Mycol. Res. 97 (6): 679-682 (1993) Printed in Great Britain 6 79

Endophytic fungi of Eucalyptus globulus: a preliminary study

LINA BETTUCCI A N D MARISABEL SARAVAY Department of Botany, Facultad de Ciencias, Tristan Narvaja 1674, Montevideo, Uruguay

Forty-one endophytic fungi were isolated from stem and leaves of young shoots of sprouting stumps and seedlings of Eucalyptus globulus. Plectosphaera eucalypti was the main species colonizing seedlings, shoot stems, mature leaves and to a lesser extent, xylem. Cytospora spp. and Microsphaeropsis sp. appeared confined to the xylem and Coniella minima to seedling stems. Young and mature leaves also showed differences in fungal colonizers: the former were essentially infected by C. minima and the latter by P. eucalypti and Harknessia hawaiiensis. A correspondence analysis performed on all taxa from stem samples has revealed three distinct clusters, corresponding to seedling, shoot xylem and complete shoot stems, respectively. Percentages of minimal similarity indexes among populations from xylem, complete sprouting stem and seedling stem were very low which also demonstrated tissue-specific differences in the composition of fungal endophyte populations. Several isolates of basidiomycetes with positive oxidative extracellular enzymes were present in all three stem units. They also overgrew and replaced the more frequent endophytes under laboratory conditions.

Fungal endophytes have been isolated from a wide range of evergreen, deciduous and coniferous plants (Carroll ef al., 1977; Carroll & Carroll, 1978; Cabral, 1985; Carroll, 1986; Petrini, 1986; Fisher & Petrini, 1987, 1990; Petrini & Fisher, 1987, 1988; Bertoni & Cabral, 1988; Sieber, 1989). These fungi can live for a certain period as neutral endophytes and produce symptoms only after appropriate ecological and physiological conditions occur (Carroll, 1986; Chapela & Boddy, 1988).

In the last 20 yr Eucalyfpus spp. have been planted throughout the world as fuel and pulp sources. E. globulus is currently being planted intensively in U ~ g u a y (Ministerio de Ganaderia, Agricultura y Pesca, 1991), Argentina and Brazil. This preliminary investigation was performed to detect potentially pathogenic endophytes in seedlings and young shoot stems from tree stumps, with emphasis on wood- rotting Basidiomycetes.

MATERIALS A N D METHODS

During autumn 1991 sprouting stumps and seedlings of Eucalyptus globulus were sampled from plantations situated on sandy soils in Maldonado on the east coast of Uruguay.

Four I - to 2-yr-old shoots from four sprouting stumps and 40 seedling stems, 5-30 cm in length, were taken to the laboratory in paper bags and processed within 24 h. Segments of 0.5 cm were cut from several internodes of each shoot. The number of segments varied from 3 to 9 according to the length of the shoots. Seedling stem segments were obtained

from three zones: near the root, in the middle part and near the leaves. In the half of the segments from shoots the bark was stripped off. Surface sterilization was performed using sodium hypochlorite according to Fisher & Petrini (1987). Each shoot segment was dissected in 6-8 chips which were placed separately onto 9 cm Petri dishes containing 2 % malt extract agar. Plates were incubated at room temperature up to 6 wk. Each different colony was transferred to fresh media to allow identification. Near-uv-light was used to induce sporulation in some cultures. Those that failed to sporulate after 6 wk (culture media were exhausted) were considered sterile.

Foliar fungal colonizers were recorded by incubating 10 surface-sterilized leaves of I-yr-old seedlings and 5-10 leaves of 2- to 4-month-old seedlings, per plant, on glass slides in Petri dishes with moist cotton at room temperature. Fruit bodies of fungi developing from inner leaf tissues were identified and the area occupied by them determined approximate1 y.

Production of extracellular oxidative enzymes was tested in basidiomycete isolates following Stalpers (1978). When overgrowth of one species occurred, discs of mycelium (3-4) from interface and overgrown zones were transferred to fresh media. Basidiomycete isolates were identified by means of cultural characteristics, according to Stalpers (1978).

Density of colonization was calculated as the percentage of chips infected by a given taxon from the total number of chips of each tissue plated out. Populations resulting from xylem, complete shoot stems and seedling stems were compared using the percentage of minimal similarity (PMS)

Lina Bettucci and Marisabel Saravay 680

(Krebs, 1989). Differences in populations of endophytes from these materials were examined by correspondence analysis subroutine from STAT-ITCF written by the Service des Etudes Statistiques, Institut Technique des CCrCales et des Fourrages (ITCF), France. Relative values (e.g. relative densities) can be used in this software package. All the analyses were carried out on all species isolated.

RESULTS

Forty-one taxa from 168 shoot stem chips and 40 discs from seedling stems were isolated. The number of taxa in each tissue was similar, ranging between 16 and 19. Chips were frequently colonized by two taxa, while colonization by more than two fungi appeared to be rare. Table 1 shows relative colonization densities in percentages, total number of taxa and total number of isolates for each material.

The fungal community of E. globulus shoot xylem appeared to be dominated by Cyfospom spp., Plectosphaera eucalypti and Microsphaeropsis sp. Basidiomycetes (Coriolus versicolor, Phanerochaete puluerulenfum and unidentified clamped isolates 18/22, 48) colonized 20.2% of the chips.

Complete shoot stems were essentially dominated by Plectosphaera eucalypfi (97.6%) that formed fruit bodies only on plant tissues. Basidiomycetes (Pycnoporus sanguineus and an unidentified clamped isolate 72) occurred in lower percentages than in xylem, infecting only 6 % of chips.

Seedling stems were mainly colonized by Plectosphaera eucalypti, Pestalofiopsis guepini and Coniella minima. The two former were dominant in 20-30 crn plants and the latter in 5-7 cm plants. An unidentified cord-forming basidiomycete (isolates 27/30) infected 7.5 % of discs belonging to the upper half of the stem in 10-15 cm seedlings.

All basidiomycete isolates produced laccase and peroxidase, except Ph. pulverulentum (Table 2).

Leaves from seedlings were colonized only by Coniella minima, while mature leaves produced P. eucalypfi and Harknessia hawaiiensis. P. eucalypti was mainly found close to the veins from the petiole up to the apex and on the blade margins, occupying nearly 40% of leaf surface. H. hawaiiensis was present in the remainder part of the blade surface.

All basidiomycetes overgrew P. eucalypfi. Basidiomycete 18 also overgrew Cytospora sp. 15 and it was in turn partially overgrown by T. longibrachiaturn. The overgrowing fungi were the only species that grew when discs of the interwoven mycelia from the original plates were transferred to fresh media.

The PMS ranged from 20% (xylem-seedling stem) and 21 % (xylem-complete stem) to 34 % (complete-seedling stem). The primary species responsible for these values was P. eucalypti. The correspondence analysis comparing the endophyte communities of xylem, complete shoot and seedling stems showed that the two first axes accumulate 100% of the inertia. The principal-coordinate axes 1-2 separate three distinct clusters of closely grouped species (Fig. I). The first (X) axis (58.9%) separates xylem from seedling and complete shoot stems. Although the complete stem contains xylem, the three most important species (Cytospora sp. 6, Mi~rosphaero~sis sp. and P. dendriticum) were only isolated from xylem (Table I).

Table 1. Endophytic fungi of Eucalyptus globulw. Relative colonization densities in percentages

Arthrinium phaeospermum (Corda) Ellis 2.4 Cytospora sp. 6 6 4 3 Microsphaeropsis sp. 405 Pellionella aff. deformans Penz. & Sacc. 1.2 Penicillium dendriticum Pitt 15.5 Trichoderma hamatum (Bon.) Bain. 5.9 Trichoderma longibrachiatum Rifai 4.8 Coriolw versicolor (L.: Fr.) QuCI. 2.4 Phanerochaete pulverulentum Novobranova 9.5 Basidiomycete 18/22 4.7 Basidiomycete 48 3.6 Sterile mycelium 19 1.2 Unidentified sp. 17 2.4 Aspergillw niger van Tieghem 1.2 Aureobasidium pullulans (de Bary) Amaud 2.4 Curvularia pallescens Boedijn 4.8 Cytospora sp. 56 3.6 Harknessia hawaiiensis Stev. & Young 5.9 Kendrickomyces indicus Rao & Mhaskar 7 1 Trichodem koningii Oud. 3.6 Pycnoporw sanguinew (L.: Fr.) Mum. 2.4 Basidiomycete 72 3.6 Sterile mycelium 29 1.2 Acremonium sp. 2.5 Chaetomium globosum Kunze: Fr. 2.5 Chaetomium sp. 13 5.0 Chaetomium sp. 28 7.5 Coniella minima Sutton & Thaung 10.0 Hainesia lythri (Desm.) Hohn. 5.0 Penicillium sp. 2.5 Phoma sp. 2.5 Trichosphaeria sp. 2.5 Basidiomycete 27/30 7.5 Cytospora sp. 15 28.6 3.6 Epicoccum purpurascens Ehrenb.: Schlecht. 1.2 7.1 Gliocladium roseum Bain. 2.4 13.1 Alternaria alternata (Fr.) Keissl. 2.4 7.5 Pestalotiopsis guepini (Desm.) Stey. 2.4 20.0 Phoma lingam Tode: Schw. 4.8 7 5 Nigrospora sphaerica (Sacc.) Mason 3.6 1.2 5.0 Plectosphaera eucalypti Thiessen 40.5 9 7 6 35.0

Total number of taxa 19 17 16 Total number of isolates 199 139 49 Total number of segments 84 84 40

Symbols used indicate: X, xylem; C, complete stem; S, seedling stem. Each chip was colonized by more than one species, thus percentages add

up to more than 100.

Table 2. Extracellular oxidative enzyme production

C. versicolor P. sanguinew Ph. pulverulentum Basidiomycete 18 Basidiomycete 22 Basidiom~cete 31 Basidiomycete 48a Basidiomycete 48b Basidiomycete 72

La, laccase; Lg, laccase g ; Es, esterase; Ph, phosphatase; Pe, peroxidase; Ty, tyrosinase. 0, negative; +, light; 1-4, positive, increasing enzyme reaction.

Endophytic fungi of E. globulus 68 1

Fig. 1. Correspondence analysis. Ordination of xylem and complete shoot stem of sprouting stump and seedling on the two first axes. Variables are the isolation densities of colonization (percentage of

2

0.5 --

-1 4 . 5

MIC ART

C6 001 Sl9

TRH PEL COR TRL

B18 PDE UNI 4 . 5 5

B48

chips infected by a given taxon from the total number of chips plated out). The first (X) axis separates xylem from seedling and from complete shoot stem. The second (Y) axis shows differences in fungal

ASP

AUR CUR TRK S29

GLI EP1 C56 pyc HAR 0°2 KEN B72

PLE

0.5 1

PHL

C13

NIG C28 TRI

PEN CON

B27 0°3 PES ALT

PHO HA1 ACR

communities between seedling and complete stem Symbols used indicate: the provenance of the species 001, xylem;

002, complete shoot; 003, seedling, and the species: Acremonium sp., ACR; Alfernaria alfernafa, ALT; Arfhriniurn phaeospermum, ART; Aureobasidium pullulans, AUR; Aspergillw niger, ASP; basidiomycete 18/22, B18; basidiomycete 27/30, 27; basidiomycete 48, 848; basiodiomycete 72, B72; Chaetomium globosum, CHA; Chaetomium sp. 13, C13; Chaefomium sp. 28, C28; Coniella minima, CON; Coriolus versicolor, COR; Curvularia pallescem, CUR; Cyfospora sp. 6, C6; Cytospora sp. 15, C15; Cyfospora sp. 56, C56; Epicoccum purpurascens, EPI; Gliocladium ruseum, GLI; Hainesia lythri, HAI; Harknessia hawaiiensis, HAR; Kendrickomyces indicw, KEN; Microsphaeropsis sp., MIC; Nigrospora sphaerica, NIG; Pellionella aff. deformam, PEL; Penicillium dendrificum, PDE; Penicillium sp., PEN; Pesfalotiopsisguepini, PES; Phanerochaefe pulverulenfum, PHA; Phoma lingam, PHL; Phoma sp., PHO; Plectosphaera eucalypti, PLE; Pymoporus sanguineus, PYC; sterile mycelium 19, S19; sterile mycelium 29, S29; Trichoderma hamafum, TRH; Trichodema koningii, TRK; Trichodema longibra- chiafum, TRL; Trichosphaeria sp., TRI; unidentified sp. 17, UNI.

The second (Y) axis (41.1%) evidences differences in fungal composition between seedlings and complete stems. P. eucalypti is present in both types of tissue. The results of the correspondence analysis confirm those obtained by the PMS, but a better separation of fungal endophytic populations from each material is achieved.

DISCUSSSION

Relative colonization densities for each species, PMS, and correspondence analysis demonstrated that tissue-specific preferences characterize the endophytic fungal communities.

A comparison between the endophytes of xylem and

complete shoot stems shows that only five species are present in both types of tissue. Some species such as P. eucalypfi, colonizing xylem and complete stems, display the ability to penetrate deeply into host tissues. The density of this taxon was consistently higher for complete stems than for xylem. Thus P. eucalypfi is apparently better adapted to live within superficial tissues as well as being able to colonize the xylem, as pointed out for several endophytes of other hosts by Petrini & Fisher (1988).

On the other hand the fact that Cytospora spp. were consistently isolated from xylem and scarcely from complete stem suggests that this species usually inhabits xylem. Cytospora spp. are well known tree pathogens (Sinclair, Lyon & Johnson, 1987) and were found to be important pathogens of Eucalyptm spp. in Argentina (Sarasola & Sarasola, 1959).

The major species colonizing seedlings apparently adopt different strategies during plant development. P. eucalypti seems to remain a successful endophytic colonizer while Pesfalotiopsis guepini tends to diminish, and Coniella minima to be replaced by other endophytes being restricted to young plants.

Differences between basidiomycete species (or isolates) colonizing shoot and seedling stems could be due to the temporal evolution of the fungal community within the stem (Rayner, Boddy & Dowson, 1987). In turn, species (or isolates) of wood-rotting fungi showed a similar tissue specificity as the other endophytes, probably related to differences in water activity between xylem and bark of shoot stem (Boddy & Griffith, 1989). It is possible that secondary growth and development also imposes a certain specificity.

The absence of records of endophytic basidiomycetes, in the literature, could be due to the real absence of these fungi as endophytes in other tree species studied or to the method used to sample for wood endophytes. We have probably been able to isolate wood-rotting basidiomycetes because we split wood segments into small chips. Alternatively, sprouting stumps may be a reservoir of wood-rotting basidiomycetes for new shoot stems.

C. minima in leaves of seedlings, and Harknessia hawaiiensis and P. eucalypti in those of mature trees, were the only species present at the same time in stems and leaves. This fact confirms Sieber's (1989) assumption that endophytic fungi also show a tendency for organ specificity.

The authors are very grateful to Dr I. Chapela, Sandoz Pharma Ltd, for his valuable suggestions and critical reading of the manuscript and to SAREC for the financial support of this work.

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(Accepted 9 October 1992)