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JOHN M. McPARTLAND1AND MARC A. CUBETA2
"Vermont Alternative Medicine}AMRITA,53 Washington Street, Middlebury, VT 05753,U.S.A.
#North Carolina State University,Department of Plant Pathology,Box 7616,Raleigh,NC 27695–7616,U.S.A.
Micropeltopsis cannabis sp. nov. and Orbilia luteola (Roum.) comb. nov. are proposed. New Cannabis host associations include
binucleate Rhizoctonia spp., Curvularia cymbopogonis,Sphaerotheca macularis,Glomus mosseae, and Pestalotiopsis sp. The geographic
ranges of Pseudoperonspora cannabina,Septoria neocannabina and Fusarium graminearum are expanded.
A 12-yr study of fungi associated with Cannabis has revealed
that many taxa cited in the Cannabis literature are based on
misdeterminations or synonyms of species with wide host
ranges (McPartland, 1992, 1994b, 1995b). New species have
been discovered – Phomopsis ganjae (McPartland, 1983),
Schiffnerula cannabis (McPartland & Hughes, 1994 a), Fusicoccum
marconii,Leptodothiorella marconii (McPartland, 1994c), and
Ascochyta arcuata (McPartland, 1994d). Taxonomic changes
include Phoma cannabis (L. A. Kirchn.) McPartl. (McPartland,
1994d) and Septoria neocannabina McPartl. (McPartland,
1995a). New host associations include Botryosphaeria obtusa,
Lasiodiplodia theobromae (McPartland, 1994c), Colletotrichum
dematium,Diaporthe arctii,Leptosphaeria acuta and
Leptosphaerulina trifolii (McPartland, 1995b).
In this final report of the 12-yr study, a new species is
described, a combination made, five new Cannabis associations
described, and three Cannabis pathogens described from new
locations.
MATERIALS AND METHODS
Methods for downloading taxonomic literature and locating
type materials are described previously (McPartland, 1994b).
Common names of diseases follow those adopted by the
American Phytopathological Society (McPartland, 1991).
Fruiting structures from stems and leaves were removed
from host tissue, rehydrated in 3% KOH and either hand-
sectioned with a razor blade or teased apart with needles
under a dissecting microscope at 4¬. Fungal tissue was
stained with either cotton blue in lactic acid, phloxine in water
or acid fuchsin in lactic acid. Root tissues with mycorrhizas
were fixed in 4% glutaraldehyde (in 0±1cacodylate buffer),
dehydrated in a graded ethanol series, infused with paraffin,
sectioned longitudinally at 20 µm thickness with a sliding
microtome, and stained with either periodic acid–Schiff reagent
(Nemec, 1981) or trypan blue (Phillips & Hayman, 1970).
Curved spores were measured along their circumference and
widths measured at their widest part. Measurements are listed
as a range; for ample material (n&50), mean measurements
are given with extremes in brackets.
Root tissues with Rhizoctonia were washed and plated on
1±5% water agar amended with streptomycin and tetracycline.
Isolates were transferred to potato dextrose agar (PDA),
stained with DAPI (Martin, 1987) and Safranin O (Yamamoto
& Uchida, 1982) to determine nuclear condition of hyphal
cells. Isolates were assigned to a specific anastomosis group
(AG) by pairing with a tester strain representing each
binucleate Rhizoctonia AG (Burpee et al., 1980; Ogoshi et al.,
1983) following the procedure of Kronland & Stanghellini
(1988).
Genomic DNA was extracted from each isolate following
a modification of the procedure described by Cubeta et al.
(1991). Briefly, lyophilized, buffered mycelia were extracted
twice with phenol–chloroform–isoamyl alcohol
(25:24:1 v}v), treated with RNAase (1 mg ml−", DNase free,
Sigma), extracted twice in 250 µl of chloroform–isoamyl
alcohol (24:1), adjusted to 3 sodium acetate (pH 5±0) and
precipitated for 24 h in 2 vol. of 95 % EtOH at ®20°. The
genomic DNA pellet was washed with 80% EtOH, dried
under vacuum, and dissolved in TE buffer (10 mTris-HCl,
1mNa#EDTA). Extracted DNA was purified by electro-
phoresis in a 0±8% low melting agarose gel (Sea Plaque,
FMC) in TBE buffer (100 mTris-HCl, 20 mNa#EDTA,
100 mBoric acid). Escherichia coli (strain HB101) DNA was
used to estimate the concentration of DNA for each isolate.
Genomic DNA was amplified with two oligonuclotide
primers (LROR ¯5«-ACCCGCTGAACTTAAGC-3«and LR7
¯5«-TACTACCACCAAGATCT-3«). LROR and LR7 are
homologous to a region in 25S ribosomal DNA (rDNA) from
base position 17 to 1448. PCR reactions were conducted with
Mycol.Res.101 (7): 853–857 (1997) Printed in the United Kingdom
New species, combinations, host associations and location
records of fungi associated with hemp (Cannabis sativa)
Cannabis fungi 854
Amplitaq DNA polymerase (U.S. Biochemicals, Cleveland,
OH) in 50 µl volumes. Thirty PCR cycles were conducted on
an automated thermocycler (Perkin–Elmer–Cetus, Norwalk,
CT). The following protocol was used; 1 min denaturation at
94°, annealing at 50°for 45 s, 50–72°gradual increase for
1 min and primer extension at 72°for 1 min. To avoid
possible contamination, PCR experiments were conducted in
accordance with the stringent procedures described by White
et al. (1990). Also, tubes without DNA template were included
in each experiment (negative control). After amplification, a
3µl aliquot from each sample was subjected to electrophoresis.
Lamda DNA digested with EcoRI}Hind III was used to
determine size of PCR products. Gels were stained with
ethidium bromide and photographed over a uv transil-
luminator to record results.
Amplified PCR products were extracted in chloroform}
isoamyl alcohol (24:1), precipitated in 3 Na acetate and
95% EtOH at ®20°for 24 h, washed with 80% EtOH, dried
under vacuum and resuspended in TE buffer. For restriction
analysis, each PCR product was divided into equal aliquots
and digested with either Hha I, Hpa II, Sau3A I, or Taq I
(Promega). After digestion, samples were subjected to
electrophoresis in a 4% agarose gel (NuSieve, FMC
Bioproducts). ¬174 DNA digested with Hae III was used as
a molecular weight standard to determine size of restriction
fragments. Gels were stained with ethidium bromide and
photographed as described above.
RESULTS AND DISCUSSION
Micropeltopsis cannabis McPartl., sp. nov. (Figs 1–3)
Ascomata catathecioidea, 45–130 µm diam., complanata, ampul-
liformia, 25–46 µm alta, Brunnea vel nigra, ostiolata, margine
integro; paries superior ex radialiter dispositis cellulis quadratis,
peridia textura prismatica; paries basilaris similiter sed pallidior.
Ostiolum centrale, elevatum, compositum e parvis cellulis et coronam
gerens setarum divergentium. Setae rectae, subulatae, crassitunicatae,
non-septatae, laeves, brunneae, 12–22 µm longae. Asci bitunicati,
ovoidei ad obclavati, 4–8-spori, 21–40¬4–9 µm. Ascosporae hyalinae,
guttulatae, ellipsoideae, septo unico in medio quaeque indutae,
nonsetulatae, 11–12¬2±5–3±0µm. Paraphyses non visae.
In caulibus emortuis Cannabis sativae. Holotypus : France: Villernur,
autumnus 1880 (Fungi Gallici exsiccati, Centuria 27, No. 1671),
BPI No. 802622 A et B.
Ascomata catathecioid, 45–130 µm diam., flattened ampulli-
form, 25–46 µm high, dark brown to black, ostiolate, margin
entire ; upper layer composed of radially arranged quadrangular
cells, peridium textura prismatica; basal layer of similar
construction to upper layer but paler. Ostiole central, raised,
composed of small cells and bearing a crown of divergent
setae forming an inverted cone of the ostiole. Setae straight,
subulate, thick-walled, non-septate, smooth, dark brown,
12–22 µm long. Asci bitunicate, ovoid to obclavate, 4–8-
spored, 21–40¬4–9 µm. Ascospores hyaline, guttulate, el-
lipsoid, with a single median septum, nonsetulate,
11–12¬2±5–3±0µm.
This fungus occurs on the BPI exsiccatus of Calloria luteola
Roumegue
're. It does not appear on BR or CUP exsiccati.
M.cannabis resembles M.palustris (J. P. Ellis) Spooner & P. M.
Kirk, a parasite of Diatrypaceae on dead culms of Phalaris
arundinacea L. It differes by the smaller ascomata, asci and
ascospores, and M.cannabis ascospores lack setulae. If the lack
of setulae weighs as a genus-delimiting character (as questioned
by Spooner & Kirk, 1990), then this species belongs in
Chaetothyriopsis.
Orbilia luteola (Roum.) McPartl., comb. nov. (Fig. 1)
3Calloria luteola Roum., Rev.Myc.3(12):7 (1881).
Apothecia superficial on stems, sessile, waxy translucent yellow-
orange when hydrated, margin entire and round to ellipsoidal,
up to 0±5 mm diam. and 100 µm thick. Excipulum consists of
hydaline thin-walled textura globulosa.Asci small, cylindrical,
8-spored, 26±0¬4±5µm. Praphyses hyaline, filiform, slightly
enlarged at the apex. Ascospores hyaline, single-celled, fusiform,
indistinctly guttulate 6±5¬1±5µm.
Collections examined: Holotype. France: Villernur, leg : H. Garonne,
det : Roumegue
're (Fungi Gallici exsiccati, Centuria 27, No. 1671), stems
of Cannabis sativa L., fibre variety – automme 1880 (BR). Isotypes
examined: BPI no. 802622A, CUP.
Only Dr Garonne has found this fungus on Cannabis, but
he found enough to distribute between 60 and 100 specimens
in Roumegue
're’s Fungi Gallici exsiccati. Most Orbilia spp.
occur on wood or herbaceous stems, as saprotroph. Benny,
Samuelson & Kimbrough (1978) observed pockets of blue-
green algae in O.luteorubellas (Nyl.) P. Karst. They propose
transferring this association to the lichens. No cyanobacterial
cells were found in O.luteola.
Binucleate Rhizoctonia spp.
Hyphae without clamp connections, at first colourless, but
rapidly becoming brown; branches form geometrically at 45°
or 90°angles from parent hyphae, constricted slightly at the
branching point: a septum always forms near the base of the
branch, 4–7 µm diam.
Collection examined: Holland: Amsterdam, leg : McPartland, on roots
of C.sativa L., euphoriant variety – 5±1993 (BPI no. 802758, ATCC
culture no. 96145).
Staining with DAPI and Safranin O demonstrated a
binuclear hyphal condition. This isolate fused (followed by
death of hyphal cells at the fusion point) only with binucleate
Rhizoctonia spp. anastomosis group G (AGG) tester strain of
Ogoshi et al. (1983).
After PCR amplification of rDNA, a 1±4 kB product was
obtained. No PCR products were obtained in any of the
controls. After digestion of amplified rDNA with four different
restriction endonucleases, the restriction phenotype of the
Cannabis isolate was identical to the AGG tester strain and
possessed all restriction fragments described by Cubeta et al.
(1991).
Binucleate Rhizoctonia spp. are morphologically similar to
Rhizoctonia solani J. G. Ku
$hn, but have thinner hyphae (4–
7µm), and usually only possess two nuclei per hyphal cell.
Unlike R.solani, binucleate Rhizoctonia spp. often produce
J. M. McPartland and M. A. Cubeta 855
Figs 1–8. Photomicrographs of fungi associated with Cannabis. Scale bars, 38 µm. Fig. 1. Caulicolous habit of Orbilia luteola (large
white apothecia) and Micropeltopsis cannabis (small black ascocarps). Fig. 2. M.cannabis, exterior aspect of ascocarp with setae. Fig. 3.
M.cannabis, sectioned ascocarp with asci. Fig. 4. Curvularia cymbopogonis conidia. Fig. 5. Sphaerotheca macularis conidiophores and
conidia. Fig. 6. Photomicrograph by E. G. Arzberger ca 1925, labelled ‘ Endotrophic mycorrhiza on Cannabis sativa’, unknown
magnification. Fig. 7. Glomus mosseae, intercellular hyphae, tight hyphal coils, and H-connections. Fig. 8. Pestalotiopsis sp., conidium.
Ceratobasidium teleomorphs. In this study, we were unable to
produce the teleomorph of our strain of binucleate Rhizoctonia.
Curvularia cymbopogonis (C. W. Dodge) J. W. Groves &
Skolko, Canadian Journal of Research 23:96 (1945)
(Fig. 4)
3Helminthosporium cymbopogonis [as cymbogoni] C. W. Dodge,
Annals of the Missouri Botanical Garden 29: 139.
Teleomorph: Cochliobolus cymbopogonis J. A. Hall & Sivan.,
Transactions of the British Mycological Society 59:315
(1972).
Conidiophores simple, septate, brown, up to 300 µm long.
Conidia acropleurogenous, smooth, straight or curved, clavate
to ellipsoidal, obconical at the base with a protuberant hilum,
4 (sometimes 3) septate, middle cells dark brown with end
cells paler, averaging 40–50¬12–15 µm (slightly smaller than
cited by Sivanesan, 1984).
Collection examined: Nepal: 2 km north of Pokhara, leg: McPartland,
on seeds of Cannabis indica Lam., euphoriant variety – 10±1986 (BPI
no. 802117).
Non-germinating seeds began sporulating with C.cymbo-
pogonis when placed in a humidity chamber. The fungus was
isolated on potato dextrose agar. Other seeds (from a fibre
variety of C.sativa in Illinois) fulfilled Koch’s postulates.
Babu et al. (1977) cite another Curvularia sp. on Cannabis,
C.lunata (Wakker) Boedijn. Litzenberger, Farr & Lip (1963)
describe a ‘Curvularia sp.’ causing leaf spots on Cambodian
Cannabis. Although they claim representative specimens were
deposited at BPI, none was located.
C.cymbopogonis occurs on dicotyledons, monocotyledons
and gymnosperms around the world. The fungus often causes
seed and seedling blights, but also arises in leaf spots. The
homothallic pseudothecia have only been seen in culture.
Cannabis fungi 856
Sphaerotheca macularis (Wallr.: Fr.) Lind, Danish Fungi:
160 (1913) (Fig. 5)
3Erysiphe macularis (Wallr.) Fr., Systema Mycologium
3: 237 (1829).
¯Sphaerotheca humuli (DC.) Burrill, Bulletin of the Illinois State
Laboratory of Natural History 2: 400 (1887)
Anamorph: Oidium sp.
Superficial hyphae flexuous, branched, with inconspicuous
appressoria, cell diam. 4–7 µm, length (37–) 64±5 (–80) µm.
Conidiophores upright, simple, hyaline, 50–100 µm high.
Conidia produced in chains, hyaline (turning brown with age),
containing fibrosin bodies (which disappear with age), ovate to
barrel-shaped, single-celled, averaging 30±2¬14±0µm.
Collection examined. U.S.A.: Illinois, Champaign, leg: Sebastian, det :
McPartland, on leaves of C.sativa, euphoriant variety – 8.1981 (BPI
no. 802123).
The anamorph of this powdery mildew was previously
described (McPartland, 1983). No teleomorph developed.
Recent attention has focused on the taxonomic significance of
erysiphacous anamorphs. ‘Imperfect keys’ for powdery
mildews have gained a measure of reliability. Using a key by
Boesewinkel (1980), the Oidium sp. is easily identified as
S.macularis.
S.macularis occurs worldwide and commonly parasitizes
hops, Humulus lupulus L., a member of the Cannabidaceae
(Miller, Weiss & O’Brien, 1960). Doidge et al. (1953) report an
unidentified Oidium sp. attacking Cannabis in South Africa,
and Hirata (1986) notes Oidium sp. on Russian and Italian
hemp.
Glomus mosseae (T. H. Nicolson & Gerd.) Gerd. & Trappe,
Mycological Memoirs 5:40 (1974) (Fig. 7)
External hyphae grow in septate, stolon-like strands along
surfaces of roots and extend into soil. External hyphae
contiguous with internal hyphae, via penetration points in
endodermis. Internal hyphae in cortex grow longitudinally in
roots, rarely radially or circumferentially, inter- and in-
tracellular, septate, narrower than external hyphae, but always
"1µm diam., uncommonly producing H-connections, hyphal
loops, tight hyphal coils and vesicles; neither arbuscules nor
sporulating structures seen.
Collection examined: U.S.A.: Illinois, Hanna City, leg : McPartland,
from roots of Cannabis sativa, fibre variety – 9.1985 (BPI).
Feeder roots from a naturalized stand of hemp were
harvested, sectioned and stained. They revealed mycorrhizal
hyphae, but no sporulating structures. Abbott & Robson
(1978) devised a key to VA mycorrhizal fungi based on
infection morphology, in the absence of sporulating structures.
The characteristics described above are typical for Glomus
spp., and closely resemble G.mosseae (Carling & Brown,
1982).
Mosse (1961) produced an artificial mycorrhizal relationship
in C.sativa, utilizing ‘an Endogone species’ (prior to 1974, all
VA mycorrhizas were labelled Endogone sp.). Her description
of infection morphology matches that of the Illinois my-
corrhizal fungus: longitudinally oriented intercellular hyphae,
tight hyphal coils, H-connections, and no arbuscules. Mosse
(1961) goes on to describe spore morphology. Her Endogone
sp. spores resemble those of G.mosseae, according to Trappe’s
(1982) synoptic key. Mosse’s Cannabis fungus also colonized
apple, clover, onion, strawberry and tomato; G.mosseae
occurs on these hosts (Gerdemann & Trappe, 1974).
Evidence of VA mycorrhizal association with C.sativa
proceeds Mosse’s study. Archives at BPI contain a glass-plate
negative labelled ‘Endotrophic mycorrhiza on Cannabis
sativa’. The undated photomicrograph (of unknown magn-
ification) was taken by E. G. Arzberger, who conducted his
research in the 1920s and 1930s. It is reproduced in Fig. 6.
Pestalotiopsis sp. (Fig. 8)
Acervuli epiphyllous, circular to oval in outline, dark brown,
up to 280–480 µm diam. Conidiophores cylindrical, septate,
occasionally branched, hyaline, up to 10 µm in length.
Conidiogenous cells holoblastic, annellidic, hyaline, cylindrical.
Conidia fusiform, 4-septate, smooth, averaging 25¬5±6µm;
basal cell hyaline, with a simple hyaline appendage averaging
6±6µm in length; three median cells umber to olivaceous
brown, thick-walled, slightly collapsed between the septa ;
apical cell hyaline, conical, with appendages. Appendages
(setulae) tubular, flexuous, with three or less commonly two
branches, averaging 17±1µm in length.
Collection examined: Nepal: 2 km north of Pokhara, leg: McPartland,
on leaves and stems of Cannabis indica euphoriant variety – 10.1986
(BPI no. 802681).
Using keys in Guba (1961), the fungus can be placed
in section Quinqueloculatae, Non-spathulatae, Vesicolorae,
Umberae-Olivea. It could be any one of 40 fungi in this section.
Of the species described by Nag Raj (1993) this Pestalotiopsis
sp. resembles Pestalotiopsis karstenii (Sacc. & Syd.) Steyaert,
except P.karstenii lacks basal appendages. Paulsen (unpublished
1971 report, Kansas State University) isolated a Pestalotia sp.
from a naturalized stand of fibre-variety Cannabis near
Lawrence, but the fungus was not described and no voucher
specimens retained.
Expanded geographic ranges
Pseudoperonospora cannabina (G. H. Otth) Curzi is one of two
fungi causing downy mildew on Cannabis. A distribution map
published by the Commonwealth Mycological Institute (1971)
includes Europe, Kazakhstan, Pakistan, India and Japan.
Waterhouse & Brothers (1981) expand the range to other
parts of the former U.S.S.R. and China. A collection of
P.cannabina on wild hemp in Illinois (Hanna City) expands the
fungus range to the Western Hemisphere.
Septoria neocannabina has previously been reported in New
York (Peck, 1884). The taxon Septoria cannabis var. microspora
Briosi & Cavara is identical to S.neocannabina (McPartland,
1995a). This synonymy expands the range of S.neocannabina
to the Eastern Hemisphere.
The anamorph of Gibberella zeae (Schwein. : Fr.) Petch,
Fusarium graminearum Schwabe, has previously been described
from hemp in Germany (Wollenweber & Reinking, 1935) and
Romania (Ceapiou, 1958). A collection of F.graminearum
collected from wild hemp near Hanna City, Illinois, expands
J. M. McPartland and M. A. Cubeta 857
the geographic range on this host to the Western Hemisphere.
The Illinois strain is homothallic and readily produces
perithecia. A culture is deposited at the Fusarium Research
Center (accession number R-8965), The Pennsylvania State
University.
Curators of the following herbaria are acknowledged for
lending collections: BPI, BR, CUP, FH, MICH and PAV. P.
Nelson at the Fusarium Research Center (State College, PA,
U.S.A.) confirmed the identification of G.zeae. This work was
partly supported by a grant from Hortapharm, B.V., The
Netherlands.
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