AFPP – 9ème CONFÉRENCE INTERNATIONALE SUR LES MALADIES DES PLANTES TOURS – 8 ET 9 DÉCEMBRE 2009
ENDOPHYTIC MYCOFLORA OF ATLAS CEDAR (CEDRUS ATLANTICA MAN.) IN BELEZMA MASSIF (AURES, ALGERIA) D. HARZALLAH (1), O. A. BENSSACI (2) and S. BOUHARATI (1) (1,3) Faculté des sciences, département de biologie, Université Ferhat Abbas, Sétif, 19000, (2) Faculté des sciences, département d’Agronomie, Université ElHadj Lakhdar, Batna, 05000, Algérie.
[email protected] SUMMARY Endophytic fungi were detected, isolated and collected from needles of Atlas cedar (Cedrus atlantica Man.), from three separated locations in Belezma massif (Aurès, Algeria). More than 20 fungal genera were obtained. The range of fungal taxa is dominated by Mitosporic group. The lower endophyte diversity in BOUMERZOUG site is eventually related to the degraded physiological status of Cedrus as a result of forest decline phenomena occurred recently in Belezma forests. The composition of studied fungal endophytes in Cedrus atlantica is mostly represented by some common genera such as Lophodermium, which is common in conifers. The genera Fusarium, Cylindrocarpon, Verticillium and Phomopsis were potential pathogens, while the presence of entomopathogenic fungus Beauveria is a sign of a probably protecting roles against phytophagos insects in sampled trees. Key words: Endophytic fungi , Cedrus atlantica, Bélezma, colonization frequency, biodiversity RÉSUMÉ LA MYCOFLORE ENDOPHYTE DU CÈDRE DE L’ATLAS (CEDRUS ATLANTICA MAN.) DANS LE MASSIF DE BELEZMA (AURÈS, ALGÉRIE) Les champignons endophytes ont été détectés et isolés à partir de la trame foliaire de cèdre de l’Atlas (Cedrus atlantica Man.) au niveau de trois sites du massif de Bélezma (Aurès, Algerie). Plus de 20 mycotaxons endophytes ont été identifiés, dont la grande part taxinomique est prédominée par le groupe des champignons Mitosporiques. La diversité peu signifiante des mycoendophytes au niveau de la station de Boumerzoug est attribuée éventuellement aux conditions phytosanitaires de la cédraie majoritairement affectée par le phénomène de Dépérissement que connaissent les peuplements de cèdre dans le Bélezma. La composition de la mycoflore endophyte étudiée chez le cèdre de l’Atlas exhibe certains Genres communs tels que, Lophodermium, lié aux Conifères. D’autres genres ; Fusarium, Cylindrocarpon,Vereticillium et Phomopsis sont considérés potentiellement pathogènes. La présence de Beauveria à confirme le switch du comportement trophique de certains champignons endophytes. Mots-clés : Champignon endophyte, Cedrus atlantica, Bélezma, fréquence de colonisation, biodiversité.
INTRODUCTION Endophytic fungi form an ecologically important component of mycobiota in forest ecosystems. This fungal group plays important roles in forestlands diversity and functions (Stone et al., 1996). They contribute by several ways in plant protection against various phytophagous insects, phytopathogenic microbes and in abiotic stress tolerance (Carroll, 1986 in Deckert, 2000; Clay, 1999; Wilson, 1997; Azevedo et al., 2000). Endophytic Fungi are also reputed by their contributions in plant nutrition and synthesis of plant growth hormones (Malinowsky and Belesky, 2000; Tan and Zou, 2001). Atlas cedar (Cedrus atlantica Man.) is one of the most conifer tree species of North African landscape. C. atlantica Man. native of Algerian and Moroccan Atlas mountains. Early data concerning C. atlantica repartition in Algeria, mentioned by Boudy (1952) in Bentouati and Oudjhih (1999) showed that this Pinaceae occupy approximately an area of 33000 ha, of which the most area is located in Aurès mountains (17000 ha). However, it is too imperative to indicate that these data should be taken with prudence because of the lack of sufficient information, especially after Atlas cedar decline phenomena. Endophytic fungi of conifer trees, including Pinus, Picea and Abies species, have been examined in various studies (Sieber-Canavesi et al., 1991; Petrini et al., 1992; Wilson, 1997; Sieber et al., 1999; Ganley and Newcombe, 2006). Nothing is known however, about this microbial group in Cedrus atlantica. The main objectives of this work are to demonstrate, for the first time, the occurrence of endophytic fungi in healthy needles of C. atlantica in Belezma National Park, BNP, (east of Algeria). A study was designed to examine the endophytic fungal species composition and frequencies in healthy needles of Atlas cedar in three sites of BNP. Species distribution and specificity of colonisation patterns have been studied and compared between sites. MATERIALS & METHODS SITE CHARACTERISTICS For Belezma National Park, Three sites have been chosen for C. atlantica needles sampling (1) BOUMERZOUG; (2) TALMET and (3) THOUGGAR . Theses sites (table I) exhibits generally identical vegetation but different edaphic and microclimate conditions. The woody species are C. atlantica, Quercus ilex (Fagaceae), Juniperus oxycedrus (Cupressaceae) and Fraxinus xanthoxylloides (Oleaceae). Table I: Sites characteristics Caractéristiques des sites Site BOUMERZOUG TALMET THOUGGAR
Altitude (m) 1510 1740 1480
Latitude 35° 35’ 32’’ N 35° 35’ 21’’ N 35° 34’ 38’’ N
Longitude 6° 05’ E 6° 02’ E 6° 03’ E
NEEDLE COLLECTIONS 10 trees of C. atlantica are randomly selected at each of the three sites. With different ages (20 years to more than 150 years). For each tree, 5 branches are chosen randomly, they must have two needles categories: young- and old or adult healthy needles. It is assumed that endophyte colonisation is more important in oldest needles (Stone, 1986; Johnston and Whitney, 1992 in Wilson, 1997). Needle samples were stored at 4° C and processed within 24- 48 h after collection. For each age category, 10 rosettes are mixed; we proceed after that to needle separation in laboratory.
SURFACE-STERILIZATION Needles were surface-sterilized by soaking first for 2 min in 95% (v/v) ethanol, then for 3 min in a solution of sodium hypochlorite (NaOCl) and finally for 30 s in 95% (v/v) ethanol. Each treatment is followed by a rinsing in sterile distilled water. Once dried, each C. atlantica needle were cut aseptically into 2 to 4-mm-long segments and put into Petri dishes (90 mm diameter) containing a PDA (Potato Dextrose Agar) medium, prepared after Smith and Onion protocol (1994), and amended by 50 mg l-1 Tetracycline® to inhibit Bacteria. Each Petri dish contains 20 needle-segments from the same age class. Then, we have finally 400 segments by site (1200 segments in total). Petri dishes were incubated at 24°C in the dark and examined daily during colony development. Pure cultures were prepared from the original mycelia which grew from the needle segments by transferring them to Petri dishes with PDA. Colonisation frequency (%) was calculated after Fisher and Petrini (1987) as the number of colonized needle segments divided by the number of total examined needles multiplied by 100. Fungal taxa are identified after microscopic observations, by means of morphological features (Lanier et al., 1976, 1978). If sporulation failed, sterile forms were isolated and categorized as Mycelium Sterile (MS) based on cultural characteristics such as texture and hyphal pigmentation. DATA ANALYSIS Relative percentage of occurrence (RPO) of a fungal group was calculated using the following formula: PRO = FCs / FCt x 100. FCs is the colonisation frequency of fungal group, while FCt represents colonisation frequency of all groups of fungi. SIMPSON (λ) and SHANNON-W EAVER (H’) diversity indices were calculated to measure species diversity of endophytic mycobiota of Atlas cedar needles, using the method of Ludwig and Reynolds (1988) in Barengo et al., (2000) using Colwell’s Estimates software v. 7.5 (Colwell, 2005). Analyse of variance (ANOVA) was effected to show effect of needle age and sites in endophyte colonization using Statistica 5.1 software. RESULTS According to our results, all trees hosted endophytes. More than one third of needle segments of C. atlantica are infected by endophytic fungi in the three sites. Colonization frequency is 57% in BOUMERZOUG; 64.75% in Talmet and 65% in THOUGGAR site. No differences between sites are detected regarding overall colonization by endophytic fungi (P< 0.05), this indicates that site do not determine numerical aspects of needles infection by endophytes, for the average age (F = 2.23, p =0.12), young needles (F = 0.62, p = 0.54) or adult needles (F = 1.20, p = 0.31). Obtained results demonstrate that endophytic colonization frequency of C. atlantica needles changes according to needle age. In fact, it seems that aged needles are more densely infected compared to young needles. This difference – statistically significant – is signalled for both studied sites (table II). Table II: Colonization frequency (%) of C. atlantica needles by endophytic fungi Fréquence de colonisation (%) des aiguilles de C. atlantica par champignons endophytes
Sites Boumerzoug Talmet Thouggar
Young needles 28 ± 6.28 34 ± 5.15 36 ± 4.20
Colonization frequency (%) Adult needles Average 86 ± 7.14 57 95.5 ± 3.02 64.75 94 ± 2.21 65
Analysis of variance and Newman-Keuls test (5%) illustrate a significant difference which scoring the endophyte infection of Atlas cedar needles according to age class. Thus, there is a general tendency for a more importantly colonisation spectrum for adult needles, this increased colonisation is probably linked to morphological and physiological development of C. atlantica needles. Twenty-two endophytic fungal taxa were detected and isolated from the 1200 needle segments examined; most for these fungi are detected from C. atlantica for the first time, such as: Verticillium sp.; Alternaria alternate; Aureobasidium pullulans; Epicoccum nigrum; Bipolaris sp. and Beauveria sp. the endophytic mycoflora of C. atlantica needles is predominated by four taxa: Aspergillus sp.; Alternaria alternate; Epicoccum nigrum and Lophodermium sp. which occurred in 4 % of more of the needle segments. Specific composition of fungal endophytes isolated differs clearly between three sites. Most endophytic fungi were recorded in TALMET, with 19 taxa, 14 fungal taxa were detected in THOUGGAR site, in BOUMERZOUG site only 09 taxa were recorded. Most endophytes are belonged to Mitosporic fungi, especially to Hyphomycetes: Alternaria, Aureobasidium, Epicoccum, Aspergillus, Cladosporium, Curvularia, Bipolaris, Penicillium, Fusarium, Beauveria, Gliocladium, Arthrinium and Verticillium. The Coelomycetes records two genera: Phomopsis and Phyllosticta. Two other taxa are ranged to Ascomycota: Lophodermium and Chaetomium. Mucor is the unique genus recorded among Zygomycota phylum (table III). Table III: General composition of fungal endophytes isolated from Atlas cedar needles in National ParK of Belezma. La composition générale des champignons endophytes isolée des aiguilles du Cèdre de l’Atlas dans le Parc National de Belezma. Mycotaxons BOUMERZOUG TALMET THOUGGAR Hyphomycetes Alternaria alternata + + + Alternaria sp. + Aureobasidium pullulans + + + Epicoccum nigrum + + + Aspergillus sp. + + + Cladosporium sp. + + Curvularia sp. + + Bipolaris sp. + + Penicillium sp. + + Fusarium sp. + + Beauveria sp. + Cylindrocarpon sp. + + Gliocladium sp. + + Arthrinium sp. + Verticillium sp. + Coelomycetes Phomopsis sp. + Phyllosticta sp. + Ascomycota Lophodermium sp. + + + Chaetomium sp. + Zygomycota Mucor sp. + STERILE MYCELIA MSI (colored by TB) + + MSII(not colored by TB) + + + +: present, -: absent, MS: mycelium sterile, TB: trypan blue.
Relative percentage of occurrence (RPO) for each of the most recorded endophytic fungi is presented in table IV. Table IV: Relative percentage of occurrence recorded for most mycoendophyte taxa Pourcentage relatif d’occurrence (PRO) calculé pour la mycoflore endophyte Fungal taxa Aspergillus sp. Alternaria alternata Lophodermium sp. Epicoccum nigrum
Young needles 59.75 28.11 10.23 31.58
Adult needles 61.41 41.77 13.45 19.82
Average 60.58 34.94 11.84 25.7
SHANNON-W EAVER diversity index (H’) records for TALMET site has the maximum value (0.47), although there is no significant difference compared to THOUGGAR site (0.40), in contrast to BOUMERZOUG site where we have recorded the most lower value of H’ (0.33). Talmet site records a small value of SIMPSON index (λ) with 0.19. It is possible to deduce that colonization frequency is not linked to endophytic diversity. Fungal endophyte distribution along C. atlantica needles is not static according to such spatial and temporal scales. Our results showed differences between the number of taxa, and in fact, at the level of diversity of endophytes between sites. The followed dendrogram shows the most fungal groups from Atlas cedar needles and their classification according to occurrence in sites (figure 1). Figure 1: Most fungal groups from Atlas cedar needles and their classification according to occurrence in sites Classification de la majorité des groupes des champignons du cèdre de l’Atlas selon leur présence sur sites Dist. Euclidiennes ALTALT ALTSP AUREOB EPICOCCU ASPERGIL CLADOSPO CULVULAR BIPOLARI PENICILL FUSARIUM CYLINDRN GLIOCLAD BEAUVERI VERTICIL PHYLLOST MUCOR MS1 ARTHRINI PHOMOPSI CHAETOMU LOPHODER MS2 0
5
10
15
20
Dist. d'Agrégat.
ALTALT : Alternaria alternata, ALTSP : Alternaria sp., AUREOB : Aureobasidium pullulans, EPICOCCU : Epicoccum sp., ASPERGIL : Aspergillus sp., CLADOSPO : Cladosporium sp., CURVULAR : Curvularia sp., BIPOLARI : Bipolaris sp., PENICILL : Penicillium sp. FUSARIUM : Fusarium sp., CYLINDRN : Cylindrocarpon sp., GLIOCLAD : Gliocladium sp., BEAUVERI : Beauveria sp., PHYLLOST : Phyllosticta sp., VERTICIL : Verticillium sp., MUCOR : Mucor sp., MS1 : Mycelium sterile I, ARTHRINI : Arthrinium sp., PHOMOPSI : Phomopsis sp., CHAETOMU : Chaetomium sp., LOPHODER : Lophodermium sp., MS2 : Mycelium sterile II.
Dendrogramm shows three principal groups of endophytic fungi in C. atlantica needles for three sites: the first group is essentially composed by those taxa which are recorded in two sites or all sites (Alternaria alternata, Aureobasidium pullulans, Epicoccum nigrum, Aspergillus sp., Cladosporium sp., Curvularia sp., Bipolaris sp., Penicillium sp., Fusarium sp., Cylindrocarpon sp., Gliocladium sp., Lophodermium sp., as well as both sterile forms. The second group includes fungal taxa isolated from TALMET site, represented by Alternaria sp., Beauveria sp., Phyllosticta sp., Verticillium sp. and Mucor sp. The third group is constituted by fungi which are detected from BOUMERZOUG site: Arthrinium sp., Phomopsis sp. and Chaetomium sp. DISCUSSION This present study demonstrates for the first time, the occurrence of endophytic fungi of Cedrus atlantica phyllosphere. The overall frequency of colonization by fungal endophytes is marked by such differences between sites, even there seems to be little considered, there may be attributed to several factors which are in the majority of the site origins such as relief, altitude of site, hygrometry, as well as edaphic condition, which determinates source of primary endophytes inoculums. For other Pinaceae trees, such similar results were obtained (Carroll and Carroll, 1978; Petrini et al., 1981; TODD, 1988; Legault et al., 1989 in Deckert, 2000; Sieber et al., 1999; Jurc et al., 2000). Other site characteristics may be implicated in needles infection variations, such as tree population density. THOUGGAR site recorded an important population density of C. atlantica especially in sampling points where Atlas cedar forest is dominated by young and middle age class trees. BOUMERZOUG site (with CF = 57%) is marked by a heavy decline of Atlas cedar estimated to 90 – 95%, only few individuals dwelling yet healthy, but very scattered in the North versant and at low altitude. Needle age is a determinant factor of endophyte infection copiousness for all sites and adult needles seems to be more densely infected by fungi. The age of tissues clearly had a great influence on endophyte frequency. The influence of the age of tissues on frequency and species composition of endophyte assemblages has been reported in several studies (Espinosa-Garcia & Langenheim, 1990; Sieber-Canavesi & Sieber, 1993). The needles of Pinaceae trees which are belonged to several age classes, varies in respect to exposition time to a so called the “optimum fungal inoculum charge” from which establishment of the symbiosis held. This bioecological characteristic is more linked to physical and chemical components of needles (Arnold & Herre, 2003). Leaf age is a good predictor of endophyte infection levels, which seem to increase universally with the age of the leaf (Bernestein & Carroll, 1977; Stone, 1987; Hata & Futai, 1993 in Deckert, 2000). This approach suggests in an intuitive sense, that foliar part of C. atlantica had a tendency to accumulate endophyte colonization with needle age, in a parallel to leaf morphological and physiological development and growth. Similar approach was adopted for Pinus strobes and P. monticola and Abies alba (Stone, 1987; Sieber-Canavesi & Sieber, 1993 Deckert, 2000)). We can explain the concentrated endophyte infection of old needles of C. atlantica at both anatomical and physiological levels by the deterioration of physical barriers integrity of needles such as waxed and cuticle layers (Miller & Roy, 1982, Kierstiens, 1996). In the other hand, physical factors can’t exclusively explain the low colonisation levels of young needles of Atlas cedar in BNP, especially after a chronologically large exposition to aerial inoculum. Physical and / or biochemical factors could be also considered (Wilson, 1997; Deckert, 2000). It is possible that a relatively few endophytic mycotaxa are detected (Carroll & Carroll, 1978; Petrini et al., 1992; Johnson & Whitney, 1992). This indicates a remarkable dominance of some fungal species which can eliminate and reduce consequently other fungal taxa. Laboratory conditions as well as sampling and culture methods are probably implicated (Petrini et al., 1982, 1992 in Wilson, 1997). The general composition of endophytic mycobiota of C. atlantica needles in BNP shows between site differences. While BOUMERZOUG site knows a severe decline of Atlas cedar forest since 2002/2003, there have been a massive degradation of “Cedrus” landscape, and
even our sampled trees appears healthy in our point of view, then there can be affected by this phenomenon in future. It seems difficult to categorize the general phytosanitary state of trees because our data are based on simple diagnostic description. BOUMERZOUG site recorded the poor endophyte diversity testifying a declining in physiological status which induces indirectly to a loss in endophyte community. Natural or induced alteration of conifer needles provokes an entry to a senescent phase implicating slight or radical changes in endophyte composition (Wilson, 1993, 2000 in Deckert, 2000). C. atlantica needles in TALMET site - with a good phytosanitary and ecological conditions - shelter more diverse fungal endophytes, Wilson (2000) in Deckert, (2000) demonstrates that Pinus strobes needles harbour a great number of fungal endophyte species in well structured sites. In this work, most mycoendophyte groups belonging to Mitosporic fungi. Several works report the specific dominance of this group in Conifer needles (Sieber-Canavesi & Sieber, 1993; Stone et al., 2004). Sexual fructifications of this artificially introduced assemblage (TAYLOR et al., 2004) were never observed on needles, implying other means of in vivo propagation suggesting in the same time a loss of sexual reproduction as a strategy of coevolution with C. atlantica in Belezma Mountains. But Wilson (1997); Ganley & Newcombe (2006) demonstrates Ascomycota dominance of endophytic fungi in Pinus resinosa and P. monticola, respectively. In our point of view, the dominance of mitosporic fungi among endophytes is probably attributed to the fact that the endophytism as a symbiotic strategy with plant partner requires a resort to asexual reproduction parallel to permanent presence of the evergreen host as an ecological niche. The presence of entomopathogenic fungus Beauveria sp. under endophytic form in C. atlantica needles is of a great importance; this suggests a possible compatibility of association between this entomopathogenic organism and Atlas cedar as plant partner. Endophytic Beauveria have been recently isolated from Pinus resinosa (Ganley & Newcambe , 2006), this same genus have also been detected as endophyte with two other entomopathogenic fungi: Lecanicilium dimorphum and L. c. f. psalliotae in date palm leaves (Gómez-Vidal et al., 2006). Lophodermium (anamorph = Leptostroma and Leptostromella) is commonly detected in C. atlantica needles for all sites. It is more frequently occurred under sexual form of in letters and senescent needles. Lophodermium is commonly associated to Conifer species (Lanier et al., 1978; Wilson, 1997) and it was reported in several Pinaceae needles: Pinus strobes, P. resinosa, P. strobes, P. mugo, Picea glauca, Abies alba and A .balsamea (Sieber-Canavesi & Sieber, 1991; Wilson, 1997; Sieber et al., 1999 Deckert & Peterson, 2000; Ganley & Newcombe, 2006). Lophodermium cedrinium was reported in C. atlantica in Algeria (Maire, 1912 in Spaulding, 1961). The life cycle of Lophodermium is well documented (Minter & Millar, 1980; Stone, 1988; Osorio & Stephan, 1991; Johnston, 1994; Deckert, 2000). Suryanarayanan & Thennarasan (2004) showed that RPO is less important for plants harbouring maximum fungal diversity. This phenomenon can be explained by the occupation patterns of tissues (Yang et al., 2000; Schulz et al., 2002). The value of RPO at TALMET site (29.89%) indicates that endophytes recovery for the most four isolated taxa is insignificant, in the same site we have recorded maximum diversity of endophytic fungi. By reading H’ and λ indices, we deduce that fungal endophytes diversity in TALMET site indicates an ecological balance and good functioning ecosystem especially when considering seasonality effects. Between-site differences among fungal communities were already evident. TALMET and BOUMERZOUG sites were characterized by a special assemblage suggesting site-specific assemblages. A similar pattern of endophyte colonization was observed also on other conifers (Sieber, 1988; Sieber et al., 1999). This study demonstrates that specificity of endophyte infection can be influenced by a combination of genetic predisposition and some environmental factors, but geographically determined also. This study provided us with information about quantitative and qualitative composition of Endophyte mycobiota in Atlas cedar phyllosphere at different locations, and confirmed that Atlas cedar represents a reservoir for a relatively broad range of fungal endophytes including potential pathogens, saprotrophytes as well as some biological control agents. The species composition depends on tissue age. We assume that species composition and the formation
of distinctly different endophyte associations is due to site-specific conditions. Future research should concentrate on finding the mechanism of fungal infection and the switch from different biotic regimes (asymptomatic to symptomatic colonization) that would permit an eventual use of endophytic fungi as biocontrol agents and in reforestation (Boddy & Griffith, in Shamoun & Sieber, 2000). ACKNOWLEDGMENTS We thank Dr. Thomas N. Sieber (Department of Forest and Wood Sciences, Forest Pathology and Dendrology, Swiss Federal Institute of Technology, Zürich, Switzerland) and Mrs. Laabed A. and Boukarkar H. (Belezma National Park, Batna, Algeria) for technical help. REFERENCES ARNOLD A. E. & HERRE E. A. 2003. Canopy cover and leaf age affect colonization by tropical fungal endophytes: ecological pattern and process in Theobroma cacao (Malvaceae). Mycologia 95(3): 388-398. AZEVEDO J. L., MACCHERONI JR. W., PEREIRA J. O. & DE ARAUJO W. L. 2000. Endophytic microorganisms: a review on insect control and recent advances on tropical plants. Electronic Journal of Biotechnology 3 (1): 40-65. BENTOUATI A. & OUDJHIH B. 1999. Premières etudes de la croissance et de la productivité du cèdre de l’Atlas (Cedrus atlantica Manetti) dans le massif de Bélezma (Aurès). CARROLL G. C. & CARROLL F. E. 1978. Studies on the incidence of coniferous needle endophytes in the Pacific Northwest. Canadian Journal of Botany 56: 3034-3043. CLAY K. & HOLAH J. 1999. Fungal endophyte symbiosis and plant diversity in successional fields. Science 285: 1742-1744. COLWELL R. K. 2005. EstimateS : Statistical estimation of species richness and shared species from samples. Version 7.5. DECKERT R. J. 2000. Structural and ecological aspects of the relationship of phyllosphere fungi with their host, Pinus strobus L. PhD. thesis University of Guelph (Canada). 172 pp. ESPINOSA-GARCIA F. J. & LANGENHEIM J. H. 1990. The endophytic fungal community in leaves of a coastal redwood population – diversity and spatial patterns. New Phytologist 116: 89-97. FISHER, P.J., & PETRINI, O. 1987. Location of fungal endophytes in tissues of Suaeda fruticoa: a preliminary study. Mycological Research 89: 246-249. GANLEY R. J. & NEWCOMBE G. 2006. Fungal endophytes in seeds and needles of Pinus monticola. Mycological Research 110: 318-327. GOMEZ-VIDAL S., LOPEZ-LLORCA L. V., JANSSON H.-B. & SALINAS J. 2006. Endophytic colonization of date palm (Phoenix dactylifera L.) leaves by entomopathogenic fungi. Article in press. Micron xxx: 9 pp. JOHNSON J. A. & W HITNEY N. J. 1992. Isolation of fungal endophytes from black spruce (Picea mariana) dormant buds and needles from New Brunswick, Canada. Canadian Journal of Botany 70: 1754-1757. JURC D., JURC M, SIEBER T. N. & BOJOVIC S. 2000. Endophytic Cenangium ferruginosum (Ascomycota) as a reservoir for an epidemic of Cenangium dieback in Austrian pine. Phyton 40 (4): 103-108. KIERSTIENS G. 1996. Signalling across the divide: a wider perspective of cuticular structure function relationship. Trends in Plant Science 1: 125-129. LANIER L., JOLY P., BONDOUX P. & BELLEMERE A. 1978. Mycologie et Pathologie Forestière, Volume I : Mycologie forestière. Edition Masson. 487 pp. LANIER L., JOLY P., BONDOUX P. & BELLEMERE A. 1976. Mycologie et Pathologie Forestière, Volume II : Pathologie forestière. Edition Masson. 478 pp. MALINOWSKI D. P. & BELESKY D. P. 2000. Adaptations of endophyte-infected cool-season grasses to environmental stresses: mechanisms of drought and mineral stress tolerance. Crop Science 40 (4): 923-940.
MILLER W. A. & ROY K. W. 1982. Mycoflora of soybean leaves, pods, and seeds in Mississippi. Canadian Journal of Botany 60: 2716-2723. PETRINI O., SIEBER T. N., TOTI L. & VIRET O. 1992. Ecology, metabolite production, and substrate utilization in endophytic fungi. Natural Toxins 1: 185-196. Schulz B., Boyle C., Draeger S., Römmert A.-K. , KROHN K. 2002. Review: Endophytic fungi: a source of biologically active secondary metabolites. Mycological Research 106: 996-1004. Shamoun S. F. & Sieber T. N. 2000. Colonisation of leaves and twigs of Rubus parviflorus and R. spectabilis by endophytic fungi in a reforestation site in British Columbia. Mycological Research 104 (7): 841-845. Sieber T. N., Rys J. & Holdenrieder O. 1999. Mycobiota in symptomless needles of Pinus mugo ssp. Uncinata. Mycological Research 103 (3): 306-310. Sieber T. N. 1988. Endophytische Pilze in gesunden und geschädigten Fichten (Pica abies (L.) Karst.). European Journal of Forest Pathology 18: 321-342. Sieber-Canavesi F., Petrini O. & Sieber T. N. 1991. Endophytic Leptostroma species on Picea abies, Abies alba, and Abies balsamea: a cultural, biochemical, and numerical study. Mycologia 83 (1): 89-96. Sieber-Canavesi F. & Sieber T. N. 1993 Succesional patterns of fungal communities in needles of European silver fir (Abies alba Mill.). New Phytologist 125: 149-161. Smith D. & Onions A. H. S. 1994. IMI Technical Handbooks No. 2: The preservation and maintenance of living fungi. CAB International. Wallingford. 122 pp. Stone J. K. 1987. Initiation and development of latent infections by Rhabdocline parkeri on Douglas-fir. Canadian Journal of Botany 65: 2614-2621. Stone J. K. 1988. Fine structure of latent infections by Rhabdocline parkeri on Douglas-fir with observations on uninfected epidermal cells. Canadian Journal of Botany 66: 45-54. Stone J. K., Sherwood M. A. & Carroll G. C. 1996. Canopy microfungi: function and diversity. Northwest Science 70: 37-45. Stone J. K., Polishook J. D. & White J. F. 2004. Endophytic fungi. In Mueller G. M., Bills G. F. & Foster M. S. (eds.) Biodiversity of Fungi: inventory and monitoring methods. Elsevier Academic Press. pp 241-269. Suryanarayanan T. S. & Thennarasan S. 2004. Temporal variation in endophyte assemblages of Plumeria rubra leaves. Fungal diversity 195-202. Tan R. X. & Zou W. X. 2001. Endophytes: a rich source of functional metabolites. Nat. Prod. Rep. 18: 448-459. Taylor J. W., Spatafora J., O’Donnell K., Lutzoni F., James T., Hibbett D. S., Geiser D., Bruns T. D. & Blackwell M. 2004. The fungi. In CRACRAFT J. & DONOGHUE M. J. (eds.) Assembling the Tree of Life. Oxford University Press. pp 171-194. Wilson R. 1997. Endophytic fungi from four tree species in New Brunswick and a comparison of two methods of identification of Leptostroma isolates of Pinus resinosa: morphology and molecular probing. PhD. thesis. University of New Brunswick (Canada). 131 pp.
