Persoonia 24, 2010: 1–11 www.persoonia.org RESEARCH ARTICLE doi:10.3767/003158510X486568 Calonectria species associated with cutting rot of Eucalyptus L. Lombard1, X.D. Zhou1,4, P.W. Crous3, B.D. Wingfield2, M.J. Wingfield1 Key words Abstract Decline in the productivity of Eucalyptus hybrid cutting production in the Guangdong Province of China is linked to cutting rot associated with several Calonectria spp. The aim of this study was to identify these fungi using Australia morphological and DNA sequence comparisons. Two previously undescribed Calonectria spp., Ca. pseudoreteaudii Calonectria sp. nov. and Ca. cerciana sp. nov. were identified together with Ca. pauciramosa. Calonectria pseudoreteaudii China resides in the Ca. reteaudii complex and Ca. cerciana is closely related to Ca. morganii. Connected to the dis- Cylindrocladium covery of Ca. pseudoreteaudii, species in the Ca. reteaudii complex were re-considered and the group is shown Eucalyptus to accommodate two cryptic species. These originate from Australia and are described as Ca. queenslandica sp. systematics nov. and Ca. terrae-reginae sp. nov. Article info Received: 30 June 2009; Accepted: 17 November 2009; Published: 12 January 2010. INTRODUCTION 2002). Of these, leaf blight is most devastating in the tropical regions of South East Asia and South America and is particularly Species of Calonectria (Ca.), and their Cylindrocladium (Cy.) serious on Eucalyptus spp. (Booth et al. 2000, Crous & Kang anamorphs, are important plant pathogens worldwide (Crous 2001, Crous 2002, Rodas et al. 2005). 2002). Past taxonomic studies on these pathogens have fo- Decline in Eucalyptus hybrid cutting production due to cutting cused on morphology and sexual compatibility to delimit new rot has recently been observed in a commercial forest nursery species (Peerally 1991, Crous & Wingfield 1994). More recently, in the Guangdong Province of China. Initial investigations in- DNA sequence comparisons have resulted in the recognition of dicated that the causal agents were unknown Calonectria spp. several species complexes in Calonectria (Schoch et al. 1999, that represented species in the Ca. reteaudii and Ca. morganii Crous et al. 2004, 2006a). complexes (Zhou et al. 2008). The aim of this study was to iden- One of the newly recognised groups in Calonectria is the Ca. tify these Calonectria spp. using morphological characteristics reteaudii complex (Crous & Kang 2001, Kang et al. 2001). The and phylogenetic inference. In addition, the taxonomic status complex encompasses several Calonectria spp. with Cylindro- and circumscription of species in the Ca. reteaudii species cladium anamorphs morphologically similar to the Ca. reteaudii complex were re-considered. anamorph state, having clavate vesicles with multiseptate macroconidia. These include Cy. angustatum, Cy. hurae, Ca. Materials AND METHODS leguminum and Ca. rumohrae (Crous 2002). The Ca. morganii species complex (Crous et al. 1993, Schoch Isolates et al. 2001) includes Ca. insularis, Ca. morganii and Cy. Hybrid clonal Eucalyptus cuttings showing symptoms of cutting hawksworthii (Schoch et al. 1999, 2001, Crous 2002). Species rot were collected in the nursery of the China Eucalypt Research in this complex are characterised by ellipsoid to obpyriform to Centre (CERC) in Guangdong Province, China. Diseased cut- clavate vesicles, 1-septate conidia and orange to red perithecia tings were placed in moist chambers and incubated for 48 h at producing 1-septate ascospores (Peerally 1991, Schoch et al. room temperature to induce sporulation. Direct isolations were 1999, Crous 2002). made onto malt extract agar (2 % w/v; MEA; Biolab, Midrand, Species in both the Ca. reteaudii and the Ca. morganii com- South Africa) and cultures were incubated for 7 d at 25 °C plexes are responsible for a wide variety of disease symptoms under continuous near-ultraviolet light. For each isolate, single on several plant hosts in subtropical and tropical regions of the conidial cultures were prepared on MEA and representative world (Bolland et al. 1985, Peerally 1991, Sharma & Mohanan strains are maintained in the culture collection (CMW) of the 1991, Booth et al. 2000, Crous 2002, Rodas et al. 2005). Dis- Forestry and Agricultural Biotechnology Institute (FABI), Uni- ease symptoms include leaf blight (Sharma & Mohanan 1991, versity of Pretoria, Pretoria, South Africa, the Centraalbureau Booth et al. 2000, Rodas et al. 2005) and cutting rot (Sharma & voor Schimmelcultures (CBS), Utrecht, The Netherlands, and Mohanan 1982, Sharma et al. 1984, Schoch et al. 1999, Crous the China Eucalypt Research Centre (CERC), ZhanJiang, China. Isolates of Ca. reteaudii were obtained from the culture 1 Department of Microbiology and Plant Pathology, Tree Protection Co- collection of CBS, including representative isolates used in the operative Programme, Forestry and Agricultural Biotechnology Institute, study of Kang et al. (2001). University of Pretoria, Pretoria 0002, South Africa; corresponding author e-mail: [email protected]. DNA sequence comparisons 2 Department of Genetics, Forestry and Agricultural Biotechnology Institute, University of Pretoria, Pretoria 0002, South Africa. Calonectria isolates were grown on MEA for 7 d. Mycelium was 3 CBS-KNAW Fungal Biodiversity Centre, Uppsalalaan 8, 3584 CT Utrecht, then scraped from the surfaces of Petri dishes, freeze-dried, The Netherlands. and ground to a powder in liquid nitrogen, using a mortar and 4 China Eucalypt Research Centre (CERC), Chinese Academy of Forestry (CAF), ZhanJiang 524022, Guangdong Province, P.R. China. pestle. DNA was extracted from the powdered mycelium as © 2010 Nationaal Herbarium Nederland & Centraalbureau voor Schimmelcultures You are free to share - to copy, distribute and transmit the work, under the following conditions: Attribution: You must attribute the work in the manner specified by the author or licensor (but not in any way that suggests that they endorse you or your use of the work). Non-commercial: You may not use this work for commercial purposes. No derivative works: You may not alter, transform, or build upon this work. For any reuse or distribution, you must make clear to others the license terms of this work, which can be found at http://creativecommons.org/licenses/by-nc-nd/3.0/legalcode. Any of the above conditions can be waived if you get permission from the copyright holder. Nothing in this license impairs or restricts the author’s moral rights. 2 Persoonia – Volume 24, 2010 M.J. Dudzinski C. L. Schoch A.C. Alfenas A.C. — M.J. Dudzinski A.C. Alfenas A.C. P.W. Crous P.W. E.C.Y. Liew E.C.Y. R.M. Leahy P.W. Crous P.W. K.M. Old M.J. Wingfield Collector M.J. Dudzinski A. Peerally M.J. Wingfield M.J. Wingfield M.J. Wingfield & X.D.Zhou R.M. Leahy N.E. El-Gholl M.J. Wingfield Alfenas A.C. Krugner T.L. B. Brown — Crous P.W. M.J. Dudzinski M.B. Figueiredo M.J. Wingfield & X.D.Zhou M.J. Wingfield & X.D.Zhou M.J. Wingfield & X.D. Zhou M.J. Wingfield & X.D. Zhou M.J. Wingfield & X.D. Zhou M.J. Wingfield & X.D. Zhou M.J. Wingfield & X.D. Zhou M.J. Wingfield & X.D. Zhou Vietnam Madagascar Brazil Indonesia Vietnam Brazil Madagascar China USA USA South East Asia South East Africa South Australia Indonesia Vietnam Mauritius Colombia Ecuador Origin China USA USA Indonesia Brazil Spain Australia Thailand Madagascar Vietnam Brazil China China China China China China China China cutting cutting cutting cutting cutting cutting cutting cutting cutting E. grandis × E. grandis E. grandis E. grandis E. grandis E. grandis E. grandis E. grandis E. grandis × × × × × × × × sp. sp. sp. sp. sp. sp. Host E. camaldulensis Soil Soil Hibiscus sabdariffa E. camaldulensis Rumohra adiantiformis Soil Soil Arachnoides adiantiformis Tillandsia capitata Tillandsia E. grandis E. pellita E. urophylla E. camaldulensis Nelumbo nucifera Soil Soil T. capitata T. Leucothoë axillaris E. urophylla Eucalyptus urophylla E. urophylla E. urophylla Argyreia Eucalyptus Eucalyptus E. urophylla E. urophylla E. urophylla E. urophylla E. urophylla E. urophylla Pinus Eucalyptus Eucalyptus E. camaldulensis Annona squamosa α FJ918536 GQ240675 FJ918555 FJ918564 FJ918567 FJ918569 FJ918540 FJ918538 FJ918548 FJ918556 AY725709 FJ918563 FJ918560 FJ918552 FJ918566 FJ918544 GQ240676 GQ240681 FJ918539 FJ918567 FJ918558 GQ267288 AY725712 FJ918570 FJ918542 GQ267289 GQ267290 FJ918562 FJ918559 FJ918541 FJ918565 FJ918551 FJ918553 FJ918543 FJ918535 FJ918537 FJ918547 Translation Translation elongation factor -1 DQ190660 GQ240659 FJ918525 DQ190680 DQ190719 FJ918533 DQ190673 FJ918518 DQ190728 FJ918526 AY725660 DQ190679 FJ918529 DQ190695 FJ918532 DQ190667 GQ240660 GQ240665 DQ190663 DQ190720 DQ190649 GQ267243 AY725663 FJ918534 FJ918520 GQ267244 GQ267245 AF348241 FJ918528 FJ918519 FJ918531 DQ190656 FJ918523 FJ918521 DQ190659 DQ190661 DQ190654 Histone H3 GenBank accession no. -tubulin β GQ240642 GQ240643 AF210862 DQ190579 DQ190611 FJ918516 AF389836 AF389831 AF333408 AF210861 AY725618 DQ190578 FJ918511 DQ190593 FJ918515 AF389834 GQ240644 GQ240649 AF389830 AF232857 AF333407 GQ25288 AY725620 FJ918517 FJ918505 GQ267205 GQ267206 FJ918513 FJ918510 FJ918504 FJ918514 AF207543 FJ918508 AF389835 DQ190573 AF389833 AF389837 T T T 1 used in the phylogenetic study. T T T T T T T Isolate number CBS 112144 CBS 112143 CBS 113582 CBS 114559 CBS 111871 CBS 109065 CBS 111478 CMW 25311 CBS 582.50 CBS 112153 CBS 114551 CBS 114558 CBS112744 CBS 111793 CMW 25290 (= CBS 123695) CBS 109169 CMW 30823 = CMW 30603 (= CBS 112155 CPC 3210 = DFRI00172) CBS 113583 CMW 18446 CMW 25309 (=