# Seedlings responses to arbuscular mycorrhizal inoculation of the Atlas Cypress, *Cupressus atlantica* Gaussen, from various Moroccan Western High Atlas origins

Youssef SFAIRI¹

Mohamed NAJIB AL FEDDY²

Issam JADRANE³

Abdelaziz ABBAD⁴

Lahcen OUAHMANE³

¹ Direction Régionale des Eaux et Forêts et de la lutte contre la Désertification du Haut Atlas Marrakech Maroc

² Institut National de Recherche Agronomique CRRA Plant protection research unit Phytobacteriology laboratory Marrakesh Maroc

³ Cadi Ayyad University Faculty of Sciences Semlalia Laboratory of Ecology and Environment Marrakesh Maroc

⁴ Cadi Ayyad University Faculty of Sciences Semlalia Laboratory of Biotechnology, plant protection Marrakesh Maroc

**Auteur correspondant /**
**Corresponding author:**
Ouahmane Lahcen - l.ouahmane@uca.ac.ma

<figure><img src="image_1.png" /><figcaption>**Photo 1.**
Monumental Atlas Cypress Tree in Mzouzit station. Photo L. Ouahmane.</figcaption></figure>

DOI: 10.19182/bft2018.337.a31634 - Droit d'auteur © 2018, Bois et Forêts des Tropiques - Date de soumission : 25 janvier 2018 ; date d'acceptation : 29 mai 2018 ; date de publication : 1er juillet 2018.

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cirad

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**Citer l'article / Cite the article**

Sfairi Y., Najib Al Feddy M., Jadrane I., Abbad A., Ouahmane L., 2018. Seedlings responses to arbuscular mycorrhizal inoculation of the Atlas Cypress, *Cupressus atlantica* Gaussen, from various Moroccan Western High Atlas origins. Bois et Forêts des Tropiques, 337: 27-38. DOI: https://doi.org/10.19182/bft2018.337.a31634

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Licence Creative Commons : Attribution - Pas de Modification 4.0 International. Attribution-NoDerivatives 4.0 International (CC BY-ND 4.0)
## RÉSUMÉ

Réponses des plantules du cyprès de l'Atlas,
*Cupressus atlantica* Gaussen, à l'inoculation
de mycorhizienne en provenance de diverses
origines de l'ouest du Haut Atlas au Maroc

Le cyprès de l'Atlas, *Cupressus atlantica* Gaussen, est endémique stricte de l'ouest du Haut Atlas marocain. Cette espèce joue un rôle écologique et socio-économique très important dans son aire de distribution : pour produire du bois de feu, aider à protéger les sols contre l'érosion, et comme plante médicinale chez les populations locales. Malheureusement, subissant diverses pressions, principalement le surpâtage et les activités humaines excessives, l'espèce souffre d'une détérioration alarmante et de perte de territoire. La variabilité des champignons mycorhiziens arbusculaires et sa réponse à l'inoculation mycorhizienne des plantules sous serre ont été étudiées pour différentes provenances. Douze populations naturelles représentant l'aire de distribution du cyprès de l'Atlas ont été sondées, à Adebdi, Aghbar, Allous, Idni, Ighil, Ighzer, Mzouzit, Rikt, Tiouna, Taghzout, Talat N'os and Taws. L'analyse des variations des champignons mycorhiziens associés aux racines de cyprès de l'Atlas ont révélé la présence d'au moins sept morphotypes appartenant aux genres *Rhizophagus*, *Glomus*, *Gigaspora* et *Acaulospora*. Le taux de colonisation mycorhizienne dans les racines du cyprès de l'Atlas a été examiné dans son milieu naturel, montrant une variation de 25 % à 45 %. Dans les conditions sous serre, la croissance des plantules, avec ou sans inoculation avec un complexe mycrohizien, a été surveillée pour les plantules provenant des douze lieux échantillonnés. Les résultats montrent qu'en absence des champignons bénéfiques dans le substrat de culture, la croissance et la nutrition des plantules ont été hautement dépendantes de l'origine des graines. En revanche, en présence du champignon dans le substrat de culture, les paramètres de croissance et de nutrition des plantules des douze provenances ne différaient pas de manière significative. Deux groupes principaux ont été identifiés par l'analyse en composantes principales et la classification ascendante hiérarchique basées sur les caractères analysés. Le premier groupe comprend les populations de la vallée de N'Fis, à savoir Allous, Idni, Ighil, Ighzer, Mzouzit, Rikt, Talat N'os et Taws, alors que le second groupe est composé des populations du côté sud, Aghbar, Adebdi, Taghzout et Tiouna.

**Mots-clés :** *Cupressus atlantica*, *Rhizophagus*, *Glomus*, *Gigaspora*, *Acaulospora*, cyprès de l'Atlas, spores de champignons mycorhiziens, colonisation des racines, croissance des plants, Maroc.

## ABSTRACT

Seedling responses to arbuscular mycorrhizal inoculation of the Atlas cypress,
*Cupressus atlantica* Gaussen, from various
Moroccan Western High Atlas origins

The Atlas cypress, *Cupressus atlantica* Gaussen, is a strict endemic of the Moroccan Western High Atlas. The species plays a very important ecological and socio-economic role in its area of distribution: it is used in the production of firewood, helps to protect soils against erosion and is also used as a medicinal plant by the local population. Unfortunately, due to various pressures, mainly overgrazing and overuse by humans, the species is experiencing alarming deterioration and loss of territory. The variability of arbuscular mycorrhizal fungi in Atlas cypress of different origins and the response of its seedlings to mycorrhizal inoculation in greenhouse conditions were studied. Twelve natural populations representing the distribution area of the Atlas cypress were surveyed, at Adebdi, Aghbar, Allous, Idni, Ighil, Ighzer, Mzouzit, Rikt, Tiouna, Taghzout, Talat N'os and Taws. The analysis of variations in the mycorrhizal fungi associated with the roots of the Atlas cypress has shown the presence of at least seven morphotypes belonging to the *Rhizophagus*, *Glomus*, *Gigaspora* and *Acaulospora* genera. The rates of mycorrhizal colonization in the roots of the Atlas cypress examined in its ecosystem varied from 25% to 45%. In greenhouse conditions, the growth of seedlings from seeds originating in the twelve locations was monitored with and without inoculation of a mycorrhizal fungal complex. The results show that in the absence of these beneficial fungi in the culture substrate, the growth and nutrition of the seedlings was highly dependent on the origins of the seeds. However, in the presence of mycorrhizal fungi in the culture substrate, the growth and nutrition parameters for the seedlings from the twelve populations did not show significant differences. Two main groups were identified by principal component analysis and hierarchical classification based on all the characters analyzed. The first comprises the populations of the N'Fis valley, namely Allous, Idni, Ighil, Ighzer, Mzouzit, Rikt, Talat N'os and Taws, while the second group is made up of the populations from the southern side, namely Aghbar, Adebdi, Taghzout and Tiouna.

**Keywords:** *Cupressus atlantica*, *Rhizophagus*, *Glomus*, *Gigaspora*, *Acaulospora*, *Atlas* cypress, mycorrhizal fungi spores, root colonization, seedling growth, Morocco.

## RESUMEN

Respuesta a la inoculación de micorriza
arbuscular en el plantío de ciprés del Atlas,
*Cupressus atlantica* Gaussen, proveniente
de varios lugares del alto Atlas occidental
marroquí

El ciprés del Atlas, *Cupressus atlantica* Gaussen, es estrictamente endémico del alto Atlas occidental marroquí. La especie juega un rol ecológico y socioeconómico muy importante en esta área de distribución: se utiliza en la producción de leña, contribuye a la protección de suelos frente a la erosión y la población local también la utiliza como planta medicinal. Por varios motivos, principalmente el sobrepastorio y la actividad humana excesiva, la especie sufre un deterioro alarmante y una pérdida de territorio. Se estudió la variabilidad del hongo mycorrhiza arbuscular para diferentes orígenes del ciprés del Atlas, así como la respuesta de las plantaciones a la inoculación de micorriza en condiciones de invernadero. Se realizó un seguimiento de doce poblaciones naturales que representan el área de distribución del ciprés del Atlas: Adebdi, Aghbar, Allous, Idni, Ighil, Ighzer, Mzouzit, Rikt, Tiouna, Taghzout, Talat N'os y Taws. El análisis de la variación del hongo mycorrhiza associado a las raíces del ciprés del Atlas ha mostrado la presencia de al menos siete morfotipos pertenecientes a los géneros *Rhizophagus*, *Glomus*, *Gigaspora* y *Acaulospora*. Las tasas de colonización de micorriza en las raíces examinadas del ciprés del Atlas en su ecosistema varían entre el 25 % y el 45 %. En condiciones de invernadero, el crecimiento de los plantíos de semillas originales de las doce ubicaciones estudiadas se monitorizó con o sin inoculación de complejo fúngico micorrífico. Los resultados muestran que, en ausencia de estos hongos benéficos en el substrato de cultivo, el crecimiento y nutrición del plantío tenía una fuerte dependencia con el origen de las semillas. Sin embargo, con la presencia de hongos micorrícicos en el substrato de cultivo, los parámetros de crecimiento y nutrición entre plantíos originarios de las doce poblaciones no mostró diferencias significativas. El análisis del componente principal y la clasificación basada en todos los caracteres analizados permitieron identificar dos grupos principales. El primer grupo incluye las poblaciones del valle N'Fis, Allous, Idni, Ighil, Ighzer, Mzouzit, Rikt, Talat N'os y Taws; mientras que el segundo grupo incluye las poblaciones del lado sur, concretamente Aghbar, Adebdi, Taghzout y Tiouna.

**Palabras clave:** *Cupressus atlantica*,
*Rhizophagus*, *Glomus*, *Gigaspora*,
*Acaulospora*, ciprés del Atlas, esporas de
hongos micorrínicos, colonización de raíces,
crecimiento de plantío, Marruecos.
Introduction

Mycorrhizal symbioses are key elements in natural sys-
tems (Brundrett et al., 1991). They play a major role in water
absorption as well as in the uptake of important nutrients
(Kormanik et al., 1982; Fontana, 1985; Strullu, 1990). They
also contribute to the plants' protection against microbial
pathogens (Whipps, 2004; Pozo et al., 1999) while impro-
ving multi-trophic interactions within the soil. The symbiotic
relationship between trees and their mycorrhizal fungi thus
contributes to the establishment of the forest ecosystem and
to its longevity. The climate in the Mediterranean regions is
characterized by long periods of drought and hot summers;
precipitations are rare, irregular and often times torren-
tial (López-Bermúdez and Albaladejo, 1990; Vallejo et al.,
1999). These conditions render Mediterranean ecosystems
particularly precarious and highly sensitive to degradation;
this in turn causes the loss or reduction of mycorrhizal diver-
sity in the soil and the decrease of the mycorrhizal potential
in the affected areas (Mc Lellan et al., 1995). As a result, the
restoration of native plants in these regions is compromised
and subject to serious limitations (Requena et al., 2001).
The importance of myccorhizal fungi in the preservation of
biodiversity in forest ecosystems is indeed undeniable (Van
der heijden et al., 1998). Their ability to control the produc-
tivity and diversity of the seedlings has a direct effect on the
diversity of plant communities (Burrows and Pfleger, 2002;
Kernaghan et al., 2003). This mutual influence between the
plant and fungal communities can play a fundamental role
in the determination of the existing diversity of both plants
and fungi. Mycorrhizal symbioses affect the maintenance
of plant density in natural habitats (Requena et al., 2001;
Peterson et al., 1985) and modify the structure and function
of plant communities (Douds and Millner, 1999); they are
essential indicators of the evolution of ecosystems.

Mycorrhizal fungi play the role of “ecosystem engineers”
and can be used for the improvement of the productivity of
natural and anthropized ecosystems. However, the produc-
tion of high quality plants with high nutritional status and
resistance to biotic and abiotic stressors through the intro-
duction of selected mycorrhizal fungi can be challenging. To
circumvent these limitations, mycorrhizal fungi can be used
in combination with culture substrate in forest nurseries
which would allow the production of vigorous forest seedlings
capable of resisting in reforestation perimeters and withstan-
ding transplantation choc (Caravaca et al., 2005; Rincón et al.,
2006; Ouahmane et al., 2006). Indeed, in the Atlas Cypress,
the rates of reintroduction of young plants are mediocre,
which leads to increased costs of reforestation. These refores-
tation costs are further exacerbated by soil constraints: the
soils are highly calcareous, with low mineral concentration
and very little water content. These difficult environmental
conditions have a direct impact on the Atlas Cypress seed-
ling mortality. To alleviate these constraints, multiple strate-
gies can be employed to improve success rates, one of which
would be the mycorrhization of the Atlas Cypress seedlings in
forest nurseries. However, to ensure the success of the use
of beneficial fungi in an artificial inoculation program, the

characterization and proper selection of the mycorrhizal fungi
is of upmost importance (Francis and Thornes, 1990; Vallejo
et al., 1999). Therefore, as a first step in the controlled mycor-
rization of the Atlas Cypress, the population of mycorrhizal
fungi associated with the Atlas Cypress in its natural habitat
is analyzed in order to select the fungi capable of improving
the quality of Cypress seedlings. The study of the mycorhi-
zal fungi associated with the Atlas Cypress will focus on the
evaluation of the mycorrhizal infectivity potential of the soils
and the identification of the different species encountered.
The characterization of these populations is essential to the
implementation of a plant improvement program in nurseries
(Borchers and Perry, 1990; Miller et al., 1992; Roth and Berch,
1992; Ouahmane, 2007).

This study had the following objectives: (i) to estimate
the diversity of mycorrhizal fungi associated with the roots of
*Cupressus atlantica* in twelve different soils where the plant
is naturally found; (ii) to estimate the variability in forest nur-
series of the plants’ response to mycorrhizal inoculation of
young seedlings from seeds collected from the twelve diffe-
rent origins.

<figure><img src="image_6.png" /><figcaption>**Photo 2.**
Spores of Arbuscular Mycorrhizal fungi in soil under the Atlas Cypress tree.
Photo L. Ouahmane.</figcaption></figure>

<figure><img src="image_7.png" /><figcaption>**Photo 3.**
Atlas Cypress roots colonized by Arbuscular
Mycorrhizal Fungi.
Photo L. Ouahmane.</figcaption></figure>
# Materials and methods

## Seed collection

Atlas Cypress cone collection was conducted for 12 natural populations, representative of the natural distribution, in September 2010. Composite samples of cones from terminal branches of 10 different trees were randomly selected in each of the 12 origins: Adebdi, Aghbar, Allous, Idni, Ighil, Ighzer, Mzouzit, Rikt, Tiouna, Taghzout, Talat N'os and Taws (table I).

## Soil sampling

For each origin, 10 kg of rhizospheric soil were randomly collected at depths varying between 10 cm and 40 cm. In total, 10 samples were collected for each origin of the Atlas Cypress. The diversity and abundance of mycorrhizal fungi associated with the Atlas Cypress were evaluated. In the same samples, roots were examined in order to estimate the extent of root infection by the mycorrhizal fungi.

## Extraction and identification of the populations of mycorrhizal fungi in the soil

The extraction of spores from the collected soils was conducted in two steps; the first involves the isolation of spores from the soil through a water sieving and decantation technique (Gerdemann and Nicholson, 1963) whereas the second involves the use of centrifugation in a 65% saccharose solution to isolate the spores (Brundrett et al., 1985).

<table><caption>**Table I.**
Geographical characteristics of the different Atlas Cypress provenances.</caption><thead><tr><td>Collection site</td><td>Latitude/ Longitude</td><td>Altitude (m)</td></tr></thead><tbody><tr><td>Adebdi</td><td>N 30°50' / W 08°29'</td><td>1300</td></tr><tr><td>Aghbar</td><td>N 30°55' / W 08°22'</td><td>1750</td></tr><tr><td>Alouss</td><td>N 30°59' / W 08°19'</td><td>1650</td></tr><tr><td>Idni</td><td>N 30°54' / W 08°17'</td><td>1600</td></tr><tr><td>Ighil</td><td>N 30°57' / W 08°15'</td><td>1315</td></tr><tr><td>Ighzer</td><td>N 30°55' / W 08°17'</td><td>1551</td></tr><tr><td>Mzouzit</td><td>N 30°57' / W 08°14'</td><td>1280</td></tr><tr><td>Rikt</td><td>N 30°59' / W 08°11'</td><td>1173</td></tr><tr><td>Tiouna</td><td>N 30°52' / W 08°53'</td><td>1581</td></tr><tr><td>Taghzout</td><td>N 30°54' / W 08°51'</td><td>1839</td></tr><tr><td>Talat N'os</td><td>N 31°50' / W 08°08'</td><td>1150</td></tr><tr><td>Taws</td><td>N 30°56' / W 08°16'</td><td>1391</td></tr></tbody></table>

## Spores isolation

After thorough homogenization of each soil specimen, 100 g were screened through a series of sieves (800 µm and 50 µm) under water jets. The resulting sample was transferred to a 200 mL beaker, the beaker was filled with water and the solution was allowed a 15 min. sedimentation time after which the supernatant and floating particles were eliminated. The soil-water solution was centrifuged at 3000 rpm for 5 min., the supernatant was removed and replaced by a 65% saccharose solution (w:v) and the resulting mixture of sediments and saccharose was once again centrifuged at 1000 rpm for 3 min. The supernatant was filtered under vacuum filtration using Wathman filters. The spores were then isolated using a thin brush under a binocular magnifier and carefully washed then preserved in physiological liquid. The obtained spores were examined and counted under a binocular magnifier at 40x and the different morphotypes were classified and identified at the species level and for some, at the genus level. The isolated spores were conserved in polyvinyl-lacto-glycerin (PVLG) which allows the preservation of the spores' initial features, their long term conservation and better visualization of the spore walls (Estaun et al., 1997).

## Identification of arbuscular mycorrhizal fungi

The spores were separated under binocular magnification according to the phenotypic characteristics: color, shape, consistency, attachment to hyphae. The diameters of the spores were measured under a microscope at 40x magnification along with the thickness of the wall and the attached hyphae. The measurements were conducted with a microscope connected to digital image processing software and the essential characteristics of the spores and associated hyphae were evaluated: spore wall thickness, lamellar structure, shape of the attachment zone between the spore and hypha, thickness of the hyphae and its wall structure. Direct count of the number of spores in 100 g of soil was also conducted in order to estimate their abundance in the soil as well as the relative abundance of each spore type in relation to the total number of spores.

In order to identify the fungi, about 20 spores of each morphotype recovered from the collected soils were placed on a microscope slide with a cover slip. Half was placed in a 1:1 volume mixture of PVLG and Melzer reagent, while the other half was placed in PVLG. The original descriptions of the species as well as the descriptions available on the INVAM website (1997) served as a reference for species identification. The morphological characteristics of the spores were compared to the type specimens and reference strains.

## Determination of the infection rate of the Atlas Cypress roots by mycorrhizal fungi

In order to clearly determine the presence of endomycorrhizal structures, the cellular contents of the roots were eliminated and stained with a specific coloring agent such as acid Fuchsin or Trypan blue. The finest roots from each origin were mixed and distributed into four batches that would each be used to prepare one slide. To test for the presence of an endomycorrhizal infection, the roots of the collected
plants were colored according to the Phillips and Hayman method (1970). For each origin of the Atlas Cypress, fine roots were collected from 10 randomly selected trees at 2 m from the trunk basis, the minimal distance between selected trees was fixed beforehand to at least 100 m. The Atlas Cypress roots were easily recognized thanks to their very high abundance and their horizontal distribution under the tree canopy. After thorough washing, the roots were placed in 10% Potassium hydroxide solution (KOH) for 60 min. at 90°C; this allows the plant cells to be rid of their cytoplasm and pigments. The roots were then washed, placed in 5% lactic acid solution in order to neutralize the remaining potassium hydroxide solution then rewashed and rinsed in room temperature water. The roots were then washed with distilled water and placed in 0.05% Trypan blue solution diluted in lactoglycerol (1/3 water, 1/3 glycerol, 1/3 lactic acid) for 15 min. at 90°C. After cooling, the roots were rinsed then conserved in lactoglycerol solution. Fungal structures such as arbuscules, vesicles and mycelia, were then blue colored. Roots were cut into about 1 cm in length fragments and two squash slides were prepared from 40 randomly selected fragments. The slides were then observed under a microscope at 40x. By counting the number of fragments displaying arbuscules and/or vesicles in relationship to the total number of fragments, infection rate was estimated. In order to estimate the endomycorrhizal colonization of the roots, the method described by Trouvelot *et al.* (1986) was employed. Overall, we have estimated infection parameters such as mycorrhizal infection frequency, defined as the percentage of mycorrhized root fragments relative to the total number of observed fragments, and the intensity of the infection developed by the entire root system.

## Production of the inoculum

The arbuscular mycorrhizal inoculum production was conducted in a glasshouse. Maize (Zea mays L.), a highly mycrotrophic plant, was used as an endophyte for the production of endomycorrhizal inoculum for the indigenous mycorrhizal complex naturally associated with the Atlas Cypress. Maize seeds were sterilized in 30% oxygenated water for 30 min. then rinsed multiple times with distilled water. They were then set for germination in plastic pots containing 1.5 kg of rhizospheric soil collected around the roots of Atlas Cypress in the 12 origins, the pots were watered regularly. The mycorrhization status of the trapped plants was then verified under a binocular magnifier through nondestructive observation at 40x.

## Seed pregermination and seedlings inoculation

The Atlas Cypress cones (Cupressus atlantica Gaussen) were collected from the 12 origins during the month of September. The seeds were then extracted and separated according to their origin, disinfected in 90% bleach solution diluted to 50% for 3 to 4 min., thoroughly rinsed, then placed in sterile water for 30 min. They were then subjected to scarification pretreatment. The pretreated seeds were then placed in Petri dishes (30 seeds per dish) containing

filter paper washed with 5 mL of sterile water. The petri dishes were then incubated for 10 to 15 days in the dark at 20°C, which is considered to be the species' optimal temperature for germination (Bechir, 2004). After germination had occurred and rootlets were visible, the pregerminated seeds were planted in pots. After 3 months of growth, the Maize roots were harvested and disinfected in a mixture of chlorine T (2%) and streptomycin 0,2 g/L (Mosse, 1973) then rinsed with sterile distilled water. They were then cut into 1 cm fragments and used as inoculum for young seedlings of the Atlas Cypress. The Maize root fragments used for inoculation had a 75% mycorrhization rate according the method developed by Trouvelot *et al.* (1986) and contained approximately 250 vesicles/cm. Once the rootlets had reached length of 1 to 2 cm, the seeding was conducted in plastic pots (1 L volume) containing a mixture of sand and peat with a 2:1 volume ratio. The sand was run through a screen with a mesh size of 5 mm then covered with water to which 1 L of commercial bleach solution was added and left for 3 days. The sand was then thoroughly rinsed then sterilized for 3 hours in an autoclave at 120°C; the peat was also autoclaved for 3 hours at the same temperature. The inoculation of pregerminated seeds was done with the addition of 1 g of Maize root fragments in the dip (1 cm x 5 cm) where the pregerminated seeds were sown. The control plants that did not receive the inoculum and the pregerminated seeds were placed directly on the substrate with the addition of fungi. The seedlings were grown in a greenhouse at a temperature of 27 ± 2°C, a 65% relative humidity and a photoperiod of 16/8 (day/night). They were watered regularly without fertilizers addition. The trials were conducted using a randomized block design with 40 repetitions per treatment.

## Dendrometric parameters

During seedlings growth under greenhouse conditions, dendrometric parameters were measured: the height of the different resulting seedlings from the bottom of the crown to the apex, and the diameter of the stem. After six months of culture, the shoot dry biomass (SDB) and the root dry biomass (RDB) were reported. The dry weights were measured after the drying process was conducted in a heat chamber at 65°C for 72 hours.

## Mineral nutrition of the Atlas Cypress seedlings: N, P, K

Three mineral elements were used in the analysis of the nutritional effect of the mycorrhizal fungi on the growth of the Atlas Cypress. Considering its importance as limiting factor in plant production, the total phosphorus in the seedlings was measured by applying a colorimetric method after calcination of the Atlas Cypress plants using Barton reagent containing ammonium molybdate and ammonium metavanadate (NH₄VO₃). The reading was done using a spectrophotometer at 820 nm with the use of a standard range using Potassium dihydrogen phosphate (KH₂PO₄). The total nitrogen was measured using the Kjeldahl method and the potassium was measured in the mineral solution obtained from the calcination using a colorimetric technique and flame emission spectrometry.
## Statistical analysis

In order to compare the heterogeneity of the different parameters measured in the population, a variance analysis (ANOVA) was conducted for each factor using Windows SPSS software (version 12.0). The averages were compared using the Newman Keuls test with 0.05 probability threshold for each variance analysis series. For each morphological characteristic, the correlations between the averages of the populations were analyzed using a correlation table. In order to understand the overall relationships within the population, a principal component analysis (PCA) was conducted associated with an Ascending Hierarchical Classification (AHC). A Pearson 'chi-squared test' was used to check the dependence between Mycorrhizal frequency and Mycorrhizal colonization of roots from different origins.

## Results

### Diversity of mycorrhizal fungi

In the analysis of the diversity of mycorrhizal fungi associated with the Atlas Cypress in the twelve surveyed origins, seven taxa were identified. *Rhizophagus aggregatus* was the dominant species in this study followed by *Rhizophagus fasciculatus*, *Rhizophagus intraradices*, and *Rhizophagus manihotis* (*Rhizophagus clarus*), three other unidentified species belonging to the *Glomus*, *Gigaspora* and *Acaulospora* genera were also encountered. The number of spores per 100 g of soil varied between 100 spores at the Talat N'os station and 608 spores at the Mzouzit station (table II). Additionally, it was determined that each of the

six sites Aghbar, Allous, Idni, Ighil, Ighzer and Mzouzit, were home to seven species, the Rikt site to six species whereas, the Taghzout and Tiouna sites were home to five species. Four mycorrhizal species were found at the Adebdi and Talat N'os sites and only three species were found in the Taws site which had the lowest number of mycorrhizal species encountered in this study (table III). Dominance analysis had shown that the species relative importance was similar in soils of the major of provenances except for the Mzouzit station which presents in addition the higher total number of spores/100 g soil (table III). Furthermore, this same result was noted when analyzing the equitability that concerned distribution of individuals per species (table III).

### Root mycorrhizal colonization

Examination of the roots of Atlas Cypress trees from the different locations has shown that 35% of the examined roots from the Tiouna site display mycorrhizal infections, at the Allous site, 70% of the roots were infected. On average, 55% of the examined roots from all origins showed signs of mycorrhizal infection. In order to estimate the total volume occupied by the fungi in the roots, mycorrhizal infection intensity was estimated through microscopic observation of the specifically stained root fragments. The results indicate that roots from the Talat N'os sites show 20% colonization intensity, whereas roots from the Rikt and Ighil sites had a colonization intensity of 45%, the average value for all 12 sites was 32.2%. Nevertheless the Pearson 'chi-squared test' had shown no dependence between Mycorrhizal frequency and Mycorrhizal colonization of roots from different origins (table IV).

<caption>**Table II.**</caption>

Abundance and diversity of arbuscular mycorrhizal fungi in the rhizospheric soils of the Atlas Cypress trees from twelve provenances. In the same column, numbers followed by the same letter are not considered to be significantly different according to the Newman and Keuls method with a 0.05 threshold.

<table><thead><tr><th>Sites</th><th>Rhizophagus aggregatus</th><th>Rhizophagus fasciculatus</th><th>Rhizophagus intraradices</th><th>Rhizophagus clarus</th><th>Glomus sp.</th><th>Gigaspora sp.</th><th>Acaulospora sp.</th><th>Total number of spores/100g soil</th></tr></thead><tbody><tr><td>Adebdi</td><td>64<sup>d</sup></td><td>92<sup>a</sup></td><td>0<sup>f</sup></td><td>0<sup>d</sup></td><td>160<sup>a</sup></td><td>0<sup>d</sup></td><td>4<sup>b</sup></td><td>320</td></tr><tr><td>Aghbar</td><td>24<sup>e</sup></td><td>104<sup>a</sup></td><td>36<sup>d</sup></td><td>8<sup>c</sup></td><td>48<sup>b</sup></td><td>48<sup>a</sup></td><td>8<sup>b</sup></td><td>276</td></tr><tr><td>Allous</td><td>96<sup>c</sup></td><td>48<sup>c</sup></td><td>24<sup>e</sup></td><td>20<sup>b</sup></td><td>32<sup>b</sup></td><td>52<sup>a</sup></td><td>8<sup>b</sup></td><td>280</td></tr><tr><td>Idni</td><td>124<sup>c</sup></td><td>92<sup>a</sup></td><td>52<sup>c</sup></td><td>24<sup>b</sup></td><td>24<sup>b</sup></td><td>36<sup>a</sup></td><td>12<sup>a</sup></td><td>364</td></tr><tr><td>Ighil</td><td>112<sup>c</sup></td><td>80<sup>b</sup></td><td>72<sup>b</sup></td><td>24<sup>b</sup></td><td>8<sup>c</sup></td><td>12<sup>c</sup></td><td>8<sup>b</sup></td><td>316</td></tr><tr><td>Ighzer</td><td>44<sup>d</sup></td><td>52<sup>c</sup></td><td>136<sup>a</sup></td><td>32<sup>a</sup></td><td>28<sup>b</sup></td><td>32<sup>a</sup></td><td>16<sup>a</sup></td><td>340</td></tr><tr><td>Mzouzit</td><td>424<sup>a</sup></td><td>32<sup>c</sup></td><td>60<sup>b</sup></td><td>44<sup>a</sup></td><td>32<sup>b</sup></td><td>4<sup>c</sup></td><td>12<sup>a</sup></td><td>608</td></tr><tr><td>Rikt</td><td>172<sup>b</sup></td><td>76<sup>b</sup></td><td>44<sup>c</sup></td><td>12<sup>c</sup></td><td>8<sup>c</sup></td><td>8<sup>c</sup></td><td>0<sup>c</sup></td><td>320</td></tr><tr><td>Taghzout</td><td>52<sup>d</sup></td><td>40<sup>c</sup></td><td>32<sup>d</sup></td><td>52<sup>a</sup></td><td>0<sup>d</sup></td><td>8<sup>c</sup></td><td>0<sup>c</sup></td><td>184</td></tr><tr><td>Talat N'os</td><td>16<sup>e</sup></td><td>32<sup>c</sup></td><td>36<sup>d</sup></td><td>16<sup>c</sup></td><td>0<sup>d</sup></td><td>0<sup>d</sup></td><td>0<sup>c</sup></td><td>100</td></tr><tr><td>Taws</td><td>52<sup>d</sup></td><td>28<sup>c</sup></td><td>0<sup>f</sup></td><td>0<sup>d</sup></td><td>0<sup>d</sup></td><td>24<sup>b</sup></td><td>0<sup>c</sup></td><td>104</td></tr><tr><td>Tiouna</td><td>104<sup>c</sup></td><td>88<sup>a</sup></td><td>16<sup>e</sup></td><td>44<sup>a</sup></td><td>8<sup>c</sup></td><td>0<sup>d</sup></td><td>0<sup>c</sup></td><td>260</td></tr><tr><td>Total Number of fungal spores /100 g soil</td><td>1284</td><td>764</td><td>508</td><td>276</td><td>348</td><td>224</td><td>68</td><td>3472</td></tr></tbody></table>
<table><caption>**Table III.**

Diversity analysis of arbuscular mycorrhizal fungi from twelve different origins of the Atlas Cypress.</caption><thead><tr><th rowspan="2"></th><th colspan="10">Sites</th></tr><tr><th>Adebdi</th><th>Aghbar</th><th>Allous</th><th>Idni</th><th>Ighil</th><th>Ighzer</th><th>Mzouzit</th><th>Rikt</th><th>Taghzout</th><th>Talat N'os</th><th>Taws</th><th>Tiouna</th></tr></thead><tbody><tr><th>Total number of<br>morphotypes per origin</th><td>4</td><td>7</td><td>7</td><td>7</td><td>7</td><td>7</td><td>7</td><td>6</td><td>5</td><td>4</td><td>3</td><td>5</td></tr><tr><th>Total number of<br>spores/100 g soil</th><td>320</td><td>276</td><td>280</td><td>364</td><td>316</td><td>340</td><td>608</td><td>320</td><td>184</td><td>100</td><td>104</td><td>260</td></tr><tr><th>Dominance (species<br>relative importance)</th><td>0.3728</td><td>0.2287</td><td>0.2078</td><td>0.2199</td><td>0.2501</td><td>0.2269</td><td>0.5073</td><td>0.3669</td><td>0.2391</td><td>0.2832</td><td>0.3757</td><td>0.3079</td></tr><tr><th>Shannon indice</th><td>1.082</td><td>1.66</td><td>1.731</td><td>1.692</td><td>1.559</td><td>1.713</td><td>1.09</td><td>1.256</td><td>1.487</td><td>1.319</td><td>1.038</td><td>1.313</td></tr><tr><th>Simpson indice</th><td>0.6272</td><td>0.7713</td><td>0.7922</td><td>0.7801</td><td>0.7499</td><td>0.7731</td><td>0.4927</td><td>0.6331</td><td>0.7609</td><td>0.7168</td><td>0.6243</td><td>0.6921</td></tr><tr><th>Equitability (Distribution<br>of individual/Species)</th><td>0.7802</td><td>0.8528</td><td>0.8893</td><td>0.8697</td><td>0.8009</td><td>0.8801</td><td>0.5604</td><td>0.7007</td><td>0.9236</td><td>0.9513</td><td>0.945</td><td>0.8155</td></tr></tbody></table>

<table><caption>**Table IV.**

Mycorrhizal frequency and roots colonization intensity from the twelve provenances of the Atlas Cypress. In the same column, numbers followed by the same letter are not considered to be significantly different according to the Newman and Keuls method with a 0.05 threshold.</caption><thead><tr><th>Sites</th><th>Frequency %</th><th>Root Colonization %</th></tr></thead><tbody><tr><td>Adebdi</td><td>65<sup>c</sup></td><td>35<sup>b</sup></td></tr><tr><td>Aghbar</td><td>50<sup>d</sup></td><td>35<sup>b</sup></td></tr><tr><td>Allous</td><td>70<sup>b</sup></td><td>30<sup>c</sup></td></tr><tr><td>Idni</td><td>60<sup>d</sup></td><td>35<sup>b</sup></td></tr><tr><td>Ighil</td><td>55<sup>d</sup></td><td>45<sup>a</sup></td></tr><tr><td>Ighzer</td><td>85<sup>a</sup></td><td>30<sup>c</sup></td></tr><tr><td>Mzouzit</td><td>65<sup>c</sup></td><td>35<sup>b</sup></td></tr><tr><td>Rikt</td><td>45<sup>e</sup></td><td>45<sup>a</sup></td></tr><tr><td>Taghzout</td><td>45<sup>e</sup></td><td>35<sup>b</sup></td></tr><tr><td>Talat N'os</td><td>45<sup>e</sup></td><td>25<sup>d</sup></td></tr><tr><td>Taws</td><td>40<sup>f</sup></td><td>20<sup>e</sup></td></tr><tr><td>Tiouna</td><td>35<sup>f</sup></td><td>35<sup>b</sup></td></tr><tr><td>Average</td><td>55</td><td>32,25</td></tr></tbody></table>

### Growth parameters

In the analysis of the effect of the seed origin on growth parameters in the absence of any other treatment, it was observed that the height and shoot dry biomass (SDB) were the two parameters influenced by the origin. The height of the plants varied between 8.12 cm at the Ighzer site and 14.37 cm at the Adebdi site, whereas the shoot dry biomass

varied between 0.72 mg per plant for the seedlings from the Tiouna and Taghzout sites and 1.57 mg per plant for the seedlings from the Aghbar site. The analysis of nutritional parameters (total nitrogen and phosphorus in the plants) has shown significant differences between plants resulting from seeds of different origins. The total nitrogen content of the seedlings varied between 0.65 mg per plant in the Taws site and 1 mg per plant for most other sites. On the other hand, the total phosphorus content of the seedlings varied between 0.3 mg per plant for the Adebdi and Aghbar sites and 0.75 mg for the Ighil, Ighzer and Taghzout sites.

Growth parameters of the six-month-old greenhouse plants resulting from seeds that were inoculated by the functional complex isolated from the rhizospheric soil of the Atlas Cypress in the twelve sites had shown that the effect of the origin was not significant for all the analyzed parameters (table V). In fact, in the presence of mycorrhizal fungi following inoculation in the greenhouse cultures, the observed differences between seedlings of different origins were no longer present (table V). The parameters that showed the most significant effect were the shoot wet biomass and the shoot dry biomass whereas, the height and stem diameter were not influenced by the origin of the seeds in the presence of mycorrhizal fungi (table V). Additionally, it was shown that phosphorus content varied from one origin to another whereas the nitrogen content was less influenced.

### Mycorrhization parameters

After six months growth under greenhouse conditions, seedlings from the 12 different origins, displayed that mycorrhization frequency, root colonization and mycorrhizal dependence varied greatly from one origin to another. The observation of the root system of the seedlings has shown that over 60% of the examined root fragments presented signs of mycorrhizal infection. In the seedlings grown from seeds collected in Allous, Ighzer and Mzouzit, all the analyzed fragments have displayed at least one sign of the presence of mycorrhizal structures. The average root
<caption>**Table V.**</caption>

Effects of the Mycorrhizal inoculation on the growth and nutrition status of seedlings from the 12 provenances of *Cupressus atlantica* after 6 months of growth under greenhouse conditions. My = Mycorrhizal inoculation, NMy = No Mycorrhizal inoculation, H: Height (cm), D = Stem diameter (cm), WAGB = Wet above ground Biomass, WRB = Wet root Biomass, DAGB = Dry above ground Biomass, DRB = Dry root Biomass, N = Total Nitrogen (mg/plant), P = Total Phosphorus (mg/plant). Numbers followed by the same letter are not considered to be significantly different according to the Newman and Keuls method with a 0.05 threshold.

<table><thead><tr><th>Sites</th><th>Treatment</th><th>H</th><th>D</th><th>WAGB</th><th>WRB</th><th>DAGB</th><th>DRB</th><th>N</th><th>P</th></tr></thead><tbody><tr><td rowspan="2">Adebdi</td><td>My</td><td>16.75<sup>a</sup></td><td>3.06<sup>a</sup></td><td>2.8<sup>a</sup></td><td>3.87<sup>a</sup></td><td>1.92<sup>a</sup></td><td>1.8<sup>a</sup></td><td>1.25<sup>a</sup></td><td>0.52<sup>a</sup></td></tr><tr><td>Nmy</td><td>14.37<sup>b</sup></td><td>2.8<sup>b</sup></td><td>2.52<sup>b</sup></td><td>3.32<sup>b</sup></td><td>1.55<sup>b</sup></td><td>1.45<sup>b</sup></td><td>0.7<sup>b</sup></td><td>0.3<sup>b</sup></td></tr><tr><td rowspan="2">Aghbar</td><td>My</td><td>13.12<sup>a</sup></td><td>2.9<sup>a</sup></td><td>2.8<sup>a</sup></td><td>3.15<sup>a</sup></td><td>1.85<sup>a</sup></td><td>1.37<sup>a</sup></td><td>0.85<sup>a</sup></td><td>0.75<sup>a</sup></td></tr><tr><td>Nmy</td><td>12.37<sup>b</sup></td><td>2.4<sup>b</sup></td><td>2.75<sup>a</sup></td><td>2.25<sup>b</sup></td><td>1.57<sup>b</sup></td><td>1.17<sup>b</sup></td><td>0.67<sup>b</sup></td><td>0.3<sup>b</sup></td></tr><tr><td rowspan="2">Allous</td><td>My</td><td>14.5<sup>a</sup></td><td>3.05<sup>a</sup></td><td>2.47<sup>a</sup></td><td>3.25<sup>a</sup></td><td>1.95<sup>a</sup></td><td>2.47<sup>a</sup></td><td>1.75<sup>a</sup></td><td>0.7<sup>a</sup></td></tr><tr><td>Nmy</td><td>11.25<sup>b</sup></td><td>2.86<sup>a</sup></td><td>2.22<sup>b</sup></td><td>3.17<sup>b</sup></td><td>0.95<sup>b</sup></td><td>1.82<sup>b</sup></td><td>1<sup>b</sup></td><td>0.37<sup>b</sup></td></tr><tr><td rowspan="2">Idni</td><td>My</td><td>16<sup>a</sup></td><td>2.67<sup>a</sup></td><td>2.72<sup>a</sup></td><td>2.6<sup>a</sup></td><td>2.37<sup>a</sup></td><td>1.72<sup>a</sup></td><td>2<sup>a</sup></td><td>1<sup>a</sup></td></tr><tr><td>Nmy</td><td>8.12<sup>b</sup></td><td>2.58<sup>b</sup></td><td>1.82<sup>b</sup></td><td>2.52<sup>a</sup></td><td>1.35<sup>b</sup></td><td>1.17<sup>b</sup></td><td>0.62<sup>b</sup></td><td>0.6<sup>b</sup></td></tr><tr><td rowspan="2">Ighil</td><td>My</td><td>15.6<sup>a</sup></td><td>2.9<sup>a</sup></td><td>3.05<sup>a</sup></td><td>3<sup>a</sup></td><td>2.8<sup>a</sup></td><td>1.85<sup>a</sup></td><td>1.55<sup>a</sup></td><td>0.87<sup>a</sup></td></tr><tr><td>Nmy</td><td>12.8<sup>b</sup></td><td>2.7<sup>a</sup></td><td>2.32<sup>b</sup></td><td>2.45<sup>b</sup></td><td>1.8<sup>b</sup></td><td>1.25<sup>b</sup></td><td>0.85<sup>b</sup></td><td>0.75<sup>b</sup></td></tr><tr><td rowspan="2">Ighzer</td><td>My</td><td>16<sup>a</sup></td><td>2.67<sup>a</sup></td><td>2.72<sup>a</sup></td><td>2.6<sup>a</sup></td><td>2.37<sup>a</sup></td><td>1.72<sup>a</sup></td><td>1.75<sup>a</sup></td><td>0.8<sup>a</sup></td></tr><tr><td>Nmy</td><td>8.12<sup>b</sup></td><td>2.58<sup>a</sup></td><td>1.82<sup>b</sup></td><td>2.52<sup>b</sup></td><td>1.37<sup>b</sup></td><td>1.17<sup>b</sup></td><td>1<sup>b</sup></td><td>0.75<sup>a</sup></td></tr><tr><td rowspan="2">Mzouzit</td><td>My</td><td>14.6<sup>a</sup></td><td>2.8<sup>a</sup></td><td>2.2<sup>a</sup></td><td>2.95<sup>a</sup></td><td>1.95<sup>a</sup></td><td>1.42<sup>a</sup></td><td>1.35<sup>a</sup></td><td>0.75<sup>a</sup></td></tr><tr><td>Nmy</td><td>8.5<sup>b</sup></td><td>2.17<sup>b</sup></td><td>1.07<sup>b</sup></td><td>1.37<sup>b</sup></td><td>0.95<sup>b</sup></td><td>1<sup>b</sup></td><td>0.82<sup>b</sup></td><td>0.37<sup>b</sup></td></tr><tr><td rowspan="2">Rikt</td><td>My</td><td>15.25<sup>a</sup></td><td>3.5<sup>a</sup></td><td>2.9<sup>a</sup></td><td>2.15<sup>a</sup></td><td>1.67<sup>a</sup></td><td>0.82<sup>a</sup></td><td>1.55<sup>a</sup></td><td>0.75<sup>a</sup></td></tr><tr><td>Nmy</td><td>12<sup>b</sup></td><td>3.29<sup>b</sup></td><td>2.3<sup>a</sup></td><td>2<sup>b</sup></td><td>0.75<sup>b</sup></td><td>0.67<sup>b</sup></td><td>1<sup>b</sup></td><td>0.35<sup>b</sup></td></tr><tr><td rowspan="2">Taghzout</td><td>My</td><td>13.25<sup>a</sup></td><td>2.87<sup>a</sup></td><td>2.2<sup>a</sup></td><td>2.35<sup>a</sup></td><td>0.84<sup>a</sup></td><td>0.55<sup>a</sup></td><td>1.82<sup>a</sup></td><td>1<sup>a</sup></td></tr><tr><td>Nmy</td><td>12.25<sup>b</sup></td><td>2.57<sup>b</sup></td><td>1.8<sup>a</sup></td><td>2.15<sup>b</sup></td><td>0.72<sup>b</sup></td><td>0.45<sup>a</sup></td><td>0.87<sup>b</sup></td><td>0.75<sup>b</sup></td></tr><tr><td rowspan="2">Talat N'os</td><td>My</td><td>14.25<sup>a</sup></td><td>2.57<sup>a</sup></td><td>2.85<sup>a</sup></td><td>2.8<sup>a</sup></td><td>1.2<sup>a</sup></td><td>0.95<sup>a</sup></td><td>1.82<sup>a</sup></td><td>0.77<sup>a</sup></td></tr><tr><td>Nmy</td><td>12<sup>b</sup></td><td>2.51<sup>a</sup></td><td>2.18<sup>b</sup></td><td>2.57<sup>b</sup></td><td>0.8<sup>b</sup></td><td>0.67<sup>b</sup></td><td>1<sup>b</sup></td><td>0.35<sup>b</sup></td></tr><tr><td rowspan="2">Taws</td><td>My</td><td>13.25<sup>a</sup></td><td>2.58<sup>a</sup></td><td>2.15<sup>a</sup></td><td>2.8<sup>a</sup></td><td>1.2<sup>a</sup></td><td>0.97<sup>a</sup></td><td>2<sup>a</sup></td><td>1.25<sup>a</sup></td></tr><tr><td>Nmy</td><td>12<sup>b</sup></td><td>2.51<sup>a</sup></td><td>1.8<sup>b</sup></td><td>2.57<sup>b</sup></td><td>0.8<sup>b</sup></td><td>0.67<sup>b</sup></td><td>0.65<sup>b</sup></td><td>0.55<sup>b</sup></td></tr><tr><td rowspan="2">Tiouna</td><td>My</td><td>14<sup>a</sup></td><td>2.9<sup>a</sup></td><td>2.7<sup>a</sup></td><td>2.35<sup>a</sup></td><td>0.86<sup>a</sup></td><td>0.55<sup>a</sup></td><td>1.35<sup>a</sup></td><td>0.85<sup>a</sup></td></tr><tr><td>Nmy</td><td>12.25<sup>b</sup></td><td>2.6<sup>b</sup></td><td>2.2<sup>b</sup></td><td>2.15<sup>b</sup></td><td>0.72<sup>a</sup></td><td>0.35<sup>b</sup></td><td>1<sup>b</sup></td><td>0.55<sup>b</sup></td></tr></tbody></table>

mycorrhizal colonization intensity for all examined seedlings has a value of 46.6%, it varied between 28.5% for Taws originated seedlings and 59.1% for the Allous originated seedlings. Mycorrhizal dependence expressed by the seedlings from the difference origins had an average value of 37.2%; it varied between 16% for the Tiouna origin and 71% for the Mzouzit origin (table VI).

## Global analysis of the response to natural or artificial mycorrhizal inoculation of the Atlas Cypress

Principal component analysis (PCA) of the effect of seeds origin and mycorrhization on growth parameters, nutritional status, root colonization by mycorrhizal fungi and mycorrhizal dependence of the seedlings from different origins (figure 1) allowed the classification of the provenances into two main groups. The first group was made of the following locations: Taws, Taghzout, Tiouna, Talat N'os, Adebdi

and Aghbar, whereas the Rikt, Ighzer, Ighil, Idni, Allous and Mzouzit provenance made up the second group. In fact, mycorrhizal dependence, which indicates the overall mycorrhization effect on growth and biomass production, showed a clear distinction between the two groups: mycorrhizal dependency varied between 15 and 33% in the first group and 43 and 71% in the second group (table VI).

In order to classify the different provenances based on their response to growth in a forest greenhouse, a hierarchical data analysis based on the similarities between the different regions was conducted. The results allowed the establishment of two distinct groups. The first group was made of the Tiouna, Aghbar, Taghzout and Adebdi provenances which correspond, from a geographic perspective, mainly to the high N'Fis valley. The second group correspond to the low and middle valley of N'Fis and is made of the Mzouzit, Allous, Idni, Talat N'os, Taws, Ighzer, Ighil and Rikt locations (figure 2).
<caption>**Table VI.**</caption>

Mycorrhization frequency, root colonization and mycorrhizal dependence in 12 provenances of *C. atlantica* after six months growth under greenhouse conditions. Numbers followed by the same letter are not considered to be significantly different according to the Newman and Keuls method with a 0.05 threshold.

<table><thead><tr><th>Sites</th><th>Mycorrhizal frequency (%)</th><th>Root colonization (%)</th><th>Mycorrhizal dependence (%)</th></tr></thead><tbody><tr><td>Adebdi</td><td>85<sup>d</sup></td><td>45<sup>c</sup></td><td>19.2<sup>f</sup></td></tr><tr><td>Aghbar</td><td>80<sup>e</sup></td><td>47.5<sup>c</sup></td><td>15.1<sup>g</sup></td></tr><tr><td>Allous</td><td>100<sup>a</sup></td><td>59.12<sup>a</sup></td><td>51<sup>c</sup></td></tr><tr><td>Idni</td><td>90<sup>c</sup></td><td>45.5<sup>c</sup></td><td>43<sup>d</sup></td></tr><tr><td>Ighil</td><td>85<sup>d</sup></td><td>52<sup>b</sup></td><td>53<sup>b</sup></td></tr><tr><td>Ighzer</td><td>100<sup>a</sup></td><td>46.75<sup>c</sup></td><td>43<sup>d</sup></td></tr><tr><td>Mzouzit</td><td>100<sup>a</sup></td><td>38<sup>d</sup></td><td>71<sup>a</sup></td></tr><tr><td>Rikt</td><td>95<sup>b</sup></td><td>61<sup>a</sup></td><td>55<sup>b</sup></td></tr><tr><td>Taghzout</td><td>70<sup>g</sup></td><td>52<sup>b</sup></td><td>14<sup>g</sup></td></tr><tr><td>Talat N'os</td><td>75<sup>f</sup></td><td>29<sup>e</sup></td><td>33<sup>e</sup></td></tr><tr><td>Taws</td><td>60<sup>h</sup></td><td>28.5<sup>e</sup></td><td>33<sup>e</sup></td></tr><tr><td>Tiouna</td><td>60<sup>h</sup></td><td>55<sup>b</sup></td><td>16<sup>g</sup></td></tr><tr><td>Average</td><td>83.34</td><td>46.61</td><td>37.19</td></tr></tbody></table>

<figure><img src="image_8.png" /><figcaption>**Figure 2.**</figcaption></figure>

Hierarchical Analysis of growth parameters, nutritional status and mycorrhizal root colonization of the seedlings from 12 origins of *Cupressus atlantica* after six months of growth under greenhouse conditions.

## Discussion

The analysis of the diversity of mycorrhizal fungi associated with the Atlas Cypress in the twelve locations representative of the geographic distribution of the species has shown considerable variation in their abundance from one population to another. The seven described species coexist in six of the twelve different populations that were studied with a higher dominance observed at the Mzouzit site due to the high concentration of spores seen at this location. *Rhizophagus aggregatus* was the dominant species in this study followed by *Rhizophagus fasciculatus*. This result does not corroborate previous study at the Idni location (Ouahmane et al., 2007) where *Rhizophagus fasciculatus* was the dominant species. Nonetheless, the number of collected spores per 100 g of rhizospheric soil of the Atlas Cypress at the different locations was comparable to the results obtained by Ouahmane et al. (2006). In this study, the Talat N'os and Taws stations were the poorest in terms of mycorrhizal fungi spores which is seemingly due to the steep gradient at both sites. Indeed, erosion phenomena decrease the quality of the soils and lead to the formation of a deeper root system and taproots unfit for mycorrhizal development. On the other hand, the Mzouzit site was the richest in terms of spore numbers which might be due to its flat terrain, well-formed soil and denser root system. The observation of the roots from the different Atlas Cypress sites has shown that the Talat N'os and Taws sites have the lowest colonization intensity relatively to the other populations. This difference to the previously addressed conditions at those sites, the two populations grow on rocky substrates with a very high gradient. The highest levels of colonization were observed for the more

<figure><img src="image_9.png" /><figcaption>**Figure 1.**</figcaption></figure>

Principal component analysis (PCA) of growth parameters, nutritional status of the seedlings from the 12 origins after 6 months of growth in forest greenhouse. A: Inoculated seedling with Mycorrhizal fungi; B: Non inoculated seedling.
<figure><img src="image_10.png" /><figcaption>**Photo 4.**
Atlas Cypress seedlings under greenhouse conditions.
Photo L. Ouahmane.</figcaption></figure>

<figure><img src="image_11.png" /><figcaption>**Photo 5.**
Atlas Cypress trees in Idni Station.
Photo L. Ouahmane.</figcaption></figure>

stable populations where alluvium accumulation is the highest thus allowing a finer, more fasciculate and more superficial root system that is in constant contact with surface fungal mircoflora. The different populations have consequently shown significant statistical differences for the studied parameters. These results related to composition of fungal soil communities with plant abundance and geographic origin are well documented (Reininger *et al*., 2015; Thioye *et al*., 2017).

In order to further characterize the diversity of the Atlas Cypress populations, the physiological and nutritional behavior of the seedlings from the different locations was studied through forest greenhouse cultures. The first analysis of the differences between the greenhouse-grown seedlings focused on the effect of the origin of the seeds in the absence of any treatment. The results show a significant effect on all growth and nutritional parameters: plant height, stem diameter, above ground and root wet and dry biomass and nitrogen and phosphorus content of the plant tissues. An important height variation was observed between the Ighzer and Adebdi locations where the height of the plant increased by a factor of 1.8. The shoot dry biomass also increased by a factor of 2 when

going from the Tiouna and Taghzout locations to the Aghbar location. Based on these dendrometric measurements, the plants from the Talat N'Os, Taws, Tiouna, Taghzout and Rikt provenances were the poorest in above ground and root biomass. The second series of seedlings in this study came from seeds from different origins of *C. atlantica* that were inoculated at an early stage by a mixture of arbuscular mycorrhizal fungi. The seedlings were first compared to inoculated seedlings from the same origin after six months of growth under greenhouse conditions, the results showed a huge improvement in the growth and nutrition of the inoculated plants for all origins. This further confirms results obtained by Ouahmane et al. (2007) where a strong dependence of the Atlas Cypress on mycorrhizal symbiosis was noticed. Similarly, when the inoculated seedlings from the different origins were compared to each other after 6 months of greenhouse culture, the origin of the seeds was shown to be less significant for all analyzed parameters. The above ground biomass was the only parameter that varied from one location to another. All the mycorrhized plants in this experience had a large size and had similar morphologies and the statistical analysis did not show any significant differences between the mycorrhized plants for all the examined parameters. In fact, the more spectacular effects of the mycorrhizal inoculation on the growth and nutrition of the plants have already been shown for many species in Morocco (Ouahmane, 2007; El Mrabet et al., 2012; Abbas et al., 2013) and also in other Mediterranean countries (Plenchette and Strullu, 1996; Azcón and Barea, 1997; Azcon-Aguilar et al., 2003; Bonfonte and Genre, 2010). In this study, the results have clearly shown that the growth of the Atlas Cypress and its nitrogen and phosphate nutrition were greatly improved by mycorrhizal fungi. However, this high level of mycorrhizal dependency displayed by *C. atlantica* has shown great variation from one origin to another. The seedlings from seeds collected from the Taghzout, Tiouna and Aghbar provenances were found to be least dependent on mycorrhizal symbiosis.

In order to synthetize all the differences observed for the different parameters in the field and in greenhouse cultures, a principal component analysis of the effect of the origin and mycorrhizal inoculation on the plant's growth and nutrition parameters, root colonization by mycorrhizal fungi and mycorrhizal dependency was conducted. The analysis has shown that plants obtained from seeds originating from the different provenances showed important variation and allowed the establishment of two main groups. The first group is made of the Taws, Taghzout, Tiouna, Talat N'os, Adebdi and Aghbar provenances and the second is made of the Rikt, Ighzer, Ighil, Idni, Allous and Mzouzit provenances. Mycorrhizal dependency differed greatly between the two groups. The hierarchical analysis of the data has also allowed the establishment of two distinct groups. The first group is made of Tiouna, Aghbar Taghzout and Adebdi whereas the second one is made of the Mzouzit, Allous, Idni, Talat N'os, Taws, Ighzer, Ighil and Rikt sites. Geographically, the first group corresponds to the high N'Fis valley and the Souss plains whereas the second group corresponds to the low and middle N'Fis valleys. The differences in the geographic and bioclimatic conditions between the different populations seem to have a direct effect on the physiological behavior of the seeds and seedlings of the Atlas Cypress.
Conclusion

The soils hosting the Atlas Cypress, *Cupressus atlantica*, were poor in terms of mycorrhizal fungi spores probably due to the harsh environmental conditions occurring in these areas like drought, strong slope, rocky and superficial soils, and strong water erosion. Nevertheless, mycorrhizal fungi are associated with the roots even at an advanced age, the strength of this association could contribute to the protection of the Atlas Cypress trees from the environmental constraints occurring in their habitat such as drought and fungal pathogens. The Atlas Cypress is thus heavily dependent on its fungal partnerships for its development in all studied provenances. Under nursery conditions without mycorrhizal fungi inoculation, the high variability among different origins was directly tied to genetic variation between and among the different populations. When mycorrhized plants were compared to each other, the growth and nutrition response of the Atlas Cypress is hence dependent on its origin and on the interaction between the different provenances and the fungal symbiont. The combination of strong, resilient genotypes and powerful mycorrhizal partners could guarantee the wider distribution and longevity of the Atlas Cypress. The mycorrhization of the Atlas Cypress seedlings in nurseries before their transfer to reforestation perimeters could contribute to a greater success of reforestation programs based on *Cupressus atlantica*.

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