Reclassification of the butternut canker fungus, Sirococcus clavigignenti-juglandacearum, into the genus Ophiognomonia

f u n g a l b i o l o g y 1 1 5 ( 2 0 1 1 ) 7 0 e7 9

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Reclassification of the butternut canker fungus, Sirococcus clavigignenti-juglandacearum, into the genus Ophiognomonia K. D. BRODERS*, G. J. BOLAND School of Environmental Sciences, University of Guelph, Guelph, ON N1G 2W1, Canada

article info

abstract

Article history:

Sirococcus clavigignenti-juglandacearum (Sc-j), which causes a canker disease on butternut, is

Received 8 July 2010

largely responsible for the decline of this tree in the United States and Canada. The original

Received in revised form

description of the species was based on anamorphic characters because the teleomorph is

22 October 2010

unknown. Recent phylogenetic investigations have found that Sc-j is not a member of the

Accepted 26 October 2010

genus Sirococcus, and accurate taxonomic classification is required. The objective of this

Available online 31 October 2010

study is to use sequence data to determine the phylogenetic placement of Sc-j within the

Corresponding Editor:

Gnomoniaceae, Diaporthales. Isolates were recovered from infected Juglans ailantifolia

Joseph W. Spatafora

var. cordiformis (heartnut), Juglans cinerea (butternut), and Juglans nigra (black walnut) in

Keywords:

internal transcribed spacers 1 and 2, and the translation elongation factor 1-alpha from

Ontario and the eastern United States. The genes coding for b-tubulin, actin, calmodulin, Ascomyceta

28 isolates of Sc-j and representatives of the major lineages within the Gnomoniaceae

butternut canker

were evaluated. There was no difference in the sequences of the five genes among the iso-

forest pathology

lates of Sc-j studied, indicating a recent introduction followed by asexual reproduction and

Gnomoniaceae

spread via conidia. The phylogenetic analyses demonstrate this fungus does not belong to

Juglandaceae

the genus Sirococcus, and provides strong support (99 % MP and 100 % NJ bootstrap values,

multilocus phylogenetics

and 100 % Bayesian posterior probabilities) for its inclusion in the genus Ophiognomonia, thereby supporting a reclassification of the butternut canker fungus to Ophiognomonia clavigignenti-juglandacearum. ª 2010 The British Mycological Society. Published by Elsevier Ltd. All rights reserved.

Introduction The genus Sirococcus is comprised of asexually reproducing fungi, which have no known sexual state. The genus includes several important pathogens, including those which cause shoot blight and tip dieback of conifers and perhaps the most notorious Sirococcus clavigignenti-juglandacearum N.B. Nair, Kostichka & J.E. Kuntz, the cause of butternut canker. In a comprehensive review of the coelomycetes, Sutton (1980) only mentions two species, Sirococcus spiraea (Lebedeva) Petr. and the type specimen for Sirococcus, Sirococcus strobilinus Preuss, now referred to as Sirococcus conigenus (D.C.) Cannon & Minter (1983).

In contrast to other members of the genus, S. clavigignentijuglandacearum is a pathogen of the deciduous host, butternut (Juglans cinerea L.), which is a medium-sized hardwood tree that is native to the eastern North American forests. In recent years, the tree has been decimated by this fungal pathogen, with reports indicating declines of as much as 70e90 % in some regions (Anderson & LaMadeleine 1978). Butternut canker was first reported from southwestern Wisconsin in 1967 (Renlund 1971) and the pathogen causing the disease was first described in 1979 (Nair et al. 1979). The pathogen grows under the bark layer after the spores have found an entry point, where it forms a thin, black stromatic tissue under the bark, and eventually produces a number of stromatal

* Corresponding author. Tel.: þ1 519 824 4120x54458; fax: þ1 519 837 0442. E-mail address: [email protected] 1878-6146/$ e see front matter ª 2010 The British Mycological Society. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.funbio.2010.10.007

Reclassification of the butternut canker fungus

columns composed of interwoven mycelia, which lifts the dying bark layers of the tree until it ruptures (Tisserat & Kuntz 1984). The pathogen is primarily spread via rain splash and wind dispersal (Tisserat & Kuntz 1983). Long distance movement of the pathogen likely takes place by insect transmission (Katovich & Ostry 1998; Halik & Bergdahl 2002), and/ or within infected seed of butternut and black walnut (Juglans nigra L.) (Innes & Rainville 1996); evidenced by the fact that isolated butternuts are often infected. Sirococcus clavigignenti-juglandacearum is also able to infect and colonize other hardwood species. Ostry and Moore (2007) found in greenhouse inoculations that the pathogen was able to colonize several genera of hardwoods including Carya, Castenea, Juglans, Prunus, and Quecus spp., and several important commercial Persian walnut (Juglans regia L.) cultivars were moderately to highly susceptible. It is unclear how long butternut canker disease has existed in North America, but research suggests that S. clavigignenti-juglandacearum was recently introduced into North America (Furnier et al. 1999). Since its initial report in 1967, butternut canker was subsequently reported in Canada in Quebec in 1990, in Ontario in 1991 (Davis et al. 1992), and in New Brunswick in 1997 (Harrison et al. 1998) where it was thought to have been present for at least 7 y. The rapid spread of the pathogen into Canada, combined with the devastating effect of this disease has led to the butternut being designated as an endangered species in Canada in Nov. 2003 (Neilson et al. 2003). The devastating effect of this pathogen on butternut and the potential danger of an introduction into the commercial walnut growing regions of CA demonstrate the importance of understanding as much as possible about the evolutionary history of this pathogen. An accurate name that reflects phylogeny may provide valuable ecological information about a plant-associated fungus, including its potential pathogenicity, host range and appropriate control measures (Rossman & Palm-Hernandez 2009). Therefore, placing this pathogen in the proper genus will imply valuable information about the biological characteristics of this, and other species included in the genus. Several recent studies into the phylogenetic relationships among members of the Family Gnomoniaceae, Order Diaporthales have observed the distant relationship between S. clavigignenti-juglandacearum and other species of Sirococcus (Green & Castlebury 2007; Konrad et al. 2007; Mejia et al. 2008; Sogonov et al. 2008). In these studies it was determined that S. clavigignenti-juglandacearum was more closely related to species in the genus Gnomonia. However, a comprehensive study of the leaf-inhabiting genera of Gnomoniaceae found that many of the species classified as Gnomonia were rather members of the genus Ophiognomonia (Sogonov et al. 2008). This review also suggested the inclusion of S. clavigignentijuglandacearum as a member of Ophiognomonia. However, only a single isolate was used in this analysis and a detailed classification of the species was not completed at the time. The objective of this study was to use sequence data from five genes to determine the phylogenetic placement of S. clavigignenti-juglandacearum among the Gnomoniaceae, thereby, proposing a reclassification of the species into the genus Ophiognomonia.

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Material and methods Fungal material Diseased plant tissues from Juglans ailantifolia var. cordiformis Carr. (heartnut), Juglans cinerea and Juglans nigra were collected from various locations in Ontario from Mar.eAug. 2009. In addition, several isolates (Table 1) from diverse locations in the United States were provided from Mike Ostry at the Northern Research Station, USDA, St. Paul, MN. Diseased plant tissue was surface disinfected for 1 min in a 0.6 % sodium hypochlorite solution, rinsed in deionized water, allowed to dry on sterile paper towels, plated onto acidified potato dextrose agar (PDA) (0.75 ml 50 % lactic acidL1 PDA), and incubated at room temperature for 5e7 d. Putative cultures of Sirococcus clavigignenti-juglandacearum were transferred to fresh plates of PDA. Single spore cultures were obtained by placing conidia, from pycnidia growing in vitro, into sterile deionized water and then spread on PDA, allowed to incubate at room temperature for 48 h, and individual germinating conidia were transferred to fresh plates of PDA using a stereomicroscope. Twenty-eight isolates of S. clavigignenti-juglandacearum from several locations in North America, as well as different Juglans species, were studied (Table 1). The isolates recovered from J. cinerea tissues were isolated from infected buds, twig cankers, leaf lesions, and trunk cankers. Isolates from J. nigra were recovered from stem and seed pericarp lesions, and isolates recovered from J. ailantifolia var. cordiformis were recovered from infected buds. In addition to the isolates of S. clavigignenti-juglandacearum, three species of Sirococcus and other closely related species were studied. To identify species closely related to S. clavigignenti-juglandacearum, pair-wise sequence comparisons were conducted through GenBank with BLASTn (Altchul et al. 1990) using default settings. GenBank numbers for all taxa included in this study are presented in Table 1. Data associated with all specimens examined are listed following each taxon.

DNA extraction and PCR amplification For DNA extraction, isolates were grown on cellophane-covered PDA for 7e10 d, and mycelia were collected and DNA was extracted using the MoBio Power Soil DNA extraction kit (Mo Bio Laboratories Inc., Carlsbad, CA.). The genes coding for b-tubulin, actin, calmodulin, the internal transcribed spacer regions 1 and 2, including the 5.8S rDNA (ITS), and a fragment of the translation elongation factor 1-alpha (tef1-a) containing introns 4 and 5 were amplified. Gene fragments were amplified in a total volume of 50 ml reaction consisting of 10 ml of 5 Green GoTaq reaction buffer (Promega Corp., Madison, WI), 5 ml of 25 mM MgCl2, 1 ml containing 10 mM each dNTP, 0.25 ml of GoTaq Taq polymerase, 5 ml each of 5-mM concentrations of each primer, 2 ml of DNA at a concentration of 10 ng ml1, and 21.75 ml of sterile deionized water. PCR parameters were 95  C for 5 min; followed by 35 cycles of 95  C for 1 min, 54  C for 1 min, 72  C for 1 min; and completed with 72  C for 5 min followed by 4  C. PCR products were purified using Qiaquick spin columns (Qiagen Inc., Valencia, CA). For sequencing, 2 ml of primer at 5 pmoles ml1 were added to 2 ml of purified DNA (20 ng ml1) product. Amplified products were sequenced with

72

Table 1 e Isolates in the phylogenetic analyses of Ophiognomonia clavigignenti-juglandacearum and related members of the Gnomoniaceae, culture numbers, country, host, and accession numbers for actin, calmodulin, ITS, b-tubulin, and tef1-a. New sequences from this study are in bold. Taxon

CBS 109747 1155-2 AR 3640 CBS 911.79 CBS 121077 CBS 109763 CBS 109771 CBS 199.53 CBS 121227 CBS 121226 CBS 121263 CBS109773 CBS 109744 CBS 782.79 CBS 121266 ATCC 36642 SCJ2 SCJ3 P005 WB22 GA1-1 GA5-1 BUD2-3 P013 P017 P019 P029 P034 P037 P043 P045 1210-10 1235-4 1339-13 1343-408-1 1365-4 1375-4A 1378-3 1382-1B HN-1 HN-2 70-BW2 BW10-2

Country Switzerland Canada USA: WI Switzerland Austria Austria USA: WA Italy USA: VA USA: MD USA: TN Austria Canada Switzerland Canada: BC USA: WI Canada: ON Canada: ON Canada: ON Canada: ON Canada: ON Canada: ON Canada: ON Canada: ON Canada: ON Canada: ON Canada: ON Canada: ON Canada: ON Canada: ON Canada: ON USA: NY USA: NY USA: MN USA: WI USA: MN USA: IN USA: IN USA: WI Canada: ON Canada: ON Canada: ON Canada: ON

Host

Actin

Calmodulin

Fagus sylvaticum Castanea dentata Aesculus hippocastanum Acer pseudoplatanus Alnus incana Betula pendula Cornus florida Corylus avellana Liquidiambar styraciflua Fragaria vesca Quercus alba Alnus viridis Alnus rubra Alnus viridis Populus balsamifera Juglans cinerea Juglans cinerea Juglans cinerea Juglans cinerea Juglans cinerea Juglans cinerea Juglans cinerea Juglans cinerea Juglans cinerea Juglans cinerea Juglans cinerea Juglans cinerea Juglans cinerea Juglans cinerea Juglans cinerea Juglans cinerea Juglans cinerea Juglans cinerea Juglans cinerea Juglans cinerea Juglans cinerea Juglans cinerea Juglans cinerea Juglans cinerea Juglans ailantifolia var. cordiformis Juglans ailantifolia var. cordiformis Juglans nigra Juglans nigra

DQ313631 GU989031 DQ313616 DQ313617

DQ313603 GU993762 DQ313587 DQ313588

EF512485

EF512506

GU989001 GU989002 GU989003 GU989006 GU989004 GU989005 GU989007 GU989008 GU989019 GU989020 GU989021 GU989022 GU989023 GU989024 GU989025 GU989026 GU989009 GU989010 GU989011 GU989012 GU989015 GU989016 GU989017 GU989018 GU989027 GU989028 GU989029 GU989030

GU993734 GU993735 GU993736 GU993737 GU993738 GU993739 GU993740 GU993741 GU993742 GU993743 GU993744 GU993745 GU993746 GU993747 GU993748 GU993749 GU993750 GU993751 GU993752 GU993753 GU993754 GU993755 GU993756 GU993757 GU993758 GU993759 GU993760 GU993761

ITS

b-tubulin

tef1-a

DQ313525 GU993820 DQ313557 DQ313549 EU199184 EU199180 EF512464 EU254779 EU254748 EU254824 EU254839 DQ323523 EU199197 EU254864 EU254870 GU993705 GU993706 GU993707 GU993708 GU993709 GU993710 GU993711 GU993712 GU993713 GU993714 GU993715 GU993716 GU993717 GU993718 GU993719 GU993720 GU993721 GU993722 GU993723 GU993724 GU993725 GU993726 GU993727 GU993728 GU993729 GU993730 GU993731 GU993732

EU219134 GU993733 EU219107 EU219177 EU219127 EU219105 EU219092 EU219148 EU219184 EU219144 EU219218 EU219102 EU219103

DQ313565 GU993791 DQ313558 DQ313559 EU221891 EU221884 EU221897 EU221885 EU221898 EU221961 EU221939 EU221896 EU221991 EU221974 EU221955 GU993792 GU993793 GU993794 GU993795 GU993796 GU993797 GU993798 GU993799 GU993800 GU993801 GU993802 GU993803 GU993804 GU993805 GU993806 GU993807 GU993808 GU993809 GU993810 GU993811 GU993812 GU993813 GU993814 GU993815 GU993816 GU993817 GU993818 GU993819

EU219221 GU993763 GU993764 GU993765 GU993766 GU993767 GU993768 GU993769 GU993770 GU993771 GU993772 GU993773 GU993774 GU993775 GU993776 GU993777 GU993778 GU993779 GU993780 GU993781 GU993782 GU993783 GU993784 GU993785 GU993786 GU993787 GU993788 GU993789 GU993790

K. D. Broders, G. J. Boland

Apiognomonia errabunda Cryphonectria parasitica Cryptodiaporthe aesculi Cryptodiporthe hystrix Cryptosporella suffusa Cryptosporella betulae Discula destructiva Gnomonia gnomon Gnomonia petiolorum Gnomoniopsis fructicola Gnomoniopsis paraclavulata Melanconis alni Melanconis marginalis Ophiognomonia alni-viridis Ophiognomonia balsamiferae Ophiognomonia claviginenti-juglandacearum Ophiognomonia claviginenti-juglandacearum Ophiognomonia claviginenti-juglandacearum Ophiognomonia claviginenti-juglandacearum Ophiognomonia claviginenti-juglandacearum Ophiognomonia claviginenti-juglandacearum Ophiognomonia claviginenti-juglandacearum Ophiognomonia claviginenti-juglandacearum Ophiognomonia claviginenti-juglandacearum Ophiognomonia claviginenti-juglandacearum Ophiognomonia claviginenti-juglandacearum Ophiognomonia claviginenti-juglandacearum Ophiognomonia claviginenti-juglandacearum Ophiognomonia claviginenti-juglandacearum Ophiognomonia claviginenti-juglandacearum Ophiognomonia claviginenti-juglandacearum Ophiognomonia claviginenti-juglandacearum Ophiognomonia claviginenti-juglandacearum Ophiognomonia claviginenti-juglandacearum Ophiognomonia claviginenti-juglandacearum Ophiognomonia claviginenti-juglandacearum Ophiognomonia claviginenti-juglandacearum Ophiognomonia claviginenti-juglandacearum Ophiognomonia claviginenti-juglandacearum Ophiognomonia claviginenti-juglandacearum Ophiognomonia claviginenti-juglandacearum Ophiognomonia claviginenti-juglandacearum Ophiognomonia claviginenti-juglandacearum

Culture

EU219201 EU219128 EU219141 EU219143

EU219179 EU219183 EU219222 EU219197 EU219212

EU254873 EU254903 EU254910 EU254918 DQ323534 EU199192 EU254923 EU254936 EU254941 EU254961 EU254977 EU254997 EF512481 EF512474 EF512478

EU219223 EU219176 EU219196

EU222008 EU222005 EU221996 EU221944 EU221949 EU221946 EU222004 EU221956 EU221941 EU221942 EU221999 EU221947 EF512543 EU221923 EF512540

Reclassification of the butternut canker fungus

73

the BigDye version 3.1 ready reaction kit (Applied Biosystems) on an ABI 3730 automated sequencer at the University of Guelph Genomics Facility. Internal transcribed spacer regions 1 and 2, including the 5.8s rDNA, were amplified and sequenced using primers ITS1 and ITS4 (White et al. 1990). A region of the tef1-a gene was amplified using primers EF1-728F (Carbone & Kohn 1999) and EF1-1567R (Rehner 2001), and the resulting fragment was sequenced using primers EF1-983F (Rehner 2001) and EF1-1567R. The b-tubulin, calmodulin, and actin fragments were amplified and sequenced with primers T1 and T2 (Odonnell & Cigelnik 1997), Cal228F and Cal737R (Carbone & Kohn 1999), and Actin512F and Actin783R (Carbone & Kohn 1999), respectively.

Ophiognomonia intermedia Ophiognomonia ischnostyla Ophiognomonia leptostyla Ophiognomonia micromegala Ophiognomonia nana Ophiognomonia padicola Ophiognomonia pseudoclavulata Ophiognomonia rosae Ophiognomonia sassafras Ophiognomonia setacea Ophiognomonia vaslijevae Plagiostoma barriae Sirococcus conigenus Sirococcus piceicola Sirococcus tsugae

CBS 119194 NA CBS 844.79 CBS 121910 CBS 833.79 CBS 854.79 CBS 121236 CBS 121263 CBS 121243 CBS 121256 CBS 121253 CBS 121249 CBS 101225 CBS 119621 CBS 119627

United Kingdom USA: ME Swizterland USA: DC Finland Switzerland USA: PA USA: ME USA: PA USA: NC USA: TN USA: WA Austria Switzerland USA: OR

Betula alba Betula papyrifera Juglans regia Carya tomentosa Betula nana Prunus padus Carya tomentosa Rosa sp Sassafras albidum Plantanus occidentalis Juglans nigra Acer macrophyllum Picea abies Picea abies Cedrus deodara

EF512502 EF512495 EF512499

EF512522 EF512515 EF512519

Sequence analysis Raw sequences were manually adjusted and edited using BioEdit and were deposited in GenBank as listed in Table 1. Two datasets were prepared. The first alignment consisted of ITS, b-tublin, and tef1-a sequences from representatives of the major lineages within the Gnomoniaceae with nine species representing Ophiognomonia, three species representing the Sirococcus genus, and Melanconis and Cryphonectria species used as the outgroup taxa. This alignment was similar to that of Sogonov et al. (2008) with the exception that ITS and b-tubulin sequences were used instead of nrLSU and rpb2 gene sequences. The second alignment consisted of actin, calmodulin, ITS, b-tublin, and tef1-a of all available isolates of Sc-j with outgroup species identified from the previous dataset as closely related or of taxonomic importance and easily alignable, including Sirococcus tsugae Castl., D.F. Farr & Stanosz, S. conigenus, Sirococcus piceicola Castl., D.F. Farr & Stanosz, Discula destructiva Redlin, Apiognomo€ hn, Apiognomonia hystrix nia errabunda (Roberge ex Desm.) Ho (Tode) Sogonov and Cryptodiaporthe aesculi (Fuckel) Petr. Sequences used in each alignment were concatenated using the Combine function in SNAP Workbench (Price & Carbone 2005). The phylogenetic and molecular analyses were completed using MEGA version 4.0 (Kumar et al. 2004). Alignments were completed using ClustalW and trees were inferred with the following methods: the neighbour-joining (NJ) method (Kimura two-parameter distance calculation) and maximum parsimony (MP) using the max-mini branch-and-bound search. For the parsimony and neighbour-joining analyses, gaps were treated as missing data with missing or ambiguous sites ignored for the affected pair-wise comparison. All positions were included in the analyses and relative support for the branches was estimated with 1 000 bootstrap replications (Felsenstein 1985) for NJ and MP analyses. Analysis of polymorphic sites was done using DnaSP (Rozas et al. 2003). Phylogenetic trees were also inferred using Bayesian inference as implemented in MrBayes v. 3.1.2 (Huelsenbeck & Ronquist 2001). A Bayesian analysis using the general time reversible (GTR) model was selected for the entire unpartitioned alignment, with likelihood parameters settings (lset) number of substitution types (nst) ¼ 6, with a proportion of sites invariable and the rest drawn from the gamma distribution (rate ¼ invgamma). Four independent analyses, each starting from a random tree, were run under the same conditions for the combined gene alignment. Three hot and one cold chain Markov Chain Monte Carlo with 1 000 000 generations with

74

sampling every 100 generations was used for the analysis. The first 250 000 generations were discarded as the chains were converging (burnin period).

Results Fungal material Twenty-eight isolates of Sirococcus clavigignenti-juglandacearum from 21 locations throughout North America were evaluated in this study, including isolates from Juglans ailantifolia var. cordiformis, Juglans cinerea, and Juglans nigra (Table 1). Sequence data for all isolates were deposited in GenBank and accession numbers are provided in Table 1.

DNA analyses The final edited sequences for Sirococcus clavigignenti-juglandacearum contained 559 bp for the ITS, 816 bp for the b-tubuin, 267 bp for the actin, 474 bp for the tef1-a, and 445 bp for the calmodulin gene regions. No differences in the sequences of the five genes among the 28 isolates were observed. This provides a strong indication for a recently introduced organism with a primarily asexual reproductive lifecycle. The combined alignment 1 consisted of sequences of ITS, b-tubulin, and tef1-a genes from 22 members of the Gnomoniaceae, with 1 259 total positions of which 754 were constant, 355 were parsimony informative, and 150 were variable but not parsimony informative. MP phylogenetic analysis for the combined sequences resulted in three equally parsimonious trees (Length ¼ 1 538, CI ¼ 0.531, RI ¼ 0.612, RCI ¼ 0.326). The majority-rule consensus tree from the Bayesian analysis was included in Fig 1 with Bayesian posterior probabilities, and maximum parsimony and neighbour-joining bootstrap values included at each node. The results of the phylogenetic analysis (Fig 1) demonstrate that S. clavigignenti-juglandacearum, hereafter referred to as Ophiognomonia clavigignenti-juglandacearum (Oc-j ), forms a monophyletic group (100 % posterior probability, 99 % MP and 100 % NJ bootstrap values) within the Gnomoniaceae with other members of the genus Ophiognomonia including Ophiognomonia padicola (Lib.) M. Monod, Ophiognomonia rosae (Fuckel) Kirschst., Ophiognomonia sassafras (Ellis & Everh.) M. Monod, Ophiognomonia setaceae (Pers.) Sogonov, Ophiognomonia micromegala (Ellis & Everh.) Sogonov, Ophiognomonia balsamifera Sogonov, Ophiognomonia ischnostyla (Desm.) Sogonov and Ophiognomonia leptostyla (Fr.) Sogonov (Fig 1). There was also strong support (100 % posterior probability, 73 % MP and 96 % NJ bootstrap values) for inclusion in a larger group including members of the Ophiognomonia, Gnomonia € t., Discula destructiva, Ambarignomonia gnomon (Tode) J. Schro petiolorum (Schwein.) Sogonov 2008, Pleuroceras tenellum (Ellis & Everh.) M.E. Barr 1978, Cryptosporella suffusa (Fr.) L.C. Meijia € hn, Apiogno& Castl., Apiognomonia veneta (Sacc. & Speg.) Ho monia errabunda, Apiognomonia hystrix, Plagiostoma barriae Sogonov, and Cryptodiaporthe aesculi. The other group with 100 % posterior probability and BS support included Sirococcus conigenus, Sirococcus tsugae, Sirococcus piceicola, Gnomoniopsis paraclavulata Sogonov, and Gnomoniopsis fructicola

K. D. Broders, G. J. Boland

(G. Arnaud) Sogonov. The two species that were most similar to Oc-j in both the Bayesian and NJ analyses were O. balsamifera, a recently described species found on overwintered petioles of Populus balsamifera L. (Sogonov et al. 2008), and O. ischnostyla, found on overwintered leaves of Alnus, Betula, Carpinus, Juglans, and other hardwoods in Europe and North America (Sogonov et al. 2008). The combined alignment 2 consisted of sequences of actin, calmodulin, ITS, b-tubulin, and tef1-a genes from 28 isolates of Oc-j as well as S. tsugae, S. conigenus, S. piceicola, D. destructiva, A. errabunda, A. hystrix, C. aesculi, and Cryphonectria parasitica (Murrill) M.E. Barr as the outgroup. Of the 1 809 total sites, 1 084 were constant, 486 were parsimony informative, and 239 were variable but not parsimony informative. MP phylogenetic analysis for the combined sequences resulted in 146 most parsimonious trees. Fig 2 shows the tree generated using the NJ method for the combined alignments with MP bootstrap values above and Bayesian posterior probabilities below the branches. Results show that there was no variation among the 28 isolates of Oc-j, despite their wide geographic distribution and different host tissue (Fig 2). The results also demonstrate Oc-j is more closely related to other members of the Gnomoniaceae than to species in the genus Sirococcus.

Taxonomy Ophiognomonia clavigignenti-juglandacearum (Nair, Kostichka, & Kuntz) Broders & Boland, comb. nov. Basionym: Sirococcus clavigignenti-juglandacearum Nair, Kostichka, & Kuntz, Mycologia 71: 641e646. 1979. Habitat: While O. clavigignenti-juglandacearum was originally described as being isolated only from Juglans cinerea, it has since been isolated from both Juglans ailantifolia var. cordiformis and Juglans nigra by the authors as well as Ostry (1997) and Ostry et al. (1997). Distribution: Canada (Ontario, Quebec, New Brunswick) and U.S.A. (AK, CT, IN, MI, MN, MO, NC, NH, NY, OH, TN, VT, WI). Specimens examined: Canada, Ontario, Guelph, 14 Apr. 2009, K. D. Broders, Hilton Centre, University of Guelph Arboretum; Ontario, Cambridge, 08 Jul. 2009, K. D. Broders, RARE Conservation Authority; Ontario, York, 2007, R. Wilson; Ontario, Simco Lake, 18 Jul. 2008, R. Wilson; Ontario, Guelph Lake, 08 Aug. 2008, R. Wilson; Ontario, Conestoga Lake, 14 Aug. 2008, R. Wilson; Ontario, Bradford, 26 Aug. 2008, R. Wilson; Ontario, Brockville, 28 Aug. 2008, R. Wison; Ontario, Charleston Lake, 11 Sep. 2008, R. Wilson; Ontario, Big Rideau Lake, 16 Sep. 2008, R. Wilson. U.S.A. NY, Ulster Co., 30 Jun. 1993, M. Ostry; NY, Essex Co., 19 Aug. 1993, M. Ostry; IN, Martell, 12 Aug. 2008, M. Ostry; IN, Hoosier National Forest, 14 Aug. 2008, M. Ostry; MN, Hay Creek, 03 Apr. 2001, M. Ostry; MN, Rum River State Forest, 13 Apr. 2006, M. Ostry; WI, Mauston, 2009, M. Ostry; WI, White Water, 13 Feb. 2002, M. Ostry; WI, Arlington, 1978, V.M.G Nair (ATCC36624). Notes: In addition to the detailed description by Nair et al. (1979), O. clavigignenti-juglandacearum causes discrete dark circular lesions on the pericarp of seeds of both J. cinerea and J. nigra (Fig 3A). The fungus has also been isolated from dark red to black elongate lesions on the petioles of J. nigra

Reclassification of the butternut canker fungus

75

Fig 1 e The majority-rule consensus tree from the Bayesian analysis of three loci (ITS, b-tubulin, and tef1-a) of 25 members of Gnomoniaceae using species of Melanconis and Cryphonectria as outgroup taxa. Bayesian posterior probabilities, maximum parsimony, and neighbour-joining bootstrap values are displayed next to node in this order with (-) indicating a support value less than 70 % or the branch was resolved alternatively in the analysis.

(Fig 3B,C). Infection of terminal buds of J. ailantifolia var. cordiformis and J. cinerea was observed in late spring as healthy buds begin to swell and open, however, infected buds fail to open and begin to turn dark reddish brown and then black and infection progresses down the stem turning it a reddish brown (Fig 4). In addition to the description given by Nair et al. (1979) of the fungus in culture, the authors have observed three other frequently recovered phenotypes (Broders & Boland 2010). The four phenotypes observed were: A) light coloured mycelium, concentric rings, sporulating, and no pigmentation of the agar (L, R, Sp); B) sectored (both dark and

light mycelium), concentric rings, sporulating, and no pigmentation of the agar (Sec, R, Sp); C) dark mycelium, concentric rings, sporulating, and dark pigmentation of the agar (Dk, R, Sp,); or D) dark, slow growing with thin or diffuse mycelium, and dark pigmentation of the agar (Dk, D). Phenotype C was the culture morphology described by Nair et al. (1979).

Discussion The maximum parsimony, neighbour-joining, and Bayesian analyses (Figs 1 and 2), based on five loci (actin, calmodulin,

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K. D. Broders, G. J. Boland

Fig 2 e Neighbour-joining phylogenetic tree based on five loci (actin, calmodulin, ITS, b-tubulin, and tef1-a) for 28 isolates of Ophiognomonia clavigignenti-juglandacearum including the type specimen ATCC 36624. Maximum parsimony bootstrap values greater than 70 % are shown above the branch and Bayesian posterior probabilities greater than 95 % are shown below each branch.

b-tubulin, elongation factor1-a, and ITS), placed the butternut canker fungus into the genus Ophiognomonia with strong bootstrap and posterior probability support. The results demonstrate that, while this fungus is a member of the Gnomoniaceae, it clearly does not belong to the genus Sirococcus (Figs 1 and 2). Although the teleomorph is unknown, molecular data support the reclassification of the butternut canker fungus to Ophiognomonia clavigignenti-juglandacearum. These analyses verify previous observations that O. clavigignenti-juglandacearum is not a member of the genus Sirococcus (Konrad et al. 2007), but rather should be included with members of the genus Ophiognomonia (Green & Castlebury 2007; Konrad et al. 2007; Rossman et al. 2008; Sogonov et al. 2008). Even when Nair et al. (1979) described this fungus they had doubts based on host range experiments, as other members of the genus Sirococcus are pathogenic on coniferous hosts and no members of the genus were reported to be associated

with deciduous hosts. They concluded that ‘It is therefore considered necessary to name a new species, and the name Sirococcus clavigignenti-juglandacearum is proposed, based on the imperfect state. To date, the perfect state of the fungus has not been reported.’ The genus Ophiognomonia is based on Gnomoniella subgenus Ophiognomonia Sacc. for species having elongate, often septate ascospores. The genus remained relatively obscure, with only the two endophytic species, Ophiognomonia cryptica D. Wilson & M.E. Barr (Wilson et al. 1997) on Quercus emoryi and Ophiognomonia elasticae (Koord.) M. Monod (Paulus et al. 2006) on Ficus pleurocarpa, mentioned in recent publications. The recent review of leaf-inhabiting genera of the Gnomoniaceae by Sogonov et al. (2008) described several new species, and many species previously regarded as belonging to Gnomonia were placed in Ophiognomonia. Sogonov et al. (2008) demonstrated that host specificity is an important character in

Reclassification of the butternut canker fungus

Fig 3 e Symptoms of Ophiognomonia clavigignenti-juglandacearum infection on (A) pericarp of butternut (Juglans cinerea) seeds and (B & C) black walnut (Juglans nigra) petioles.

Fig 4 e Dieback symptoms and cross-sections of an infected terminal bud caused by Ophiognomonia clavigignenti-juglandacearum on butternut (Juglans cinerea).

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circumscription of genera and species of the Gnomoniaceae. Species of Ophiognomonia are most frequently recovered from members of the Fagales including the Betulaceae, Fagaceae, and Juglandaceae, but some species also have been reported from other plant families. This would seem ecologically appropriate for O. clavigignenti-juglandacearum, which has been recovered from J. ailantifolia var. cordiformis, J. cinerea, and J. nigra, as opposed to the ecology of other members of Sirococcus, which are pathogens of coniferous hosts. The symptoms of O. clavigignenti-juglandacearum on J. nigra are typical of other species of the Gnomoniaceae, which occur most commonly on fallen or still attached, overwintered leaves including petioles or herbaceous stems. The sequences from the five genes were identical for all 28 isolates of O. clavigignenti-juglandacearum examined, even though the isolates originated from different locations, hosts, and collection dates. This supports current hypotheses that O. clavigignenti-juglandacearum was recently introduced into North American and primarily reproduces asexually (Innes & Rainville 1996; Furnier et al. 1999; Halik & Bergdahl 2002). While the teleomorph of O. clavigignenti-juglandacearum has not been observed, this should not impede the reclassification of this fungus. Morphology has not always been reliable because of the large amount of variation in phenotypic features and the great number of related asexual forms. As independent characters, molecular sequence data have been applied to resolve many evolutionary history and taxonomic problems, which are difficult to solve using morphology alone. Oc-j is not the only species to reproduce asexually and lack a known sexual state, as is often the case for many plant-associated fungi (Rossman 1993). Another member of the Gnomoniaceae, Discula destructiva, the cause of dogwood anthracnose, is such a species for which a sexual state is unknown. Both Zhang & Blackwell (2001) and Castlebury et al. (2002) were unable to determine the sexual state of D. destructiva with the use of molecular data. However, they were able to demonstrate the relationship of D. destructiva to other members of the Gnomoniaceae and that the genus Discula is not monophyletic. Accurate scientific names should provide as much information as possible about a given organism, including its evolutionary history. Knowledge of the relationship of the asexual species to sexual species may assist in discovering the sexual state, thereby completing knowledge of the life history of a fungus. For many asexual fungi a sexual stage may not exist, but knowledge of the phylogeny allows scientists to make predictions about the biology, especially ecology and host range, of these species. While a teleomorph for the butternut canker fungus has not been observed, there is evidence (unpublished data) for recombination from linkage disequilibrium analysis and Hudson’s RM (Hudson & Kaplan 1985), which is a based on a four-gamete test to estimate the minimum number of recombination events, using SNP makers developed for Oc-j (Broders et al. 2010), suggesting limited sexual reproduction may be taking in place in nature. However, when, on which host(s), and on which host tissue(s) this is taking place are still to be determined. With the revelation that the butternut canker fungus is more closely related to other members of the Ophiognomonia rather than true

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Sirococcus, we can begin to hypothesize where, when, and on what substrates to search for the sexual structure of this fungus. Evidence would suggest that we may find the sexual stage on overwintered leaves, petioles or other plant tissue of members of the Juglandaceae or perhaps other members of the Fagales.

Acknowledgements We would like to thank Richard Wilson, Ontario Ministry of Natural Resources (OMNR) for assistance in locating infected trees in Ontario, OMNR and the Natural Sciences and Engineering Research Council (NSERC) of Canada for funding, the R. J. Hilton Centre of the University of Guelph Arboretum and the RARE Charitable Research Reserve for access to butternut trees, and Dr. Mike Ostry at the Northern Research Station, USDA, St. Paul, MN for access to isolates of S. clavigignentijuglandacearum from across the United States.

references

Altchul SF, Gish W, Miller W, Myers EW, Lipman DJ, 1990. Basic local alignment serach tool. Journal of Molecular Biology 215: 403e410. Anderson RL, LaMadeleine LA, 1978. The Distribution of Butternut Decline in the Eastern United States, Forest Survey Report. USDA Forest Service, p. 5. Broders KD, Boland GJ, 2010. Development of a molecular diagnostic assay for detection of the butternut canker pathogen Sirococcus clavigignenti-juglandacearum. Plant Disease 94: 952e958. Broders KD, SanMiguel PJ, Westerman RP, Woeste K, Boland GJ, 2010. Discovering single nucleotide polymorphisms (SNPs) in an uncharacterized fungal genome using EagleView to evaluate 454 sequencing data. Phytopathology 100: S17. Cannon PF, Minter DW, 1983. The nomenclatural history and typification of Hypoderma and Lophodermium. Taxon 32: 572e583. Carbone I, Kohn LM, 1999. A method for designing primer sets for speciation studies in filamentous ascomycetes. Mycologia 91: 553e556. Castlebury LA, Rossman AY, Jaklitsch WJ, Vasilyeva LN, 2002. A preliminary overview of the Diaporthales based on large subunit nuclear ribosomal DNA sequences. Mycologia 94: 1017e1031. Davis CN, Myren DT, Czerwinski EJ, 1992. First report of butternut canker in Ontario. Plant Disease 76: 972. Felsenstein J, 1985. Confidence limits on phylogenies: an approach using the bootstrap. Evolution 6: 227e242. Furnier GR, Stolz AM, Mustaphi RM, Ostry ME, 1999. Genetic evidence that butternut canker was recently introduced into North America. Canadian Journal of Botany 77: 783e785. Green S, Castlebury LA, 2007. Connection of Gnomonia intermedia to Discula betulina and its relationship to other taxa in Gnomoniaceae. Mycological Research 111: 62e69. Halik S, Bergdahl DR, 2002. Potential beetle vectors of Sirococcus clavigignenti-juglandacearum on butternut. Plant Disease 86: 521e527. Harrison KJ, Hurley JE, Ostry ME, 1998. First report of butternut canker caused by Sirococcus clavigignenti-

K. D. Broders, G. J. Boland

juglandacearum in New Brunswick, Canada. Plant Disease 82: 1282. Hudson RR, Kaplan NL, 1985. Statistical properties of the number of recombination events in the history of a sample of DNA sequences. Genetics 111: 147e164. Huelsenbeck JP, Ronquist F, 2001. MRBAYES 3: Bayesian phylogenetic inference under mixed models. Bioinformatics 19: 1572e1574. Innes L, Rainville A, 1996. Distribution and detection of Sirococcus clavigignenti-juglandacearum in Quebec. Phytoprotection 77: 75e78. Katovich SA, Ostry ME, 1998. Insects associated with butternut and butternut canker in Minnesota and Wisconsin. The Great Lakes Entomol 31: 97e108. Konrad H, Stauffer C, Kirsits T, Halmschlager E, 2007. Phylogeographic variation among isolates of the Sirococcus conigenus P group. Forest Pathology 37: 22e39. Kumar S, Tamura K, Nei M, 2004. MEGA3: integrated software for molecular evolutionary analysis and sequence alignment. Briefings in Bioinformatics 5: 150e163. Mejia LC, Castlebury LA, Rossman AY, Sogonov MV, White JF, 2008. Phylogenetic placement and taxonomic review of the genus Cryptosporella and its synonyms Ophiovalsa and Winterella (Gnomoniaceae, Diapothales). Mycological Research 112: 23e35. Nair VMG, Kostichka CJ, Kuntz JE, 1979. Sirococcus clavigignentijuglandacearum: an undescribed species causing canker on butternut. Mycologia 71: 641e646. Neilson C, Cherry M, Boysen B, Hopkin A, McLaughlin J, Beardmore T, 2003. COSEWIC Status Report on the Butternut Juglans cinerea in Canada in COSEWIC Assessment and Status Report on the Butternut Juglans cinerea in Canada. Committee on the Status of Endangered Wildlife in Canada, Ottawa, pp. 1e32. Odonnell K, Cigelnik E, 1997. Two divergent intragenomic rDNA ITS2 types within a monophyletic lineage of the fungus Fusarium are nonorthologous. Molecular Phylogenetics and Evolution 7: 103e116. Ostry ME, 1997. Sirococcus clavigignenti-juglandacearum on heartnut (Juglans ailantifolia var. cordiformis). Plant Disease 81: 1461. Ostry ME, Katovich S, Anderson RL, 1997. First report of Sirococcus clavigignenti-juglandacearum on black walnut. Plant Disease 81: 830. Ostry ME, Moore M, 2007. Natural and experimental host range of Sirococcus clavigignenti-juglandacearum. Plant Disease 91: 581e584. Paulus B, Gadek P, Hyde K, 2006. Successional patterns of microfungi in fallen leaves of Ficus pleurocarpa (Moraceae) in an Australian tropical rain forest. Biotropica 38: 42e51. Price EW, Carbone I, 2005. SNAP: workbench management tool for evolutionary population genetic analysis. Bioinformatics 21: 402e404. Rehner S, 2001. Primers for Elongation Factor 1-a (EF1-a). Available from: http://ocid.nacse.org/research/deephyphae/EF1primer.pdf Renlund DW, 1971. Forest Pest Conditions in Wisconsin, Ann. Rep. Wisconsin Department of Natural Resources, Madison WI, 53 p. Rossman AY, 1993. Holomorphic hypocrealean fungi: Nectria sensu stricto and teleomorphs of Fusarium. In: Reynolds DR, Taylor JW (eds), The Fungal Holomorph: Mitotic, Meiotic, and Leomorphic Speciation in Fungal Systematics. CAB International, Wallingford, pp. 149e166. Rossman AY, Castlebury LA, Farr DF, Stanosz GR, 2008. Sirococcus conigenus, Sirococcus peceicola sp. nov. and Sirococcus tsugae sp. nov. on conifers: anamorphic fungi in the Gnomoniaceae, Diaporthales. Forest Pathology 38: 47e60.

Reclassification of the butternut canker fungus

Rossman AY, Palm-Hernandez ME, 2009. Systematics of plant pathogenic fungi: why it matters. Plant Disease 92: 1376e1386. Rozas JC, S.-D.J., Messenguer X, Rozas R, 2003. DnaSP, DNA polymorphism analyses by the coalescent and other methods. Bioinformatics 19: 2496e2497. Sogonov MV, Castlebury LA, Y.R.A., Meija LC, White JF, 2008. Leafinhabiting genera of the Gnomoniaceae, Diapothathales. Studies in Mycology 62: 1e79. Sutton BC, 1980. The Coelomycetes. Fungi Imperfecti with Pycnidia, Acervuli, and Stromata. Commonweatlh Mycological Institute, Kew, Surrey, UK. Tisserat N, Kuntz JE, 1983. Dispersal gradients of conidia of the butternut canker fungus in a forest during rain. Canadian Journal of Forest Research 13: 1139e1144.

79

Tisserat N, Kuntz JE, 1984. Butternut canker: development on individual trees and increase within a plantation. Plant Disease 68: 613e616. White TJ, Bruns T, Lee S, Taylor J, 1990. Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In: Innis MA, Gelfand DH, Sninsky JJ, White TJ (eds), PCR Protocols: A Guide to Methods and Applications. Academic Press, New York, pp. 315e322. Wilson D, Barr ME, Faeth SH, 1997. Ecology and description of a new species of Ophiognomonia endophytic in the leaves of Quercus emoryi. Mycologia 89: 537e546. Zhang N, Blackwell M, 2001. Molecular phylogeny of dogwood anthracnose fungus (Discula destructiva) and the Diaporthales. Mycologia 93: 355e365.