Diversity of plant-parasitic nematode communities ...

Applied Soil Ecology 124 (2018) 7–16

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Applied Soil Ecology journal homepage: www.elsevier.com/locate/apsoil

Diversity of plant-parasitic nematode communities associated with olive nurseries in Morocco: Origin and environmental impacts

T

⁎

Mohamed Aït Hamzaa,b, , Abdelmajid Moukhlic, Zahra Ferjid, Odile Fossati-Gaschignardb, Johannes Tavoillotb, Nadine Alib,e, Hassan Boubakerf, Abdelhamid El Mousadika,1, Thierry Mateilleb,1 a

Université Ibn Zohr, Faculté des Sciences d’Agadir, Laboratoire LBVRN, BP 8106, 80000 Agadir, Morocco CBGP, IRD, CIRAD, INRA, Montpellier SupAgro, Univ. Montpellier, Montpellier, France c INRA, CRRA, BP 513, Menara, Marrakech, Morocco d Institut Agronomique et Vétérinaire Hassan II, Campus d’Agadir, Département de Protection des Plantes, BP 18/S, 80000 Agadir, Morocco e Tishreen University, Faculty of Agriculture, Plant Protection Department, PO Box 2233. Latakia, Syrian Arab Republic f Université Ibn Zohr, Faculté des Sciences, Laboratoire LBMPV, BP 8106, 80000 Agadir, Morocco b

A R T I C L E I N F O

A B S T R A C T

Keywords: Biodiversity Nursery Olive Plant-parasitic nematode Soil ecology

Plant-parasitic nematodes (PPN) are key impediments to efficient global crop production and impair the quality of susceptible plants in nurseries as well. In this context, nematode communities were determined in 305 solid substrate samples collected from 25 olive (Olea. europaea. subsp. europaea) nurseries in Morocco. Taxonomical and functional diversity as well as the structures of PPN communities were described and then compared between regions, cultivars as well as according to biotic and abiotic factors. A high diversity of PPN was observed, with the detection of 63 species and 26 genera. The most dominant taxa detected were spiral nematodes (Helicotylenchus spp. and Rotylenchus spp.), stunt nematodes (Tylenchorhynchus spp.), grazer nematodes (Tylenchus spp.) and root-knot nematodes (Meloidogyne spp.). Hoplolaimidae nematodes (Helicotylenchus spp. and Rotylenchus spp.) and Tylenchus spp. were better adapted to rainy conditions that prevailed in the northern regions of Morocco. Multiblock analyses demonstrated that functional diversity (cp and trophic groups) was more affected by the environment than taxonomical diversity (total number, species richness, locale diversity and evenness). They also indicated that PPN communities were more impacted by climatic variables (rainfall and minimum temperature) and nursery substrate origins than by soil physic-chemical factors. Nevertheless, a coinertia analysis showed that N, P and K amendments in olive nurseries enhanced the development of harmful PPN, especially root-knot nematodes.

1. Introduction Intensification of cropping systems increases practices such as the addition of synthetic compounds (e.g., pesticides, fertilizers), the manipulation of organic residues and the disturbance of the soil itself (e.g., through cultivation, substrate preparation, plant import with substrates, seed-bed preparation) (Ruthenberg, 1980; Giller et al., 1997). While short-term productivity gains are generally emphasized, longterm production sustainability is now seen as a necessity (Phillips, 1984; Altieri, 1991). Research has shown that plant production is highly subject to soil quality, especially to soil health (Pankhurst et al.,

1995) and that soil biodiversity within communities seems to be essential in terms of quality (functions) and quantity. Soil physical and chemical parameters such as texture, nutrient status, pH, moisture, etc., are well understood and commonly used as indicators of soil quality by scientists and stakeholders (Barrios et al., 2006). However, there is still a lack of knowledge regarding the distribution of soil organisms and the impact of biotic and abiotic factors on them (anthropogenic or natural constraints). In addition, land uses affect soil invertebrate communities (Lavelle et al., 2006). Accordingly, studies of community profile data offer good tools for assessing interactions between organisms as well as the effects of different environmental factors on community

⁎

Corresponding author at: Université Ibn Zohr, Faculté des Sciences d’Agadir, Laboratoire LBVRN, BP 8106, 80000, Agadir, Morocco. E-mail addresses: [email protected], [email protected] (M. Aït Hamza), [email protected] (A. Moukhli), [email protected] (Z. Ferji), [email protected] (O. Fossati-Gaschignard), [email protected] (J. Tavoillot), [email protected] (N. Ali), [email protected] (H. Boubaker), [email protected] (T. Mateille). 1 Co-leaders of the publication. http://dx.doi.org/10.1016/j.apsoil.2017.10.019 Received 31 May 2017; Received in revised form 11 October 2017; Accepted 16 October 2017 Available online 31 October 2017 0929-1393/ © 2017 Elsevier B.V. All rights reserved.


M. Aït Hamza et al.

soils, or loamy cropped soils (Table 1), supplemented with different proportions of sand, peat fertilizer and animal manure, and irrigated by sprinklers. Five olive plantlets (Olea europaea subsp. europaea) grown in plastic bags were sampled for each variety from each nursery. Information regarding substrates and cultivars was recorded. In total, 305 olive root cuttings were carried to the laboratory and kept under greenhouse conditions.

composition (Yang and Crowley, 2000). Moreover, the functional diversity, which reflects the functional differences among the species in a community (Tilman, 2001), is a major determinant of ecosystem processes (Chapin et al., 2000; Loreau et al., 2001). Soil nematodes are allocated to different trophic groups: bacterivores, fungivores, carnivores, and herbivores (Yeates et al., 1993), and consequently occupy key positions in soil food webs such as the decomposition of organic matter and nutrient recycling (Ferris et al., 2004; Briar et al., 2007). Their functional diversity has also been used as a bio-indicator of soil quality (Bongers, 1990) and habitat stability (Wasilewska, 1994). Consequently, any disturbance to plant physiology or species composition, soil texture, chemistry and climatic factors (rainfall and temperature) may alter nematode species and the diversity of their functional groups (Whitford et al., 1982; Wall and Virginia, 1999). Nevertheless, at the same time, plant-parasitic nematodes (PPN) are one of the main biotic stresses on crops. Annual losses caused by them are estimated at 8.8 to 14.6% (100–157 billion USD/year) of the world crop production (Nicol et al., 2011). On cultivated olive (Olea europaea L. subsp. europaea), PPN are able to reduce tree growth (Nico et al., 2003) and may be responsible for 5 to 10% yield losses (Koenning et al., 1999). Their impact is strengthened in intensive cultivation systems (high-density cultivation) and in nurseries where irrigation conditions favor the development of roots and, therefore, nematode reproduction (Nico et al., 2002; Castillo et al., 2003, 2010). Current PPN data on olive trees worldwide show that 153 species belonging to 56 genera have been detected on olive, including orchards, nurseries and a few wild areas (Ali et al., 2014). In Morocco, the substrates used in olive nurseries are mainly taken from either alluvial sandy soils or loamy fields and forest soils. Planting material comes from several nurseries distributed throughout the oliveproducing areas. The olive plantlets are certified pathogen-free (e.g., Verticillium dahlia) and parasite-free (e.g., PPN). However, standard health practices are not applied in all Moroccan nurseries and seasonal and informal nurseries coexist. Consequently, the quality of plantlets is not guaranteed. Considering the extension of the olive-producing areas planned, this program will enhance the production of nursery plants, and non-healthy nurseries will then be a major source for PPN introduction into orchards by transplanting infested soil and rooted plants. Considering that PPN are major parasites on olive, especially in Mediterranean nurseries (Castillo et al., 2010), and that the PPN fauna and its distribution were totally unknown in Moroccan olive nurseries, the aim of this study was (i) to characterize PPN communities in Moroccan olive nurseries where no information has been available up until now; (ii) to assess the response of nematode diversity to environmental factors (soil parameters, substrate origins and climate); and (iii) to discuss the PPN invasive risk that could affect the orchards after transplanting trees from infested nurseries.

2.2. Nematode extraction and quantification A 250 mL substrate subsample made with several randomized aliquots taken from the rhizosphere of each olive plantlet was used for nematode extraction according to the Oostenbrink (1960) elutriation procedure (ISO (2007)). PPN belonging to the Aphelenchida (fungalfeeding nematodes that can alternatively feed on plants), Dorylaimida, Triplonchida and Tylenchida orders were identified as to genus level using dichotomous keys (Mai and Mullin, 1996) and enumerated in 5 mL aliquots sampled from 25 mL suspensions (Merny and Luc, 1969) under a stereomicroscope (×60 magnification). Genus levels were expressed as the number of nematodes per litre of fresh soil. Nematodes were then killed with hot formaldehyde (4%) and fixed in De Grisse solution (De Grisse, 1969), and specimens were subsequently prepared using the glycerin-ethanol method. One hundred specimens at least were mounted onto slides (Van Bezooijen, 2006) and identified as to species. Root-knot nematodes (Meloidogyne spp.) were identified as to species level by biochemical (esterase patterns) and molecular (SCAR markers) techniques (Ali et al., 2016). 2.3. Diversity indices Taxonomical diversity of the PPN communities was assessed by: (i) the total number of PPN in a community (N); (ii) the species richness (S) that represents the total number of species in a community; (iii) the Shannon-Wiener diversity index H’ (H’ = −∑pi ln pi, where pi is the proportion of individuals in each species i that quantifies the local diversity (H’ ranges from 0 to ln(S)); and (iv) the evenness (E = H’/lnS) that quantifies the regularity of species distribution within the community (E varies between 0 and 1). PPN genera detected in communities were broken down into life strategy groups according to the colonizer/persister value (cp-value) to which they belong (Bongers, 1990). The diversity of the communities was described by calculating: (i) the plant-parasitic index (PPI = ∑cpini/N), which quantifies the plant-feeding diversity of the communities; and (ii) the relative mean abundance (%) of each cp-value class in a community calculated as follows: Rcpi = cpini/N, (Bongers, 1990). PPN species were also assigned to the trophic groups according to their feeding habits (Wasilewska, 2006): obligate plant feeders (OPF), facultative plant feeders (FPF) that alternatively feed on fungi, and fungal feeders (FF) that alternatively feed on plants. Trophic diversity was then described by the relative mean abundance (%) of individuals within each trophic group. The dominance of the PPN genera was estimated by modeling their abundance (A = mean number of nematodes in the samples where the genus was detected) and their frequency (F = % of samples where the genus was detected) according to Fortuner and Merny (1973).

2. Materials and methods 2.1. Site description and olive plantlet sampling Twenty-five commercial olive nurseries were selected in the main olive production regions in Morocco: Jbala, Guerouane, Haouz and Souss (Fig. 1 and Table 1). They were selected for (i) their plant production and the diversity of the varieties cultivated; (ii) the diversity of their solid growth substrates; and (iii) their geographic distribution. The regions studied were labeled according to the Emberger diagram (Emberger, 1930), modified by Stewart (1975), which takes into account the annual rainfall and the minimum average temperatures of the coldest month (MACM) during the last twelve months before the survey, defining specific topoclimatic areas (Fig. 2). Olive plants are grown in 2–3 L plastic bags filled with solid substrates containing either sandy alluvial river bank soils, organic forest

2.4. Soil physico-chemical analyses Physico-chemical soil analyses were performed by the Soil Laboratory of the IAV (Institut Agronomique et Vétérinaire “Hassan II”, Agadir, Morocco) on dry and sieved (2 mm) substrate material: proportion of clay (0–2 μm), fine (2–20 μm) and coarse (20–50 μm) silts, and fine (50–200 μm) and coarse (200–2000 μm) sands using the sedimentation method (Hedges and Oades, 1997); carbon using the method described by Allison (1960), making it possible to calculate 8



Fig. 1. Distribution of the olive nurseries surveyed in Morocco. See Table 1 for more information.

2.5. Data analyses

organic carbon content (OM = 1.724 × C); pHH2O (ISO (2005)); assimilated phosphorus was analyzed using the method of Olsen et al. (1986); nitrogen and ammonia were determined using the Kjeldahl nitrogen method (Barbano and Clark, 1990); copper, iron, magnesium, manganese, potassium, sodium and zinc were all analyzed by atomic absorption spectrophotometry (Lindsay and Norvell, 1978; Sims et al., 1991), and the substrate salinity was analyzed by measuring conductivity (μS/cm) (Richards, 1954).

Community patterns for PPN diversity indices and genera were explored through a Principal Component Analysis (PCA). Region grouping was tested using Monte-Carlo tests on PCA eigenvalues (randtest {ade4). A k + 1 multivariate method, MultiBlock Partial Least Squares (MBPLS, mbpls {ade4} (Bougeard et al., 2011), was used to interpret patterns in relation to substrate origins and soil characteristics. A Co-Inertia Analysis (CIA) (Dolédec and Chessel, 1994; Dray

Table 1 Location and characteristics of the Moroccan olive nurseries surveyed. Geographic region

City

No. of nurseries

Origin of the substrates

Olive variety

No. of samples

Jbala

Ouazzane

1

Guerouane

Meknes

4

River sand Forest soil Crop soil River sand Crop soil

Haouz

Marrakech

5

River sand Forest soil Crop soil

El Kelaa Des Sraghna

3

Forest soil Crop soil

Sidi Abdellah Ghiat

1

5 5 5 5 5 20 20 5 15 10 10 5 25 25 10 25 10 10 15 15 5

Agadir

8

Forest soil River sand River sand Crop soil

Picholine marocaine Haouzia Menara Arbequina Arbosana Haouzia Menara Picholine Languedoc Picholine marocaine Picual Arbequina Arbosana Haouzia Menara Picholine Languedoc Picholine marocaine Haouzia Menara Picholine Languedoc Picholine marocaine Picholine marocaine

Khmiss Aït Amira Biougra

2 1

Haouzia Menara Picholine marocaine Picholine marocaine Menara

5 20 15 10 5

Souss

Crop soil Forest soil Crop soil

9



Fig. 2. Bioclimatic diagram of the areas sampled according to Emberger (1930) and Stewart (1975).

3.2. Plant-parasitic nematode diversity in communities

et al., 2003) was applied between the PPN genus abundances and physicochemical soil factors. In order to ovoid outlier patterns just for genera, the scarcest genera (total abundance less than 1%) were excluded from the genus dataset prior to running analyses. Multivariate analyses were performed using the R language (Core Team, 2016); package: readxl (Wickham, 2016); and base; {ade4} (Chessel et al., 2004; Dray and Dufour, 2007).

There was no evidence of any olive genetic diversity (cultivars) effect on the PPN diversity (data not shown). The MBPLS loading plot of the PPN diversity data (Fig. 4A) showed that the functional diversity (cp and trophic groups) contributed more to the analysis than the taxonomical diversity (N, S, H’, E). The first MBPLS axis indicated contrasted positions of the obligate parasites (OPF), most of which are cp-3 nematodes, and the facultative plant-feeders (FPF), most of which are cp-2 nematodes. Cp-5 (most of which are fungal feeders) and cp-4 nematodes contributed less (i.e., higher contribution to the second axis). The plant-parasitic index (PPI) was generally associated with OPF and FF nematodes. The loading plot of the environmental factors (Fig. 4B) indicated a contrasted position of the rainfall (RF) and the minimum temperature (MACM) on the first axis. The origins of the substrates were less contributive variables (such as Crop and River), except for the Forest variable that was significant on the second axis. The MBPLS indicated a north-south opposition of the Jbala and Guerouane nurseries (positive values) and the Haouz and Souss nurseries (negative values) (Fig. 4C). The obligate nematodes (OPF, cp-3, high PPI) occurred more often in the Souss and the Haouz nurseries impacted by high MACM that used mainly forest substrates, except for two Haouz nurseries (14 and 19) that used only riverbank substrates and that are located in cold areas. Facultative plant-feeders (FPF, cp-2) occurred significantly more often in the northern nurseries (Guerouane and Jbala) that are impacted by high annual rainfall (RF).

3. Results 3.1. Taxonomical diversity of plant-parasitic nematodes PPN were detected in all of the soil samples. Sixty-three species belonging to 26 genera were found to be associated with olive plantlets in Moroccan nurseries (Table 2). They belonged to one family of each order Aphelenchida, Dorylaimida and Triplonchida, and 10 families of Tylenchida. Hoplolaimidae and Telotylenchidae were the most diversified families, including 19 and 11 species, respectively. Seven genera (Gracilacus, Helicotylenchus, Meloidogyne, Merlinius, Paratylenchus, Tylenchorhynchus and Tylenchus) were dispersed throughout the regions sampled. In contrast, five genera (Aorolaimus, Cacoporus, Ditylenchus, Filenchus and Telotylenchus) were localized in only one region. The Haouz region hosted more species than the others. When modeling PPN dominance (Fig. 3), 80.8% of the genera were found to be less frequent (F < 30%) and 53.8% to be occasional (F < 5%) according to the model (Fortuner and Merny, 1973). Only 15.38% of the PPN genera were highly abundant according to the abundance threshold defined by the model (200 nematodes/dm3 of soil). Four genera were found to be dominant (F ≥ 30% and A ≥ 200 nematodes/ dm3 of soil): Helicotylenchus spp. that prevailed in all the regions, Tylenchus spp. in the Jbala and Guerouane regions, and Tylenchorhynchus spp. in the Haouz region (Table 2). No Rotylenchus spp. was noticed in the Souss region, whereas it was found in the others. Less abundant populations of root-knot nematodes (Meloidogyne spp.) were detected in 50% of the samples (Fig. 3), but they occurred in all of the regions. Globally, the more frequent the genera were, the more abundant they were.

3.3. Correspondences between plant-parasitic nematode genus patterns and environmental variables The MBPLS loading plot of the PPN taxa (Fig. 5A) indicated that Meloidogyne, Criconema, Merlinius and Zygotylenchus genera were related to the positive values of the first axis, while Filenchus, Paratrophurus and Tylenchus genera were related to its negative values. Helicotylenchus and Tylenchorhynchus genera were related to the second axis (positive and negative values, respectively). The loading plot of the 10



Table 2 Plant-parasitic nematode taxa detected in the olive nurseries surveyed in Morocco. Trophic groups (FF: fungal feeders; FPF: facultative plant feeders; OPF: obligate plant feeders). Orders and families (cp value)

Species (trophic groups)

Authors

Geographic regions Jbala

Aphelenchida Aphelenchoididae (2) Dorylaimida Longidoridae (5)

Triplonchida Trichodoridae (4)

Aphelenchoides sp. (FF) Longidorus sp. (OPF) Xiphinema pachtaicum (OPF) X. turcicum (OPF) Xiphinema sp. (OPF)

Micoletzky, 1922 Tulaganov, 1938 Luc and Dalmasso, 1964 Cobb, 1913

Ditylenchus emus (FPF) D. equalis (FPF) Ditylenchus sp. (FPF)

Khan et al., 1969 Heyns, 1964 Filipjev, 1936

Criconematidae (3) Heteroderidae (3)

Criconema sp. (OPF) Heterodera riparia (OPF) Heterodera sp. (OPF)

Hofmänner and Menzel, 1914 Subbotin et al., 1997 Schmidt, 1871

+

Hoplolaimidae (3)

Aorolaimus sp (OPF) Helicotylenchus canadensis (OPF) H. crassatus (OPF) H. crenacauda (OPF) H. digonicus (OPF) H. dihystera (OPF) H. exallus (OPF) H. minzi (OPF) H. pseudorobustus (OPF) H. tunisiensis (OPF) H. varicaudatus (OPF) H. vulgaris (OPF) Helicotylenchus sp. (OPF) Hoplolaimus sp. (OPF) Rotylenchus buxophilus (OPF) R. goodeyi (OPF) R. pumilus (OPF) R. robustus (OPF) Rotylenchus sp. (OPF)

Sher, 1963 Waseem, 1961 Anderson, 1973 Sher, 1966 Perry, 1959 Cobb, 1893 Sher, 1966 Sher, 1966 Steiner, 1914 Siddiqi, 1964 Yuen, 1964 Yuen, 1964 Steiner, 1945 Von Daday, 1905 Golden, 1956 Loof & Oostenbrink, 1958 Perry, 1959 de Man, 1876 Filipjev, 1936

+ + +

Meloidogyne arenaria (OPF) M. incognita (OPF) M. javanica (OPF) Cacopaurus sp. (OPF) Paratylenchus (Gracilacus) sp. (OPF) Paratylenchus (P.) microdorus (OPF) P. (P.) sheri (OPF) Paratylenchus (Paratylenchus) sp. (OPF)

Neal, 1889 Chitwood, 1949 Treub, 1885 Thorne, 1943 Raski, 1962 Andrássy, 1959 Raski, 1973 Micoletzky, 1922

Pratylenchoides hispaniensis (OPF) Pratylenchoides sp. (OPF) Pratylenchus neglectus (OPF) P. pinguicaudatus (OPF) Pratylenchus sp. (OPF) Zygotylenchus guevarai (OPF)

Troccoli et al., 1997 Winslow, 1958 Rensch, 1924 Corbett, 1969 Filipjev, 1936 Tobar Jiménez, 1963

Rotylenchulus sp. (OPF) Merlinius brevidens (OPF) M. microdorus (OPF) Merlinius sp. (OPF) Paratrophurus sp. (OPF) Telotylenchus paaloofi (OPF) T. ventralis (OPF) Trophurus sculptus (OPF) Trichotylenchus sp (OPF) Tylenchorhynchus clarus (OPF) T. crassicaudatus (OPF) Tylenchorhynchus sp. (OPF)

Linford & Oliveira, 1940 Allen, 1955 Geraert, 1966 Siddiqi, 1970 Arias, 1970 Kleynhans 1975 Loof, 1963 Loof, 1956 Whitehead, 1960 Allen, 1955 Williams, 1960 Cobb, 1913

Filenchus filiformis (FPF) F. misellus (FPF) Filenchus sp. (FPF) Tylenchulus sp. (FPF) Tylenchus elegans (FPF) Tylenchus sp. (FPF)

Bütschli, 1873 Andrássy, 1958 Andrássy, 1954 Cobb, 1913 De Man, 1876 Bastian,1865

Paratylenchidae (2)

Pratylenchidae (3)

Rotylenchulidae (3) Telotylenchidae (3)

Tylenchidae (2)

Souss

+

+

+

+

+

+

+

+ +

+

Cobb, 1913

Meloidogynidae (3)

Haouz

Fischer, 1894

Trichodorus sp. (OPF)

Tylenchida Anguinidae (2)

Guerouane

11

+

+

+ + +

+ +

+

+ +

+ + + + + + +

+ +

+

+ + + + + + +

+ + + + + + + +

+ + +

+

+ + +

+ +

+

+

+ + + +

+

+ + +

+

+

+

+

+

+

+ +

+ +

+ + + + + + + + +

+ + + + +

+ + + + +

+ + +

+ +

+

+

+ +

+ + + + + +

+ + + + +

+ + +



Table 3 Variables analyzed and corresponding codes. Variables

Code

Nematode genera Aorolaimus Aphelenchoides Cacoporus Criconema Ditylenchus Filenchus Gracilacus

Aor Aph Cac Cri Dit Fil Gra

Helicotylenchus

Hel

Heterodera

Het

Hoplolaimus Longidorus

Hop Lon

Meloidogyne Merlinius Paratrophorus Paratylenchus Pratylenchus

Mel Mer pTro pTyl Pra

Rotylenchulus

Rol

Rotylenchus

Rot

Telotylenchus Trichodorus Trichotylenchus

Tel Trd Trt

Tylenchorhynchus

Tyo

Tylenchulus Tylenchus

Tys Tyu

Xiphinema Zygotylenchus

Xip Zyg

Variables Geographic regions Jbala Guerouane Haouz Souss Climate minimum temperature of the coldest month (°C) annual rainfall (mm) Origins of the substrates cropped fields forests river banks Diversity indices total number of nematodes species richness local diversity evenness obligate plant feeders facultative plant feeders fungal feeders abundanceof cp groups

Code

Variables

Code

Soil characteristics J G H S

Coarse sand Fine sand Coarse silt Fine silt Clay Nitrogen Copper

cSa fSa cSi fSi Cla N Cu

MACM

Iron

Fe

RF

Magnesium

Mg

Manganese Phosphorus

Mn P

Crop Forest River

Potassium Sodium Zinc pH Conductivity

K N Zn pH Con

N

Organic matter

OM

S H’ E OPF FPF FF cp2 to cp5

Fig. 4. Multiblock analysis between nematode diversity indices, substrate origins and environmental factors. (A) PCA loading plot of the diversity indices. (B) PCA loading plot of the environmental factors. (C) Score plot for the olive nurseries sampled. Encoding for taxa and environmental variables is listed in Table 3.

Fig. 3. Dominance diagram of the nematode genera detected in the olive nurseries surveyed in Morocco. Dotted lines indicate delineation between low and high abundances (A) or frequencies (F), as described in Fortuner and Merny (1973). Encoding for taxa is listed in Table 3.

that occurred in the southern nurseries (Souss and Haouz) and substrates of forest origin to a lesser extent. The second PPN group (Filenchus, Paratrophurus Tylenchus, Ditylenchus, Paratylenchus and Rotylenchus genera) corresponded mainly to rainy regions (Jbala and Guerouane) and substrates from crop origins.

environmental variables (Fig. 5B) clearly opposed the two climatic variables (MACM and RF). Substrates made with soil material of crop origin were also very discriminant. The second axis was related to substrates made with forest material and to high rainfall. The substrates made with riverbank material did not contribute to the analysis. The loading plot of the nurseries (Fig. 5C) clearly identified a north-south distinction of the nurseries (first axis) that were secondarily separated on the second axis. The first PPN group (Meloidogyne, Criconema, Merlinius and Zygotylenchus genera) was mostly associated with high MACM

3.4. Correspondences between plant-parasitic nematode genus patterns and physico-chemical soil parameters We observed a lower contribution of the physico-chemical soil characteristics (Fig. 5B). However, considering that PPN spend a large part of their life cycle in the soil and that organic and mineral 12



4. Discussion Biodiversity results from interactions between processes that deal with evolution and ecology, in changing physicochemical and biogeographical environments (Huston, 1997). Nowadays, biodiversity is on the decline because of continuous human intervention, including habitat destruction, pollution, climate change, biopiracy and the spread of non-native species (Chapin et al., 2000; Willis and Bhagwat, 2009; Barnosky et al., 2011). Because of this, the intensification of the agriculture component in the “Green Morocco Plan” program could induce the emergence of plant pathologies, including PPN. 4.1. Taxonomical and functional diversity of plant-parasitic nematodes The present study indicates that more than 65% of the olive planting stocks sampled were infected by PPN. That confirms that non-sanitized nurseries are able to encompass a high PPN richness, and that most of the taxa detected here correspond to those recorded in association with olive trees worldwide (Palomares-Rius et al., 2015; Castillo et al., 2010; Ali et al., 2014), including Morocco (Aït-Hamza et al., 2015; Ali et al., 2017). Moreover, most of the dominant taxa detected in nurseries (especially Helicotylenchus spp., Tylenchorhynchus spp., Meloidogyne spp. and Rotylenchus spp.) correspond to the most widespread taxa on olive trees around the Mediterranean Basin (Nico et al., 2002; Castillo et al., 2010; Ali et al., 2014). Because these taxa are both frequent and abundant, they can be considered to be well adapted to the olive tree and especially to the growing conditions of the nursery. It was clearly established that native substrates, even from ecosystems (riverbanks and forests), are infested with PPN that are later widely spread in nurseries. However, fungal feeders (FF) and facultative plant feeders (FPF) that are involved in microbial activities for organic matter degradation (Villenave et al., 2009) were rare, as opposed to obligate parasites (OPF). Because FF and FPF nematodes are involved in complex trophic chains, their low occurrence in nursery substrates would make the organic nutrients less available for olive plantlets over a long period. Since PPN were introduced with native substrates and then mixed in highly disturbed final substrates, this could explain why the diversity indices (N, S, H’, E) did not significantly differ between the regions surveyed and did not change with nursery conditions. We must consider that the substrate production processes lead to soil destructuration and to a very strong disturbance of the biotic niches. This situation could be compared with microcosm experiments that consider mixed inoculum of PPN. Indeed, they cannot reflect the distribution of PPN species observed in fields. However, several parameters can be found to induce differences in the reproductive rates of several PPN species (Villenave et al., 1997). In this study, the final structure of the PPN fauna derived from nursery substrates was dominated by Helicotylenchus spp., which represented more than 80% of the individuals. A similar dominance of PPN, regardless of environmental impacts, was observed by Villenave et al. (1997) who processed experimental assemblages of PPN communities. It can be hypothesized that, in olive nurseries, trade-offs between PPN species and other soil-inhabiting organisms are completely destroyed, and this situation might allow to the most aggressive PPN (high fitness with olive) to rapidly develop.

Fig. 5. Multiblock analysis between plant-parasitic nematode communities, substrate origins and environmental factors. (A) PCA loading plot of the nematode genera. (B) PCA loading plot of the environmental factors. (C) Score plot for the olive nurseries sampled. Encoding for taxa and environmental variables is listed in Table 3.

amendments are usually added in substrates, we investigated the impact of the substrate physico-chemical parameters on PPN diversity patterns. The contribution of PPN and soil factors to the COA analysis (Fig. 6) indicated that Meloidogyne spp., Tricotylenchus spp., Paratrophurus spp. and Telotylenchus spp. were dominant in substrates characterized by intermediate-size particles (fine sands and coarse silts −20–200 μm) and high NPK amounts. Most of the other taxa, especially Tylenchorhynchus spp. Tylenchus spp. and Helicotylenchus spp., were more consistent with clayey and organic soils with concentrated soil nutrient solutions (=soil conductivity).

4.2. Responses of plant-parasitic nematode communities to climatic conditions Climate is the main force that determines the structure of the PPN communities in nurseries since the annual rainfall and the temperature (especially MACM) significantly impact both the taxonomical and the functional pattern of the PPN communities: (i) facultative plant-feeder and colonizer nematodes (cp-2) such as Anguinidae (Ditylenchus spp.) and Tylenchidae (Filenchus spp., Tylenchulus spp., Tylenchus spp.) were enhanced in the less arid regions such as Jbala (sub-humid climate) and 13



Fig. 6. Score plot of the eigenvalues for their contribution to the COI analysis between physico-chemical soil factors (COISF) and plant-parasitic nematodes (COIPPN). Circles and squares represent positive and negative correlations, respectively. Their size is proportional to the strength of the correlation. Encoding for soil characteristics and nematode genera is listed in Table 3.

habitat origin of the native substrates is of great importance in structuring the PPN communities. Substrates from different origins could be considered as reservoirs of high diversity where a part remains unknown (Christensen and Emborg, 1996). The very high heterogeneity of the PPN population levels observed in the nurseries surveyed refers to two main reasons: first, the spatial distribution of the nematodes in the native soils is aggregated like in all soils (Hoschitz and Kaufmann, 2004), so soil sampling can lead to heterogeneous data. Second, their distribution and development depend on their environment: (i) soil climate; (ii) local soil texture and structure; (iii) microbial competition (Piśkiewicz et al., 2007) etc.. Interestingly, the substrates taken from cropped fields specifically brought more facultative fungal feeders and fast colonizer PPN (cp-2) (Anguinidae and Tylenchidae) than obligate and cp-3 parasites. These crop substrates generally came from vegetable fields heavily treated with nematicides, which could explain the poverty of these substrates with the most pathogenic PPN. Surprisingly, these taxa were instead imported with substrates taken from forests, which were, moreover, inhabited by more diversified PPN communities than crop substrates. This is consistent with other ecological observations that showed that lowly disturbed ecosystems mainly host more diverse communities of soil organisms, as demonstrated for earthworms (Fragoso et al., 1997), for PPN (Cadet, 1998) and for other soil biota (Postma-Blaauw et al., 2010, 2012). It is the complete opposite in cultivated areas (e.g., Souss) where the PPN communities are lowly diversified. In cultivated areas, human practices lead to species decline, as was already observed with bees, birds, plants (Yamaura et al., 2012), soil microfauna (PostmaBlaauw et al., 2010, 2012) and nematodes (Pan et al., 2012). It appeared evident that the substrates taken from riverbanks were less infested with PPN, thus constituting a lower risk of invasion. These impacts on communities could be related to the biological characteristics of the nematodes, leading them to respond differently to environmental perturbations and climate changes. Nursery conditions induce favorable environments for PPN multiplication, especially irrigation, which enhances the development of plant roots (Nico et al., 2002; Castillo et al., 2010). This was consistent with other observations

Guerouane (semiarid climate); (ii) obligate plant-feeders that correspond to high fitness PPN (high PPI and cp-3 nematodes such as Meloidogynidae (Meloidogyne spp.), Criconematidae (Criconema spp.), Telotylenchidae (Merlinius spp.) and Pratylenchidae (Zygotylenchus spp.)) were enhanced by warmer climates that impact the Souss (infraMediterranean area) and the Haouz (warm thermo-Mediterranean) regions, even though they are more arid. The most likely consequences of climate change are shifts in the geographical distribution of plant hosts and pathogens and altered crop losses, caused in part by changes in the efficacy of control strategies (Coakley et al., 1999). Such a change does not exclude the threat of the emergence of new pests, including nematodes (Nicol et al., 2011). Carter et al. (1996) predicted the increase of RKNs as the climate changes because of additional pathogen generation per year in a warmer climate. This is especially important since most nematode life processes have thermic optima that determine their ideal geographic ranges (Luc et al., 2005). Boag et al. (1991) suggested a close association between mean July soil temperature and nematode distribution. Based on this observation, they predicted that climate change could result in increased nematode populations. Moreover, Neilson and Boag (1996) estimated that a 10 °C warming would theoretically allow the nematode species to migrate over a long distance northward. Changes in precipitation could influence nematode distribution on a large scale, although previous findings had suggested that soil moisture would not affect nematode distribution in most agricultural soils in northern Europe (Boag et al., 1991; Neilson and Boag, 1996; Coakley et al., 1999). Agroecosystems are highly complex, with a myriad of interacting factors determining system behavior. Man alters system behavior by periodically disrupting relationships among soil organisms. The degree of disruption depends on the specific agroecosystem, with the most disruption occurring in highly mechanized and energy-intensive agriculture (Freckman and Caswell, 1985). Although nematodes migrate very slowly, humans are credited with efficiently disseminating them. Nematode spread into new regions could put a wide range of crops at risk. Additionally, introduction of new crops into a region could also expose them to infestation by nematode species already present. The 14



References

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Acknowledgements This research was supported by a Ph.D. grant from the IRD (Institut de Recherche pour le Développement, Marseille, France). It was also funded by the PESTOLIVE project: Contribution of olive history for the management of soil-borne parasites in the Mediterranean Basin (ARIMNet action KBBE 219262); and by the BIONEMAR project: Development of fungal bionematicides for organic production in Morocco (PHC-Toubkal action 054/SVS/13). 15



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