Mycologia

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Batrachochytrium dendrobatidis gen. et sp. nov., a

chytrid pathogenic to amphibians

Joyce E. Longcore, Allan P. Pessier & Donald K. Nichols

To cite this article: Joyce E. Longcore, Allan P. Pessier & Donald K. Nichols (1999)
Batrachochytrium�dendrobatidis gen. et sp. nov., a chytrid pathogenic to amphibians, Mycologia,
91:2, 219-227, DOI: 10.1080/00275514.1999.12061011

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Mycologia, 91 (2), 1999, pp. 219-227.
© 1999 by The Mycological Society of America, Lawrence, KS 66044-8897
Issued 15 March 1999
Batrachochytrium dendrobatidis gen. et sp. nov.,
a chytrid pathogenic to amphibians
Joyce E. Longcore 1
Department of Biological Sciences, University of Maine,
Orono, Maine 04469-5722
Allan P. Pessier2
Donald K. Nichols
Department of Pathology, National Zoological Park,
Smithsonian Institution, Washington, DC 20008
Abstract: Captive and wild frogs from North and
Central America and Australia recently have died
with epidermal infections by chytridiomycete fungi.
We isolated a chytridiomycete into pure culture from
a captive, blue poison dart frog that died at the Na-
tional Zoological Park in Washington, D.C. Using this
isolate, we photographed developmental stages on
nutrient agar, examined zoospores with transmission
electron microscopy, and inoculated test frogs. This
inoperculate chytrid develops either monocentrically
or colonially and has thread-like rhizoids that arise
from single or multiple areas on the developing zoo-
sporangium. The taxonomically important features
of the kinetosomal region of the zoospore indicate
that this chytrid is a member of the Chytridiales but
differs from other chytrids studied with transmission
electron microscopy. Its microtubule root, which be-
gins at kinetosome triplets 9-1 and extends parallel
to the kinetosome into the aggregation of ribosomes,
is distinctive. Histologic examination of test frogs re-
vealed that the pure culture infected the skin of test
frogs, whereas the skin of control frogs remained free
of infection. The fungus is described as Batrachochy-
trium dendrobatidis gen. et sp. nov.
Key Words: Chytridiales, chytridiomycosis, Chytri-
diomycota, frogs, fungus, ultrastructure, zoospore
INTRODUCTION
During 1996-1998, juvenile blue poison dart frogs
(Dendrobates azureus), green-and-black poison dart
frogs (D. auratus), and White's tree frogs (Litoria ca-
erulea) at the National Zoological Park (NZP) died
Accepted for publication November 9, 1998.
1 E-mail: longcore@maine.maine.edu
2 Current address: Department of Pathology, Zoological Society of
San Diego, San Diego, CA 92112-0551
219
Published online 04 Jun 2019
with a unique skin disease associated with the pres-
ence of spherical eukaryotic organisms located in the
epidermis. Electron microscopy of the affected epi-
dermis revealed that the intracellular organisms in
the skin produced zoospores characteristic of mem-
bers of the Chytridiomycota (Pessier et al 1999). His-
tologically similar chytrids had been observed in the
skin of frogs from NZP as early as 1988 in a single
White's tree frog. Initially and in a subsequent report
of an infection in arroyo toads (Nichols et al 1996)
the infective organism was not recognized as a chy-
trid. Chytrids have also been observed histologically
in the skin of frogs that died at several other US zoos
and research institutions (Nichols et al 1998) and
have been hypothesized to be the proximate cause of
declines of wild populations of amphibians in Central
America and Australia (Berger et al 1998). Although
zoosporic members of the Kingdom Fungi are well
known as parasites of protozoans and invertebrates,
none previously had been reported from tissue of liv-
ing vertebrates (Sparrow 1960, Powell1993).
We have isolated the infective organism from the
epidermis of a blue poison dart frog and herein de-
scribe its morphology and the ultrastructural features
of its zoospores. Because thalli of many chytridiomy-
cetes are simple and provide few characters, ultra-
structural features of the asexually produced zoo-
spores are evaluated to make taxonomic decisions
(Barr 1990). The development of the thallus and the
ultrastructure of the zoospores of the frog pathogen
differ distinctly from those of other chytridialean
genera and species; therefore, we describe this or-
ganism as a new genus and species.
MATERIALS AND METHODS
Histology in the host.--Complete necropsies were performed
on dead frogs; tissues were processed for histologic exami-
nation as previously described (Pessier et al 1999). Mter
paraffin embedding, 5-Q JLm sections were stained with he-
matoxylin and eosin.
Isolation and culture.-Initial attempts to bait for the fungus
by placing snakeskin with water and debris from the con-
tainers in which infected frogs had lived were unsuccessful
and the snakeskin rapidly become overgrown with oomy-
cetes. One hind leg from an infected blue poison dart frog
that had recently died was double bagged in plastic and
shipped overnight on ice packs to J. Longcore's laboratory.
220 MYCOLOGIA
Pieces of loose epidermis were removed from between the
toes of the frog and placed on culture dishes containing
PmTG nutrient agar (1 g peptonized milk, 1 g tryptone, 5
g glucose, 10 g agar, 1 L distilled water, with 400 mg strep-
tomycin sulfate and 200 mg penicillin-G added after auto-
claving (Barr 1986, 1987). While viewing with X30 stereo
magnification and substage lighting, small pieces (0.5-1
mm2 ) of infected skin were dragged through agar with a
sterile needle to remove bacteria and yeast cells. Cleaned
pieces of skin were transferred to a plate of PmTG nutrient
agar and incubated at room conditions (20-23 C). Sporan-
gia developed on the frog skin but did not release zoo-
spores or continue development of a colony. A group of
these sporangia was added to PmTG broth. Mter 16 d the
broth was opalescent from the growth of the fungus. Since
the isolation of the culture used in this study other cultures
have been isolated by transferring groups of wk-old sporan-
gia that formed on the original isolation plate onto TGhL
agar (16 g tryptone, 4 g gelatin hydrolysate, 2 g lactose, 12
g agar, 1 L distilled water). To produce zoospores for TEM
and to grow zoosporangia for light microscopy, we spread
approximately 1 mL of broth containing the fungus onto
culture plates containing 2% tryptone agar or TGhL agar.
Maximum temperature for growth was determined by in-
oculating flasks containing 50 mL of TG broth (1% tryp-
tone and 0.3% glucose; Gauriloff et al1980) with 0.5-1 mL
of broth containing isolate L-197 grown at room tempera-
ture. Controls were maintained at room temperature. Thalli
at different developmental stages were photographed with
a Nikon Labophot-2 microscope using phase lenses and Ko-
dak Tri-X Pan 400 film.
Sterile, distilled water was added to cultures of isolate L-
197 growing on 20 agar plates to induce discharge of zoo-
spores. The liquid containing the zoospores was pooled and
zoospores were prepared for transmission electron micros-
copy (TEM) with a sequential glutaraldehyde-osmium te-
troxide method (Barr 1981, Longcore 1992). Serial sections
were cut with a diamond knife and placed on carbon-sta-
bilized, pioloform-coated slots. Sections were viewed on a
Philips CM-10 microscope at 80 kV.
Infection of frogs.-To attempt to fulfill Koch's postulates,
five 2-3 mo-old blue-and-yellow poison dart frogs (Dendro-
bates tinctorius) were obtained from a dealer who did not
have a history of chytridiomycosis or skin disease in his
frogs. Frogs were separated into 2 groups; 2 frogs were in-
oculated daily for up to 30 d with 0.1 mL of swirled, pure
broth culture of isolate L-197, applied to the hind legs.
Three frogs were used as controls and were inoculated sim-
ilarly with 0.1 mL of sterile broth. Frogs that died or were
euthanized were processed for histology as above. The fun-
gus was isolated from a test frog using the isolation protocol
above.
TAXONOMY
Batrachochytrium Longcore, Pessier et Nichols gen.
nov.
Thalli monocentrici aut colonici. Ex una ad complures
rhizoides stirpes; rhizoides similes filae. Quodque segmen-
tum colonici thalli efficiens zoosporangium. Zoosporangia
cum una aut compluribus inoperculatis fluxis papillis; na-
tantes zoospores sphaericas aut paene ovatas. Ribosomata
aggregata. Numerus guttularum olei; assuescata ad mem-
branam microcorporis atque nexa ad peripheram riboso-
mati aggregati. Kinetosomata radicula facta de aggregatis
microtubulis; assuescata ad (microtubula) triplica 9-1 atque
extentia parallela ad kinetosoma in ribosomatum aggrega-
tionem. Kinetosoma annexa ad centriolam non-flagellatam
cum innexis fibris. Egens rumposomato et occluso regionis
transitantis. Habitatum: Efficiens monocentrica et colonica
sporgangia in strato corneo amphibianum.
TYPE. Batrachochytrium dendrobatidis Longcore,
Pessier et Nichols.
Thalli monocentric or colonial. One to several rhi-
zoidal axes; rhizoids thread-like. Each segment of co-
lonial thallus forms a zoosporangium. Zoosporangia
with one or more inoperculate discharge papillae;
swimming zoospores spherical or slightly ovate. Ri-
bosomes aggregated. Numerous lipid globules; asso-
ciated with sheet of microbody and nested in periph-
ery of ribosomal mass. .Kinetosomal root consisting
of a group of microtubules; arising near triplets 9-1
and extending parallel to kinetosome into ribosomal
core. Kinetosome attached to nonflagellated centri-
ole with overlapping fibers. Lacking rumposome and
transition zone plug.
Habit. Forming monocentric and colonial sporan-
gia in epidermis of amphibians.
Batrachochytrium dendrobatidis Longcore, Pessier et
Nichols sp. nov. FIGS. 2-30.
Descriptio ad genus. Sporangia in purum cultum angu-
lata ad sphaerica; ad 40 IJ.m diametra ex una ad complures
papillas. Longitudo flagelli 19-20 IJ.m.
TYPE. FIGS. 2-30.
Description as for genus. Sporangia on nutrient
agar up to 40 f.Lm diam with 1-several discharge pa-
pillae. Flagellum 19-20 f.Lm long.
Diagnosis based on isolate I.rl97 from a blue poi-
son dart frog, NZP #97432, 14 Sep. 1997. Also ob-
served: isolate L-198 from a black-and-green poison
dart frog, NZP #97471, 3 Oct. 1997; isolate L-206
from a White's tree frog, NZP #98149, 11 May 1998
and isolate L-203 from a false tomato frog (Dyscophus
guinetz), Bronx Zoo, New York, #C6-98, 20 Jan. 1998.
Etymology. Greek; batracho = frog and chytr =
earthen pot. The species name is from "Dendrobates,"
the genus of frogs in which the organism was respon-
sible for high mortality in a captive population and
from which the culture was isolated to make the type
photographs.
RESULTS
Observations in the host.-The stratum corneum (su-
perficial epidermal layer) was thickened, up to 2-5
 LoNGCORE ET AL: B.
FIG. 1. Haemotoxylin and eosin stained section of Ba-
trachochytrium dendrobatidis in epidermis of naturally in-
fected blue poison dart frog. Arrow indicates open sporan-
gium, arrowheads indicate colonial sporangia; top of photo
is surface of skin; younger stages of the fungus are in the
deeper layers.
times normal. Within the thickened stratum corne-
um were large numbers of spherical (7.0-15.0 j.Lm
diam) chytrid thalli (FIG. 1) (Pessieretall999). Thal-
li were most numerous in the skin of the ventral ab-
domen, hind limbs and feet. Several profiles of thalli
were observed within the skin including monocentric
thalli, colonial thalli with thin interior walls and zoo-
sporangia with prominent discharge papillae (FIG. 1).
One of the 2 frogs that were inoculated with isolate
L-197 died on the 23rd d of inoculation and the sec-
ond experimental frog was found dead on the 31st d
of the experiment. Two control frogs (the third con-
trol frog died of metabolic problems unrelated to the
experiment on the lOth dafter inoculations began)
were euthanized 5 dafter the last experimental frog
died. Complete necropsies performed on all frogs re-
vealed that only the 2 frogs that were inoculated with
B. dendrobatidis had histologic evidence of skin dis-
ease and chytrid thalli within the epidermis. We iso-
lated B. dendrobatidis from one of the artificially in-
fected frogs.
Morphology in pure culture.-Motile zoospores are
nearly spherical or somewhat ovate, vary in size (com-
monly 3-5 IJ.m diam) and lack prominent lipid glob-
ules (FIG. 2). Zoospores may become elongate and
DENDROBATIDIS GEN. ET SP. NOV. 221
are frequently elongate (FIG. 3) when first released
from the zoosporangium. Frequently one to many cy-
toplasmic extensions project and retract from motile
zoospores (FIG. 4). A posterior flagellum (19-20 j.Lm
long), dark bodies, which are the lipid globules and
microbody, and a shadowy area, which is the aggre-
gation of ribosomes, are visible with phase micros-
copy, particularly when zoospores are flattened on a
layer of agar under a coverslip (FIGs. 4, 5).
On PmTG or 2% tryptone nutrient agars most en-
cysted zoospores do not form cell walls; they lyse rath-
er than developing into sporangia. On TGhL agar,
however, some zoospores form germlings with fine,
thread-like rhizoids. Rhizoids may extend from a sin-
gle area (FIG. 6) of a developing zoosporangium, or
from several areas (FIG. 7). On nutrient agar, zoospo-
rangia increase in size over the next 4-5 d (FIGs. 8-
10) and when mature have one to several prominent
discharge papillae, depending on the size of the spo-
rangium (FIGs. 10, 11). Rhizoids are thread-like (FIG.
8) and may be short and bushy or sparsely branched
and extend long distances from the sporangium. Dis-
charge papillae form during the growth of the spo-
rangium, and walls at the tips of papillae break down
and form plugs that deliquesce and release zoospores
(FIGs. 10-12). The wall at the edge of the papillar
opening often appears slightly thickened (FIG. 13).
Most thalli develop in the described manner, how-
ever, during early development some thalli form in-
ternal septa (FIGS. 14, 15). Each cell formed by these
septa develops as do the zoosporangia described
above and forms its own discharge papilla. Thus, a
group of sporangia can grow from a single zoospore.
This type of development is most similar to the cat-
egory of development referred to as "colonial" by
Barr (1990), and we refer to it by that name. The
colonial thalli that develop on nutrient agar are anal-
ogous to the thalli with internal walls that form when
the fungus develops in epidermal cells of the host
(FIG. 16). Resting spores were neither produced on
nutrient agar nor seen in host tissue.
Isolation and culture.-Single zoospores usually failed
to develop on the nutrient agar that we used, how-
ever, groups of sporangia deposited onto agar from
broth culture readily formed colonies, even on nu-
trient agar that did not support the growth of single
zoospores. This "group effect" also occurred during
isolation attempts. Single or small groups of sporan-
gia that developed on cleaned pieces of skin failed
to continue development, whereas larger groups of
sporangia continued growth and formed colonies. In
broth culture isolate L-197 develops fastest at 23 C.
Growth occurs at 28 C, although at a slower rate than
at lower temperatures. Broth cultures that failed to
222 MYCOLOGIA

FIGs. 2-16. Development of isolate L-197 of Batrachochytrium dendrobatidis in pure culture. 2. Shape of motile zoospore.
3. Elongate amoeboid zoospore. 4. Fine cytoplasmic extension (arrow) from surface of zoospore. 5. Zoospore flattened on
agar surface under coverslip; black dots are probably lipids and microbody and gray shadowy area is the aggregation of
ribosomes. 6. Germling with rhizoids from single area of thallus. 7. Germling with 3 rhizoidal axes. 8 and 9. Thread-like
rhizoids on developing sporangia. 10. Large zoosporangium with 2 discharge papillae (arrows). 11. Wall of tip of papilla has
begun to deliquesce. 12. Zoospores discharging through 2 papillae (arrows). 13. Nearly empty zoosporangium showing slight
thickening of wall at tip of papilla. 14. Group of thalli; one thallus with internal septation (arrow). 15. Colonial thallus with
several walls (arrows). 16. Appearance of isolate L-197 in skin of blue-and-yellow dart frog. Colonial thallus (arrow) and
monocentric zoosporangium (arrowhead) in epidermis of host that was experimentally infected. Scale bar = 10 IJ.m for all.
 LONGCORE ET AL: B. DENDROBATIDIS GEN. ET SP. NOV.
make substantial growth after 2 wk at 29 C grew well
after flasks were placed at 23 C. PmTG and reduced-
strength TGhL have been used successfully as isola-
tion media, and TGhL has been the best growth me-
dium. During attempts to isolate Batrachochytrium
from 5 frogs (4 from NZP and one from the Bronx
Zoo), other fungi also grew on cleaned pieces of skin.
These included hyphomycetes, a spherical, multi-
pored species of Rhizophydiurn:, and the oomycete
Aphanomyces.
Zoospore ultrastrudure.-The ultrastructural view of
the zoospore is dominated by a core of aggregated
ribosomes and a single posterior flagellum (FIG. 17).
Endoplasmic reticulum more or less surrounds the
ribosomal mass and also is found within it (FIGS. 18,
19); a cone of ribosomes surrounded by ER extends
to the kinetosome (FIGs. 17-19). Lipid globules and
microbodies lie just outside the ribosomal core and
often are nested partly within it (FIGs. 17, 18). A
sheet of microbody, which is usually linear in outline
(FIGS. 17, 18), abuts or partly wraps lipid globules,
but seldom entirely encloses them. One or more
groups of lipid globules and microbody may lie any-
where around the periphery of the ribosomal core
(FIGS. 17, 18). Cisternae of endoplasmic reticulum do
not consistently surround or abut lipid globules.
Nine lipid globules, organized into two clumps, were
in one zoospore that was analyzed from serial sec-
tions. Mitochondria are scattered around the edge of
the ribosomal aggregation (FIGS. 17-20), and may be
nested partly or completely within it. Some mito-
chondria abut lipid globules and microbody (FIG.
17). The nucleus is cupped towards, and nested in,
the mass of ribosomes (FIGs. 17-19). Often, small
clumps of ribosomes enclosed by ER lie on the outer
side of the nucleus (FIG. 17).
The cytoplasm that is exterior to the core of ribo-
somes and major organelles contains clear and
dense-cored vesicles (FIGS. 17, 19) and a single Golgi
apparatus (FIGS. 18, 19), which is not in any fixed
area of the spore. The cytoplasmic extensions that
can be seen with light microscopy also are seen with
TEM (FIGs. 18-20). These extensions contain dense
cytoplasm with few vesicles or other substructure
identifiable with our fixation (FIG. 20).
Flagellar apparatus.-The kinetosome from which
the flagellar axoneme extends lies in the posterior of
the zoospore and connects to the plasma membrane
with 9 interconnected props (FIGS. 17, 19, 21, 24, 25)
similar to those in other members of the Chytridi-
omycota (Barr and Hadland-Hartmann 1978, Barr
1992). A nonflagellated centriole (nfc) lies parallel
to the kinetosome (FIGs. 21, 27, 28), and two of its
triplets are connected to triplets 6 and 7 of the ki-
223
netosome with overlapping connecting fibers (FIGs.
27, 28) for most of the length of the nfc. Fibrillar
material also extends from triplet 5 but does not con-
nect directly to a triplet of the nfc. The transition
zone of the kinetosome contains concentric fibers
(FIGS. 23, 24), transitional fibers (FIG. 23) and a ter-
minal plate (FIG. 19) but no transition zone plug. A
root consisting of a group of microtubules arises at
the side of the kinetosome near kinetosome triplets
9 and 1 (FIGs. 27, 28) and extends toward the inte-
rior of the cell, parallel with the kinetosome (FIGs.
21, 27-30). Two-8 microtubules have been identified
in different series of sections. The microtubule root
is embedded in a cone of ribosomes surrounded by
ER and extends into the mass of densely packed ri-
bosomes (FIGs. 18, 21, 29, 30). Because of the diffi-
culty in distinguishing microtubules among ribo-
somes, we could not determine the exact number of
micro tubules or the ending point of the root. FIGURE
31 presents a schematic view the ultrastructure of the
zoospore.
DISCUSSION
We first identified Batrachochytrium as a chytridiomy-
cete from electron photomicrographs of zoosporan-
gia containing zoospores within the skin of a White's
tree frog (Pessier et al 1999). Our identification was
based on the presence of flagellar props, discoid cris-
tae in the mitochondria and aggregated rather than
dispersed ribosomes. This conclusion has been
strengthened by the more thorough examination of
the zoospores from pure culture. Characters associ-
ated with the flagellar apparatus are the most reliable
of the ultrastructural characters for grouping chytrid
taxa at the ordinal and genus level (Barr 1990, 1992).
The flagellar apparatus of Batrachochytrium has a
nonflagellated centriole that is parallel and attached
to the kinetosome, and a microtubule root that orig-
inates from the side of the kinetosome. These char-
acters are diagnostic of the order Chytridiales. Barr's
emendation of the Chytridiales (1980: 2388) states
" ... zoospore microtubules extend from one side of
the kinetosome in a parallel array, they usually ex-
tend to the side of a rumposome which is on the lipid
globule surface." The microtubule root in Batrach-
ochytrium is similar to microtubule roots in other chy-
tridialean zoospores in that it arises near kinetosome
triplets 9-1 (Barr and Desaulniers 1988). It differs,
however, in that other chytridialean roots that arise
from this position leave the kinetosome at nearly
right angles and extend to a part of the microbody-
lipid globule complex (MLC) (Powell 1978, Powell
and Roychoudhury 1992). In Batrachochytrium, the
microtubule root leaves the kinetosome in a parallel
224 MYCOLOGIA

FIGS. 17-20. TEM of zoospores of Batrachochytrium dendrobatidis. Abbreviations used: ce, cytoplasmic extension; ER,
endoplasmic reticulum; G, Golgi apparatus; K, kinetosome; L, lipid globule; M, mitochondrion; mb, microbody; mt, mi-
crotubules; nfc, nonflagellated centriole; N, nucleus; P, prop; tp, terminal plate; Va, vacuole; Ve, vesicle. 17. Longitudinal
section of zoospore with posterior flagellum attached to kinetosome; showing arrangement of lipid globules and associated
microbody, mitochondria and nucleus around mass of ribosomes. 18. Lipids and microbody near kinetosome; cone of
ribosomes surrounded by ER around microtubule root. 19. Longitudinal section of zoospore showing Golgi apparatus in
cytoplasm outside of mass of ribosomes and cytoplasmic extension. 20. Cytoplasmic extension. Scale bars = 1 JLm. Bar for
17 also for 19.
 LONGCORE ET AL: B. DENDROBATIDIS GEN. ET SP. NOV. 225

FIGS. 21-30. Kinetosomal area of B. dendrobatidis. 21. Longitudinal section of parallel kinetosome and nfc showing over-
lapping connecting fibers (arrow) and microtubule root (arrowhead) extending into ribosomes. 22-30. Serial sections 1-8
and 12. 22. Axoneme where 2 central microtubules begin. Doublets are numbered following the convention of Barr and
Desaulniers (1988). 23. Concentric fiber (arrow) and transitional fibers (arrowhead); asterisk indicates microtubule doublet
or triplet #I. 24. Connection of props (arrow) to plasmalemma. 25. Props (arrow). 26. Beginning of triplets of kinetosome.
27. Fibrillar connection from kinetosome triplets 6 and 7 to nonflagellated centriole; beginning of root microtubules (arrows)
near triplets 9, and 1. 28. Ribosomes surrounding root microtubules near tripletS 8-2. 29. microtubules (arrows) in cone of
ribosomes. 30. Microtubules within cone of ribosomes surrounded by ER. Scale bar = 0.5 11m for all.

direction and extends into the aggregation of ribo-
somes. Although the zoospores of Lacustromyces hie-
malis Longcore (1993) and Rhizophlyctis harderi Ueb-
elmesser (Roychoudhury and Powell 1992) contain
microtubule roots that extend into the core of ribo-
somes, neither of these roots arises from the triplet
9-1 position. Rather, in both of these species, the mi-
crotubule root that arises from the 9-1 position ex-
tends toward the MLC.
An MLC containing a group of lipid globules and
no rumposome rather than a single large lipid glob-
ule with a rumposome, although unusual for the Chy-
tridiales, is also characteristic of Lacustromyces, which
also was placed in the Chytridiales (Longcore 1993).
226 MYCOLOGIA
FIG. 31. Summary diagram of Batrachochytrium zoo-
spore with flagellum and kinetosomal area enlarged. Ab-
breviations used: ce, cytoplasmic extension; ER, endoplas-
mic reticulum; G, Golgi apparatus; K, kinetosome; L, lipid
globule; M, mitochondrion; mb, microbody; nfc, nonflag-
ellated centriole; N, nucleus; P, prop; Va, vacuole. Organ-
elles surrounding the ribosomal mass and cytoplasmic ex-
tensions are not consistently found in any particular part of
the cell.
We do not infer, however, that possession of this fea-
ture indicates that the two genera are closely related.
Their kinetosomal characters suggest that they are
not. The distinctive overlapping of the kinetosome
and nfc connective fibers, and the origin of the con-
nective fibers from triplets 6 and 7 of the kinetosome
are features that occur in species with a Rltizophydium
sub-type of zoospore (Barr 1980, Barr and Desaul-
niers 1988). These features and the lack of a transi-
tion zone plug, which is also absent in the Rltizophy-
dium zoospore, suggest that Batrachochytrium may be
related to species with a Rltizophydium subtype of zoo-
spore. Batrachochytrium and Rltizophydium, however,
have dissimilar MLCs, and no species of Rltizophy-
dium is known to have colonial thalli.
When grown on agar, Batrachochytrium thalli de-
velop endogenously. The nucleus remains in the en-
cysted zoospore and the encysted zoospore enlarges
to form the zoosporangium. When rhizoids extend
from only one area of the sporangium, the organism
looks like a species of Rltizophydium (Sparrow 1960,
1973); however, when sporangia develop with several
rhizoidal axes, the classical genus would be Rltizo-
phlyctis. Rltizophlyctis spp., however, do not have
thread-like rhizoids. We have not yet traced the in-
fection process in the skin cells of frogs, but if the
zoospore encysts on the outside of the cell and the
nucleus and contents enter the cell via a germ tube,
as it does in other endobiotic species (endogenous
development), then the classical genus would be En-
tophlyctis. Although the thallus of B. dendrobatidis has
few invariable morphological features, this species is
not likely to be confused with any species of Ento-
phlyctis, Rltizophydium, or Rltizophlyctis because its
zoospore is distinctive even at the light microscopic
level. In addition, some Batrachochytrium thalli are
colonial, both in the host and in pure culture. Care-
ful observation is necessary, however, so that colonial
development is not confused with development of
thalli from zoospores that do not exit the parent zoo-
sporangium. Classical chytridialean genera, which
rely heavily on type of development, have little pre-
dictive value for phylogeny (Barr 1990, Longcore
1995). Consequently, ultrastructural characters are
needed to describe new genera in the Chytridiales.
Based on its capability to form colonial thalli and its
distinctive zoosporic ultrastructure, a new genus is
appropriate for this fungus.
Chytrids that are histologically indistinguishable
from B. dendrobatidis occur on a range of amphibian
hosts in North American zoos (Pessier et al 1999,
Nichols et al 1998), in wild populations of amphibi-
ans in Central America and in captivity and in the
wild in Australia (Berger et al 1998). Whether the
populations of Batrachochytrium that affect amphibi-
ans from these widespread areas will be attributable
to the same species is yet to be determined. Berger
et al (1998) studied the zoospore ultrastructure of
the chytrid from frog tissue from Central America
and Australia. They found no microtubule root as-
sociated with the kinetosome and presented a zoo-
spore diagram that portrays the nonflagellated cen-
triole at an angle and unconnected to the kineto-
some. Although these are important taxonomic char-
acters, we do not interpret the differences between
their finding and ours as a difference in actual ultra-
structure. The zoospores studied by Berger et al
(1998) were within zoosporangia and were fixed with-
in the frog tissue. Thus the zoospores may have been
immature and not optimally fixed. In addition, serial
sections were not examined. These differences in
methods most likely account for Berger et al not ob-
serving a microtubule root or the connection and
parallel orientation of the kinetosome and the non-
flagellated centriole. The zoospores studied by Ber-
ger et al were otherwise similar to those of B. den-
drobatidis. Because zoospore ultrastructure is not
consistently useful at the species level and scant mor-
 LONGCORE ET AL: B. DENDROBATIDIS GEN. ET SP. NOV.
phology exists on which to base species differences
in this organism, differences among species may
need to be detected using molecular methods.
Batrachochytrium seems to be a widespread and
ecologically significant genus of chytrids. Important
questions remain about how the amphibian patho-
gen persists in nature, and whether it lives outside of
a host. We have not seen resting spores in the labo-
ratory, although they may occur in nature. Our ability
to culture Batrachochytrium suggests that it may be
capable of growing in nature as a saprobe. Also, de-
velopment of the fungus does not cease when the
host dies. In the laboratory at least one generation
of sporangia has developed on skin removed from
dead frogs, before bacteria and oomycetes overran
the skin. When in pure culture, Batrachochytrium
grows on boiled snakeskin (keratin), and the fungus
may be able to live saprobically on keratin in nature
if other components of the ecosystem limit the
growth of bacteria and oomycetes.
ACKNOWLEDGMENTS
We thank Drs. Tracey McNamara and Michael Linn of the
Wildlife Conservation Society for providing the infected
false tomato frog. Mr. Paul Miles, owner of The Boa Barn,
graciously donated the poison dart frogs used in the rein-
fection study. We gratefully acknowledge Dr. Tina Passman,
chair of the Department of Modern Languages and Classics,
University of Maine for translating the Latin diagnosis. A
Senior Post-doctoral Fellowship (97-3545A) from Friends of
the National Zoo (FONZ) supported Dr. Pessier.
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