molecular characterization of dematiaceous root endophytes

1397

S. K. HARNEY*, S. O. ROGERS AND C. J. K. WANG

Faculty of Environmental and Forest Biology, State University of New York, College of Environmental Science and Forestry, Syracuse, New York

13210-2788, U.S.A.

Sterile dematiaceous fungi are commonly isolated from plant roots. They are often assigned to Mycelium radicis atrovirens, a name

originally proposed for black, sterile, fast-growing, pseudomycorrhizal fungi. Dematiaceous fungi isolated from roots may be

mutualists, commensalists, or pathogens and, in the absence of sporulation, identification is not possible. Forty-six isolates of

dematiaceous fungi from the roots of different hosts and locations were characterized using restriction site mapping of polymerase

chain reaction amplified nuclear ribosomal DNA internal transcribed spacers. The restriction site maps were compared to identified

dematiaceous mycorrhizal and pseudomycorrhizal fungi. Computer generated trees (UPGMA and parsimony analysis) characterized

two unknown isolates as Phialophora finlandia, an ectendomycorrhizal fungus. The majority of the isolates were characterized as

Phialocephala fortinii-like. Phialocephala fortinii has been reported as both pathogenic and non-pathogenic in a number of hosts. There

was variation within the P. fortinii-like group suggesting intraspecific variation or a species complex.

Sterile, dematiaceous fungi are commonly isolated from roots

and often remain unidentified (e.g. Melin, 1923 ; Haselwandter

& Read, 1980 ; Currah et al., 1988 ; Stoyke & Currah, 1991 ;

O’Dell & Trappe, 1992). In a recent study examining the

mycorrhizas of outplanted white pine seedlings, a large

number of isolates were non-sporulating and dark (Wilcox,

Wang, Prabhu, LoBuglio & Harney, unpublished), of the sort

commonly assigned to Mycelium radicis atrovirens (Mra).

Culture manipulation allowed Wang & Wilcox (1985) to

induce three Mra-like isolates to sporulate : Chloridium

paucisporumC. J. K. Wang & H. E. Wilcox, Phialophora finlandia

C. J. K. Wang & H. E. Wilcox, and Phialocephala fortinii C. J. K.

Wang & H. E. Wilcox. Chloridium paucisporum (Wilcox &

Ganmore-Neumann, 1974 ; Wilcox et al., 1974) and P. finlandia

(Wilcox & Wang, 1987a, b) formed ectendomycorrhizas with

Pinus resinosa Ait. and mainly ectomycorrhizas with Picea

rubens Sarg. Phialocephala fortinii formed pseudomycorrhizal

(pathogenic) associations with Picea mariana (Mill.) B.S.P.

(Richard & Fortin, 1974), P. resinosa and P. rubens (Wilcox &

Wang, 1987a ; Wang & Wilcox, 1985). Phialophora finlandia

and C. paucisporum, morphologically similar to the cosmo-

politan ectomycorrhizal fungus Cenococcum geophilum, have

never been reported elsewhere.

Phialocephala fortinii has been isolated from orchid roots

(Currah et al., 1987), alpine plant roots (Ericaceae, Rosaceae)

(Stoyke & Currah, 1991) and lupin roots (O’Dell & Trappe,

1992). Its role in orchidaceous and ericaceous plants is

uncertain, although it does not appear to cause serious

* Current address : Biology Department, San Diego State University, SanDiego, CA 92182, U.S.A.

damage (e.g. Stoyke & Currah, 1991). In lupin (Lupinus

latifolius Agardh.) and Pinus contorta Dougl. there is evidence

that it lives within the root without penetrating the vascular

tissue or forming any mycorrhizal structures, and is neither

beneficial nor pathogenic (O’Dell et al., 1993).

Because the role of dematiaceous endophytes is variable,

accurate identification of root isolates is the first step in

eliciting their ecological role. Culture manipulation is time

consuming, and often unsuccessful. PCR (polymerase chain

reaction) amplification and restriction site mapping of the

internal transcribed spacer (ITS) region have previously been

utilized to characterize and identify ectomycorrhizal fungi

(Gardes et al., 1991 ; Gardes & Bruns, 1993). We evaluated

restriction site mapping and phenetic analysis of PCR amplified

ribosomal DNA (rDNA) ITS regions as a means to characterize

a number of Mra-like isolates.

MATERIALS AND METHODS

Forty-six isolates were used in this study (Table 1). Cultures

were maintained on 2% malt extract agar (MEA). All

unknown isolates were sterile and chosen either because they

resemble P. finlandia or P. fortinii (Mra-like), or randomly from

a selection of dematiaceous isolates.

Isolates were incubated at room temperature in 2% liquid

malt extract broth, either in static or agitating conditions on

a rotating shaker, for DNA extraction. Mycelium was

harvested by vacuum filtration over filter paper (Whatman no.

1), frozen at ®80 °C and lyophilized. Lyophilized mycelium

was stored at ®20°.Total DNA was extracted using a CTAB

(cetyltrimethylammonium bromide) micropreparation method

Mycol. Res. 101 (11), 1397–1404 (1997) Printed in the United Kingdom

Molecular characterization of dematiaceous root endophytes

Dematiaceous root endophytes 1398

Table 1. List of 46 dematiaceous isolates used in phylogenetic analysis

Isolate number* Isolate name Substrate Location

S8-1 Cenococcum geophilum Fr. Picea glauca (Moench) Voss Wanakena, NY

BDD22-extype Chloridium paucisporum Pinus resinosa Ait. Syracuse, NY

P30, P31, P109, P136 Phialocephala dimorphospora W. B. Kendr. Utility pole Chester, NJ

DAOM165556a P. dimorphospora Decayed stump Canada

FAP7-extype Phialocephala fortinii Pinus sylvestris L. Finland

R151, R152 P. fortinii P. resinosa Warrensburg, NY

UAMH5452 P. fortinii Calypso bulbosa (L.) Oakes Grass Lake, Alberta

UAMH5628 P. fortinii Platanthera dilata (Pursh) Lindl. Castle River, Alberta

UAMH6677 P. fortinii Luetkea pectinata (Pursh) Kuntze Outpost Lake, Alberta

SE24 P. fortinii Lupinus latifolius Agardh. Oregon

MTD313-20 Phialophora cyclaminis Beyma Sclerotinia minor Jagger Maryland

MTD632-11

FAG15-extype Phialophora finlandia Pinus sylvestris Finland

† FAF8B, FAG4, FAP8 Unknown P. sylvestris Finland

†M436, M450, M458, M467, M479, Unknown P. strobus L. Warrensburg, NY

M503, M504, M523, M545, M630,

M843, M1014

† F1, F3, F5 Unknown Picea rubrens Sarg. Whiteface Mtn, NY

† F2 Unknown P. rubrens Hubbard, NH

† F4 Unknown P. rubrens Kussuthe, ME

†K92 47, K93 270 Unknown P. abies (L.) Karst. Germany

†K92 90, K92 68 Unknown P. abies Switzerland

†K93 312, K93 313 Unknown Fagus sylvatica Ehrh. Germany

†K93 246 Unknown Pinus sylvestris Germany

†K93 202, K93 187 Unknown Abies alba Mill. Switzerland

* MTD isolates were obtained from M. Dunn, UAMH isolates from Lynne Sigler, SE24 from J. Trappe (A. Jumpponen), F isolates from P. Wargo and K

isolates from K. Ahlich.

† Indicates rows of isolates which are Mra-like (Mycelium radicis atrovirens).

(Rogers & Bendich, 1985, 1994 ; Rogers et al., 1989). The

nuclear rDNA ITS (internal transcribed spacer) region was

amplified on a Programmable Thermal Controller (MJ

Research) (30 cycles of : 1 min at 90°, 2 min at 50° and 2 min

at 72°). Primer pairs used were prITS4 with prITS5, prITS1

with prITS4, prITS1 with prITS2, and prITS3 with prITS4

(White et al., 1990). Reactions were overlaid with 35 µl of

light mineral oil (Sigma). Amplification was confirmed by

electrophoresis on 1±5% agarose gels.

The restriction endonucleases used for RFLP analysis were

Alu I, Ava II, BamH I, Cfo I, Cla I, Dde I,, EcoR I, Hae III, Hinf I,

Msp I, Rsa I, Sau3A I, Sau96A I, and Taq I (Promega, WI). The

reactions consisted of 2 µl amplified DNA, 1 µl (buffer

supplied by the manufacturer), 6±5 µl H#O, and 0±5 µl enzyme

(1–10 units). The reactions were incubated overnight at 37°(except Taq I which was incubated at 65° for 6–10 h). An

additional 0±5 µl of enzyme was added after several hours.

The products were run on 2% agarose gels, the band size

recorded, and restriction site maps constructed. Mapping was

performed using double digests of selected enzymes as well as

from prITS1}2 and prITS3}4 primer amplifications digested

with each enzyme.

A Jaccard similarity matrix of restriction enzyme sites, and

the presence or absence of an intron, was generated using the

computer program SIMQUAL of NTSYS (Numerical Tax-

onomy System of Multivariate Statistical Programs) (Rohlf,

1988). A cladogram was generated by analysis of the matrix

using UPGMA (unweighted pair-group method using arith-

metic means) clustering with the computer program SAHN

(sequential, agglomerative, hierarchical and nested clustering

method). The accuracy of fit was computed using cophenetic

correlation computer programs COPH and MXCOMP.

Parsimony trees were generated using PAUP (phylogenetic

analysis using parsimony, version 3±0o) (Swofford, 1990).

Bootstrap analysis (100 replications) using the heuristic search

strategy was performed to determine the support for the

internal branches of the trees.

RESULTS

In general, analysis of restriction enzyme patterns produced

three clusters, with some exceptions (Figs 1, 2). Three distinct

clusters were produced in UPGMA similarity analysis (Fig. 1),

with a number of isolates showing 100% similarity within the

clusters. The cophenetic correlation value of 0±98 indicates a

high goodness of fit for the cluster analysis (Rohlf, 1988).

Phialophora finlandia (FAG15), and Chloridium paucisporum

(BDD22), both ectendomycorrhizal on pine, were 100%

similar to the ectendomycorrhizal fungus FAG4 and the

unknown M479 isolated from white pine roots (cluster I)

(Fig. 1).

Phialocephala dimorphospora isolates clustered together in

each analysis (cluster II) (UPGMA and PAUP) (Figs 1, 2).

Isolates P30 and P31, isolated from utility poles, were

identical, as were P109 and P136, also isolated from utility

poles. Isolate DAOM165556a, isolated from decaying wood,

was closely allied. The second distinct group within cluster II

consisted of the P. dimorphospora isolates, P30 and P31 (100%

similar) which were 92% similar to P109 and P136 (100%

similar to each other), and DAOM165556a, 97% similar to

P109 and P136.

Isolates in the third cluster (cluster III) were morphologically

S. K. Harney, S. O. Rogers and C. J. K. Wang 1399

BDD22 Chloridium paucisporum – extypeFAG4FAG15 Phialophora finlandia – extypeM479P30 Phialocephala dimorphospora

FAP7 Phialocephala fortinii – extypeDAOM165556a P. dimorphospora

P31 Phialocephala dimorphosporaP109 Phialocephala dimorphosporaP136 Phialocephala dimorphospora

R151 Phialocephala fortiniiUAMH6677 Phialocephala fortiniiK93 187K93 246K93 312M450M458M467M630SE24 Phialocephala fortiniiUAMH5452 Phialocephala fortiniiK93 202M843FAF8BK92 47F1F4K92 68M523

FAP8R152 Phialocephala fortinii

R503 Phialocephala fortiniiR504 Phialocephala fortinii

M436

M1014K93 270K92 90K93 313

F2

F3F5M545

S8-1 Cenococcum geophilum

UAMH5628 Phialocephala fortinii

MTD313-20 Phialocephala cyclaminisMTD632-11 Phialocephala cyclaminis

0 20 40 60 80 100

Percent similarity

I

II

III

Fig. 1. UPGMA cluster diagram (NTSYS) of relationships among dematiaceous isolates based on similarity coefficients of rDNA

restriction enzyme sites. The cophenetic correlations is 0±98. Unnamed isolates were sterile in culture.

Mra-like (Figs 1, 2). All were from roots and included several

P. fortinii cultures : FAP7 (extype culture), R151 and R152,

UAMH6677, SE24, UAMH5452, and M503 and M504.

Support for individual groups was high, but in general,

dropped between groups. Isolates did not cluster according to

geographic origin.

The third cluster included almost all the Mra-like isolates

and could be divided into smaller clusters. A number of

isolates clustered at 100% similarity : FAP7, R151,

UAMH6677, K93 187, K93 312, M450, M458, M467 and

M630; SE24, UAMH5452, and K93 202 ; FAF8B and K92 47 ;

F1 and F4 ; R152, M436, M503, M504 and M1014 ; K92 90

and K93 313 ; F3 and F5. The FAP7 group and SE24 group

clustered at 97% similarity, with the SE24 group showing

93% similarity to M843.

Isolates with similar colony morphology did not closely

cluster within the Mra-like group (cluster III) (Figs 1, 2).

Isolates K92 90, K92 270 (both from Picea abies), K93 313

(from Fagus sylvatica) and F1, F3 and F5 (from Picea rubrens)

loosely grouped with the Mra-like cluster but with low

percent similarity. Isolate UAMH5628 (from Platanthera dilata),

identified as P. fortinii, did not cluster with the Mra-like

isolates.

The remaining clusters could not be easily interpreted, since

they exhibited low percent similarities. Several individual

isolates (F2, M545, S8-1 and UAMH5628) did not closely

group with any other isolates. The P. cyclaminis isolates

(MTD313-20 and MTD632-11) showed a low percent

similarity to all isolates examined.

The phylogramgenerated using the heuristic search strategy

of PAUP (Fig. 2) was similar to the UPGMA tree in Fig. 1. Not

all isolates are included in the phylogram to increase clarity.

Representatives from each cluster are presumed. The P.

cyclaminis isolate (MTD313-20) was used as the outgroup.


100

55

80

56

60

FAG15 Phialophora finlandia – extype

P30 Phialocephala dimorphospora

P109 Phialocephala dimorphospora

DAOM165556a Phialocephala dimorphospora

I

FAP7 Phialocephala fortinii – extype

54SE24 Phialocephala fortinii

II

III

M523

FAP8

FAF8B

M843

K92 68

R152 Phialocephala fortinii

F1

F2

F3

K93 270

K92 90

UAMH5628 Phialocephala fortinii

M545

S8-1 Cenococcum geophilum

MTD313-20 Phialophora cyclaminis

50

Fig. 2. Bootstrap tree (PAUP) determined by a heuristic search of 100 replications. Bootstrap values greater than or equal to 50% are

indicated on the branches. Unnamed isolates were sterile in culture. The characteristics of the tree are : CI¯ 0±569, HI¯ 0±431, RI¯0±582, RC¯ 0±331.

Three groups, FAG15, P. dimorphospora and Mra-like groups

produced separate clusters. Bootstrap analysis showed 100%

support for grouping the P30 isolate with P109 and

DAOM165556a. Isolates UAMH5628, M454 and S8-1 were

not closely allied to any other group.

The restriction site matrix is shown in Table 2 and the

composite restriction enzyme map of the 3 main clusters are

shown in Fig. 3. All isolates had a length of approx. 620 bp

from primer prITS1 to prITS4 with three exceptions.

UAMH5628 had a length of approx. 680 bp and F2 was

approx. 580 bp in length. Both length mutations occurred in

the ITS1 spacer region. Cenococcum geophilum had a length of

approx. 540 bp.

Introns in the 18S gene between primers prITS1 and prITS5

were found in 12 isolates. An intron of approx. 300 bp was

present in isolates FAG4, FAG15, BDD22, P30, P31,

DAOM165556a, SE24, UAMH5452, M479, M523, F2 and

G11.

EcoR I, Cla I and Taq I sites in the 5±8S gene are conserved

in a wide range of fungi and were present in all isolates

examined. Enzymes which distinguished between P. finlandia,

P. dimorphospora, P. cyclaminis and the Mra-like group were

Alu I, Hinf I, Sau3A I, Sau96A I, Rsa I and Hae III. Ava II and

Cfo I distinguished between the P. finlandia-like group and

P. dimorphospora isolates. A BamH I site was present only in

the P. dimorphospora isolates (P30, P31, P109, P136 and

DAOM165556a). Enzymes which distinguished isolates

within the Mra-like group include Alu I, Hinf I, Ava II, Cfo I,

Sau96A I and Rsa I.

DISCUSSION

Sterile dematiaceous fungi, often isolated from a number of

substrates, may have various ecological roles. Culture

morphology has not been satisfactory for taxonomic purposes.

In the absence of distinguishing morphological characters,

molecular data can be incorporated in the identification of

sterile isolates and used to help interpret the ecological

activities.

The phylogenetic analyses produced three distinct clusters,

the Phialophora finlandia group, the Phialocephala dimorphospora

group and a looser ‘Mra-like ’ (Mycelium radicis atrovirens)

group. FAG15 was the extype culture of P. finlandia, BDD22

the extype culture of C. paucisporum, and FAG4 and M479

were unidentified. Microscopically, P. finlandia conidiophores


DAOM165556a

FAG15P30P109

FAP7SE24R152FAP8FAF8BM523M843F1F3K92 68K93 270K92 90K92 68F2M545UAMH5628MTD313-20S8-1

DAOM165556a

FAG15P30P109

FAP7SE24R152FAP8FAF8BM523M843F1F3K92 68K93 270K92 90K92 68F2M545UAMH5628MTD313-20S8-1

DAOM165556a

FAG15P30P109

FAP7SE24R152FAP8FAF8BM523M843F1F3K92 68K93 270K92 90F2M545UAMH5628MTD313-20S8-1

DAOM165556a

FAG15P30P109

FAP7SE24R152FAP8FAF8BM523M843F1F3K92 68K93 270K92 90F2M545UAMH5628MTD313-20S8-1

T1 T2 T3 T4 O1 O2 O3 O4 O5 O6 M1 M2 M3 M4 M5 M6 M7 M8M9 M10 M11 M12R

EcoR I†

Intron*Isolate

Taq I Cfo I Msp I

Table 2. Data matrix of restriction enzyme sites used in analysis. Data based on presence (1) or absence (0) of characters

Isolate D1 D2 D3 D4 D5 D6 D7 D8 D9 D10 D11 D12 L1 L2 L3 L4 L5 V1 V2 V3 V4 V5 V6Dde I Alu I Ava II

Isolate H1 H2 H3 H4 H5 H6 H7 H8 H9 H10 F1 F2 F3 F4 F5 F6 F7 F8 F9 F10

Hae III Hinf I

Isolate A1 A2 A3 A4 A5 A6 A7 A8 A9 S1 S2 S3 S4 S5 S6 B1

Rsa I Sau3A I

U1 U2 U3 U4 U5 U6 U7 U8 U9

Sau96A I

U10 C

Cla I

* + indicates presence of the intron, – indicates absence of the intron.

† 1 is the presence of a band, 0 is the absence of a band.

BamH I


prITS1F2

18S (SSU)

Cluster I

H6 O1

U2V1

D1

A1

prITS3

prITS2

S1CT1 R F1

ITS1 5.8S

O2 D2U3H1

U1V2 S2

ITS2 25S (LSU)

T2 M1 M2prITS4

Cluster II

D3B

U6H5

U4V3 O1 D1 A3 S1

CT1 R F1 O2

M5T2

U5V4 D2 S3

M6

L1U7H2

M4

D4 H6 M3

V5 D1

U9 A1

S1CT1 R F1 O2

T2

D1 D2 F3U8H1 O3 L2 A5 S2

A4 A2

100 bp

Cluster III

Fig. 3. Composite restriction enzyme site maps. Each map is from the 3« end of the 18S rRNA gene to the 5« end of the 25S gene, and

include the 5±8S gene, ITS1 and ITS2 (positions indicated at top of figure). Maps are centred with respect to the 5±8S gene. Triangles

in the 18S gene indicate intron. Each restriction site position is indicated by the appropriate letter and number (where more than one

site for that enzyme exists). Positions of primers prITS1–prITS4 are shown by half-arrows at the top of the figure. Scale is at the

bottom. Abbreviations for enzymes are : A, Rsa I, B, BamH I ; C, Cla I ; D, Dde I ; F, Hinf I ; H, Hae III ; L, Alu I ; M, Msp I ; O, Cfo I ; R,

EcoR I ; S, Sau3A I ; T, Taq I ; U, Sau96A I ; V, Ava II.

are semimacronematous and highly branched whereas

conidiophores of Chloridium paucisporum are solitary or

loosely branched. The morphological characters of phialides

and conidia of these two fungi are very different (Wilcox et al.,

1974, Wang & Wilcox, 1985). Anatomical studies show

differences in ecto- and ectendomycorrhizal development

between P. finlandia and C. paucisporum (Wilcox & Wang,

1987a). Possible explanations of the 100% similarity observed

include that they are so closely related genetically they cannot

be distinguished or that P. finlandia and C. paucisporum may be

conspecific.

The sterile isolates FAG4 and M479 are 100% identical to

P. finlandia. FAG4 and FAG15 were collected together,

formed similar mycorrhizas and were thought to be identical,

but FAG4 could never by induced to sporulate (H. E. Wilcox

& C. J. K. Wang, pers. comm.). The 100% similarity between

FAG4, FAG15 (both collected from Finland) and M479

(isolated from an outplanted white pine seedling in

Warrensburg, New York) suggests that P. finlandia is possibly

more widespread than reported. Phialophora finlandia is a

dematiaceous mycorrhizal fungus that rarely sporulates and

could be mistaken for C. geophilum, a common dematiaceous

ectomycorrhizal fungus. Morphological descriptions of C.

geophilum mycorrhizas which undergo colour change (Park,

1970 ; Giltrap, 1983) suggest that it may be a misidentified P.

finlandia (or C. paucisporum) (Wilcox & Wang, 1987a).

There is a close relationship between the P. dimorphospora

isolates but little similarity to the other groups. P30 and P31

(100% identical) were isolated from the same utility pole in

Chester, NJ (C. J. K. Wang, pers. comm.) and probably

represent the same individual. P109 and P136, also collected

from utility poles in Chester, NJ, showed a high percent

similarity with the DAOM165556a isolate from a decayed

stump in Canada, the only difference being the presence or

absence of an intron in the 18S gene. Introns are fairly

common in deuteromycetes and intraspecific variation has

been reported (Rogers et al., 1993 ; Shinohara, 1994 ; Shinohara

et al., 1996), suggesting that P109, P136 and DAOM165556a

may be identical. P30 and P31 also had introns, and only one

additional restriction site (Dde I), suggesting a close re-

lationship to the other P. dimorphospora isolates studied.

The third cluster contained the majority of Mra-like

isolates, with some variation between the groups. Isolates

which had been identified as P. fortinii based on conidiogenesis

include FAP7 (extype), R151, R152, UAMH5452,

UAMH5628, UAMH6677, SE24, M503 and M504. Several

isolates which were identified as P. fortinii had low support

using molecular analysis. This may be due to a wide variation

within the group or it may be that the isolates represent more

than one species. Phialocephala fortinii and P. dimorphospora are

fairly similar morphologically and have been confused with

each other. Molecular analysis clearly distinguished between

these two groups. This suggests that P. fortinii is a species with

a high degree of variation.

Two groups which had a high percent similarity could only

be separated based on the presence or absence of an intron

and, therefore, are probably the same. These two groups

contain five of the nine isolates which had been identified as

P. fortinii (along with sterile unknowns).

Of the isolates identified as P. fortinii (excluding

UAMH5628), FAP7 was isolated in Finland ; M503, M504,

R151 and R152 in New York ; UAMH6677 and UAMH5452

in Alberta ; and SE24 in Oregon. Phialocephala fortinii has also

been isolated from Quebec (Richard & Fortin, 1973),

Czechoslovakia (Cerny & Cudlı!n, 1989), Germany and

Switzerland (K. Ahlich, pers. comm.), suggesting that P. fortinii


has a wide geographic range, and probably remains

unidentified as it rarely sporulates. Stoyke et al. (1992)

characterized a number of sterile dematiaceous isolates from

the roots of subalpine and alpine plants as P. fortinii based on

RFLP analysis of PCR amplified rDNA. The P. fortinii isolates

did not cluster according to geographic region.

Phialocephala fortinii and the P. fortinii-like isolates show

little host specificity. The cultures of P. fortinii in this study

were isolated from Pinus sylvestris (FAP7), Pinus resinosa (R151,

R152, M503 and M504), Calypso bulbosa (UAMH5452),

Luetkea pectinata (UAMH6677) and Lupinus latifolius (SE24).

Phialocephala fortinii has also been isolated from Picea mariana

(Richard & Fortin, 1973), Picea abies (Cerny & Cudlı!n, 1989 ;

K. Ahlich, pers. comm.) and Abies alba (K. Ahlich, pers.

comm.). Host origin did not show any consistency in the

cluster analysis.

The variation within the large cluster of the Mra-like

isolates is not easily explained. Isolates F2 and M545, along

with the C. geophilum isolate S8-1, can be presumed to be

different, with the F2 and M545 isolates remaining unknown.

P. fortinii may have a large amount of intraspecific variation or

the variation may indicate a species complex, with the isolates

perhaps closely related to P. fortinii. Molecular analysis

suggests that the P. fortinii-like cluster includes different

species. However, the identification of P. fortinii from the

FAP7}SE24 groups and R152}M303}M504 group suggests

that P. fortinii might show a high degree of intraspecific

variation. Stoyke et al. (1992) showed intraspecific variation

with isolates of P. fortinii-like sterile dematiaceous fungi (using

six enzymes) and concluded the isolates were conspecific or

closely related to P. fortinii. LoBuglio et al. (1991) found a high

degree of variation between isolates of C. geophilum using

RFLP analysis suggesting C. geophilum is a heterogeneous

species or a fungal complex of broad taxonomic range.

Sequencing data later resolved some of the variation seen

within the C. geophilum isolates (Shinohara, 1994) suggesting

that the variation observed in the P. fortinii and P. fortinii

isolates may also be resolved by sequencing.

It appears that a number of the isolates can be tentatively

characterized as P. fortinii-like. Phialocephala fortinii exhibits a

large amount of variation, as indicated by the high degree of

variation between sporulating cultures of P. fortinii. The low

resolution may be the result of individual isolates which are

not similar to any other isolates present. Branch and bound

parsimony analysis (not shown) generated a total of five trees

with branching differences occurring among 4–5 of the

isolates examined. Three of these isolates loosely cluster with

the P. fortinii-like group using the heuristic approach and two

clustered using UPGMA in analyses. Sequencing may help in

clarifying some of the ambiguity.

Additional experiments examining pathogenicity of isolates

from different hosts and geographic origin are being done in

order to determine if virulence can be associated with the

different groups.

The authors would like to thank Mike Allen for critically

reviewing this manuscript and Robin Pietropaolo for technical

assistance. This research was supported by the McIntire

Stennis Cooperative Forestry Research Program of the United

States Department of Agriculture. This is part I of a dissertation

submitted by S. Harney in partial fulfilment of the Ph.D.

degree at the State University of New York, College of

Environmental Science and Forestry, Syracuse, New York.

REFERENCES

Cerny, M. & Cudlı!n, P. (1989). Micromycetes from the rhizosphere of

Norway spruce stands under different pollution stress. Agriculture,

Ecosystems and Environment 28, 49–54.

Currah, R., Hambleton, S. & Smreciu, A. (1988). Mycorrhizae and mycorrhizal

fungi of Calypso bulbosa. American Journal of Botany 75, 739–752.

Currah, R., Sigler, L. & Hambleton, S. (1987). New records and new taxa of

fungi from the mycorrhizae of terrestrial orchids of Alberta. Canadian

Journal of Botany 65, 2473–2482.

Gardes, M. & Bruns, T. D. (1993). ITS primers with enhanced specificity for

basidiomycetes – application to the identification of mycorrhizae and rusts.

Molecular Ecology 2, 113–118.

Gardes, M., White, E. J., Fortin, J. A., Bruns, T. D. & Taylor, J. W. (1991).

Identification of indigenous and introduced symbiotic fungi in

ectomycorrhizae by amplification of nuclear and mitochondrial ribosomal

DNA. Canadian Journal of Botany 69, 180–190.

Giltrap, N. (1983). Influence of irradiance and concentration of glucose in the

substrate on mycorrhizal development by Cenococcum geophilum and

Paxillus involutus in axenic culture. Transactions of the British Mycological

Society 81, 627–629.

Haselwandter, K. & Read, D. (1980). Fungal associations of roots of dominant

and sub-dominant plants in high-alpine vegetation systems with special

reference to mycorrhiza. Oecologia 45, 57–62.

LoBuglio, K., Rogers, S. & Wang, C. J. K. (1991). Variation in ribosomal DNA

among isolates of the mycorrhizal fungus Cenococcum geophilum. Canadian


Melin, E. (1923). Experimentelle Untersuchungen u$ ber die Konstitution und

O> kologie der Mykorrhizen von Pinus silvestris L. und Picea abies (L.) Karst.

In Mykologische Untersuchungen und Berichte Zweiter Band (ed. R. Fakk), pp.

222–247, Aktiengesellschaft fu$ r Druck und Verlag : Germany.

O’Dell, T., Massicotte, H. & Trappe, J. (1993). Root colonization of Lupinus

latifolius Agardh. and Pinus contorta by Phialocephala fortinii Wang and

Wilcox. New Phytologist 124, 93–100.

O’Dell, T. & Trappe, J. (1992). Root endophytes of lupin and some other

legumes in Northwestern U.S.A. New Phytologist 122, 479–485.

Park, J. (1970). A change in color of aging mycorrhizal roots of Tilia americana

formed by Cenococcum graniforme. Canadian Journal of Botany 48,

1339–1341.

Richard, C. & Fortin, J. A. (1973). The identification of Mycelium radicis

atrovirens (Phialocephala dimorphospora). Canadian Journal of Botany 51,

2247–2248.

Richard, C. & Fortin, J. A. (1974). Distribution ge! ographique, e! cologie,

physiologie, pathoge! nicite! et sporulation du Mycelium radicis atrovirens.

Phytoprotection 55, 67–88.

Rogers, S. O. & Bendich, A. (1985). Extraction of DNA from milligram

amounts of fresh, herbarium and mummified plant tissues. Plant Molecular

Biology 5, 69–76.

Rogers, S. O. & Bendich, A. (1994). Extraction of total cellular DNA from

plants, algae and fungi. In Plant Molecular Biology Manual 3rd ed. (ed. S. B.

Gelvin & R. A. Schilperoort), pp. D1 :1–8. Kluwer Academic Publishers :

Boston, MA, U.S.A.

Rogers, S. O., Rehner, S., Bledsoe, C., Mueller, G. & Ammirati, J. (1989).

Extraction of DNA from basidiomycetes for ribosomal DNA hybridizations.

Canadian Journal of Botany 67, 1235–1243.

Rogers, S. O., Yan, Z., Shinohara, M., LoBuglio, K. & Wang, C. J. K. (1993).

Messenger RNA intron in the nuclear 18S ribosomal RNA gene of

deuteromycetes. Current Genetics 23, 338–342.

Rohlf, F. (1988). NTSYS-pc : Numerical Taxonomy and Multivariate Analysis

System. Version 1.50. Exeter Publishing Ltd : New York.

Shinohara, M. (1994). Molecular evolutionary study of Cenococcum geophilum.

Ph.D. Thesis, State University of New York, College of Environmental

Science and Forestry, Syracuse, New York.


Shinohara, M., LoBuglio, K. F. & Rogers, S. O. (1996). Group-I intron family

in the nuclear ribosomal RNA small subunit genes of Cenococcum geophilum

isolates. Current Genetics 29, 377–387.

Stoyke, G. & Currah R. (1991). Endophytic fungi from the mycorrhizae of

alpine ericoid plants. Canadian Journal of Botany 69, 347–352.

Stoyke, G., Egger, K. & Currah, R. (1992). Characterization of sterile

endophytic fungi from the mycorrhizae of subalpine plants. Canadian


Swofford, D. (1990). PAUP: Phylogenetic Analysis Using Parsimony, version

3.1. Computer program distributed by the Illinois History Survey :

Champaign, Illinois.

Wang, C. J. K. & Wilcox, H. E. (1985). New species of ectendomycorrhizal

and pseudomycorrhizal fungi : Phialophora finlandia, Chloridium paucisporum,

and Phialocephala fortinii. Mycologia 77, 951–958.

White, T., Bruns, T., Lee, S. & Taylor, J. (1990). Amplification and direct

sequencing of fungal ribosomal RNA genes for phylogenetics. In PCR

(Accepted 26 February 1997)

Protocols : A Guide to Methods and Applications (ed. M. Innis, D. Gelfand, J.

Sninsky & T. White), pp. 315–322. Academic Press, Inc. & Harcourt Brace

Jovanovich Publishers : New York.

Wilcox, H. E. & Ganmore-Neumann, R. (1974). Ectendomycorrhizae in Pinus

resinosa seedlings. I. Characteristics of mycorrhizae produced by a black

imperfect fungus. Canadian Journal of Botany 52, 2145–2155.

Wilcox, H. E., Ganmore-Neumann, R. & Wang, C. J. K. (1974). Characteristics

of two fungi producing ectomycorrhizae in Pinus resinosa. Canadian Journal

of Botany 52, 2279–2282.

Wilcox, H. E. & Wang, C. J. K. (1987a). Mycorrhizal and pathological

associations of dematiaceous fungi in roots of 7-month-old tree seedlings.

Canadian Journal of Forest Research 17, 884–899.

Wilcox, H. E. & Wang, C. J. K. (1987b). Ectomycorrhizal and

ectendomycorrhizal associations of Phialophora finlandia with Pinus resinosa,

Picea rubens, and Betula alleghaniensis. Canadian Journal of Forest Research 17,

976–990.

molecular characterization of dematiaceous root endophytes

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