Fungal diversity in adult date palm (Phoenix dactylifera L.) revealed by culture-dependent and culture-independent approaches

Fungal diversity in adult date palm ( Phoenix dactylifera L.) revealed by culture-dependent and culture-independent approaches 1 5 first-author †E-mail:inesbenchobba@yahoo.fr †E-mail:inesbenchobba@yahoo.fr inesbenchobba@yahoo.fr 1 2 1 3 4 1 1 5 Laboratoire de Biotechnologies Végétales Appliquées à l’Amélioration des Cultures LBVAAC, Faculté des Sciences de Sfax, B.P. 1171, 3000 Sfax, Tunisia Laboratoire de Biotechnologies Végétales Appliquées à l’Amélioration des Cultures LBVAAC, Faculté des Sciences de Sfax, B.P. 1171, 3000 Sfax, Tunisia Laboratoire de Microorganismes et de Biomolécules, Centre de Biotechnologie de Sfax, Université de Sfax, Route Sidi Mansour, B.P. 1177, 3018 Sfax, Tunisia Laboratoire de Microorganismes et de Biomolécules, Centre de Biotechnologie de Sfax, Université de Sfax, Route Sidi Mansour, B.P. 1177, 3018 Sfax, Tunisia Laboratoire de Phytopathologie Oasienne, Centre Régional de Recherches en Agriculture Oasienne à Degache, Degache 2260, Tunisia Laboratoire de Phytopathologie Oasienne, Centre Régional de Recherches en Agriculture Oasienne à Degache, Degache 2260, Tunisia Plateforme Séquençage-Génotypage SFR “Montpellier Environnement Biodiversité”, ISEM, Université de Montpellier 2, 34095 Montpellier, France Plateforme Séquençage-Génotypage SFR “Montpellier Environnement Biodiversité”, ISEM, Université de Montpellier 2, 34095 Montpellier, France UMR 5119, Université de Montpellier 2, Sciences et Techniques, pl E. Bataillon, CC093, 34095 Montpellier, France UMR 5119, Université de Montpellier 2, Sciences et Techniques, pl E. Bataillon, CC093, 34095 Montpellier, France Endophytic flora plays a vital role in the colonization and survival of host plants, especially in harsh environments, such as arid regions. This flora may, however, contain pathogenic species responsible for various troublesome host diseases. The present study is aimed at investigating the diversity of both cultivable and non-cultivable endophytic fungal floras in the internal tissues (roots and leaves) of Tunisian date palm trees ( Phoenix dactylifera ). Accordingly, 13 isolates from both root and leaf samples, exhibiting distinct colony morphology, were selected from potato dextrose agar (PDA) medium and identified by a sequence match search wherein their 18S–28S internal transcribed spacer (ITS) sequences were compared to those available in public databases. These findings revealed that the cultivable root and leaf isolates fell into two groups, namely Nectriaceae and Pleosporaceae. Additionally, total DNA from palm roots and leaves was further extracted and ITS fragments were amplified. Restriction fragment length polymorphism (RFLP) analysis of the ITS from 200 fungal clones (leaves: 100; roots: 100) using Hae III restriction enzyme revealed 13 distinct patterns that were further sequenced and led to the identification of Alternaria , Cladosporium , Davidiella ( Cladosporium teleomorph), Pythium , Curvularia , and uncharacterized fungal endophytes. Both approaches confirmed that while the roots were predominantly colonized by Fusaria (members of the Nectriaceae family), the leaves were essentially colonized by Alternaria (members of the Pleosporaceae family). Overall, the findings of the present study constitute, to the authors’ knowledge, the first extensive report on the diversity of endophytic fungal flora associated with date palm trees ( P. dactylifera ). Date palm tree ( Phoenix dactylifera ) Endophytic cultivable fungi rDNA internal transcribed spacer (ITS) Phylogenetic analysis Total DNA diversity analysis Introduction 1 The date palm ( Phoenix dactylifera ) is a multi-purpose plant species typically grown in the arid and semi-arid regions of the world and is globally valued for its nutritional and health-promoting fruit. Current estimates indicate that Tunisia is the world’s tenth largest producer and first African exporter of date palms in value. It has an average annual production of over 100 000 t, of which more than 30% are exported. The Deglet Nour variety dominates, accounting for about 60% of the total production. Constituting about 13% of all Tunisian exports, date palm cultivation is a major agricultural and socio-economic activity in Tunisia. The cultivation of date palms is, however, often hampered by several constraints, particularly its susceptibility to a wide range of damaging diseases caused by different pathogens living in the same environment. In fact, date palms have been cultivated with in vitro conditions for several years (Drira and Benbadis, 1985 1985 ). Further knowledge about the fungal flora that may naturally be associated with healthy palm trees is, however, necessary to (1) improve yield, (2) facilitate the occupation of the ecological “niche” and avoid the random colonization of young plantlets by potential fungal pathogens, and (3) help further plantlet acclimation to arid soil conditions. In general, at least part of the endophytic flora of a plant, namely the fungal flora, can be cultivated. If the cultivatable fungi have a reasonable potential of young plantlet colonization, they could be easily used as inoculum for in vitro plantlet producing or, later, in plant nurseries. However, to the authors’ knowledge, no systematic study has so far been performed on the total endophytic flora associated with date palms. Accordingly, the present study was undertaken to extensively assess both the cultivable and non-cultivable fungal floras of this tree. In fact, a number of recent studies have shown that both approaches could bring varying, yet complementary, results. The introduction of molecular methods characterizing fungal flora led to new insights on microbial diversity (Hugenholtz et al., 1998 1998 ). The literature presents various methods for the characterization of fungal flora. These include the determination of the growth parameters (Steinberg et al., 1997a 1997a ), analysis of the diversity of β-tubulin sequences (Watanabe et al., 2011 2011 ), ribosomal genes sequencing (Kodsueb et al., 2006 2006 ; Crous et al., 2007 2007 ; Schroers et al., 2009 2009 ), intergenic 18S–28S internal transcribed spacer (ITS) sequencing (Camara et al., 2002 2002 ; Braun et al., 2003 2003 ; Pryor and Bigelow, 2003 2003 ; Kwasna et al., 2006 2006 ; Manamgoda et al., 2012 2012 ), multilocus analysis (Zhang et al., 2009a 2009a ), and single sequence repeat (SSR) genotyping (Wang et al., 2010 2010 ). Further methods include terminal restriction fragment length polymorphism (T-RFLP) (Dickie and FitzJohn, 2007 2007 ), amplified ribosomal DNA restriction analysis (ARDRA) (Sutthisa et al., 2010 2010 ), as well as ribosomal intergenic spacer analysis (RISA) (Sigler and Zeyer, 2002 2002 ). Alternatively, denaturing gradient gel electrophoresis (DGGE) (Vallaeys et al., 1997 1997 ; Ampe et al., 1999 1999 ; Coppola et al., 2001 2001 ), temperature gradient gel electrophoresis (TGGE) (Felske et al., 1999 1999 ), temporal temperature gel electrophoresis (TTGE) (Sonjak et al., 2009 2009 ), and single-strand conformation polymorphism (SSCP) (Zumstein et al., 2000 2000 ; Feurer et al., 2004 2004 ) can be used in molecular analyses. The present work, which aimed to identify and characterize the endophytic flora associated with date palms, opted for the application of the ITS sequencing technique. In fact, sequence data provide valuable grounds for comparison with similar data available in public databases and comparable data in the literature, with the potential to provide interesting geographically related information on plant species. These data can also give indications on the GRAS (generally recognized as safe) status of members of this endophytic flora that could constitute part of developed inocula. The assessment of the GRAS status (for both the plant host and the final consumer) of potential endophytic plant growth promoting fungi (PGPF) is, in fact, essential before developing industrial applications to counteract target plant pathogens. Among endophytic fungi, PGPF species are common inhabitants of a wide range of plant species and can be found in the cells (Petrini, 1991 1991 ), intercellular space, or vascular systems (Lee et al., 2009 2009 ) of the plant. However, some endophytic fungal species have been reported to be pathogenic to the plant. It is, therefore, important to characterize endophytic flora while keeping in mind their functional role. Wilson ( 1995 1995 ) provided a working definition of the term “endophyte” by analyzing the different levels of endophytic association and stated that “endophytes are fungi which, for all or part of their life cycle, invade the tissues of living plants and cause unapparent and asymptomatic infections entirely within plant tissues but cause no symptoms of the disease”. From a functional point of view, endophytic fungi are divided into two groups, non-pathogenic fungi that promote growth or suppress fungal diseases (Backman and Sikora, 2008 2008 ) and pathogenic fungi that cause disease in plants (Strobel and Daisy, 2003 2003 ). In addition to comprising species that are pathogenic to the plant, endophytes also include fungi that have diverse positive effects on their hosts. The latter include Balansia (Ascomycetes) (Bacon et al., 1977 1977 ; 1979 1979 ), though reported elsewhere to be implicated in toxicosis of cattle and humans (Groger, 1972 1972 ). Other PGPF fungi have been described to protect their hosts against insect pests, pathogens, and even domestic herbivores (Yan et al., 2011 2011 ). These include Aspergillus flavus and Penicillium sublateritium (Webber, 1981 1981 ), which live and feed on the host plant, and in turn, produce functional metabolites that enhance its fitness and resistance against stresses (Rana et al., 1997 1997 ). Last but not least, a number of species, including selected Fusarium sp. are reported to act as antagonists against plant pathogens. Accordingly, PGPF have commonly been used in practice as inocula to improve the growth of plants and suppress pathogens (Steinberg et al., 1997b 1997b ; Cao et al., 2002 2002 ; Alabouvette et al., 2009 2009 ). Considering the important opportunities that the decipherment of currently undeciphered fungal flora might bring to the field, the present study was undertaken to explore the fungal flora associated with date palms ( P. dactylifera ). In particular and for practical purposes, special focus was given to the cultivable fraction of date palm endophytes, because this fraction could be further used for the development of inocula to promote the health and growth of date palms and to conserve production potential and agro-ecosystem sustainability of Tunisian oases. The non-cultivable fraction of date palm endophytes was also investigated using molecular methods, to provide a more complete overview of the diversity of this mostly undeciphered flora. Accordingly, the diversities of the cultivable and non-cultivable endophytic populations were analyzed in both the roots and leaves of healthy Tunisian date palm trees ( P. dactylifera ), with special focus on the characterization of fungi with potential PGPF effects. Combined ITS-polymerase chain reaction (PCR), random cloning, and phylogenetic analysis were employed to explore and compare the endophytic diversity between the roots and leaves of the same plant. Materials and methods 1 Sampling of date palm 2 Oasis sampling site: the date palm tree samples employed in the present study were from an old cultivar of the “Deglet Nour” variety situated in the Jouali oasis, near Tozeur (Southwest Tunisia), on the Algero-Tunisian border. This date palm field is located in the major date palm production area of Tunisia, with a surface area of 4 719 km 2 . The sampled date palm trees were approximately 50 years old, with an average height of 7 to 8 m. For the sake of a systematic overview of date palm endophytes, both leaf and root samples were collected from a healthy tree during summer. Healthy mature leaves whose size ranged between 3.5 and 4.5 m were collected from the central crown of the tree (the most active crown). Healthy roots having an average diameter of (0.3±0.08) cm were gathered from soil surface layers ranging between 0 and 20 cm. All root and leaf samples were collected from a single date palm tree. Soil texture: the soil at the oases of Tozeur presents a typically carbonated sandy loam texture. The water pH of the oasis is 8.1, total CaCO 3 is 119 g/kg, organic matter is 12.08 g/kg, nitrogen is 0.56 g/kg, carbon is 7.03 g/kg, K 2 O is 0.18 g/kg, CaO is 49.1 g/kg, MgO is 0.51 g/kg, Na 2 O is 0.53 g/kg, and P 2 O 5 is 0.31 g/kg (Namsi et al., 2007 2007 ). Isolation of endophytic fungi 2 Endophytic fungi were isolated from the internal tissues of roots and leaves of a healthy date palm tree. The leaves and roots were washed in running tap water for 30 min. Sixteen adjacent 1 mm×2 mm segments were cut from each washed root and leaf. The samples were surface-sterilized by sequential washes in 90% ethanol for 10 min, 2.5% sodium hypochlorite for 2 min, and 80% ethanol for 3 min. They were then rinsed five times with sterile distilled water and allowed to surface-dry under sterile conditions. In fact, surface-sterilization has previously been reported to present an effective method for the removal of yeasts, fast-growing Zygomycetes, and other epiphyllous organisms from endophyte cultures (Schulz et al., 1993 1993 ; Arnold et al., 2000 2000 ). The leaf and root segments were placed on Petri dishes containing potato dextrose agar (PDA) (Collins and Lyne, 1984 1984 ) and incubated at 28 °C for up to 20 d for the selective isolation of fungal endophyte. Morphologically distinct fungal colonies were re-isolated by sub-culture on appropriate media. Collected spores were stored at −20 °C in 20% sterile glycerol until used for further analysis and molecular identification assays. Isolation of genomic DNA from isolates of endophytic fungi 2 Genomic DNA was extracted from isolates of endophytic fungi using the Wizard SV genomic DNA purification system kit (Promega, France) following the manufacturer’s instruction. DNA quality and concentration were assessed with NanoDrop (Spectrophotometer ND-1000, France) in accordance with the manufacturer’s instruction. Total DNA extraction from leaf and root samples 2 A slightly modified version of the protocol described by Dellaporta et al. ( 1983 1983 ) was employed for DNA extraction. In brief, plant tissues were pulverized with liquid nitrogen (−196 °C) to obtain a fine powder and then transferred to an organic solvent-resistant test tube. A warm 2-mercaptoethanol (2-ME)/cetyl trimethyl ammonium bromide (CTAB) extraction solution was added to the pulverized tissues with mixing. The tissues were incubated for up to 60 min at 65 °C with occasional mixing. An equal volume of 24:1 chloroform/isoamyl alcohol was added to the lysate, which was centrifuged for 5 min at 7 500× g (or 10 000 r/min in microcentrifuge for smaller samples) at 4 °C, until lysis was complete. The top (aqueous) phase was recovered. One-tenth volume of CTAB/NaCl solution heated to 65 °C was added to the recovered aqueous phase, which was followed by extraction with an equal volume of chloroform/isoamyl alcohol. The top (aqueous) phase was recovered after centrifugation. Then, one volume CTAB precipitation solution was added and centrifuged for 5 min at 500× g (or 2 700 r/min in microcentrifuge) at 4 °C. The supernatant was recovered while the pellets were resuspended in a high-salt TE buffer (0.5 to 1.0 ml/g starting material). The nucleic acids were precipitated by adding a 0.6 volume of isopropanol and then centrifuged for 15 min at 7 500× g and 4 °C. The pellets were washed with 80% ethanol, dried, and resuspended in a minimal volume of TE buffer (0.1 to 0.5 ml/g starting material). PCR amplification and sequencing of ITS rRNA genes from both cultivable endophytic fungi and clones 2 Fungal ITS rDNA fragments were amplified from the DNA extracted from strains or, alternatively, total date palm samples. PCR amplification was performed using universal fungal ITS rRNA gene-specific oligonucleotide primers ITS1 (5′-TCCGTAGGTGAACCTGCGG-3′) and ITS4 (5′-TCCTCCGCTTATTGATATGC-3′) (White et al., 1990 1990 ) with a thermal cycler (M.J. Research, Biometra, Stratagene) in a volume of 25 μl. The DNA sample was mixed with a polymerase reaction buffer, 25 mmol/L MgCl 2 , 10 mmol/L dNTPs, the above primers (20 μmol/L), and 5 U/μl Taq polymerase (Promega, France). The thermocycling conditions consisted of an initial denaturation step at 94 °C for 2 min, followed by 35 amplification cycles at 94 °C for 30 s, 60 °C for 1 min, and 72 °C for 2 min, and a final polymerization step at 72 °C for 7 min. PCR products were electrophoresed in a 1.5% agarose gel (15 g/L) and visualized by ethidium bromide staining. Cloning and sequencing of ITS rRNA gene amplified from the total DNA extracted from the leaf and root samples 2 The PCR products amplified from the total DNA extracted from the leaf and root samples using the universal external primers ITS1 and ITS4 were immediately ligated into the pCR4-TOPO vector (Invitogen, Carlsbad, CA, USA). Recombinant pCR4-TOPO plasmids were used to transform Escherichia coli TOPO10 One Shot competent cells as specified by the manufacturer (Invitrogen, France). For each DNA sample, 100 transformed clones were randomly picked for plasmid extraction (Bimboim and Doly, 1979 1979 ). Plasmids were then amplified using the primers M13Forward (M13F) and M13Reverse (M13R) (Invitrogen, France) according to the manufacturer’s instructions. The fragments obtained were then digested with Hae III (Promega, USA) (Dwivedi et al., 2012 2012 ). Clones were grouped according to RFLP banding patterns and further sequenced using primers M13F (5′-GTAAAACGACGGCCAG-3′) and M13R (5′-CAGGAAACAGCTATGAC-3′). Sequencing was performed on a 3130x1 genetic analyzer (Life Technologies Corporation, Tokyo, Japan) using the Big Dye Terminator v3.1 cycle sequencing kit (Life Technologies, USA) according to the manufacturer’s recommendations. Molecular analysis of the DNA and PCR products from cultivable isolates 2 The ITS PCR products of the DNA extracted from cultivable isolates were purified with Zymoclean™ Gel DNA purification kit to remove primer in excess following the manufacturer’s instruction, and were then sequenced using the forward and reverse primers used for ITS PCR amplification, respectively. Sequence assembly and analysis 2 Sequences were subjected to a preliminary basic local alignment search tool (BLAST) analysis (Altschul et al., 1990 1990 ) in the National Center for Biotechnology Information (NCBI) databases, and sequence matches were used to determine their identities. Multiple sequence alignment was then carried out using ClustalW (Thompson et al., 1994 1994 ) at the European Bioinformatics Institute Website ( http://www.ebi.ac.uk/clustalw/ http://www.ebi.ac.uk/clustalw/ ). Phylogenetic analyses were performed using MEGA5 (Tamura et al., 2007 2007 ) using reference sequences presented in Table 1 1 . For accuracy reasons, sequences from collection strains were preferred, when available, to infer phylogeny. Alternatively, ITS sequences from clone libraries were used. Both anamorphs and corresponding teleomorphic affiliations were used for tree labeling according to data available in databases. Obvious corrections and annotation updates were made. A phylogenetic tree was constructed by maximum parsimony (MP) analysis using the Kimura two-parameter distance method. The robustness of the inferred tree was evaluated by bootstrap (100 replications). Strain Accession number Reference Alternaria bokurai EU520199 Pryor and Bigelow, 2003 2003 Alternaria alternate AF347031 Pryor and Bigelow, 2003 2003 Alternaria japonica AF229474 Pryor and Bigelow, 2003 2003 Alternaria macrospora AF229469 Pryor and Bigelow, 2003 2003 Alternaria radicina AF229472 Pryor and Bigelow, 2003 2003 Alternaria arborescens AF347033 Pryor and Bigelow, 2003 2003 Alternaria solani AF229475 Pryor and Bigelow, 2003 2003 Alternaria infectoria AF229480 Pryor and Bigelow, 2003 2003 Embellisia proteae AY278842 Pryor and Bigelow, 2003 2003 Pleospora herbarum KC009768 Zhang et al., 2009a 2009a Pleospora herbarum JX397964 Zhang et al., 2009a 2009a Pleospora tarda AF229481 Zhang et al., 2009a 2009a Davidiella tassiana AF393705 Kwasna et al., 2006 2006 Davidiella macrospora AF297231 Kwasna et al., 2006 2006 Davidiella dianthi AF393698 Kwasna et al., 2006 2006 Fungal endophyte HM537053 Zhang et al., 2013 2013 Cladosporium australiense HM147999 Braun et al., 2003 2003 Cladosporium oxysporum AF393720 Braun et al., 2003 2003 Cladosporium herbarum AJ244227 Braun et al., 2003 2003 Cochliobolus microlaenae JN601032 Dimuthu et al., 2012 2012 Curvularia coicis AF081447 Manamgoda et al., 2012 2012 Curvularia gladioli AF071337 Manamgoda et al., 2012 2012 Curvularia gudauskasii AF071338 Manamgoda et al., 2012 2012 Preussia africana JQ031265 Zhang et al., 2012 2012 Preussia australis AY510413 Arenal et al., 2005 2005 Preussia sp. HQ607926 Rodrigues et al., 2011 2011 Massarina corticola JX421713 Zhang et al., 2009b 2009b Corynespora cassiicola FJ852657 Dixon et al., 2009 2009 Uncultured Pleosporales JQ247356 Rodrigues et al., 2013 2013 Ochrocladosporium elatum EU040233 Crous et al., 2007 2007 Leptospora rubella JQ936327 Crous et al., 2009 2009 Phoma herbarum JQ282910 Zhang et al., 2009a 2009a Fusarium equiseti AB587000 Watanabe et al., 2011 2011 Fusarium incarnatum AB586988 Watanabe et al., 2011 2011 Fusarium oxysporum AB586994 Watanabe et al., 2011 2011 Fusarium phyllophilum AB587006 Watanabe et al., 2011 2011 Pythium inflatum JQ898463 Tambong et al., 2006 2006 Pythium inflatum AY598626 Lévesque and de Cock, 2004 2004 Pythium catenulatum AY598675 Lévesque and de Cock, 2004 2004 Uncultured root fungus EU144681 Porras-Alfaro et al., 2008 2008 Fusarium sp. JX391934 This study Fusarium equiseti JX391935 This study Alternaria bokurai JX391938 This study Alternaria bokurai JX391937 This study Curvularia sp. JX391936 This study Alternaria sp. JX391940 This study Alternaria sp. JX391950 This study Curvularia sp. JX391943 This study Cladosporium sp. JX391945 This study Cladosporium herbarum JX391946 This study Pythium sp. JX391949 This study Curvularia sp. JX391944 This study Fungal endophyte JX391947 This study Alternaria alternata JX391941 This study Alternaria sp. JX391942 This study Fungal endophyte JX391948 This study Alternaria sp. JX391939 This study Alternaria sp. JX391951 This study Nucleotide sequence accession numbers 2 The nucleotide sequence data reported here were deposited in the GenBank under the accession numbers: R2 (JX391934), R3 (JX391935), Fc1s (JX391936), Fc3s (JX391937), Fc4s (JX391938), 2C2 (JX391939), 1C2 (JX391940), 2G5 (JX391941), 2F4 (JX391942), 1C12 (JX391943), 2B6 (JX391944), 1E1 (JX391945), 1G10 (JX391946), 2E6 (JX391947), 2H7 (JX391948), 2A3 (JX391949), 1C5 (JX391950), 2B8 (JX391951). Results 1 Morphological and molecular analyses of the cultivable endophytic fungi from mature palm trees 2 The emergence of diverse colony morphotypes was observed on the PDA agar media. A total of 13 fungal colonies presenting distinct morphologies could be re-isolated from the macerates of the leaves and roots grown on those media. The colonies exhibited different colors that varied from white to grey or green. Moreover, while some colonies showed fast growth, others underwent a moderately fast growth. A lifeless and powdery texture was observed. Further, the ITS rRNA genes were amplified and sequenced, and a BLAST search was performed, which led to a preliminary classification of our isolates as presented in Table 2 2 . Further, phylogenetic analysis was performed to more precisely infer isolates’ taxonomy using sequences published from collection strains, as references, during tree reconstruction. Our isolates could be clustered within two distinct groups. Group I included five strains isolated from the roots representing members of the Nectriaceae family. Two isolates presented identical ITS sequences (R3) and were related to Fusarium equiseti and three isolates were related to Fusarium sp. (R2) (Fig. 1 1 ). Group II contained eight strains isolated from the leaves representing members of the Pleosporaceae family. They consisted of five relatives of Alternaria bokurai (Fc3s: three isolates; Fc4s: two isolates) and three Curvularia sp. (Fc1s: three isolates) (Fig. 1 1 ). Palm roots were noted to harbor a number of different genera, with a prevalence of Fusarium . The leaves, on the other hand, were colonized by three major genera, with a predominance of Alternaria (Table 2 2 ). Out of 13 fungal isolates, 5 endophytic fungal taxa were represented. From a taxonomic perspective, the leaf and root samples were noted to differ in terms of endophytic fungal colonization. Interestingly, the strains assigned to Fusarium sp. were isolated from a healthy plant that did not show visible symptoms of disease, suggesting that they belonged to non-pathogenic Fusarium species. Accession numbers of our isolates (R2, R3, Fc1s, FC3s, Fc4s) are presented in Table 2 2 . 2 Taxonomic identification 2 Order 2 Strain 2 Closest BLAST match 2 Phylogenetic affiliation 3 Number of entophytic fungus isolates 2 Overlap (bp) Leaf Root Total 3 Ascomycota 3 Pleosporales Fc3s “ Alternaria sp.” Alternaria bokurai 2 0 2 565 Fc4s “ Alternaria gaisen ” Alternaria bokurai 3 0 3 553 Fc1s “ Bipolaris tetramera ” Curvularia sp. 3 0 3 562 2 Ascomycota 2 Hypocreales R2 “ Fusarium sp.” Fusarium sp. 0 3 3 552 R3 “ Fusarium lacertarum ” Fusarium equiseti 0 2 2 533 The tree was rooted with the Pythiaceae branch. Bootstraps values > 50% (100 replicates) are shown at the nods Evaluation of the total diversity of the palm tree fungal endophytic flora by ITS rRNA cloning and sequencing of whole plant DNA 2 An ITS rDNA clone library analysis was employed to investigate the biodiversity and community structure within the leaves and roots of date palms. ITS ribosomal DNA clone libraries were constructed and complete sequence assembly of 100 clones per root and leaf sample was analyzed. The RFLP analysis of the ITS fungal clones using Hae III revealed the presence of 11 different profiles for the total 200 clones analyzed. Leaves presented eight different profiles (out of 100 clones) and roots showed five different profiles (out of 100 clones) (Fig. 2 2 ). Two profiles were common to leaves and roots. Indeed, not only 2C2 (root) and 1C5 (leaf) but also 2B8 (root) and 1C2 (leaf) were identical. Sequences of corresponding 13 clones 1C2, 2B8, 2G5, 2H7, 2C2, 2F4, 1G10, 1E1, 2B6, 1C12, 1C5, 2A3, and 2E6 were assigned and deposited in GenBank (non-redundant nucleotide database). Their accession numbers are listed in Table 3 3 . The closest relative species were firstly determined for each clone sequence by direct BLAST searches in GenBank (Table 3 3 ). The results from the search for similarity matches revealed that 12 of the 13 clone sequences (1C2, 1C5, 2B8, 2G5, 2H7, 2C2, 2F4, 1G10, 1E1, 2B6, 1C12, and 2E6) showed the closest matches with Ascomycota and that one clone (2A3) showed a significant match with Oomycota. Table 3 3 shows the results of BLAST searches for clone sequences from palm leaves and roots. Their phylogenetic affiliation and relationship with other fungal strains were further determined by the construction of a phylogenetic tree, which is presented in Fig. 1 1 . For confidence reasons, final phylogenetic affiliations were inferred by similarity with the closest ITS sequences from culture collection strains, when available, after curation of inappropriate taxonomical affiliation of sequences derived from BLAST searches. Obtained sequences were classified into four groups (Fig. 3 3 ). Group I was the most represented (78% of total root and leaf clones) and consisted of members from the Pleosporaceae family. Pleosporaceae appeared dominant in both roots and leaves samples with two genera Alternaria and Curvularia represented. Clones of Group I, assigned as 1C2, 1C5, 2B8, 2G5, 2C2, and 2F4, were related to Alternaria sp. and more precisely, for given clones, to Alternaria alternata . The Alternaria genus was globally representing an overall 61% of the 200 clones assessed with similar occurrence of the genus in leaves and roots. Indeed, Alternaria genus represented 55% of the root clones and 68% of the leaf clones. However, the distribution of the different Alternaria species varied between root and leaf as shown in Fig. 2c 2c . Similar results were obtained for Curvularia , which represented an overall 17% of the total clone libraries, with 25% among leaf clones and 10% among root clones, respectively (Fig. 2c 2c ). However, as for Alternaria , root and leaf species of Curvularia differed, as suggested by the distribution of the 1C12 and 2B6 patterns among leaves and roots, respectively. Interestingly, our clones were related to obviously yet uncultivable Curvularia species in the databases (accession No. EU144681), but were also matching, with a highly significant score, a sequence obtained from one of our own cultivable isolates (Fc1s) (Fig. 1 1 ), suggesting that corresponding Curvularia species could, indeed, be cultured. Group II had a single representative of the Pythiaceae family, 2A3, distantly related to Pythium catenulatum which was further assigned as Pythium sp. (Fig. 1 1 ). It represented 10% of the root library but 0% from the leaves (Fig. 2c 2c ), representing an overall 5% of the total clones (Fig. 3 3 ). Group III consisted of members of the Davidiellaceae family and represented an average 10% of the total clones (Fig. 3 3 ). The clones of this group, represented by 1G10 and 1E1, were exclusively found among the leaf library (Fig. 2c 2c ) where they represented an overall 20% of the leaf clones. These clones were related to Cladosporium herbarum and Cladosporium sp., respectively (Fig. 1 1 ). Finally, remaining clones, related to uncultivable Pleosporales, were assembled in Group IV. These sequences represented an overall 7% of the total clones (Fig. 3 3 ). One clone, 2H7, was distantly related to Ochrocladosporium (Pleosporomycetidae). The second clone, 2E6, was also related to uncultivable Pleosporales but fell into the Sporormiaceae , as demonstrated by a significant bootstrap value of its node with Preussia and Corynespora species (Fig. 1 1 ). Clones from root tissues (2A3, 2B6, 2E6, 2G5, 2F4, 2H7, 2B8, and 2C2) (a) and leaf tissues (1C2, 1C12, 1E1, 1C5, and 1G10) (b) of Phoenix dactylifera . Bands 1–13: 2C2, 2B8, 2G5, 2F4, 2B6, 2A3, 2E6, 2H7, 1C2, 1C12, 1G10, 1E1, and 1C5, respectively. (c) Distribution of clones among patterns shows a peaked distribution For confidence reasons, the final phylogenetic affiliation was derived by similarity with the closest culture collection isolates, when available, after curation of inappropriate taxonomical position of sequences obtained from the non-redundant nucleotide database Clone Fragment size (bp) Closest BLAST match Phylogenetic affiliation * * Phylum Family %identity Leaf  1C2 580 “ Alternaria arborescens ” Alternaria sp. Ascomycota Pleosporaceae 98  1C5 570 “ Alternaria gaisen ” Alternaria sp. Ascomycota Pleosporaceae 98  1C12 570 “ Bipolaris tetramera ” Curvularia sp. Ascomycota Pleosporaceae 99  1E1 548 “ Davidiella tassiana ” Cladosporium sp. Ascomycota Davidiellaceae 99  1G10 546 “ Cladosporium sp.” Cladosporium herbarum Ascomycota Davidiellaceae 99 Root  2A3 573 “ Pythium sp.” Pythium sp. Oomycota Pythiaceae 99  2B6 570 “ Bipolaris tetramera ” Curvularia sp. Ascomycota Pleosporaceae 99  2E6 575 “Fungal endophyte” Fungal endophyte Ascomycota Sporomiaceae 100  2G5 583 “ Alternaria abutilonis ” Alternaria alternata Ascomycota Pleosporaceae 99  2F4 579 “ Alternaria gaisen ” Alternaria sp. Ascomycota Pleosporaceae 98  2H7 563 “Fungal endophyte” Fungal endophyte Ascomycota Pleosporomycetidae 98  2C2 570 “ Alternaria gaisen ” Alternaria sp. Ascomycota Pleosporaceae 98  2B8 570 “ Alternaria arborescens ” Alternaria sp. Ascomycota Pleosporaceae 98 Overall, both the ITS rDNA gene identification from clones amplified from total palm DNA and sequencing of ITS rDNA from cultivable strains yielded concordant results as the dominant species, Alternaria and Curvularia , were recovered by both methods. However, Fusarium species, that constituted the dominant flora among the cultivable fraction, were not detected by the cloning strategy. Conversely, some dominant species among the clones were not represented among the cultivable flora, such as Cladosporium (or its teleomorph Davidiella ), but also Pythium . Of course, sequences related to uncultivable fungal endophytes were detected only by molecular techniques, except for Curvularia sp. (Table 4 4 ). Globally, the diversity recovered by the global DNA approach was broader than that obtained by the cultivable method. +: species found; −: species not found Strain/clone Closest phylogenetic relatives Cultivable fraction ITS rDNA  R2 Fusarium sp. (Nectriaceae) + −  R3 Fusarium equiseti (Nectriaceae) + −  Fc1s Curvularia sp. (Pleosporaceae) + +  Fc3s Alternaria bokurai (Pleosporaceae) + −  Fc4s Alternaria bokurai (Pleosporaceae) + −  1C2 Alternaria sp. (Pleosporaceae) − +  1G10 Cladosporium rbarum (Davidiellaceae) − +  1E1 Cladosporium sp. (Davidiellaceae) − +  1C12 Curvularia sp. (Pleosporaceae) + +  1C5 Alternaria sp. (Pleosporaceae) − +  2H7 Fungal endophyte (Pleosporomycetidae) − +  2C2 Alternaria sp. (Pleosporaceae) − +  2F4 Alternaria sp. (Pleosporaceae) − +  2B6 Curvularia sp. (Pleosporaceae) + +  2E6 Fungal endophyte (Sporomiaceae) − +  2A3 Pythium sp. (Pythiaceae) − +  2B8 Alternaria sp. (Pleosporaceae) − +  2G5 Alternaria alternata (Pleosporaceae) − + Discussion 1 The present study constitutes the first attempt to extensively characterize the endophytic fungal flora associated with the date palm tree ( P. dactylifera ). Its aim was to develop an overview of the diversity of this mostly undeciphered flora, which may lay the ground for the construction of a more comprehensive description that covers other date palm varieties and field conditions. Our study shows that both DNA-based approaches and traditional cultivation techniques should be used to elucidate the diversity of the endophytic fungal flora associated with mature date palm trees, such as the Deglet Nour variety investigated in this work, which accounts for 70 years of Tunisian date palm production. Both of our approaches (by culture and molecular techniques) demonstrated a high diversity of palm tree associated fungal flora in both root and leaf tissues of date palm ( P. dactylifera L.). Overall, 11 sequence types (species equivalent) were recorded from the palm tree tissues. Six sequence types were obtained in the leaf samples among which five were obtained using the molecular approach ( Alternaria alternata , Alternaria sp., Curvularia sp., Cladosporium herbarum , and Cladosporium sp.) and two using the culture approach ( Alternaria bokurai and Curvularia sp.), Curvularia being common to clones and isolates. Ten sequence groups were detected in the palm roots with eight sequences identified from DNA cloning strategy ( Alternaria alternata , three different Alternaria sp., Curvularia sp., and Pythium sp., and two types of uncultivable Pleosporales) and two from cultivation strategy ( Fusarium equiseti and Fusarium sp.). Most of these fungi have been already reported as endophytes in other plants (Cao et al., 2002 2002 ; Gond et al., 2007 2007 ; Khan et al., 2007 2007 ), including wheat (Larran et al., 2002a 2002a ), soybeans (Larran et al., 2002b 2002b ), bananas (Pocasangre et al., 2000 2000 ; Cao et al., 2002 2002 ), and tomatoes (Larran et al., 2001 2001 ). Several endophytic fungi have recently been described from oil palms (Rungjindamai et al., 2008 2008 ), such as Trametes elegans and Fomitopsis pinicola . Fusarium solani and Fusarium oxysporum f. sp. albedinis were early reported in date palm trees (Lamberti, 1988 1988 ; Hatimi, 1989 1989 ; Fernandez et al., 1995 1995 ). The endophytic fungi were not evenly distributed, with Pleosporaceae (not only Alternaria sp. and its teleomorph species Bipolaris sp., but also Curvularia sp.) constituting the dominant group in both the leaves and roots of the date palm tree. Indeed, Alternaria sp. constituted over 40% of the cultivable isolates and 61% of the clones with three species being identified ( Alternaria alternata and two Alternaria sp. sequence types). To a lesser extent, Curvularia species represented 23% of the isolates and 17% of the total clone sequences. Several previous studies reported the occurrence of those species in other plants, including banana (Cao et al., 2002 2002 ), Aegle marmelos (Gond et al., 2007 2007 ), Coffea Arabica L. (Fernandes et al., 2009 2009 ), tangerine and lemons (Dini-Andreote et al., 2009 2009 ), Deschampsia antarctica Desv. (Rosa et al., 2009 2009 ), Huperzia serrata (Wang et al., 2011 2011 ), Bletilla ochracea (Tao et al., 2008 2008 ), wheat (Smit et al., 1999 1999 ), Atriplex canescens (Lucero et al., 2011 2011 ), and Artemisia species (Huang et al., 2009 2009 ). The group of Pleosporaceae represented by Alternaria species consists mainly of saprophytes that are commonly found in soil or on decaying plant tissues; however, some of them were reported to acquire pathogenic traits (Thomma, 2003 2003 ). Species of Alternaria are prevalent in the mycoflora that infests and often parasitizes the seeds of a wide variety of food crops (Schroeder and Cole, 1977 1977 ). Here, endophytic Alternaria and cultivable and uncultivable Curvularia species were not only isolated from tissue samples but also directly amplified by PCR amplification. The fact that these strains and clone sequences were isolated in 2010 from a healthy tree and still remained viable in 2012 (unpublished results) suggests, indeed, their GRAS status. The second dominant group of endophytes, assigned as Nectriaceae (38.5%), was observed to be present exclusively in root tissues of date palm and in the cultivable fraction. Two species were identified, namely Fusarium equiseti and Fusarium sp. Those species are, in fact, reported to constitute the most important endophytes in a number of plant species, including Aegle marmelos (Gond et al., 2007 2007 ), banana (Cao et al., 2002 2002 ), Apodytes dimidiate (Shweta et al., 2010 2010 ), tomato (Hibar et al., 2007 2007 ), and maize (Gherbawy et al., 2002 2002 ). Pathogenic Fusaria such as F. oxysporum has long been known to be the causal agent of the Bayoud disease (El Hassni et al., 2007 2007 ). However, the Fusarium isolates from this study originated from a long-lasting healthy tree, suggesting their GRAS status. The lack of sequences belonging to Fusaria among the clones could however result from the difficulty to lyse the Fusarium cells (Vallaeys, unpublished), and reflect the inadequacy of plant DNA purification methods to assess the diversity of endophytic flora and further, underline the epidemiologic risk to underestimate or neglect given fungi populations, especially when referring to pathogens. Furthermore, in this study, Cladosporium and its teleomorphic species Davidiella represented 10% of the clones. Although commonly cultivable, this genus was only detected in this study by clone sequencing and appropriate media should be optimized to isolate and culture this genus in date palm. Cladosporium species are commonly found in many other plants (both herbaceous and woody) and in soil (Arx, 1949 1949 ; Fisher et al., 1995 1995 ; Taylor et al., 1999 1999 ; Bensch et al., 2010 2010 ), including wheat (Smit et al., 1999 1999 ), pharmaceutical plants ( Heterosmilax japonica ) (Gao et al., 2005 2005 ), and Atriplex canescens (Lucero et al., 2011 2011 ) where they constitute an extremely large and important genus of plant pathogens. However, Cladosporium also includes cosmopolitan and ubiquitous saprotrophs. Here again, the fact that Cladosporium- related sequences were obtained from an old-aged healthy tree suggests their non-pathogenic status. Finally, our molecular approach allowed us to enlighten the occurrence of uncultivable endophytes. In root samples, this firstly included Pythium (5% of the total clone sequence only), a common endophytic fungi, mainly reported as pathogenic, which can infect various hosts but remains undetectable using conventional culture techniques. Members of this genus act as pathogens towards a range of organisms, including plants, algae, fish, shrimp, mosquito larvae (Saunders et al., 1988 1988 ), and even humans (de Cock et al., 1987 1987 ). Some species of the genus can, however, be beneficial, functioning as biological control agents protecting against pathogenic fungi (Abdelghani et al., 2004 2004 ; Paul, 2004 2004 ). Few species, such as Pythium mycoparasiticum (Jones and Deacon, 1995 1995 ), Pythium contiguanum (Paul, 2004 2004 ), and Pythium paroecandrum (Abdelghani et al., 2004 2004 ), have been reported to behave as mycoparasites. However, although modern taxonomy is widely developed for Pythium (Lévesque and de Cock, 2004 2004 ), palm-derived sequences seem to belong to novel species, which do not allow for delineation of pathogenic versus GRAS or pathogenic status. Furthermore, our molecular analysis data showed that palm tissues were also colonized by two sequence types hitting up to now uncultivable Pleosporales (6% of total clone libraries). Indeed, one clone showed distant similarity with Preussia sp. (Sporormiaceae) which comprises species from soil, wood, and plant debris (Cain, 1961 1961 ; Arenal et al., 2007 2007 ). However no cultivable close relatives were available for this clone sequence. A clone sequence type, absent from the cultivable group, was affiliated distantly to Ochrocladosporium . Interestingly, another sequence types, presenting significant BLAST hits with given up to now uncultivable root fungal sequences, were detected both among our isolates and among our clones. This demonstrated that it could be cultured on traditional PDA agar. As previously reported, this sequence belonged to the dominant Pleosporales and thus appeared as a novel Curvularia species that could easily be further characterized. This result constitutes major progress in our knowledge of the palm-associated fungal flora. Further, the preliminary characterization of endophytic fungi in this study revealed a variation in the type of indigenous fungi isolated from roots and leaves. Indeed, some genera were recorded in the leaf tissues but not registered in the root tissues collected from the same tree, which could be attributed to the different environments involved, namely the leaves, representing a source of nutrients, and the roots, representing a nutrient sink (Lim et al., 2003 2003 ). Among these, Fusarium sp. and Fusarium equiseti were recorded in the root but not in leaf tissues of the plant. Further comparisons between the endophytic communities obtained from the traditional and the molecular techniques indicated that the indigenous fungi isolated from the roots and leaves were different. In fact, some species, such as Cladosporium sp. (1G10 and 1E1), were only recorded in leaf tissues. Conversely, other species, such as Pythium sp. (2A3), were recorded in root but not in leaf tissues. These results suggest that the endophytic population of date palm varies depending on the plant organ analyzed. Nevertheless, some species, namely Alternaria sp. and Curvularia sp., were recorded in both tissues. The comparison between the findings obtained for the endophytic fungal assemblages within P. dactylifera from the traditional and the culture independent molecular techniques suggests that some endophytic fungi could not be isolated from plant tissues using traditional cultural methods, presumably due to inherent limitations in those techniques. Conversely, the direct detection and identification of endophytic fungi from plant tissues using molecular techniques presents a number of limitations, such as those already suggested for Fusaria . In addition to lysis difficulties, as only sparse hyphae may exist within the plant tissues, underrepresented fungal DNA may be lost during the PCR process. This can be explained by competition with plant DNA present in excess in the sample, as universal primers also amplify the plant ITS. This results in numerous plant-associated sequences in the clone library. Conversely, the primers may not completely match with some fungal template DNA. Another equally important point concerns the treatment of the host tissue (Guo et al., 2001 2001 ). It is important to thoroughly clean the surface of the host tissue (with ethanol, detergents, and reducing agents) to remove potential phylloplane fungi and other organisms present on the host surface. Though presenting a number of limitations, molecular techniques appear to offer a complementary method for the detection of endophytic fungi, particularly those that cannot be incubated on artificial media and can be directly amplified from plant tissues. With the improvement of DNA extraction and PCR molecular techniques, this method could yield rapid and accurate tools for the direct detection and identification of endophytic fungi within plant tissues. In short, the present study demonstrated the occurrence and diversity of cultivable and uncultivable endophytes in date palm ( P. dactylifera L.). The roles of these endophytes in this habitat remain to be elucidated. Further studies, some of which are currently underway, are needed to gain more comprehensive data with regards to the biodiversity of the fungal endophyte population in date palm suffering from troublesome diseases, such as the brittle leaf disease, and to compare them with data pertaining to healthy fungal endophyte populations. More generally, this study underlines the necessity to more extensively investigate the fungal diversity of the Mediterranean environment which, despite its importance, has not been subjected to exhaustive studies. Indeed, only a few disperse examples are addressed in the literature (Fisher et al., 1992 1992 ; Peláez et al., 1998 1998 ; Girlanda et al., 2002 2002 ), which clearly means, as underlined by this study, that in the Mediterranean environment, a large number of new species are likely to still be awaiting discovery. References EY Abdelghani , K Bala , B Paul . 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This article does not contain any studies with human or animal subjects performed by any of the authors. 10.1631/jzus.B1200300 Project supported by EGIDE (No. 18470SA), CMCU (No. 08G908), and the Tunisian Ministry of Higher Education 2012-11-05 2013-11-24 2013-03-05 2022-01-05 Copyright © Zhejiang University and Springer-Verlag Berlin Heidelberg 2013 2013 Journal of Zhejiang University-SCIENCE B (Biomedicine and Biotechnology) 12 14