities (Ali et al., 2016). Furthermore, experiments conducted by Liébanas and Castillo (2004) showed that six crucifer species, with potential uses as intercrops in Andalusian olive orchards, are hosts for the three common root-knot nematodes M. arenaria, M. incognita and M. javanica and support moderate to high nematode reproduction. Therefore, it is better to avoid the use of intercrops with olive, or choose plants that are not hosts for nematodes pathogenic to olive. Additionally, growing legumes as intercrops when temperatures are suboptimal for root-knot nematodes might allow their use in olive orchards without increasing the population densities of Meloidogyne spp. (Liébanas & Castillo, 2004).

The abundance of Pratylenchus spp. in olive orchards under super-intensive systems without intercrops doesn’t agree with results obtained by Aїt Hamza et al. (2015) who reported that these nematodes are more prevalent in rainfed olive orchards. In addition, Ali et al. (2017) recorded that traditionally irrigated olive orchards are more conducive to the multiplication of Pratylenchus spp.. This disagreement could be explained by the differences between the Pratylenchus species identified in our study and those reported in the previous studies.

Furthermore, we have found that the relative abundance of ectoparasites, mainly Helicotylenchus spp., is more important in olive orchards under a rainfed regime (without irrigation and without intercrops). This agrees with results obtained by Aїt Hamza et al. (2015) showing that Tylenchidae, Helicotylenchus spp. and Rotylenchus spp. are more prevalent on wild olive than cultivated olive under a traditional regime. Also, Aїt Hamza et al. (2015) cited that these nematodes are better adapted to rainy conditions.

Results indicated that the type of soil affects the diversity of plant-parasitic nematode communities and that there was a strong effect of the interaction between soil type and intensification on this diversity. In fact, we noticed that sandy soils are more conducive to the multiplication of Rotylenchulus spp. in traditional olive orchards, and Meloidogyne spp. in intensive olive orchards. While clay soils increased the multiplication of Pratylenchus spp. in super-intensive systems. This agrees with previous studies reporting that the impact of soil properties on nematodes is specific to certain genera (Aїt Hamza et al., 2015; Chen et al., 2012). Furthermore, Archidona-Yuste et al. (2018) indicated the importance of soil texture in driving the distribution of root-knot nematodes, particularly for Meloidogyne species. In fact, the presence of M. incognita was highly correlated with sandy loamy soils. Indeed, Chen et al. (2012) demonstrated that the multiplication of Pratylenchus spp. is negatively correlated to the proportion of sand in the soil. In addition, Aїt Hamza et al. (2015) demonstrated that Meloidogyne spp. and Pratylenchus spp. were more prevalent in fine sand textured soils. This disagrees with our results, which suggest that Pratylenchus spp. were more prevalent in clay soils. This difference could be attributed to the Pratylenchus species (Palomares-Rius et al., 2015). The prevalence of Rotylenchulus spp. in sandy soils was also reported by Olabiyi et al. (2009) who have studied the presence of this nematode in different soils in Niger.

Palomares-Rius et al. (2015) recorded that the soil texture and variety of olive are the main factors influencing the composition of plant-parasitic nematode communities. In fact, plant-parasitic nematodes are significantly more abundant in clay loam soils while their higher diversity was reached on sandy loam soils that showed the lower nematode abundance.

More recently, Archidona-Yuste et al. (2020a, b) revealed the influence of spatial structure and soil on species richness and beta diversity in contrast to agricultural management. These studies revealed novel insights and suggest involving spatial structure on future studies concerning the diversity of plant-parasitic nematode communities.

Conclusions

This study explored for the first time the plant-parasitic nematodes infecting olive trees and their diversity in the main areas of olive production in Tunisia that included 123 olive orchards. These investigations revealed that the intensification of olive orchards with irrigation and the association of intercrops are the main agronomic factors influencing the multiplication and the diversity of plant-parasitic nematodes infecting olive trees. In particular, olive orchards under super-intensive regime are more conducive to the multiplication of Pratylenchus spp., while the presence of irrigated intercrops enhances the multiplication of Meloidogyne spp.. These two nematodes are widespread in the rhizosphere of olive (Aït Hamza et al., 2018; Ali et al., 2014; Ali et al., 2017; Archidona-Yuste et al., 2019; Palomares-Rius et al., 2015) and many species are pathogenic to olive trees and can induce significant damage in nurseries and olive orchards that can lead to tree decline (Aït Hamza et al., 2018; Ali et al., 2014; Castillo et al., 2010). Indeed, the intensification of olive orchards may cause the disturbance of the ecosystem (Loumou & Giourga, 2003) and make the olive trees more susceptible to some diseases (Trapero et al., 2009) like Verticillium wilt induced by Verticillium dahliae (Lamberti et al., 2002; Saeedizadeh et al., 2006) which was reported for the first time in Tunisia in 2006 (Triki et al., 2006). So, in real situations, when olive trees show symptoms such as wilting, chlorosis, defoliation, and dead brown branches, we must think to plant-parasitic nematodes more than soil-borne fungus. In fact, injuries induced by the penetration of plant-parasitic nematodes into roots pave the way for infection by bacteria and fungi (Sassanelli, 2009). In addition, the damage caused by Verticillium dahliae on olive trees could be enhanced in the presence of Meloidogyne spp. or Pratylenchus spp. (Castillo et al., 2010; Saeedizadeh et al., 2006).

This study demonstrated that intensified systems are more suitable for the multiplication of plant-parasitic nematodes, and in particular endoparasites. So, for the establishment of new olive orchards under intensive and super-intensive regimes, it will be essential to choose certified olive plants, which are free from diseases and pathogens, since nurseries are a primary source of contamination (Aït Hamza et al., 2018; Castillo et al., 2010; Sassanelli, 2009). Additionally, it is necessary to select as intercrops plants which have a suppressive effect on nematodes, such as plants used for biofumigation (Castillo et al., 2010).