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Vol. 34. Núm. 2.
Páginas 116-120 (Abril - Junio 2017)
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Vol. 34. Núm. 2.
Páginas 116-120 (Abril - Junio 2017)
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DOI: 10.1016/j.riam.2016.06.007
Acceso a texto completo
Endophytic fungi associated with roots of date palm (Phoenix dactylifera) in coastal dunes
Hongos endófitos asociados con raíces de palmera datilera (Phoenix dactylifera) en dunas costeras
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Fadila Mohamed Mahmouda,b,c,
Autor para correspondencia
m-fadhela@netcourrier.com

Corresponding author.
, Zoulikha Krimic,d, Jose G. Maciá-Vicenteh, Mohamed Brahim Errahmanig, Luis V. Lopez-Llorcae,f
a Department of Botany, National Superior School of Agronomy, Algiers, Algeria
b Department of Biology and Cell Physiology, Faculty of Natural and Life Sciences, Blida 1 University, BP 270, Blida, Algeria
c Laboratory of Protection and Enhancement of Agrobiological Resources, Department of Biotechnology, Faculty of Natural and Life Sciences, Blida 1 University, Blida, Algeria
d Department of Biotechnology, Faculty of Natural and Life Sciences, Blida 1 University, Algeria
e Laboratory of Plant Pathology, Department of Marine Sciences and Applied Biology, University of Alicante, Alicante, Spain
f Multidisciplinary Institute for Environmental Studies (MIES) Ramon Margalef, University of Alicante, Alicante, Spain
g Department of Chemistry, Faculty of Sciences, Blida 1 University, Algeria
h Institute of Ecology, Evolution and Diversity, Goethe Universität Frankfurt, Max-von-Laue-Str. 13, D-60438 Frankfurt am Main, Germany
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Tablas (2)
Table 1. Characteristics of sampling sites and diversity of date palm root endophytes.
Table 2. Data summary and classification of endophytic fungi colonizing roots of date palms in three dunes of SE Spain into operational taxonomic units (OTUs) and morphological taxa.
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Abstract
Background

Symbiotic interactions with fungal endophytes are argued to be responsible for the tolerance of plants to some stresses and for their adaptation to natural conditions.

Aims

In this study we aimed to examine the endophytic fungal diversity associated with roots of date palms growing in coastal dune systems, and to screen this collection of endophytes for potential use as biocontrol agents, for antagonistic activity and mycoparasitism, and as producers of antifungal compounds with potential efficacy against root diseases of date palm.

Methods

Roots of nine individual date palms growing in three coastal locations in the South-East of Spain (Guardamar, El Carabassí, and San Juan) were selected to isolate endophytic fungi. Isolates were identified on the basis of morphological and/or molecular characters.

Results

Five hundred and fifty two endophytic fungi were isolated and assigned to thirty morphological taxa or molecular operational taxonomic units. Most isolates belonged to Ascomycota, and the dominant order was Hypocreales. Fusarium and Clonostachys were the most frequently isolated genera and were present at all sampling sites. Comparisons of the endophytic diversity with previous studies, and their importance in the management of the date palm crops are discussed.

Conclusions

This is the first study on the diversity of endophytic fungi associated with roots of date palm. The isolates obtained might constitute a source of biological control agents and biofertilizers for use in crops of this plant.

Keywords:
Root-endophytic fungi
Ascomycota
Hypocreales
Fusarium
Clonostachys
Resumen
Antecedentes

Se ha propuesto que la simbiosis con hongos endófitos puede ser responsable de la tolerancia de las plantas a algunas situaciones de estrés ambiental y de su adaptación a las condiciones naturales.

Objetivos

Este estudio tiene como objetivo analizar la diversidad de hongos endófitos asociados con las raíces de palmeras datileras que crecen en sistemas de dunas costeras. La finalidad es la evaluación de un grupo de cepas fúngicas para su uso como agentes de control biológico por su actividad antagónica o micoparasitaria, o como productores de compuestos antifúngicos con potencial aplicación frente a enfermedades radiculares de la palmera datilera.

Métodos

Se muestrearon raíces de 9 palmeras que crecían en 3 localidades costeras en el Sudeste de España (Guardamar, El Carabassí y San Juan), y se aislaron sus hongos endófitos asociados. Las cepas se identificaron mediante el estudio de caracteres morfológicos y/o moleculares.

Resultados

Se aislaron 552 hongos endófitos, que se clasificaron en 30 taxones morfológicos o unidades taxonómicas operativas moleculares. La mayoría de las cepas pertenecen a la división Ascomycota; el orden dominante fue Hypocreales. Los géneros aislados con más frecuencia fueron Fusarium y Clonostachys, que estuvieron presentes en todos los sitios de muestreo. Nuestros resultados de diversidad hongos endófitos se comparan con los de otros estudios previos, y se discute su importancia para el tratamiento de cultivos de palmera datilera.

Conclusiones

Este es el primer estudio sobre la diversidad fúngica endofíticamente asociada con raíces de palmera datilera. Las cepas obtenidas son una fuente potencial de agentes de control biológico o biofertilizantes para la aplicación en cultivos de esta planta.

Palabras clave:
Hongos endófitos radiculares
Ascomycota
Hypocreales
Fusarium
Clonostachys
Texto completo

Endophytes colonize the tissues of living plants without causing symptoms.24 They have been shown to protect plants from abiotic and biotic stresses17,19 and to promote plant growth.15 Fungal endophytes associated with roots of date palm (Phoenix dactylifera L.) are a potential source of biocontrol agents of root-pathogens of this crop. Little work has been done on date palm endophytes.6–9,23 In this study, we focus on the biodiversity of endophytic fungi associated with roots of date palms growing in coastal dune systems, under conditions imposing drought and water stress. These soils are characterized by ecological factors that differ from those of large date palm plantations. Previous studies have shown that roots of plants in similar conditions harbor a large endophytic diversity.12 Moreover, coastal plants can develop symbioses with endophytes that might enhance their tolerance to stress.17 Therefore, the use of endophytes in bioremediation strategies could be valuable in arid ecosystems.4,11 The aim of this work was to isolate and characterize endophytic fungi from date palm roots in order to generate a collection of strains for future screenings for biocontrol agents and production of antifungal compounds, with potential application against root diseases of date palm.

Root samples were collected from healthy date palms growing in sand dunes at three coastal locations in Alicante Province, SE Spain: San Juan, El Carabassí, and Guardamar (Table 1). Availability and irregular dispersion of date palms in this location was the cause of our choice of the samples. We collected root samples from nine plants (1.5–5m tall), consisting of 15-cm-long fragments at a depth of 20–40cm. Root samples were surface-sterilized, cut into pieces (Table 1), and plated in a culture medium for the isolation of fungal endophytes, following previously described procedures.13 Young individual fungal colonies were sub-cultured onto potato dextrose agar (PDA, Oxoid, Hampshire, UK), and stored in the culture collection of the Laboratory of Plant Pathology at the University of Alicante (Spain). These pure fungal cultures on PDA were grouped into morphotypes according to their macroscopic and microscopic features. Cultures that developed reproductive structures were identified to genus or species level with aid of literature describing their phenotypic characteristics. Thirty nine isolates that could not be identified morphologically, were selected and processed for further molecular characterization, via sequencing of their rDNA internal transcribed spacer regions (ITS) according to the protocol described by Abdullah et al.,1 and were assigned GenBank accessions KP006331–KP006369. These strains were grouped into operational taxonomic units (OTUs) according to ITS sequence similarity of 97%, and assigned to taxa as described by Maciá-Vicente et al.13 Molecular classifications were further assessed by means of phylogenetic analyses (Supplementary Fig. S1). The remaining isolates that were not sequenced were grouped in taxa according to their morphological characters. Isolation data were compared across sampling locations using analysis of variance (ANOVA) or the Kruskal–Wallis test, followed by the Tukey Honest Significance Difference test for multiple comparisons. The diversity indicators Shannon index (H′) and Pielou's evenness index (J) were calculated for the three locations. Similarity in OTU and taxa composition of fungal communities among locations was calculated using the Jaccard index. Comparison of the occurrence of dominant fungi across locations was performed using the χ2 test, based on the counts of isolates at each site.

Table 1.

Characteristics of sampling sites and diversity of date palm root endophytes.

  All sites  San Juan  El Carabassi  Guardamar 
Coordinates  –  38.37793N
0.40830
38.22623N
0.50906
38.10356N
0.64266
Number of plants sampled 
Number of root pieces plated  630  300  180  150 
Number of isolates  552  304  153  95 
Number of taxa/OTUs  30  20  19  12 
Shannon diversity index (H′)  8.93  3.26  2.90  2.77 
Pielou's evenness (J2.22  0.75  0.70  0.77 

Incidence of endophytes was highest in San Juan, representing more than 55.5% of the total number of isolates (Table 1). This is due to the larger number of root pieces plated, and to the presence of endophytic bacteria in other samples which inhibited the fungal growth, especially in Guardamar. In our isolation-based study, the total fungal diversity detected was relatively high as compared to others studies.2,6,7 We detected a total of thirty OTUs and taxa belonging primarily to ascomycetous fungi (15 OTUs, 10 taxa), followed by Basidiomycota (2 OTUs) and Zygomycota (1 taxon). Our findings are consistent with the results of previous studies on other hosts, in which a predominance of ascomycetes has been reported as a characteristic of endophytic root and leaf communities.3,12,13,16,20,21 The Ascomycota includes fungi of economic importance ranging from virulent plant pathogens to effective agents of biological control, and from producers of antibiotics to sources of potent mycotoxins.18 Taxon 1 was the most frequently detected group, overall and across sites, and it could be ascribed to genus Fusarium by morphological means (Table 2). Fusarium species are adapted to a wide range of geographical sites, climatic conditions, ecological habitats, and host plants. The second most frequent group of isolates, OTU1, also belonged to the Hypocreales, and was related to Clonostachys rosea (Table 2). This species (syn. Gliocladium roseum22) is commonly found in several different terrestrial and freshwater environments23 and has been shown to successfully control a diversity of plant pathogens in greenhouses and in the field.25 Considering that this species was commonly found as an endophyte of healthy palm trees in all three locations sampled, it would appear to be a good candidate for further studies on its potential as a biological control agent of date palms diseases.

Table 2.

Data summary and classification of endophytic fungi colonizing roots of date palms in three dunes of SE Spain into operational taxonomic units (OTUs) and morphological taxa.

OTU/taxon  Identification methods  GenBank accession numbers of sequenced isolates/Site sampling  Closest related species (BLASTn) Query coverage and identity (%)  Classification and closest related taxa  Number of isolates
per sampling site
          S. Juan  El C.  G.  Sum 
OTU 1  Morphological and molecular  KP006352, KP006351, KP006350, KP006348 (San Juan)
KP006349, KP006356, KP006357, KP006355 (Guardamar)
KP006353, KP006354 (El Carabassi) 
Clonostachys rosea (KJ158182.1)
574/568 (99%) 
A, S, Hypocreales
Clonostachys sp. 
52  18  79 
OTU 2  Morphological and molecular  KP006338, KP006339, KP006337, KP006336, KP006340, KP006335 (Guardamar)  Fusarium equiseti (JQ936180.1)
553/553 (100%) 
A, S, Hypocreales
Fusarium sp. 
11  11 
OTU 3  Morphological and molecular  KP006342, KP006341, KP006334, KP006343 (San Juan)
KP006333 (El Carabassi) 
Fusarium solani (KC254048.1)
580/580 (100%) 
A, S, Hypocreales
Fusarium sp. 
10  11 
OTU 4  Morphological and molecular  KP006332, KP006331 (El Carabassi)  Penicillium commune (KC009833.1)
593/593(100%) 
A, E, Eurotiales
Penicillium sp. 
OTU 5  Morphological and molecular  KP006346 (Guardamar), KP006368 (El Carabassi)  Aspergillus tubingensis (EF621571.1)
607/607 (100%) 
A, E, Eurotiales Aspergillus sp. 
OTU 6  Morphological and molecular  KP006359 (San Juan)
KP006358 (El Carabassi) 
Beauveria bassiana (AJ560666.1)
576/576 (100%) 
A, S, Hypocreales Beauveria bassiana 
OTU 7  Morphological and molecular  KP006345, KP006344 (Guardamar)  Campanella olivaceonigra (JX444167.1)
451/513 (87%) 
B, Ag, Agaricales Agaricales sp. 
OTU 8  Morphological and molecular  KP006365 (El Carabassi)  Phomopsis lagerstroemiae (AY622994)
299/346 (86%) 
A, S, Diaporthales
Diaporthales sp. 
OTU 9  Morphological and molecular  KP006361 (El Carabassi)  Phomopsis lagerstroemiae (AY622994)
159/162 (98%) 
A, S, Diaporthales
Phomopsis sp. 
OTU 10  Morphological and molecular  KP006366 (El Carabassi)  Corynespora cassiicola (EU822309.1)
230/244 (94%) 
A, D, Pleosporales
Pleosporales sp. 
OTU 11  Morphological and molecular  KP006367 (El Carabassi)  Ilyonectria radicicola (AF220968.1)
551/545 (99%) 
A, S, Hypocreales
Ilyonectria sp. 
OTU 12  Morphological and molecular  KP006369 (San Juan)  Clonostachys sp. (AJ890438.1)
131/142 (92%) 
A, S, Hypocreales
Hypocreales sp. 
28  28 
OTU 13  Morphological and molecular  KP006347 (San Juan)  Aspergillus sclerotiorum (KC478519.1)
620/626 (99%) 
A, E, Eurotiales
Aspergillus sp. 
OTU 14  Morphological and molecular  KP006360 (San Juan)  Phomopsis asparagi (KC590096.1)
565/581 (97%) 
A, S, Diaporthales
Phomopsis sp. 
OTU 15  Morphological and molecular  KP006362 (San Juan)  Marasmiellus candidus (HM240532.1)
661/717 (92%) 
B, Ag, Agaricales
Marasmiaceae 
OTU 16  Morphological and molecular  KP006363 (San Juan)  Myrothecium verrucaria (AJ302003.1)
588/588 (100%) 
A, S, Hypocreales
Myrothecium sp. 
OTU 17  Morphological and molecular  KP006364 (San Juan)  Diaporthe hongkongensis (NR111848.1)
583/530 (91%) 
A, S, Diaporthales
Diaporthales sp. 
Taxon 1  Morphological  –  –  A, S, Hypocreales
Fusarium sp 
94  48  37  179 
Taxon 2  Morphological  –  –  A, S, Hypocreales
F. oxysporum 
Taxon 3  Morphological  –  –  A, E, Eurotiales
Paecilomyces sp. 
10 
Taxon 4  Morphological  –  –  A, E, Eurotiales
Penicillium sp. 
Taxon 5  Morphological  –  –  A, E, Eurotiales
Aspergillus sp. 
11  14 
Taxon 6  Morphological  –  –  A, E, Eurotiales
Aspergillus niger 
Taxon 7  Morphological  –  –  A, S, Sordariales
Chaetomium sp. 
16  11  27 
Taxon 8  Morphological  –  –  A, S, Hypocreales
Trichoderma sp. 
Taxon 9  Morphological  –  –  A, Hypocreales
Acremonium sp. 
10 
Taxon 10  Morphological  –  –  A, D, Pleosporales
Alternaria sp. 
Taxon 11  Morphological  –  –  Z, Mortierellaceae
Mortierella sp. 
Taxon 12  Morphological  –  –  Sterile mycelium  30  22  19  71 
Taxon 13  Morphological  –  –  Morphospecies  24  10  40 

A, Ascomycota; B, Basidiomycota; Z, formerly called Zygomycota; S, Sordariomycetes; E, Eurotiomycetes; D, Dothideomycetes; Ag, Agaricomycetes; S. Juan, San Juan; El C., El Carrabassi; G, Guardamar.

The occurrence of the dominant endophytic fungi (taxon 1, OTU1, taxon 13, and taxon 12; Table 2) across sampling sites was compared with the χ2 test. Significant differences were found between sites, with highest frequencies in San Juan, and lowest in Guardamar, for taxon 1 (χ2=30.65, P<0.001), OTU1 (χ2=39.06, P<0.001), and taxon 13 (χ2=13.40, P=0.001). There were no evident differences in occurrence of taxon 12 (χ2=2.73, P=0.255).

Sterile mycelia were remarkably common in this study, representing 71 isolates of the total fungal groups. However, it is difficult to determine whether these isolates are naturally sterile or if they have simply lost the ability to sporulate in culture. OTU6, related to the entomopathogenic fungus Beauveria bassiana, was found as an endophyte in date palms in dunes of San Juan and El Carabassi. Beauveria spp. have been used as biological control agents of soil and phylloplane insect pests.5 More recently, date palms worldwide are suffering from severe infestations by the red palm weevil (Rhynchophorus ferrugineus).14 Natural epizootics of B. bassiana on R. ferrugineus have been found in areas near the sites studied in this work.10 Therefore, the presence of this fungus as a natural endophyte of date palm roots is of great significance. The results of this work open the possibility of finding specific fungal isolates with the potential for biocontrol, or for the discovery of new antagonists such as mycoparasites or producers of useful secondary metabolites that could limit the damage caused by date palm diseases such as bayoud, or pests such as the red palm weevil.

Acknowledgements

The authors wish to thank the Algerian Ministry of Higher Education and Blida University the award of a scholarship to complete this research. We also thank the staff of the Laboratory of Plant Pathology, Department of Marine Sciences and Applied Biology, Multidisciplinary Institute for Environmental Studies (MIES) Ramón Margalef, University of Alicante, where the experimental work was carried out.

Appendix A
Supplementary data

The following are the supplementary data to this article:

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