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. 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