Mycol. Res. 92 (3): 322-326 (1989) Prinled in Greal Brilain 322

Endophytic fungi in twigs of healthy and diseased Norwayspruce and white fir

THOMAS N. SIEBER

Mikrobiologisches Institut, ETH-Zentrum, Ch-8092, Zurich, Switzerland

Endophytic fungi in twigs of healthy and diseased Norway spruce and white fir. Mycological Research 92 (3): 322-326 (1989).

Almost all 5- to 6-yr-old twigs of healthy and diseased white fir (Abies alba) and Norway spruce (Picea abies) were colonized by atleast one fungal taxon. The endophyte populations of both tree species were very similar but some populations differed significantlywith site, possibly due to climatic and ecological differences.

Significant differences in endophyte populations between healthy and diseased trees could only be detected at one site forNorway spruce with respect to Sirodothis sp_ and Phomopsis occulta.

Air pollutants are suspected as possible causes of changes in endophyte populations.

Key words: Picea abies, Abies alba, Endophytes, Fungi, Forest decline, Air pollutants.

Table 2. Number of trees investigated at each site

between endophyte populations of healthy and diseased treeshave been recorded.

• Mean values (1987) according to R. Gehrig, EidgenossischeMaterialpriifungsanstalt (EMPA), Diibendorf (ZH), Switzerland.

Table 1. Characterization of the sites

Uigem Alptal Davos

Grid reference 669775/ 696775/ 784450/259025 211 125 187725

Height (m.a.s.l.) 685 1185 1640Mean temperature (OC) 8-6 6'3 2'8Mean yearly rainfall (mm) 1006 1652 1007Mean relative humidity (%) 78 80 71

Air pollutants'

NO (J.1g m-3) 3'0 0'5 0'6N02 (J.1g m-3) 24 6 6S02 (J.1g m-3

) 15 5 4

HealthyDiseased

White fir

5 55 5

Not present

Healthy

610

10

Diseased

Norway spruce

41010

UigemAlptalDavos

In 1987 more than 50% of both Norway spruce (Picea abies (L)Karst.) and white fir (Abies alba Mill,) in Switzerland werereported to show more than 15 % needle loss (SanasilvaReport 1987). Air pollution, lack of nutrients, damage to therhizosphere, poor forest management or climatic changes arefrequently suspected as pOSSible causes (Bormann, 1985 ;Lichtenthaler & Buschmann, 1984; Manion, 1985; Schutt et aI.,1985; Schutt, 1985; Wargo, 1985). Other authors, however,suppose that needle loss can also be caused by viruses,bacteria and fungi (Nienhaus, 1985; Rehfuess & Rodenkirchen,1984), If this is the case these organisms must be able to infectand live endophytically in healthy host tissues, and diseasedtrees must be colonized to a greater extent by such organisms.Fungal endophytes have been isolated from a wide range ofevergreen and deciduous plants (see Petrini, 1986), and mostof the conifers examined so far host endophytic fungi (Carroll,Muller & Sutton, 1977; Carroll & Carroll, 1978; Sieber­Canavesi & Sieber, 1987). The nature of these symbioses isdiverse and can be mutualistic, neutral or antagonistic (Latch,Hunt & Musgrave, 1985; Sieber, 1985; Carroll, 1986; Petrini,1986). Fungi known to be potential pathogens can live for acertain period as neutral endophytes and cause symptomsonly after onset of appropriate ecological and physiologicalconditions (Sieber, 1985; Carroll, 1986). Some of the potentialpathogens of conifer needles could thus behave in this way.

This project has therefore been undertaken to detectpotential pathogenic endophytes in twigs of diseased Norwayspruce and white fir and to compare the endophyte populationsof healthy and diseased trees. Information about the fungalspecies involved has been collected; the influence of climaticand ecological conditions of the sites and the differences

Thomas N. Sieber 323

Table 3. Mean frequency (%) of colonization of twigs of Norway spruce and white fir by endophytic fungi

Picea abies Abies alba

Healthy Diseased Healthy Diseased

Percentage of twigs colonized by endophytic fungi 98'0 99'3 99'5 99'5Two fungal species 22'8 24'7 15'2 15'2Three fungal species 1'4 1'6 1'9 1'9

Ascomycetes

Coniochaeta velutina (Fuckel) Munk 0'2 0 2'9 0'5

Mollisia sp.• with Cystodendron-anarnorph 0'2 0 0'5 1'4

Strattonia minor Lundq. 0'8 0'4 1'9 1'9

Deuteromycetes

Coelomycetes

AgyrielJopsis caeruleo-atra Hohn. 1'2 0'4 27'1 19'0Botryodiplodia ercelsa (Karst.) Pet. & Syd. 1'4 2'0 0 0

Brunchorstia sp. 2'8 1'8 0 1'0

Coniothyrium conorum Sacco & Roum. 0'8 1'6 0 0'5

CornicularielJa abietis Karst. 10'3 9'2 1'4 0

Cryptosporiopsis abietina Petrak 6'0 2'2 1'0 0Endobotryella oblonga (Fcld) Hohn. 0 1'6 0 0Gelatinosporium sp. 15'9 15'4 10'5 20'0

Leptostroma anarn. of Lophodermium piceae (Fuckel) Hohn. 1'4 0'5 0 0

Libertella faginea Desm. 1'2 1'1 0 0

Phlyctaena sp. 4'6 2'0 0 0

Phomopsis occulta (Sacc.) Trav. 7'5 14'3 28'6 39'0

Pocillopycnis umensis (Bubak & Vleugel) Dyko & Sutton 18'7 17'2 0 0Pseudocenangium succineum (Spree) Dyko & Sutton 0'4 0'5 8'6 3'8

Rhizosphaera kalkhojfii Bubak 1'2 0'5 0 0

Sirococcus myrlilli (Feltg.) Pet. 1'4 0'7 0 0

Sirodothis sp. 25'2 37'2 8'5 1'9

Zythiostroma pinastri (Karst.) Hohn. 0 0'4 2'4 2'9

Hyphomycetes

Acremonium anam. of Nectria fuekeliana Booth 1'4 0'5 0 0

Cercoseptoria sp. 0'2 1'3 0 0Erophiala sp. 0'2 1'6 2'9 1'4

Geniculosporium anam. of Hyporylon bipapillatum Berk. 0'2 0 1'4 0Geniculosporium anam. of Hyporylon serpens (Pers.: Fr.) Kickx 0'8 1'1 1'0 1'0Hormonema dematioides Lagerberg & Melin 2'0 0'7 1'9 1'9Oedocephalum elegans Preuss anam. of Pezj:m echinospora Karst. 0'4 0'2 1'0 2'4Phialophora hoffmannii (Beyma) Schol-Schwarz 1'6 2'7 3'3 2'4

Rhizoctonia sp. 0'4 0'7 0'5 1'9

Other species' 4'2 2'7 4'3 3'8

Sterile mycelia 10'9 6'8 9'0 11'9

• Frequency of colonization by species which could be isolated only from one or two tWigs. include Acremonium kiliense Griitz. A. strictumGams. Alternaria a/ternata (Fr.) Keissler, Anthostomella conorum (Fuckel) Sacc.. Asterome/la sp., Aureobasidium pullulans (de Bary) Am., Chalaraaellaria Cooke & Ellis. Cladosporium cladosporioides (Fresen.) de Vries, C. orysporum (Link: Fr.) Berk. & Curt.. Cytospora sp.. Epicoccum nigrumLink. Geniculosporium sp., Periconiella anarn. of Biscogniauria nummularia (Bull.) O. Kuntze. Phialophora alba Beyma, Phialophora cyclaminisBeyma, Phialophora lagerbergii (Melin & Nannf.) Conant, Pithomyces sp.• Pleurocytospora sp.. Preussia f/eisehhakii (Auersw.) Cain, Ramularia sp..Rosellinia the/ena (Fr.) Rab.• Sirocoecus conigenus (DC) P. Cannon & Minter. Sporidesmium sp.. 50rdaria jimieola (Rob.) Ces. & de Not., SporormielJaintermedia (Auersw.) Ahmed & Cain. TroposporelJa fumosa Karsten. Virgariella sp., Xylaria spp.

MATERIALS AND METHODS

Twigs of white fir were collected at two and Norway spruceat three sites in Switzerland: the sites were located at differentaltitudes with different climatic and ecological conditions(Table 1). Sampling was done according to Table 2. Trees withmore than 15 % needle loss were considered diseased. The

mean tree age of both diseased and healthy tree groups wasroughly the same. The height of the trees was at least 12 m.

Seven twigs were cut from each tree at a height of 3-5 mabove ground. taken to the laboratory and processed within24 h. From each twig three 5- to 6-yr-old segments of about3 em length were surface-sterilized by washing first for 1 minin ethanol (96 %). then 5 min in sodium hypochlorite with 4 %

Endophytic fungi in Norway spruce and white fir 324

available chlorine, and rinsing again for 0'5 min in ethanol(96%). A piece of approximately 3 mm was cut from themiddle of each segment and placed on a 90 mm Petri dishcontaining 2 % malt agar (Oxoid malt extract) supplementedwith 50 mg 1-1 oxytetracycline hydrochloride. Plates wereincubated at IS DC for 30-60 d depending on the growthrates of the fungi. Fungi growing out of the segments weretransferred to I % malt extract agar slants. Most isolatessporulated within 1-6 months.

Frequency of colonization by the most commonly isolatedfungi was compared by means of discriminant analysis usingthe statistical package SPSS-X (release 2.0) on a cyber180-855 (ETH, Zurich).

Boxplots were used to compare healthy and diseased treesgraphically with respect to colonization by the most frequentlyisolated fungal taxa (Cleveland, 1985), and the differenceswere tested by means of the Mann-Whitney U test.

RESULTS

Almost all twigs examined were colonized by at least onefungal species. Nearly 25 % of the twigs of Norway spruceand IS % of the twigs of white fir were colonized by twodifferent fungi. Fifty-three species were isolated from twigs ofNorway spruce and thirty-seven from white fir. The speciesmost frequently isolated are listed in Table 3: all belong to theAscomycotina or Deuteromycotina.

Fungal populations of the twigs of Norway spruce andwhite fir were found to be very similar. All species isolatedfrom white fir were also colonizers of Norway spruce; thereverse did not always apply; however, this may be due to thesmaller number of twigs of white fir that were examined.

Corniculariella abietis, Cryptosporiopsis abietina, Gelatinosporiumsp., Phomopsis occulta, Pocillopycnis umensis and Sirodothis sp.were the main colonizers of twigs of Norway spruce (Table 3).

Significant differences could be detected between theendophyte populations at the three sites (discriminant analysis,P ~ 0'0001), as shown by the scaHerplot of the discriminantscores of each tree (Fig. I). The cluster-points of the three sitesare well separated and overlap to a certain extent only for

Fig. 1. Scatterplot of the discriminant scores for Norway spruce. D.trees at Uigern: .... trees at Alptal; 0, trees at Davos; *' groupcentroids.

Alptal and Uigem: two trees in Lagem are assigned by theanalysis to the Alptal group, whereas one tree in Alptal iscolonized by fungi otherwise typical in Lagem.

The frequency of colonization by the six most commonlyisolated endophytes appears to be related to altitude andconsequently very likely to climatic conditions of the sites. Cr.abietina and Ph. occulta occurred most frequently at Lagem, Co.abietis and Gelatinosporium sp. at Alptal and P. umensis atDavos. Sirodothis sp. was abundant in all three sites but mostfrequent at Davos, where Co. abietis, Cr. abietina and Ph. occultawere not isolated.

Differences in the number of healthy and diseased Norwayspruce twigs colonized by fungi were statistically significantonly at Lagem for Ph. occulta and Sirodothis sp. (P < 0'05),where these two fungi were more frequent in the twigs ofdiseased trees (Fig. 2). At Davos, mean colonization bySirodothis sp. was higher in diseased trees than in healthy ones,but the difference was not statistically significant.

Brunchorstia sp. (probably the anamorph of a Gremmeniellasp.), the Leptostroma-anamorph of Lophodermium piceae,Rhizosphaera kalkhoffii and Sirococcus myrtilli, which areimportant parasites of conifers (Butin, 1983; Funk 1981, 1985)were rarely isolated and more frequent in the twigs of healthytrees (Table 3).

The most frequently occurring endophytes in twigs ofwhite fir were Agyriellopsis caeruleo-atm, Gelatinosporium sp.,Ph.occulta, Pseudocenangium succineum and Sirodothis sp. (Table3).

The endophyte populations of twigs of white fir collectedat Lagem and Alptal were statistically significantly different(discriminant analysis, P ~ 0'0001). This was largely due tothe differences in number of twigs colonized by A. caeruleo­atm, Ph. occulta and Gelatinosporium sp. at the two sites (Fig.3). At Liigem colonization of twigs by Ph. occulta was almosttwenty times higher than at Alptal. On the other hand, A.caeruleo-atra and Gelatinosporium sp. were more frequent atAlptal than at Liigem. The two sites did not differ with respectto the colonization by Sirodothis sp. or Ps. succineum.

At Liigem and Alptal the number of twigs of healthy anddiseased white fir colonized by any fungus was not statisticallysignificantly different, even if some values in Table 3 wouldsuggest such a tendency, e.g. A. caeruleo-atm, Gelatinosporiumsp., Ph. occulta, Ps. succineum and Sirodothis sp.

DISCUSSION

Fungal endophyte populations of Norway spruce and white firare very similar. However, a few fungal species preferentiallycolonize one host. Agyriellopsis caeruleo-atra and Pseudocenangiu,!!succineum occur mainly in twigs of white fir. whereasCorniculariella abietis and Cryptosporiopsis abietina are morefrequent in twigs of Norway spruce (Table 3). Pocillopycnisumensis. after Sirodothis sp. the most frequent endophyte intwigs of Norway spruce, seems to be host-specific. However,this fungus was isolated mainly at Davos. where Abies alba isnot present.

Sieber (1985) demonstrated that endophytic fungi show atendency for organ specificity. The results of this study confirmthese findings for the following reasons: Sieber (1988) found

5

cc

c

cc *

c c c

-3 +--.--,----,-.--.-....,....~-r-...---.--,---+

-7 -6 -5 -4 -3 -2 -1 0 1 2 3 4Canonical discriminant function 1

6+---L.---''--...L---L.---''--...L-......L---''--...L-......L........I'--+

N 5.g~ 4

3

~:~ 2<Il

'6 0] 0

'" -1

~ -2

Thomas N. Sieber 325

A

Fig. 3. Box graph displaying selected percentiles of the number oftwigs of healthy and diseased white firs colonized by the five mostfrequently isolated endophytic fungi at (A) Lagem and (B) Alptal. Forexplanation see Fig. 2. Ac, Agyriellopsis caeruleo-atra; Ce,Gelatinosporium sp.; Po, Phomopsis occulta; Ps, Pseudocenangiumsuccineum; Si, Sirodothis sp.; D, healthy; D, diseased.

20 r----'--~~-~~'---~"---~.....,.

] 18'§ 16'0 14u 12~.~ 1~

642o i~........L----'--.J--'- .J...L.J...l._-l-L..I.--I

A

Fig. 2. Box graph displaying selected percentiles of the number oftwigs of healthy and diseased Norway spruce colonized by the sixmost frequently isolated endophytic fungi at (A) Uigem, (B) Alptaland (C) Davos. The horizontal line in the interior of the box indicatesthe median value. The upper and the lower horizontal lines of thebox represent the 75th and the 25th percentiles of the number ofcolonized twigs. If the median value falls exactly on either quartiledemarcation, the demarcation line is extra bold. The ends of thevertical bars indicate the lOth and the 90th percentiles, Points belowor above vertical bars represent values of trees outside the 80%confidence interval. If the intervals of two boxes do not overlap, thetwo corresponding populations can be considered different. Co,Cornicularie/la abietis; Cr, Cryptosporiopsis abietina; Ce, Gelatinosporiumsp,; Po, Phomopsis occulta; Pu, Pocil/opycnis umensis; Si, Sirodothis sp.;D, healthy; D, diseased.

Co Co Cr Cr GeGe Po Po Pu Pu Si Si

Si Si

Si SiPs Ps

Ps PsPo Po

Po Po

Ge Ge

Ge Ge

Ac Ac

B

'0 201l 18'2 160'0 14

~u 12.,.~ 10~ 8....0 6...E 4§ 2Z 0

Ac Ac

isolated species from twigs, and it is thus likely that themycelium of Sirodothis sp. in the needles probably originatedfrom twigs.

Sieber-Canavesi & Sieber (1987) found that Cryptoclineabietina Petrak, Gloeosporidiella sp. and Exophiala sp. are thedominant species of the endophyte populations of white firneedles. In the present investigation only Exophiala sp. couldbe isolated from twigs (Table 3).

Different climatic conditions may be responsible for thelarge differences among the populations of endophytic fungiat the three sites. Po. umensis seems to prefer the cold and dryclimate of Davos, whereas Co. abietis and Gelatinosporium sp.occurred most frequently at Alptal, characterized by atemperate, wet climate. Phomopsis occulta and Cr. abietina seembetter adapted to the rather warm and dry climate of Uigem.Only Sirodothis sp. occurred quite frequently at all sites.

It can be speculated that air pollutants may influenceendophyte populations of twigs. The differences in thefrequency of endophytes at Liigem, Alptal and Davos couldpartly be caused by air pollutants, whose concentrations arethree to six times higher in Liigem than at Alptal or Davos(Table 1).

However, the design of this project did not allow theeffects of air pollutants on endophyte populations to bequantified.

The composition of endophyte populations and thefrequency of colonization by each species are similar for bothhealthy and diseased trees. Therefore the presence ofendophytes can not be related to increased signs of disease.

Cr Cr Ge Ge Po Po Pu Pu Si Si

'0 181l 16'2 140

o ~'0 12u., 10 EJco'~ 8.... 6 1 B0... 4E 2 66E::> 0z;

Co Co CrCr GeGe Po Po Pu Pu Si Si

B

18'0 161l'2 140

12'0u10

~.~ 8

~~.... 60 4~ 2E 0::>Z

Co'Co

C

'0 18.., 16N'2 140'0 12u.,

10co.~ 8.... 60

~4

E 2::> 0Z

that the Leptostroma-anamorph of Lophoderrnium piceae andTiarosporella parca (Berk. & Broome) Withney et al. colonized5- to 6-yr-old needles of Norway spruce in Switzerland witha mean frequency of 19 % and 11 % respectively, whereas onthe twigs examined in this 'study L. piceae occurred only rarelyand T. parca not at all. Sieber (1988) also isolated Sirodothis sp.in only 1 % of the needles, mostly from the proximal parts,whereas this work showed it to be one of the most frequently

Endophytic fungi in Norway spruce and white fir

Significant differences between healthy and diseased treeswere only detected at Uigern for Sirodothis sp. and Ph. occultaon Norway spruce. One can assume that diseased Norwayspruce trees at Uigern lost their ability to prevent extensivecolonization by these two fungi. Growth of Sirodothis sp. andPh. occulta could also be enhanced. by air pollutants.

Sirodothis spp. are anamorphs of Tympanis spp.; mostTympanis spp. are known pathogens on a wide range of treespecies including conifers (Smerlis, 1970). The species ofSirodothis isolated in the present study, however is unlikely tobe a pathogen because it was more frequently isolated fromtwigs of diseased Norway spruce at only one site. Nothing isknown on the potential pathogenicity of Ph. occulta.

The author is grateful to Mr Ernst Frehner of theEidgenossische Anstalt fur das forstliche Versuchswesen atBirmensdorf for collecting the twigs in Uigern, to Mrs CarmenFischer-Hugentobler for excellent technical assistance, to DrsP. J. Fisher (University of Exeter, U.K.), 0. Petrini and F.Sieber-Canavesi for critically reading the manuscript.

This study was supported by the Swiss National ResearchCounciL Research Program NFP 14t.

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