Fungal Diversity
Pseudocercospora opuntiae sp. nov., the causal organism of
cactus leaf spot in Mexico
Victoria Ayala-Escobar1, María de Jesús Yáñez-Morales1, Uwe Braun2,
Johannes Z. Groenewald3 and Pedro W. Crous3*
1
Instituto de Fitosanidad, Colegio de Postgraduados, Km 36.5 Carr. México-Texcoco,
Montecillo, C.P. 56230, Edo de México, Mexico
2
Martin-Luther-University, Institute of Geobotany and Botanical Garden, Herbarium, Neuwerk
21, D-06099 Halle (Saale), Germany
3
Centraalbureau voor Schimmelcultures, Fungal Biodiversity Centre, Uppsalalaan 8, 3584 CT
Utrecht, The Netherlands
Ayala-Escobar, V., Yáñez-Morales, M.J., Braun, U., Groenewald, J.Z. and Crous, P.W. (2006).
Pseudocercospora opuntiae sp. nov., the causal organism of cactus leaf spot in Mexico. Fungal
Diversity 21: 1-9.
Pseudocercospora opuntiae is newly described from Opuntia spp. from Mexico, where it
causes a serious disease of this host. Although P. opuntiae is morphologically similar to other
members of the genus with pigmented conidia and conidiophores, and unthickened, not
darkened conidiogenous scars, DNA sequence data of the ITS region revealed that it clusters
distant from other species of Pseudocercospora within Mycosphaerella. These data support the
assumption that Pseudocercospora is paraphyletic within Mycosphaerella.
Key words: mitosporic fungi, Mycosphaerella anamorph, North America, Opuntia
Introduction
Species of Opuntia (cacti) are plants that grow wild in Mexico, where
they are native. Various species of this genus are important for the production
of prickly pears, or fresh edibles cladodes (pads) “nopalitos”. About 42 000 ha
of cactus pear have thus far been planted (Mondragon-Jacobo and Pérez
González, 1996). Opuntia fiscus-indica (L.) Mill. is cultivated throughout the
country, whereas O. megacantha Salm-Dyck, and O. streptacantha Lem. are
cultivated only in the Valley of Mexico, where the cv. “Reyna” is the main
plant cultivated for its green fruit. Furthermore, O. lasiacantha Pfeiff., O.
robusta H.L. Wendl., and O. tomentosa Salm-Dyck are wild cacti in some
Valley areas.
In November 2002 a new disease was observed on a cultivated Opuntia
sp. at “Ailpa Alta” in the Distrito Federal, and later in February 2003 in a stand
*
Corresponding author: Pedro Crous; e-mail: crous@cbs.knaw.nl
1
of a wild Opuntia sp. at “El Molino de las Flores” in the municipality of
Texcoco of the State of Mexico. Disease symptoms consisted of brown to
black, round lesions, approximately 2-4 cm diam, that appeared on the
cladodes. At the “Instituto de Fitosanidad, Colegio de Postgraduado” (CP) the
new disease was identified as an undescribed member of the genus
Pseudocercospora Speg. Material was sent to U. Braun at the Martin-LutherUniversity in Halle, Germany, and P.W. Crous at the Centraalbureau voor
Schimmelcultures in Utrecht, The Netherlands, for further identification. The
aim of this paper was to elucidate the taxonomy of the causal organism, and to
resolve its DNA phylogeny and generic affinity within the
Mycosphaerellaceae.
Materials and methods
Isolates
Isolates were obtained from symptomatic leaf pieces by placing
disinfested necrotic tissue fragments in moisture chambers to enhance
sporulation. Monoconidial cultures were subsequently established on wateragar (WA) (20 g agar / 1 L distilled H2O). Colonies were induced to sporulate
on Opuntia agar (OPA) (40 g of Opuntia cladodes boiled for 10 minutes, and
then blended with 20 g agar / 1 L distilled water), oatmeal-agar (OA) (15 g of
oatmeal, 20 g agar / 1 L distilled water), and potato-dextrose agar (PDA) (200
g potatoes, 20 g dextrose, 20 g agar / 1 L distilled water) (Gams et al., 1998).
Dishes of all media were point inoculated and incubated for 4 weeks at ± 24ºC
under continuous near-ultraviolet light, and inspected for sporulation at 3 day
intervals. Morphological observations in vitro were based on sporulating
cultures on host material. Thirty observations were made of each structure,
with extremes given in parentheses. Descriptions and nomenclatural details
were deposited in MycoBank (www.MycoBank.org).
DNA isolation, amplification and phylogeny
The protocol of Lee and Taylor (1990) was used to isolate genomic DNA
from fungal mycelium of a monoconidial culture grown on PDA in Petri
dishes. The primers ITS1 and ITS4 (White et al., 1990) were used to amplify
part (ITS) of the nuclear rRNA operon spanning the 3’ end of the 18S rRNA
gene, the first internal transcribed spacer (ITS1), the 5.8S rRNA gene, the
second ITS region and the 5’ end of the 28S rRNA gene. The PCR conditions,
sequence alignment and subsequent phylogenetic analysis followed the
methods of Crous et al. (2004). Newly generated sequence data were deposited
2
Fungal Diversity
in GenBank (accession numbers DQ073921–DQ073923), and the alignment in
TreeBASE (accession number SN2345).
Koch’s postulates
Pathogenicity tests were conducted on four-mo-old healthy cladodes of
cultivated Opuntia. The experiment consisted of six plants (incl. two controls)
that were inoculated (two cladodes per plant) by means of two methods.
Cladodes were either unwounded, or wounded by means of a sterile toothpick.
Method one consisted of placing colonised OPA agar disks (5 mm diam) on the
wound, and covering these with Parafilm. The second method consisted of
spraying a suspension of conidia (5000 conidia/mL, emended by means of a
haemocytometer) onto the unwounded cladode until run-off. Controls were
inoculated with a sterile agar plug, while unwounded leaves were sprayed with
sterile water. All the plants were incubated in a moist chamber at ± 90%
relative humidity for a period of two months, after which they were placed in a
shade house at ambient humidity (± 28ºC) until symptoms appeared. Reisolations were made from the margins of lesions onto PDA to confirm Koch’s
postulates.
Results
DNA phylogeny
The sequence alignment consisted of 24 ITS sequences including the
outgroup sequence and contained 506 characters (including alignment gaps)
which were used in the phylogenetic analyses. Of these characters, 106 were
parsimony-informative, 265 were constant and 135 variable characters were
parsimony-uninformative. Ten most parsimonious trees (one of which is shown
in Fig. 1) were obtained and show three main lineages, namely
Pseudocercospora (56% bootstrap support), Cercospora (100% bootstrap
support) and Passalora / Dothistroma / Pseudocercospora (99% bootstrap
support). The Cercospora and the Passalora / Dothistroma /
Pseudocercospora clades are joined with a bootstrap support value of 71%.
The tree topologies obtained using neighbour-joining analysis with the
uncorrected "p", Kimura-2-parameter and HKY85 substitution models resulted
in trees with identical topologies, but these trees differed from the parsimony
trees in that they grouped the Pseudocercospora and Cercospora clades (data
not shown). The sequence of Ps. vitis (Lév.) Speg. (type species of
Pseudocercospora) is in a well-supported clade (84% bootstrap support)
3
Mycocentrospora acerina AY266155
Pseudocercospora musae AY266148
100
Ps. musae AY266149
Ps. cruenta AY266153
Ps. eriodendri AF222840
84
56
Ps. vitis CPC 11595
Pseudocercospora
57
Ps. pseudoeucalyptorum AY725528
Ps. fijiensis var. difformis AY266150
100
Ps. fijiensis AY266152
C. canescens AY266164
C. sojina AY266158
C. nicotianae AY266159
62
C. beticola AY266165
C. apii AY266168
10 changes
71
Cercospora
100
C. kikuchii AY266161
57
Passalora arachidicola AY266154
51
Pa. personata AY266147
Pa. ampelopsidis AF362053
99
99
Ps. opuntia - wild Opuntia
Ps. opuntia Opuntia crop
63
Dothistroma sp. AY293062
53
Mycosphaerella pini AF013227
64
Passalora / Dothistroma / Pseudocercospora
C. hayi AY266163
Mycosphaerella pini AF211197
Fig. 1. One of 10 most parsimonious trees obtained from a heuristic search with 100 random
taxon additions of the ITS sequence alignment. The scale bar shows 10 changes and bootstrap
replicate values from 1000 replicates are shown at the nodes. Newly sequenced isolates are
printed in bold face and consensus branches thickened. The tree was rooted to
Mycocentrospora acerina AY266155.
4
Fungal Diversity
containing Ps. pseudoeucalyptorum Crous and Ps. eriodendri (Racib.) U.
Braun. The two isolates from Opuntia cluster together (99% bootstrap support)
in the Passalora / Dothistroma clade.
Taxonomy
Pseudocercospora opuntiae Ayala-Escobar, Braun & Crous, sp. nov.
MycoBank MB500197 (Figs. 2-11)
Etymology: Epithet referring to its host, Opuntia.
Maculae subcirculares vel saepe irregulares, 2-4 cm diam, griseo-brunneae, atrobrunneae vel sordide subnigrae. Coloniae punctiformes vel pustulatae, atro-brunneae vel
nigrae. Mycelium immersum (in vivo); hyphae 1-4 µm latae, interdum cellulis inflatis, ad 12
µm latis, pallide olivaceae vel olivaceo-brunneae, tenuitunicatae, leviae. Stromata bene
evoluta, immersa, 30-150 µm diam, ex cellulis inflatis, 2-10 µm latis, atro-olivaceo-brunneis
composita. Conidiophora numerosa, dense fasciculata, sporodochiales, erecta, recta,
subcylindrica, conica vel leviter geniculata-sinuosa, non-ramosa, 5-40 × 3-7 µm, 0-2-septata,
subhyalina, olivacea vel pallide olivaceo-brunnea, tenuitunicata, levia; cellulae conidiogenae
integratae, terminales, 5-30 µm longae; cicatrices conidiales inconspicuae, non-incrassatae,
non-fuscatae. Conidia solitaria, obclavata-cylindrica, 15-80 × 2.5-5 µm, (0-)1-7-septata,
subhyalina vel pallide olivaceo-brunnea, tenuitunicata, levia, apice obtuso, basi obconice
truncata, hila non-incrassata, non-fuscata.
Lesions subcircular to usually irregular, 2-4 cm diam, greyish-brown,
dark brown to dingy blackish (Fig. 2). Colonies punctiform to pustulate, dark
brown to blackish. Mycelium internal (developing superficial hyphae under
high humidity in a moist chamber; hyphae 1-4 µm wide, or forming swollen
hyphal cells, up to 12 µm diam, sometimes in monilioid sequences, pale
olivaceous to olivaceous-brown, thin-walled, smooth). Stromata welldeveloped, immersed, 30-150 µm diam, composed of swollen hyphal cells, 210 µm diam, dark olivaceous-brown. Conidiophores in large, dense fascicles,
forming sporodochial conidiomata, erect, straight, subcylindrical, conic to
somewhat geniculate-sinuous, unbranched, 5-40 × 3-7 µm, 0-2-septate (under
high humidity in moist chamber up to 300 µm long, strongly branched and
pluriseptate), subhyaline, olivaceous to olivaceous-brown, thin-walled, smooth.
Conidiogenous cells integrated, terminal, 5-30 µm long, proliferating
sympodially, but also percurrently; conidiogenous loci inconspicuous,
unthickened, not darkened. Conidia solitary, obclavate-cylindrical, 15-80 ×
2.5-5 µm, (0-)1-7-septate, subhyaline to pale olivaceous-brown, thin-walled,
smooth, apex obtuse, base obconically truncate, hila unthickened and not
darkened, but sometimes with a minute marginal frill if formed from a
percurrently proliferating conidiogenous cells (Figs. 7-10). Spermatogonia
intermixed with stromata, medium brown, 80-150 µm wide, giving rise to
numerous bacilliform to narrowly ellipsoid, straight to slightly curved, hyaline
spermatia, 2.5-4 × 1 µm.
5
6
Fungal Diversity
Figs. 2-11. Pseudocercospora opuntiae. 2. Symptomatic cladode. 3. Conidiophore fascicles
intermingled with spermatogonia. 4-6. Conidiophores. 7-10. Conidia. 11. Spermatia. Scale
bars: 3 = 150 µm, 4-10 = 10 µm, 11 = 1 µm.
Holotype: Mexico, Ailpa Alta, Distrito Federal, on a cultivated Opuntia sp.
(Cactaceae), 24 Nov. 2002, V. Ayala-Escobar and María de J. Yáñez-Morales (CHAPA #
167), culture ex-type CBS 117708 = CPC 11772.
Isotypes: HAL 1837 F and herb. CBS 15601.
Paratypes: Mexico, El Molino de la Flores, Texcoco, Edo. de Mexico, on a wild
Opuntia sp., Feb. 2003, María de J. Yáñez-Morales and V. Ayala-Escobar (CHAPA # 168,
HAL 1838 F).
Cultural characteristics: Colonies in OPA green-grey, reaching 3 cm
diam. within 4 weeks at 24ºC; on PDA erumpent, spreading, margins smooth,
regular, aerial mycelium moderate, surface grey-olivaceous with a thin white
margin, reverse greenish-grey; on OA smoke grey to white due to moderate
white aerial mycelium (Rayner, 1970); colonies on PDA reaching 12-16 mm
diam. after 2 wks at 24ºC. Sporulation on OPA was first observed after 4 wks.
Conidia were straight, cylindrical, subhyaline, 3-5-septate, 39-64 µm long (51
µm av.), base truncate, and apex obtuse (Figs. 7-10).
Koch’s postulates
Pseudocercospora opuntiae was successfully re-isolated from the
wounded and unwounded cladodes, using both means of inoculation (agar
plugs as well as conidial suspension). Control plants remained healthy.
Symptomatic cladodes developed stromata and spermatogonia (Fig. 3). Once
placed in moist chambers, stromata produced conidiophores within 5 d, and
conidia after 8 d.
Discussion
On the lesions in the type material, Pseudocercospora opuntiae is
associated with a dominant Asteromella state that sporulates profusely among
the fascicles. Phyllosticta concave Seaver has been considered the Asteromella
spermatial state of Mycosphaerella opuntiae (Ellis & Everh.) Dearn. Other
names that can be considered for the spermatial state of Pseudocercospora
opuntiae include Phyllosticta cacti (Berk.) Archer, P. opuntiae [Sacc. & Speg.]
var. microspora Cavara and P. opuntiicola Bubák, but the taxonomy and
generic affinity of these taxa have not yet been resolved (Archer, 1926; Aa and
Vanev, 2002), and it remains to be seen if any of them can be applied to the
Asteromella state of P. opuntiae. Although no teleomorph has yet been found,
7
it seems unlikely that P. opuntiae would be related to M. opuntiae. The latter
fungus was originally described from stems of Opuntia in the USA, and von
Arx (1984) linked this teleomorph to “Microdochium” lunatum (Ellis &
Everh.) Arx.
On account of the structure of the conidiogenous loci and conidial hila,
the new species on Opuntia clearly belongs in Pseudocercospora, and also
represents the first member of the genus described from the Cactaceae. DNA
sequence data derived from the ITS region of P. opuntiae proved interesting,
however, as it clearly represented evidence to the fact that Pseudocercospora
(typified by P. vitis (Lév.) Speg.) is paraphyletic within Mycosphaerella.
Although the genus Pseudocercospora is a well-known anamorph of
Mycosphaerella (Crous et al., 2000), some species which are
Pseudocercospora-like are probably unrelated to Mycosphaerella, as for
instance Parapithomyces clitoriae Alcorn, which has a Pseudocercospora
synanamorph (Alcorn, 1992).
Although earlier studies have proven that the genus Mycosphaerella is
monophyletic (Crous et al., 1999, 2000, 2001a, 2001b, 2004), it is becoming
evident that the same anamorph morphology has evolved more than once
within Mycosphaerella. The clustering of P. opuntiae within a clade consisting
of members of Passalora Fr. (thickened, darkened, refractive conidial scars),
and Dothistroma Hulbary (acervuli with percurrent proliferating conidiogenous
cells, giving rise to septate, hyaline conidia), clearly reiterates the fact that
within Mycosphaerella, anamorph morphology is not always phylogenetically
informative. These findings question currently accepted anamorph generic
concepts in Mycosphaerella, which rely heavily upon conidial pigmentation,
conidiomatal structure, and the nature of conidial scars (Kirschner et al., 2004;
Schubert and Braun, 2005), and poses a clear taxonomic challenge for future
studies.
Acknowledgements
Sincere thanks are due to CONACYT (from Mexico) project 38409-V (Fungal
Biodiversity) for funding this research.
References
Aa, H.A. van der and Vanev, A. (2002). A revision of the species described in Phyllosticta.
Centraalbureau voor Schimmelcultures, Utrecht, the Netherlands.
Alcorn, J.L. (1992). Parapithomyces clitoriae sp. nov. (Fungi: Hyphomycetes) and its
Pseudocercospora anamorph. Australian Systematic Botany 5: 711-715.
Archer, W.A. (1926). Mycological characters of some Sphaeropsidales in culture with
reference to classification. Annals of Mycology 24: 1-84.
8
Fungal Diversity
Arx, J.A. von (1984). Notes on Monographella and Microdochium. Transactions of the British
Mycological Society 82: 373-374.
Crous, P.W., Hong, L., Wingfield, M.J., Wingfield, B.D. and Kang, J.-C. (1999). Uwebraunia
and Dissoconium, two morphologically similar anamorph genera with distinct
teleomorph affinity. Sydowia 52: 155-166.
Crous, P.W., Aptroot, A., Kang, J.-C., Braun, U. and Wingfield, M.J. (2000). The genus
Mycosphaerella and its anamorphs. Studies in Mycology 45: 107-121.
Crous, P.W., Hong, L., Wingfield, B.D. and Wingfield, M.J. (2001a). ITS rDNA phylogeny of
selected Mycosphaerella spp. and their anamorphs occurring on Myrtaceae.
Mycological Research 105: 425-431.
Crous, P.W. Kang, J.-C. and Braun, U. (2001b). A phylogenetic redefinition of anamorph
genera in Mycosphaerella based on ITS rDNA sequence and morphology. Mycologia
93: 1081-1101.
Crous, P.W., Groenewald, J.Z., Mansilla, J.P., Hunter, G.C. and Wingfield, M.J. (2004).
Phylogenetic reassessment of Mycosphaerella spp. and their anamorphs occurring on
Eucalyptus. Studies in Mycology 50: 195-214.
Gams, W., Hoekstra, E.S. and Aptroot, A. (eds) (1998). CBS Course of mycology. 4th ed.
Centraalbureau voor Schimmelcultures, Baarn, the Netherlands.
Kirschner, R., Piepenbring, M. and Chen, C.J. (2004). Some cercosporoid hyphomycetes from
Taiwan, including new species of Stenella and new reports of Distocercospora
pachyderma and Phacellium paspali. Fungal Diversity 17: 57-68.
Lee, S.B. and Taylor, J.W. (1990). Isolation of DNA from fungal mycelia and single spores. In:
PCR Protocols: a guide to methods and applications (eds. M.A. Innis, D.H. Gelfand,
J.J. Sninsky and T.J. White). Academic Press, San Diego, USA: 282-287.
Mondragon-Jacobo, C. and Perez-Gonzalez, S. (1996). Native cultivars of cactus pear in
Mexico. In: Progress in new crops. (ed. J. Janick). ASHS Press, Arlington, VA.: 446450.
Rayner, A.W. (1970). A Mycological Colour Chart. Commonwealth Mycological Institute,
Kew.
Schubert, K. and Braun, U. (2005). Taxonomic revision of the genus Cladosporium s.l. 4.
Species reallocated to Asperisporium, Dischloridium, Fusicladium, Passalora, Pseudoasperisporium and Stenella. Fungal Diversity 20: 187-208.
White, T.J., Bruns, T., Lee, S. and Taylor, J. (1990). Amplification and direct sequencing of
fungal ribosomal RNA genes for phylogenetics. In: PCR Protocols: a guide to methods
and applications (eds. M.A. Innis, D.H. Gelfand, J.J. Sninsky and T.J. White).
Academic Press, San Diego, USA: 315-322.
(Received 25 June 2005; accepted 14 October 2005)
9