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Chapter 3 Molecular and phenotypic characterisation of three phylogenetic species discovered within the Neofusicoccum parvum / N. ribis complex
ABSTRACT
Neofusicoccum parvum and N. ribis are closely related species whose identities have often been confused. These fungal plant pathogens were recently identified as the most abundant species of Botryosphaeriaceae (Ascomycetes) isolated from native Syzygium cordatum trees in South Africa. In another study using multiple gene genealogies from five nuclear loci, three undescribed cryptic phylogenetic species, as well as N. parvum, were identified among thirty of these isolates. The aim of this study was to clarify the identity of the remaining isolates in the N. parvum / N. ribis complex from S. cordatum in South Africa, to describe newly identified cryptic species and to test their pathogenicity. Based on the RNA polymerase II subunit (RPB2) sequence comparisons the isolates were identified as N. parvum or one of three previously recognized phylogenetic species that are described here as N. cordaticola, N. kwambonambiense and N. umdonicola. These species cannot be separated a priori based on morphological characteristics, although a posteriori analysis of variance showed that the differences in conidial length and width between the species were statistically significant. The isolates of the newly described species as well as N. parvum and N. ribis were tested for pathogenicity on S. cordatum under greenhouse conditions. Isolates representing the three new species were significantly more aggressive than N. parvum and N. ribis, with N. kwambonambiense being the most aggressive. This study resolved long-standing questions of identity of species within N. parvum / N. ribis complex and lays a foundation for further studies on this group of pathogens.
INTRODUCTION
The phylogenetic species concept (PSC) (Taylor et al 2000) and genealogical concordance phylogenetic species recognition (GCPSR) have been increasingly applied in studies of species boundaries in both human and plant pathogenic fungi (e.g. Koufopanou et al 1997, Geiser et al 1998, O’Donnell et al 2000a, b, Steenkamp et al 2002, O’Donnell et al 2004, Pringle et al 2005). In these studies, using GCPSR based on concordance of multiple gene sequence genealogies, numerous cryptic species and species complexes were revealed in fungal taxa previously identified as one morphospecies. GCPSR was also used with good results in the detection of cryptic species within Botryosphaeriaceae, e.g. Diplodia scrobiculata as a sister species of D. pinea (de Wet et al 2003) and Neofusicoccum eucalypticola and N. australe as sister species of N. eucalyptorum and N. luteum respectively (Slippers et al 2004b, c). The cryptic species recognized in these studies could not have been acknowledged based on morphology or single-locus data alone, methods commonly used for identification of Botryosphaeriaceae (e.g. Jacobs and Rehner 1998, Denman et al 2000, Smith et al 2001, Zhou and Stanosz 2001, Pavlic et al 2004).
Neofusicoccum parvum and N. ribis are closely related cryptic species within the recently described genus Neofusicoccum (Botryosphaeriaceae, Ascomycetes) (Slippers et al 2004a, Crous et al 2006). Although known to develop teleomorph (sexual) structures, these fungi are commonly encountered in their anamorph (asexual) stage (Pennycook and Samuels 1985, Slippers et al 2004a, Pavlic et al 2007). The cosmopolitan distribution, sympatric occurrence on native and non-native hosts, as well as plasticity and overlap in the morphological characteristics of both their teleomorphs and anamorphs, make these species difficult to distinguish based upon morphological, ecological and geographical criteria. Consequently, these plant pathogens have often been mistaken for each other. These species could also not be separated with confidence based on ITS sequence data alone, the method most commonly used in molecular identification and phylogenetic analyses of the Botryosphaeriaceae (Smith et al 2001, Zhou and Stanosz 2001, Slippers et al 2005, Pavlic et al 2007).
Nucleotide sequence data from multiple genes were used to distinguish the identity of the type specimens of N. parvum and N. ribis (Slippers et al 2004a). However, when more isolates were included in subsequent analyses, many clustered intermediate to the type, but did not clearly cluster with either of these species (Ahumada 2002, Slippers 2003, Slippers et al 2005, Rodas et al 2009). These isolates have been referred to as the N. parvum / N. ribis complex. Isolates that belong to the N. parvum / N. ribis complex could be separated into two groups using a PCR-RFLP fingerprinting technique. They were then referred to as N. parvum sensu lato and N. ribis sensu lato (Slippers 2003). It was not clear in those studies, however, whether these groups comprise more than one cryptic species or represent inter-specific variation.
Neofusicoccum parvum sensu lato and N. ribis sensu lato were recently identified as the most abundant species of Botryosphaeriaceae isolated from native Syzygium cordatum (Myrtaceae) in South Africa (Pavlic et al 2007). In a subsequent study using multiple gene genealogies of five nuclear loci, three undescribed cryptic phylogenetic species, as well as N. parvum, were identified among these isolates (Pavlic et al 2009). None of the isolates were identified as N. ribis. In this study, we characterise a larger collection of these isolates using genotypic data and combine this with phenotypic characteristics such as conidial morphology and pathogenicity to describe the taxa. Consequently, three new phylogenetically recognised cryptic species within the Neofusicoccum parvum / N. ribis species complex are described here as N. cordaticola sp. nov. , N. umdonicola sp. nov. and N. kwambonambiense sp. nov.
MATERIALS AND METHODS
Isolates
The 103 isolates used in this study were collected during the survey of the Botryosphaeriaceae on native S. cordatum in South Africa from 2001 to 2003 (TABLE I). The collection spanned the north to south natural distribution of S. cordatum in South Africa, from Tzaneen in the Northern Province to Gonubie in the Eastern Cape Province. Isolations were made from dying twigs and asymptomatic, visually healthy twigs and leaves, as described in Pavlic et al (2007). Isolations were also made from visually healthy fruits. Fruits were washed in running tap water and surface disinfected by spraying them with 70 % ethanol and left dried on filter paper. The disinfected fruits were halved and pieces from the fruit pulp (2 mm2) were placed on 2 % malt extract agar (MEA) and incubated and maintained as described in Pavlic et al (2007). All cultures used in this study have been maintained in the culture collection (CMW) of the Forestry and Agricultural Biotechnology Institute (FABI), University of Pretoria, South Africa and representative isolates have been deposited in the collection of the Centraalbureau voor Schimmelcultures (CBS), Utrecht, The Netherlands.
DNA sequence comparisons
Thirty isolates from S. cordatum were selected and identified in a previous study (Pavlic et al 2009) as N. parvum or one of the undescribed phylogenetic species termed as Neofusicoccum sp. R1, R2 and R3. This distinction was based on multiple gene genealogies of DNA sequence data for five nuclear loci, including the internal transcribed spacer rDNA (ITS1, 5.8S, and ITS2), partial translation elongation factor 1α (EF-1α), β-tubulin-2 (βt-2a/b), a portion of the RNA polymerase II subunit (RPB2) and locus BotF15 (an unknown locus containing a simple sequence repeat), and the results were compared with a single gene sequence data. The RPB2 region was found to contain the most informative characters considering fixed single nucleotide polymorphisms (SNPs) in each species. Following the same protocol as Pavlic et al (2009), a portion of the RNA polymerase II subunit (RPB2) was sequenced for the remaining 73 isolates. The type specimens and two specimens related to the types of N. parvum and N. ribis were included for comparison. The nucleotide sequences from one strand were examined with SEQUENCE NAVIGATOR version 1.0.1. software (Perkin-Elmer Applied BioSystems, Inc., Foster City, California) and alignments were prepared online using MAFFT version 5.667 (http://timpani.genome.ad.jp/~mafft/server/) (Katoh et al 2002) to compare it to the data from Pavlic et al (2009).
Phylogenetic analyses
A maximum-parsimony (MP) tree was constructed in PAUP version 4.0b10 (Swofford 2000) using the heuristic search function, with 1000 random addition replicates and tree bisection and reconstruction (TBR) selected as branch swapping algorithm. Gaps were treated as fifth characters and all characters were unordered and of equal weight. Branches of zero length were collapsed and all multiple equally parsimonious trees were saved. To estimate branch support, maximum parsimony bootstrap values were determined using 1000 bootstrap replicates (Felsenstein 1985).
Bayesian analyses were performed using MrBayes v. 3.0b4 (Ronquist and Huelsenbeck 2003) and the best-fitting evolutionary model was estimated using MrModeltest v. 2.2 software (Nylander 2004). The Markov Chain Monte Carlo (MCMC) chains were initialised from a random tree and were run for two million generations and trees were saved every hundred generations, counting twenty thousand trees. Burn-in was set at one thousand generations, leaving just over thirty eight thousand (38002) trees from which the consensus tree was calculated. To determine the confidence of the tree topologies, values of Bayesian posterior probabilities (BPPs) (Rannala and Yang 1996) were estimated using MrBayes (Ronquist and Huelsenbeck 2003).
Morphological characteristics
In an earlier study, the 103 isolates (TABLE I) were induced to sporulate in culture as described in Pavlic et al (2007). Conidia were mounted in lactophenol on microscope slides and inspected by light microscopy. Ten measurements of conidial lengths and widths were taken for each isolate and the ranges and averages, as well as length and width ratio were calculated. Measurements were made and digital photographs taken with a HRc Axiocam digital camera and accompanying Axiovision 3.1 software (Carl Zeiss Ltd., Munich, Germany). SAS® version 8.2 undmc vm/cms statistical software was used to analyse variability in conidial lengths and widths between the isolates. Single conidial cultures grown on 2 % malt extract agar (MEA) at 25 ºC under continuous near fluorescent light were used to characterise culture morphology as described previously (Pavlic et al 2007).
Acknowledgements
Preface
Chapter 1 Botryosphaeriaceae occurring on native Syzygium cordatum in South Africa and their
potential threat to Eucalyptus
Chapter 2 Multiple gene genealogies and phenotypic data reveal cryptic species of the Botryosphaeriaceae: A case study on the Neofusicoccum parvum / N. ribis complex
Chapter 3 Molecular and phenotypic characterisation of three phylogenetic species discovered
within the Neofusicoccum parvum / N. ribis complex
Chapter 4 Cryptic diversity and distribution of species in the Neofusicoccum parvum / N. ribis
complex as revealed by microsatellite markers
Chapter 5 Seven new species of the Botryosphaeriaceae from baobab and other native trees in
Western Australia
Chapter 6 Molecular phylogenetics in the recognition of fungal species, with a particular focus on the Botryosphaeriaceae
Summary
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