Phylogeny, detection, and mating behaviour of spp. occurring on

Mahdi Arzanlou Promotoren: Prof. dr. P.W. Crous Hoogleraar in de Evolutionaire Fytopathologie Wageningen Universiteit

Prof. dr. ir. P.J.G.M. de Wit Hoogleraar in de Fytopathologie Wageningen Universiteit

Co-promotoren: Dr. L.-H. Zwiers Onderzoeker, CBS Fungal Biodiversity Centre

Dr. ir. G.H.J. Kema Senior onderzoeker, Plant Research International

Promotiecommissie Prof. dr. R.F. Hoekstra (Wageningen Universiteit) Prof. dr. H.A.B. Wösten (Utrecht Universiteit) Dr. F.T. Bakker (Wageningen Universiteit) Dr. ir. A.J. Termorshuizen (Blgg, Wageningen)

Dit onderzoek is uitgevoerd binnen de onderzoekschool Experimental Plant Sciences Phylogeny, detection, and mating behaviour of Mycosphaerella spp. occurring on banana

Mahdi Arzanlou

Proefschrift ter verkrijging van de graad van doctor op gezag van de rector magnificus van Wageningen Universiteit Prof. dr. M.J. Kropff in het openbaar te verdedigen op woensdag 28 mei 2008 des voormiddags te 11:00 in de Aula Mahdi Arzanlou (2008)

Phylogeny, detection, and mating behaviour of Mycosphaerella spp. occurring on banana

PhD thesis Wageningen University, The Netherlands With summaries in English and Dutch

ISBN 978-90-8504-800-8 To my Father, Mother and my brothers, Mohammad and Mohsen

“In generosity and helping others be like a river In compassion and grace be like the sun In concealing other’s faults be like a night In anger and fury be like the dead In modesty and humility be like the earth In tolerance be like a sea Either exist as you are or be as you look” Mevlana Jalaluddin Rumi (1207- 1273)

CONTENTS

Chapter 1 General introduction 9

Chapter 2 Multiple gene genealogies and phenotypic characters differentiate several novel of Mycosphaerella and related anamorphs on banana 23

Chapter 3 Molecular diagnostics in the Sigatoka disease complex of banana 55

Chapter 4 Phylogenetic and morphotaxonomic revision of Ramichloridium and allied genera 71

Chapter 5 Evolution of heterothallism in three major Mycosphaerella species associated with the Sigatoka disease complex of banana 129

Chapter 6 General discussion 151

Appendix Summary in English 162 Samenvatting (Summary in Dutch) 163 Acknowledgements 164 About the Author 167 List of Publications 168 Education Certificate of the EPS Graduate School 170

CHAPTER 1

General Introduction

9 Chapter 1

Speciation and species recognition in fungi

Evolution is the consequence of mutation, selection, and intraspecies or interspecies gene flow between populations. Speciation can be considered the ultimate outcome of evolutionary forces, and is defined as the splitting of an existing species into two or several new taxa, or even replacing the old species by a new one. New species emerge from ancestral species when genetic differences have accumulated among subpopulations at a level that prevent them from reproducing successfully (Zhan et al. 2002, Kohn 2005). The two main mechanisms of speciation are classified as allopatric or sympatric. In allopatric speciation the geographical isolation of populations is the main driving force for the emergence of new species. This is the main mechanism for speciation in macro-biota. Sympatric speciation involves accumulation of genetic polymorphisms within a population of a given species and is the most common mechanism among micro-biota. Evolutionary changes, which may ultimately lead to speciation among eukaryotic micro- organisms like fungi occur either by routine or by episodic selection. Routine selection is defined as the sum of selection factors, which favours the maintenance of a stable population structure over time (Brasier 1995, 2000). Populations with (i) sexual reproduction, (ii) highly polymorphic vegetative compatibility loci and (iii) many variable structural characters are considered to be subject to routine selection (Brasier 1995, 2000, McDonald et al. 2002). Episodic selection is defined as any sudden environmental disturbance that is likely to lead to a significant alteration in the population of a species; it presumably acts as a cause of sudden evolutionary developments in fungi (Brasier 1995). Disturbances, such as changes in availability of resources, exposure to a new host, arrival of a new competitor, and geographical transposition are considered as likely causes of episodic selection (Brasier 1995, 2000, Newcombe et al. 2000, 2001). There are two major routes along which episodic selection might occur among fungi. The first involves selection based on strong differences in fitness, the second involves build-up of increased variation based by interspecific hybridisation or horizontal gene transfer. Typical characteristics such as small sizes, simple structures, diverse life styles, short generation times, frequent occurrence of haploidy and asexual reproduction, anastomosis, and inter-species mycelial interactions, make fungi particularly suitable to study their microevolution (Brasier 1995). This holds also for species within the Mycosphaerella. Moreover, multiple species of the genus Mycosphaerella commonly co-occur on a single host (Crous et al. 2004, Chapter 2, this thesis). The goal of biological systematics is to recognise and describe natural groups of organisms at species and higher levels. Species recognition and determination of boundaries in fungal are crucial in order to group as many as 1.5 million fungal species (Hawksworth 1991, 2001), of which we assume that less than 10 percent have been described. Currently, there are several theoretical and operational species concepts (Mayden 1997). While theoretical species concepts are not helpful in species recognition or diagnosis, operational species concepts have both diagnostic and recognition values (Taylor et al. 2000). The main theoretical species concept is the Evolutionary Species Concept (ESC) and is defined as “…a single lineage of ancestor-descendent populations which maintains its identity from other such lineages and which has its own evolutionary fate” (Wiley 1978, Taylor et al. 2000). ESC by itself represents not a recognition criterion, and as such cannot be used for species recognition. Morphological Species Concept (MSC), Biological Species Concept (BSC) and Phylogenetic Species Concept (PSC) represent the more commonly used operational species concepts. All three are compatible with the ESC, as each one tries to define evolutionary species (Mayden 1997). The terms ‘species concept’ and ‘species recognition’ have often been used as equivalent to the theoretical

10 General introduction and outline of thesis and operational species concepts, respectively (Taylor et al. 2000). Defining morphologically distinct units is the basis for MSR (Seifert 1993, Taylor et al. 2000). The foundation for BSR is interbreeding populations, which are reproductively isolated from other groups, regardless of the absence of morphological differences (Taylor et al. 2000). PSR uses DNA sequence data to build a ‘relationship tree’ of organisms. With fungi, PSR recognises species as “… the smallest aggregation of populations with a common lineage that shares unique, diagnosable phenotypic characters” (Harrington & Rizzo 1999). Evolution is an ongoing process and it is impossible to recognise the moment that individuals in an ancestral species have splitted into progeny, using the methods of species recognition, because time is needed for changes in morphology, mating behaviour and gene sequences to occur. PSR performs more consistent with ESC than MSR and BSR, because development of a new evolutionary species out of an ancestor requires changes in gene sequences to occur before the resulting morphological traits (Taylor et al. 2000). However, it is unclear how to draw a limit for species boundaries when a gene is polymorphic within the species, or fixed for alternative alleles in more than one species. This can be avoided by applying concordance of more than one gene genealogy (Taylor et al. 2000). To avoid subjectivity, the Genealogical Concordance Phylogenetic Species Recognition (GCPSR) model was proposed, which defines a species as “a basal, exclusive group of organisms all of whose genes coalesce more recently with each other than with those of any organism outside the group, and that contains no exclusive group within it” (Baum & Donoghue, 1995, Taylor et al. 2000). When conflict occurs among lineages, the transition from concordance to conflict determines the limits of species (Taylor et al. 2000). Sequence data from various protein-coding and non-coding genes have been applied to assess levels of phylogenetic relationship among different groups of fungi. Sequence analysis of 28S nrDNA and 18S nrDNA is routinely used to resolve higher- order phylogenetic relationships within the fungal kingdom. Internal transcribed spacer (ITS) areas of the rDNA operon and housekeeping genes like the actin, translation elongation factor 1-α, β-tubulin and histone H3 genes are routinely applied to resolve phylogenetic relationships among species (Crous et al. 2001, 2004, 2006, Banke et al. 2004, Verkley & Starink-Willemse 2004, Feau et al. 2006).

Mycosphaerella, a genus of successful plant pathogens

The genus Mycosphaerella

The genus Mycosphaerella belongs to the (, Dothideomyce- tidae) (Schoch et al. 2006) and is one of the largest genera of ascomycetes, comprising several thousands of species (Crous 1998, Crous et al. 2001, Aptroot 2006). Species in this genus have a wide range of lifestyles, ranging from saprobes, plant pathogens to fungal hyperparasites (de Hoog et al. 1991, Goodwin et al. 2001, Jackson et al. 2004). Like other ascomycetes, Mycosphaerella species are haploid for the major part of their lifecycle, with a short dikaryotic and diploid phase during sexual reproduction. The dikaryotic phase followed by a diploid phase is restricted to the ascogenous hyphae, which develops into bitunicate asci containing two-celled ascospores within pseudothecial ascomata. Besides sexual reproduction, some Mycosphaerella species also produce haploid conidia in an asexual reproductive cycle. Conidia are produced in closed fruiting bodies or on free conidiophores. The taxonomy of Mycosphaerella is based on morphological characters of both anamorphs and teleomorphs (Crous 1998, Stewart et al. 1999, Crous et al. 2000). Originally, Barr (1972)

11 Chapter 1 and subsequently Crous et al. (2000) recognised six sections in the genus. Currently, the genus contains close to 3000 species (Aptroot 2006) and at least 7000 additional anamorph species (Crous et al. 2000, 2001, Crous & Braun 2003, Crous et al. 2007). In contrast to the earlier conclusion of Mycosphaerella being monophyletic based on ITS sequence data (Crous et al. 2001, Goodwin et al. 2001), recent studies employing 28S nrDNA sequence data revealed the genus to be polyphyletic, involving at least two families, namely Mycosphaerellaceae and (Schoch et al. 2006, Crous et al. 2007). Whereas the little-differentiated sexual structures are of restricted taxonomic value in distinguishing species (Crous et al. 2001, Crous & Mourichon 2002, Aptroot 2006), the morphological characters of the conidial apparatus, pigmentation, and nature of the scars and hila are successfully used to delineate species (Stewart et al. 1999, Crous et al. 2001). However, these characters do not have any true evolutionary meaning for the currently accepted anamorph-generic concepts (Stewart et al. 1999, Crous et al. 2001, Crous & Braun 2003, Crous et al. 2007). Integration of DNA phylogeny data with morphological traits facilitates the identification of species. ITS sequences and housekeeping genes like the actin, translation elongation factor 1-α, β-tubulin and histone H3 genes have frequently been applied in studying phylogenetic relationships among Mycosphaerella species and their anamorphs (Crous et al. 2001, 2004, 2006, Banke et al. 2004, Verkley & Starink- Willemse 2004, Feau et al. 2006).

Plant pathogenic Mycosphaerella species

Fungi in this genus are among the most common and destructive plant pathogens, causing considerable economic losses on a wide range of host plants worldwide, including banana, cereals, sugar beet, strawberry, soybean, , eucalypts, acacia and many others (Farr et al. 1995, Crous & Braun 2003). The most common plant pathogenic anamorphs in this complex are species of , , , , and . Plant pathogenic Mycosphaerella species are mainly foliicolous, although some are associated with stem cankers (Cortinas et al. 2006). The main damage by Mycosphaerella species is caused by defoliation or the reduction of the photosynthetic capacity of the crop due to necrosis caused by the fungal infection. Some species, such as M. citri, affect both and fruits. Mycosphaerella fijiensis attacks banana leaves, and reduces the photosynthetic capacity of the crop. However, it is also capable of inducing physiological changes, resulting in premature ripening of the fruits (reviewed in Jones 2000, Marin et al. 2003). The majority of the plant pathogenic species in this genus are host-specific (Goodwin et al. 2001, Crous & Groenewald 2005, Groenewald et al. 2006a, Stukenbrock et al. 2007), such as M. fijiensis, M. musicola and M. eumusae on banana (Jones 2003, Arzanlou et al. 2007a) and M. graminicola on wheat (Banke et al. 2004, Stukenbrock et al. 2007). In contrast, species such as M. lateralis have been reported to occur on multiple hosts (Crous et al. 2004, Jackson et al. 2004, Chapter 2, this thesis), while M. citri, a major pathogen of Citrus, has also been isolated from acacia, banana and Eucalyptus (Crous et al. 2004). Mycosphaerella disease (MLD) of Eucalyptus, caused by more than 80 species of Mycosphaerella and its sister genus , represents a serious threat to the cultivation of Eucalyptus plantations for the paper and pulp industry (Park et al. 2000, Crous et al. 2004, 2005, 2006, 2007). Among the Mycosphaerella species attacking Eucalyptus, some species such as Teratosphaeria cryptica (syn. M. cryptica) have a broad host range and cause disease on 38 species across the Eucalyptus subgenera Monocalyptus and Symphyomyrtus, while T. nubilosa (syn. M. nubilosa) shows a more narrow host range, infecting only six Eucalyptus species within the subgenus Symphyomyrtus (Park et al. 2000, Maxwell et al. 2005).

12 General introduction and outline of thesis

The Sigatoka disease complex of banana

Three Mycosphaerella species, namely M. musicola, M. fijiensis and M. eumusae are the primary agents of the Sigatoka disease complex of banana. The disease is a serious threat of banana production worldwide (reviewed in Jones 2000, 2003, Arzanlou et al. 2007a). These three species emerged on during the last century and are the main constraint to commercial banana production worldwide. The chronology of the disease record around the world suggests that South-East Asia is the centre of origin for all three species, as it is for the host genus Musa (Jones 2003, Rivas et al. 2004). Yellow Sigatoka was first described on banana in Java in 1902 and during the 1940s it reached the limits of its present distribution over the whole banana production area in Asia, Africa and South-America. In the early 1960s, M. fijiensis appeared on the Fiji islands and spread rapidly across all continents, since then replacing M. musicola in many banana-producing areas as the main disease agent, and continuing to occupy new ecological niches. Recently, it was reported from Trinidad (Fortune et al. 2005), and in a presumed pathogen-free banana-producing area of Brazil (M. Souza, pers. comm.). The presence of M. fijiensis in the Caribbean region has also been confirmed recently (Carlier & Arzanlou, unpubl. data). The third species, M. eumusae, was recognised as a new constituent of the Sigatoka complex of banana in the mid-1990s (Carlier et al. 2000, Crous & Mourichon 2002). Presently, M. eumusae is known from South-East Asia and parts of Africa, where it co-exists with the other two species. The exact distribution and pathological relevance of M. eumusae is yet unclear. Besides the three primary agents of Sigatoka leaf spot disease, numerous additional species of Mycosphaerella (or their anamorphs) have been described to occur on Musa, but their pathological relevance remains unclear (Chapter 2, this thesis). Some species have been described from diverse hosts, and appear to have hosts other than Musa, e.g. (Stewart et al. 1999, Chapter 2, this thesis). Mycosphaerella speckle of banana is caused by M. musae, whose distribution and economic relevance is also unclear. Ramichloridium musae, the causal agent of tropical speckle disease on banana, also has phylogenetic affinity with Mycosphaerella (Arzanlou et al. 2007b, Chapter 4, this thesis). The identity and distribution of the different Mycosphaerella species associated with the Sigatoka disease complex of banana is not yet fully understood. This is partly due to lack of knowledge of the species involved and the lack of useful morphological characters to distinguish different species (Arzanlou et al. 2007a, Chapter 2, this thesis). Disease control of the Sigatoka complex is mainly achieved through the application of chemical compounds (Romero & Sutton 1997, Ploetz 2000, Marin et al. 2003). Different generations of fungicides such as dithiocarbomates, benzimidazoles, azoles, and more recently strobilurins, are being used to control the Sigatoka disease in banana plantations (Romero & Sutton 1997, Marin et al. 2003). Fungicide applications are of great concern due to environmental and ecological consequences, and they greatly increase the production costs (up to 45 % in commercial plantations). The annual cost of disease control for black leaf streak on banana amounts US$ 1000 per hectare in large plantations (Arias et al. 2003), but is much higher in small plantations where aerial fungicide application is not feasible (Mobambo et al. 1993). is more serious than yellow Sigatoka, because M. fijiensis has a wider Musa host range, affecting many cultivars which show resistance to M. musicola, and attacks younger leaves, due to abundant sporulation. This means that effective control requires more frequent fungicide applications, which can reach as many as 40 applications per year (Ploetz 2000). This imposes a high selection pressure on M. fijiensis which has resulted in regular appearance of fungicide-resistant populations of this pathogen (Romero & Sutton 1997, Marin et al. 2003). A thorough understanding of the identity, genetic variation and epidemiology of the causal

13 Chapter 1 agents are required for a successful plant disease management programme. The lack of useful morphological characters for the identification of Mycosphaerella species challenges plant pathologists, and requires experienced specialists (Stewart et al. 1999). Identification of these fungi mainly relies on minute morphological differences of the anamorph morphology. These morphological features may not be always visible, as cultures sometimes become sterile soon after sub-culturing, and tend to grow very slowly on synthetic media. In addition, a large number of Mycosphaerella species known from other host plants, as well as banana, have been shown to co-occur on a single host plant, and sometimes even in a single lesion. This makes it difficult to identify the primary pathogen responsible for the disease. In recent years, PCR-based techniques have been developed as robust tools for diagnosis and detection of plant pathogenic fungi, and have contributed greatly to plant disease management (Waalwijk et al. 2004, Lievens et al. 2005). Detection tools based upon specific PCR amplification of the ITS region has already been developed to distinguish M. musicola from M. fijiensis (Johanson & Jeger 1993, Johanson et al. 1994). Since then, several other Mycosphaerella species have been reported from banana, which requires the development of new detection tools (Arzanlou et al. 2007a, Chapter 2, this thesis). Large-scale application of diagnostic PCR in suffers from serious practical drawbacks such as unpractical post-amplification procedures and the amplification efficiency which is not consistent during all PCR cycles (Winton et al. 2002, Valsesia et al. 2005). Real-time PCR alleviates some of these difficulties as it combines thermal cycling with real-time fluorescent detection of amplification. Unlike the end-point PCR, accumulation of amplicons is monitored continuously, and can therefore be used for quantification of minute quantities of DNA (pg/mL) from plant pathogens. Nowadays, real-time PCR is applied for the quantitative detection of plant pathogens, biological control agents, in plant-microbe interaction experiments (Waalwijk et al. 2004, Valsesia et al. 2005), and in population monitoring studies (Rohel et al. 2002, Hietala et al. 2003).

Sex in Ascomycetous fungi

Sexual reproduction

Ascomycetous fungi, with both a sexual and asexual reproduction cycle, are haploid for the major part of their lifecycle. During a short phase of the sexual reproduction phase they are dikaryotic and diploid (Fig. 1). Asexual reproduction generates genetically identical clones and can occur through fragmentation of hyphal cells or production of conidia in complex asexual fruiting bodies like pycnidia and acervuli. Sexual reproduction in fungi involves meiosis, which is preceded by the fusion of two cells (plasmogamy), followed by fusion of the two parental nuclei (karyogamy). Sexual reproduction together with mutation, recombination and natural selection are major forces that drive evolution. Sexual reproduction provides biodiversity in populations and might benefit the organism by purging the genome from deleterious mutations (Heitman 2006, Zhan et al. 2007). In the absence of selection pressure, asexual reproduction dominates populations. Changes in the availability of food resources, environmental conditions and other selection pressures favour a shift towards the sexual reproduction cycle (Heitman 2006, Zhan et al. 2007). In plant pathogenic fungi, sexual reproduction plays a major role in plant disease epidemiology. It contributes to the spread of the pathogen by producing airborne ascospores acting as new inoculum, and it generates variation at the population level. Detailed studies on sexual reproduction in fungi may provide better insights into genetic regulation and evolution of

14

General introduction and outline of thesis

Nuclear Fusion Cell Fusion 2N Meiosis Gametes N+N N Ascus or Meiosporangium

Ascospore Hypha or Yeast Conidia or or Meiospore Mitospores

Germination Germination

N Sexual Asexual

Fig. 1. Schematic diagram of the lifecycle and ploidy transitions within ascomycetes (adapted from the Tree of Life project, http://www.tolweb.org/Ascomycota). The ploidy is indicated with “N”.

closely related species (Turgeon 1998, MacDonald et al. 2002, Zhan et al. 2007, Chapter 5, this thesis). In higher eukaryotes, sexual dimorphism has evolved as a major genetic barrier to prevent self fertilisation. Sexual dimorphism is mainly determined by sex chromosomes. Genetic recombination between the sex chromosomes almost never occurs, ensuring exchange of genetic material between two individuals for organismal chromosomes. Sexual dimorphism does not exist in fungi, and in most of the cases, like in Neurospora crassa, individuals are hermaphrodites, i.e., they produce both male and female reproductive structures. In fungi sexual exchange of genetic material relies on the existence of simple cell recognition mechanisms that stimulate out- crossing. The term ‘mating type’ is used to define sexually compatible individuals. Blakeslee, who coined the terms homothallism and heterothallism, was the first to notice the existence of mating types in sexually reproducing fungi (Blakeslee 1904). Heterothallism (self-sterility), the most common reproduction strategy among fungi, only occurs between two fungal strains with a compatible mating system. However, even in the presence of both mating types, other genetic barriers may prevent real mating (Debuchy & Turgeon 2006). Homothallism (self-fertility) represents the situation where a single isolate is capable of completing a successful sexual cycle. However, also pseudohomothallism or secondary homothallism, has been described for some ascomycetes where self-fertile ascospores can be derived from a programmed meiotic spindle alignment, nuclear movement, and ascospore delimitation, resulting in heterokaryotic ascospores, carrying nuclei of both mating types. This phenomenon has been described for Neurospora tetrasperma, Podospora anserina and Gelasinospora tetrasperma (Merino et al. 1996) and several other species.

15 Chapter 1

Genetic regulation of fungal sex

In fungi sexual development is controlled by mating type loci. Mating type loci contain a number of genes which occupy a continuous region of the chromosome, but do not span an entire chromosome (Debuchy & Turgeon 2006). In ascomycetes, sexual development is controlled by a single mating type locus (MAT). This mating type locus is structurally unusual, because it contains one of two forms of dissimilar sequences occupying the same chromosomal position. The two non-allelic versions of the mating type locus in fungal species with heterothallic mating strategy were labelled ‘idiomorph’ (Metzenberg & Glass 1990). By convention, the mating type idiomorphs of complementary isolates are termed MAT1-1 and MAT1-2 (Turgeon & Yoder 2000). The number of genes in each idiomorph varies among different groups of fungi. However, they all contain homeodomain-encoding genes, either alpha box or high mobility group (HMG) domain transcription factors (Turgeon 1998, Debuchy & Turgeon 2006). Over the last decade, mating type genes have been cloned and characterised for several groups of ascomycetes by a variety of cloning strategies, including functional complementation, subtractive hybridisation, genomic library screening with heterologous probes, and PCR-based approaches (Arie et al. 1997, Turgeon 1998, Barve et al. 2003, Debuchy & Turgeon 2006). In recent years, the rapid expansion of fungal genome sequences becoming available has drastically increased our knowledge of the mating type locus structure and evolution. Hence, mating type loci sequences have been defined and analysed in many fungal species. Analysis of mating type loci in closely related species provides insights in the evolutionary plasticity of this unique region of the genome, as well as the evolutionary history of closely related species (Fraser et al. 2007).

Sex in Mycosphaerella

The mating strategy for the majority of Mycosphaerella species is yet unknown, as for only a few species either a heterothallic or a homothallic mating system has been described. The mechanism of sex in Mycosphaerella is still an enigma, as teleomorphs have only been discovered or induced for a few species. So far mating type idiomorphs have been characterised for M. fijiensis, the causal agent of black Sigatoka on banana, M. graminicola, the causal agent of Septoria leaf blotch on wheat, and , a pathogen of barley (Waalwijk et al. 2002, Goodwin et al. 2003, Conde-Ferráez et al. 2007). Furthermore, mating type genes of some presumed asexual species, with proven phylogenetic affinity to the genusMycosphaerella have been characterised, including several Cercospora spp., spp. and fulva (syn. fulvum) (Groenewald et al. 2006b, 2007, Stergiopoulos et al. 2007).

Outline and scope of the thesis

The research presented in this thesis provides various taxonomic aspects of the genus Mycosphaerella in general and Mycosphaerella species pathogenic on banana in particular with an emphasis on biodiversity and phylogeny. A multi-locus DNA sequence data set was established to study the biodiversity and phylogenetic relationships among the Mycosphaerella species constituting the Sigatoka disease complex of banana. Molecular tools for detection and quantification were developed for the major constituents of the Sigatoka disease complex including M. fijiensis, M. musicola and M. eumusae. Furthermore, the mating type locus structure of several Mycosphaerella species was characterised to get a better understanding of

16 General introduction and outline of thesis the evolutionary history of sex among the species associated with the Sigatoka disease complex of banana. In addition, we investigated the phylogenetic affinities of asexual genera such as Ramichloridium, Periconiella, Veronaea and Rhinocladiella to Mycosphaerella.

Chapter 1 provides an introduction to the genus Mycosphaerella, and discusses the current systematics of the genus. The Sigatoka disease complex of banana is introduced, and the problems associated with disease detection and management are summarised. A brief overview of sexual reproduction in ascomycetous fungi and Mycosphaerella in particular is provided.

In Chapter 2 the biodiversity and phylogenetic relationship among the Mycosphaerella species constituting the Sigatoka disease complex of banana is investigated. A global collection of Mycosphaerella strains isolated from banana was established as part of this study. A multi-gene phylogeny was derived from these isolates using the ITS areas of the rDNA operon, part of the actin gene, the mitochondrial small subunit ribosomal DNA, and the histone H3 gene. The phylogenetic tree obtained revealed the presence of more than 20 species of Mycosphaerella or associated anamorphs on banana, eight of which had not been described before, whereas five were shown to occur also on other host crops.

Chapter 3 describes molecular-based diagnostic tools for detection and quantification of M. fijiensis, M. musicolaand M. eumusae. TaqMan real-time PCR assays based on the β-tubulin gene were developed that could detect as little as 1 pg/µl of DNA for each Mycosphaerella species, and were validated using artificially inoculated banana leaves. The robustness of the real- time assays was confirmed using naturally infected banana leaves. Furthermore, conventional species-specific PCR primers were developed based on the actin gene that could be used to detect as little as 100, 1 and 10 pg/µl DNA from M. fijiensis, M. musicola and M. eumusae, respectively.

Chapter 4 analyses the phylogenetic position of Ramichloridium musae, the causal agent of tropical speckle disease of banana, and morphologically similar genera such as Periconiella, Veronaea and Rhinocladiella to Mycosphaerella. The phylogeny inferred from 28S nrDNA sequence data revealed the genus Ramichloridium to be heterogeneous. These data further revealed the genus Periconiella and some Ramichloridium species to be allied to Mycosphaerella. In contrast, the genera Veronaea and Rhinocladiella were shown to reside in Chaetothyriales.

Chapter 5 describes the characterisation of the mating type loci of the primary agents of the Sigatoka disease complex of banana. DNA sequences were obtained by means of chromosome walking and genomic analyses and bioinformatics were applied to define and characterise the mating type loci of Mycosphaerella species on banana. This analysis was extended to study the evolutionary history of the mating type loci in M. fijiensis, M. musicola and M. eumusae. Our data revealed a unique organisation of the mating type loci among members of Mycosphaerella. A number of new genes were shown to be present in the mating type locus, some of which were expressed, and shown to be restricted to members of Mycosphaerella.

Chapter 6 summarises the research presented in this thesis. The impact of the present study in understanding and defining the genusMycosphaerella is discussed. Furthermore, the taxonomic aspects of the Sigatoka disease complex are treated, including the evolutionary plasticity of the mating type locus of its members.

17 Chapter 1

References

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22 CHAPTER 2

Multiple gene genealogies and phenotypic characters differentiate several novel species of Mycosphaerella and related anamorphs on banana

M. Arzanlou1,2, J. Z. Groenewald1, R. A. Fullerton3, E. C. A. Abeln1,4, J. Carlier5, M. -F. Zapater5, I. W. Buddenhagen6, A. Viljoen7, P. W. Crous1,2

1CBS Fungal Biodiversity Centre, Uppsalalaan 8, 3584 CT Utrecht, The Netherlands; 2Laboratory of Phytopathology, Wageningen University, Binnenhaven 5, 6709 PD Wageningen, The Netherlands; 3The Horticulture and Food Research Institute of New Zealand, Private Bag 92169 Auckland, New Zealand; 4TNO Quality of Life, Utrechtseweg 48, 3700 AJ Zeist, The Netherlands; 5Centre de coopération internationale en recherche agronomique pour le développement (CIRAD), UMR BGPI, Montpellier, France; 6College of Agricultural and Environmental Sciences, University of California, Davis, USA; 7Department of Plant Pathology, University of Stellenbosch, P. Bag X1, Matieland 7602, South Africa.

Persoonia (2008) 20: 19–37

23 Chapter 2

ABSTRACT

Three species of Mycosphaerella, namely M. fijiensis, M. musicolaand M. eumusae are involved in the Sigatoka disease complex of bananas. Besides these three primary pathogens, several additional species of Mycosphaerella or their anamorphs have been described from Musa. However, very little is known about these taxa, and for the majority of these species no culture or DNA is available for study. In the present study, we collected a global set of Mycosphaerella strains from banana, and compared them by means of morphology and a multi-gene nucleotide sequence data set. The phylogeny inferred from the ITS region and the combined data set containing partial gene sequences of the actin gene, the small subunit mitochondrial ribosomal DNA and the histone H3 gene revealed a rich diversity of Mycosphaerella species on Musa. Integration of morphological and molecular data sets confirmed more than 20 species of Mycosphaerella (incl. anamorphs) to occur on banana. This study reconfirmed the previously described presence of Cercospora apii, M. citri and M. thailandica, and also identified Mycosphaerella communis, M. lateralis and Passalora loranthi on this host. Moreover, eight new species identified from Musa are described, namely Dissoconium musae, Mycosphaerella mozambica, Pseudocercospora assamensis, P. indonesiana, P. longispora, Stenella musae, S. musicola, and S. queenslandica.

24 Gene genealogies of Mycosphaerella species on banana

INTRODUCTION

The genus Mycosphaerella is phylogenetically heterogeneous (Crous et al. 2007a), contains more than 3000 names (Aptroot 2006), and has been linked to more than 30 well-known anamorphic genera (Arzanlou et al. 2007a, Crous et al. 2006a, b, 2007a, b). Species of Mycosphaerella inhabit different ecological niches as saprobes, plant pathogens or endophytes (Verkley & Starink-Willemse 2004, Crous et al. 2004b, 2006a, 2007a, b), and have a worldwide distribution from tropical and subtropical to warm and cool regions (Crous 1998, Crous et al. 2000, 2001). Plant-pathogenic species of Mycosphaerella are among the most common and destructive plant pathogens occurring on a wide range of hosts including trees, herbaceous plants and plantation crops. The invasion of leaf and stem tissue and concomitant distortion of the host plant physiology cause considerable economic losses (Park et al. 2000, Goodwin et al. 2001, Maxwell et al. 2004, Cortinas et al. 2006, Crous et al. 2006a, b, Hunter et al. 2006). The Sigatoka disease complex, which is the most serious and economically important leaf spot disease of banana, is attributed to species of Mycosphaerella. (anamorph Pseudocercospora musae) which causes (yellow) Sigatoka disease, M. fijiensis (anamorph P. fijiensis) which causes the black Sigatoka disease, and M. eumusae (anamorph P. eumusae), which causes eumusae leaf spot disease (reviewed in Jones 2000, 2003, Crous et al. 2002) are the major constituents of the Sigatoka disease complex. The disease reduces the photosynthetic capacity of the plant as a consequence of necrotic leaf lesions, and induces physiological alterations of the plant, resulting in reduced crop yield and fruit quality. All three species emerged on bananas during the last century, and became major constraints to commercial production worldwide. The chronology of disease records around the world and genetic structure of pathogen population suggests that South-East Asia, where the host genus Musa is indigenous, is the centre of origin for all three fungal species (Mourichon & Fullerton 1990, Carlier et al. 1996, Hayden et al. 2003, Rivas et al. 2004). Sigatoka (yellow) disease was first reported on banana in Java in 1902. The disease spread rapidly to all banana-growing regions during the following 20 years, and has since then reached the limits of its distribution worldwide (reviewed in Jones 2000, 2003). The fungus responsible for the disease was described as Cercospora musae. In 1941 Leach established the connection between C. musae and its teleomorph, Mycosphaerella musicola. Mulder (1976) validated the species descriptions, while the anamorph was transferred to Pseudocercospora as P. musae (Deighton 1976). In early 1960’s, another, even more severe leaf spot disease on banana appeared in the Fiji Islands, which Rhodes (1964) described as black leaf streak disease, and it later became known as the black Sigatoka disease. Morelet (1969) validated the species name as M. fijiensis, while Deighton (1976) placed its anamorph in Pseudocercospora as P. fijiensis. In 1974 a new variety of M. fijiensis was described from Honduras, and named as M. fijiensis var. difformis. Deighton (1979) placed both varieties in the genus Paracercospora, based on the slight thickening observed on the rims of scars and conidial hila. However, this feature was not supported by DNA phylogeny, and as there were many intermediate morphological forms, the genus Paracercospora was again reduced to synonymy under Pseudocercospora by Crous et al. (2001). Mycosphaerella eumusae was recognised as a new constituent of the Sigatoka complex of banana in the mid-1990’s (Carlier et al. 2000, Crous & Mourichon 2002, Jones 2003). Presently M. eumusae is known from parts of South-East Asia, Indian Ocean Islands and Nigeria, where it co-exists with the other two species. Besides the three primary agents of the Sigatoka disease complex, several additional species of Mycosphaerella (or their anamorphs) have been described from Musa, but for the majority of these species no culture or herbarium specimen is available, and the pathological relevance of those species remains unclear (reviewed in Jones 2000, Crous et al. 2003, Aptroot 2006).

25 Chapter 2 EU514233, EU514300, EU514353, EU514406 EU514234, EU514301, EU514354, EU514407 EU514235, EU514302, EU514355, EU514408 EU514236, —, — EU514237, —, — EU514238, —, — EU514239, —, — EU514240, EU514303, EU514356, EU514409 EU514241, EU514304, EU514357, EU514410 EU514242, EU514305, EU514358, EU514411 EU514243, EU514306, EU514359, EU514412 EU514244, EU514307, EU514360, EU514413 EU514245, EU514308, EU514361, EU514414 EU514246, EU514309, EU514362, EU514415 EU514232, EU514299, EU514352, EU514405 EU514230, —, — EU514231, EU514298, EU514351, EU514404 EU514227, —, — EU514228, —, — EU514229, —, — EU514225, EU514296, EU514349, EU514402 EU514226, EU514297, EU514350, EU514403 EU514222, —, — EU514223, —, — EU514224, —, — ank numbers (ITS, ACT, HIS, mtSS U ) ACT, Gen B ank numbers (ITS, Mauritius Mauritius Mauritius Mauritius Mauritius Mauritius Mauritius India Malaysia Malaysia Sri Lanka Thailand Thailand Vietnam Trinidad Trinidad Mozambique Africa South USA, Florida Tonga Tonga India India Western Bangladesh Western Bangladesh Western Bangladesh Western Origin Musa cultivar Musa cultivar Musa cultivar Musa cultivar Musa cultivar Musa cultivar Musa cultivar Musa cultivar Musa cultivar Musa cultivar Musa cultivar Musa cultivar Musa cultivar Musa cultivar cv. Valery: Cavaendish Valery: Musa cv. Musa cultivar — Citrus sp. AAAA SH 3436 Musa cv. AA SH 3362 Musa cv. cv. Nendran (Plantain) Musa cv. AAB Nendran (Plantain) Musa cv. AAB cv. Cavendish Musa cv. Cavendish Musa cv. Cavendish Musa cv. Source 1 or its anamorphs used for DNA analysis and morphological studies. or its anamorphs used for DNA S1030B S1037B S1037C S1037G S1037H CPC 4579 X208; CIRAD 1156; CIRAD 1157; X209; CBS 114825; CPC 4580 X865; CIRAD 535 X866; CBS 121377; CIRAD 458 X867; CBS 121378; CIRAD 459 X869; CBS 121380; CIRAD 563 X870; CBS 121381; CIRAD 485 X873; CBS 121382; CIRAD 487 X875; CIRAD 671 X1023 X24 X215 X126; CBS 122455 X742; CBS 122456 X743 X1021; CBS 122453 X1022; CBS 122454 CPC 12682; CBS 119395 CPC 12683 CPC 12684 Accession number Mycosphaerella 1. Isolates of Mycosphaerella Table Mycosphaerella eumusae Mycosphaerella Mycosphaerella communis Mycosphaerella Mycosphaerella colombiensis Mycosphaerella Mycosphaerella Dissoconium musae Cercospora apii Cercospora Species

26 Gene genealogies of Mycosphaerella species on banana EU514265, —, — EU514266, EU514324, EU514377, EU514430 EU514267, EU514325, EU514378, EU514431 EU514268, EU514326, EU514379, EU514432 EU514269, EU514327, EU514380, EU514433 EU514270, EU514328, EU514381, EU514434 EU514271, EU514329, EU514382, EU514435 EU514272, EU514330, EU514383, EU514436 EU514273, EU514331, EU514384, EU514437 EU514274, EU514332, EU514385, EU514438 EU514259, EU514320, EU514373, EU514426 EU514260, EU514321, EU514374, EU514427 EU514261, EU514322, EU514375, EU514428 EU514262, —, — EU514263, —, — EU514264, EU514323, EU514376, EU514429 EU514257, EU514318, EU514371, EU514424 EU514258, EU514319, EU514372, EU514425 EU514256, —, — EU514248, —, — EU514364, EU514417 EU514249, EU514311, EU514250, EU514312, EU514365, EU514418 EU514251, EU514313, EU514366, EU514419 EU514252, EU514314, EU514367, EU514420 EU514253, EU514315, EU514368, EU514421 EU514254, EU514316, EU514369, EU514422 EU514255, EU514317, EU514370, EU514423 ank numbers (ITS, ACT, HIS, mtSS U ) ACT, Gen B ank numbers (ITS, EU514247, EU514310, EU514363, EU514416 Cuba Islands Windward Islands Windward Australia Australia Australia Australia Australia Australia Australia Tonga Malawi Malawi Malawi Malawi Malawi Mozambique Australia Mauritius Cameroon Colombia Colombia Colombia Colombia Honduras Taiwan Ivory Coast Origin Mauritius Musa cultivar Musa cultivar Musa cultivar Williams Musa cv. Williams Musa cv. AA SH-3362 Musa cv. Lakatan Musa cv. Musa cultivar Musa cultivar Musa cultivar cv. Cavendish AAA Cavendish Musa cv. Musa cultivar Musa cultivar Musa cultivar Musa cultivar Musa cultivar Musa cultivar Musa cultivar Musa cultivar Musa cultivar Musa cultivar Musa cultivar Musa cultivar Musa cultivar Musa cultivar Musa cultivar Musa cultivar Source Musa cultivar 1 X42; CBS 116634; IMI 123823 X42; CBS 116634; X63 X67 X588 X589 X596 X602 X857; CBS 121371; UQ430 X858; CBS 121372; UQ433 X860; CBS 121374; UQ2003 X398; CBS 122458 X813 X814; CBS 122459 X818; CBS 122460 X819; CBS 122461 X879; CBS 121386; CIRAD 64 X34; CBS 122464 X884; CBS 121391; UQ438 S1024 CIRAD 86; CBS 120258; C86a X84 X92 X104 X110 X843; CIRAD 11 X847; CBS 121362; CIRAD 364 X850; CIRAD 355 Accession number X876; CBS 121383; CIRAD 744 Table 1. (Continued). Table Mycosphaerella musicola Mycosphaerella Mycosphaerella Mycosphaerella mozambica Mycosphaerella Mycosphaerella lateralis Mycosphaerella Mycosphaerella fijiensis Mycosphaerella Species

27 Chapter 2 EU514295, —, — EU514294, —, — EU514286, —, — EU514287, EU514343, EU514396, EU514449 EU514288, EU514344, EU514397, EU514450 EU514289, —, — EU514290, EU514345, EU514398, EU514451 EU514291, EU514346, EU514399, EU514452 EU514292, EU514347, EU514400, EU514453 EU514293, EU514348, EU514401, EU514454 EU514284, EU514341, EU514394, EU514447 EU514285, EU514342, EU514395, EU514448 EU514282, EU514340, EU514393, EU514446 EU514283, —, — EU514281, EU514339, EU514392, EU514445 EU514279, EU514337, EU514390, EU514443 EU514280, EU514338, EU514391, EU514444 EU514275, EU514333, EU514386, EU514439 EU514276, EU514334, EU514387, EU514440 EU514277, EU514335, EU514388, EU514441 EU514278, EU514336, EU514389, EU514442 ank numbers (ITS, ACT, HIS, mtSS U ) ACT, Gen B ank numbers (ITS, Australia India India Islands Windward Islands Windward Islands Windward Islands Windward Tonga Martinique Martinique Malaysia Malaysia Indonesia, Western Western Indonesia, Sumatra Western Indonesia, Sumatra India, Assam India, Camaroon Mozambique Windward Islands Windward Australia Mozambique Brazil Origin Musa banksii cv. Grand Nain AAA AAA Grand Nain Musa cv. (Cav.) Ravenala madagascariensis Musa cultivar Musa cultivar Musa cultivar Musa cultivar AAAA TU8 Musa cv. Musa cultivar Musa cultivar cv. Pisang Mas AA Pisang Mas Musa cv. AA Pisang Mas Musa cv. Musa cultivar Musa cultivar Musa cultivar Musa cultivar Citrus sp. Musa cultivar Musa cultivar Musa cultivar Musa cultivar Source 1 X1084; CBS 122475 X1019; CBS 122479 X1083; CBS 122468 X45 X47; CBS 122476 X55 X70; CBS 122478 X745; CBS 122477 X877; CBS 121384; CIRAD 41 X878; CBS 121385; CIRAD 56 X474; CBS 122469 X475; CBS 122470 X991; #11-5; CBS 122473 X991; #11-5; CBS 122474 X992; #11-6; X988; #9; CBS 122467 X28; CBS 122465 X138; CBS 122466 X22 X53 X882; CBS 121389; CIRAD 81 X883; CBS 121390; CIRAD 1165 Accession number

CBS: Centraalbureau voor Schimmelcultures, Utrecht, The Netherlands; CIRAD: Centre de coopération internationale en recherche agronomique pour le développement Stenella queenslandica Stenella musae Stenella musicola sp. Pseudocercospora Pseudocercospora longispora Pseudocercospora Pseudocercospora indonesiana Pseudocercospora Pseudocercospora assamensis Pseudocercospora Passalora loranthi Mycosphaerella thailandica Mycosphaerella Table 1. (Continued). Table (CIRAD), France; Montpellier, CPC: Culture collection of Pedro Crous, housed at CBS; IMI: International Mycological Institute, CABI-Bioscience, Egham, Bakeham Lane, Arzanlou, housed at CBS. Australia; X, S: Culture collection of Mahdi UK; UQ: University of Queensland, Species 1

28 Gene genealogies of Mycosphaerella species on banana

The identity and distribution of the various Mycosphaerella species associated with leaf spots of banana are not yet fully understood, which is mainly due to the difficulties experienced by scientists who have to identify them by conventional methods and without specialist taxonomic support. Furthermore, because these species are morphologically highly similar and frequently co-occur on the same lesion, pathogen recognition and subsequent disease management has proven to be rather difficult. To enable the development of specific molecular- based diagnostic tools for pathogen recognition, all related species present on the same host have to be considered. Recently, Arzanlou et al. (2007a) developed a highly sensitive set of Taqman probes to distinguish M. fijiensis from M. musicola and M. eumusae in leaf material. Little attention has been given to date, however, to other species of Mycosphaerella that occur on Musa spp. Because several Mycosphaerella species can co-occur in the same lesion (Crous 1998), it is quite possible that there may be other species of Mycosphaerella associated with the Sigatoka disease complex. The aim of the present study was, therefore, to employ a multi-gene DNA sequence typing approach on a global set of Mycosphaerella isolates to distinguish the various species occurring on banana. To this end, morphological and cultural growth data were integrated with DNA sequence data from the internal transcribed spacer region of the rDNA operon, and partial actin, histone H3, and small subunit mitochondrial ribosomal DNA gene sequences.

MATERIALS AND METHODS

Isolates

Isolates (Table 1) were obtained by isolation from infected symptomatic banana leaves, or supplied as pure cultures by the following departments and institutes: The Horticulture and Food Research Institute of New Zealand, Auckland, New Zealand; Centre de coopération internationale en recherché agronomique pour le développement (CIRAD; Montpellier, France); University of Florida, Tropical Research & Education Centre (USA); Forestry and Agricultural Biotechnology Institute (FABI, Pretoria, South Africa). Isolates were recovered from infected banana leaves as single ascospores or conidia. Germinating spores were examined 24 h after germination on 2 % malt extract agar (MEA; Sigma-Aldrich Chemie, Zwijndrecht, The Netherlands) plates under a stereomicroscope, and single-spore cultures were established on fresh MEA plates following the protocol of Crous (1998).

DNA phylogeny

Genomic DNA was isolated from fungal mycelia grown on MEA, using the FastDNA kit (BIO101, Carlsbad, CA, USA) according to the manufacturer’s protocol. The primers ITS1 and ITS4 (White et al. 1990) were used to amplify part of the internal transcribed spacer region (ITS) of the nuclear ribosomal RNA operon, including the 3’ end of the 18S rRNA gene, the first ITS region, the 5.8S rRNA gene; the second ITS region and the 5’ end of the 28S rRNA gene. A part of the actin gene (ACT) was amplified with primers ACT-512F and ACT-783R (Carbone & Kohn 1999), a part of the small subunit mitochondrial ribosomal DNA (mtSSU) with primers NMS1 and NMS2 (Li et al. 1994), and a part of the histone H3 (HIS) gene with primers CYLH3F and CYLH3R (Crous et al. 2004b). Amplification reactions were performed with each primer set in a total reaction volume of 25 µl, which was composed of 1× PCR

Buffer (Applied Biosystems, Foster City, USA), variable MgCl2 concentrations, 60 µM dNTPs,

29 Chapter 2

0.2 µM of each forward and reverse primer, 1.5 U of Taq DNA polymerase (Roche Diagnostics, Indianapolis, USA) and 1–10 ng of genomic DNA. PCR cycle conditions were 5 min of 95 °C, followed by 36 cycles of 94 °C for 30 s, 55 °C for 30 s, 72 °C for 60 s, and a final elongation at 72 °C for 7 min. Amplicons were sequenced using both PCR primers with a DYEnamic ET Terminator Cycle Sequencing kit (Amersham Biosciences, Roosendal, the Netherlands) according to the manufacturer’s recommendations, and sequences were analysed on an ABI Prism 3700 DNA Sequencer (Perkin-Elmer, Norwalk, Foster City, CA). The resulting nucleotide sequences were analysed and automatically aligned using BioNumerics v. 4.5 (Applied Maths, Kortrijk, Belgium) followed by manual improvement where necessary. Phylogenetic analyses were performed with PAUP (Phylogenetic Analysis Using Parsimony) v. 4.0b10 (Swofford 2003), using the neighbour-joining algorithm with the uncorrected (“p”), the Kimura 2-parameter and the HKY85 substitution models. Alignment gaps longer than 10 bases were coded as single events for the phylogenetic analyses; the remaining gaps were treated as missing data. Any encountered ties were randomly broken. Phylogenetic relationships were also inferred with the parsimony algorithm using the heuristic search option with simple (ITS alignment) or 100 random taxa additions (combined alignment) and tree bisection and reconstruction (TBR) as the branch-swapping algorithm; alignment gaps were treated as missing (combined alignment) or as a fifth character state (ITS alignment) and all characters were unordered and of equal weight. Branches of zero length were collapsed and all multiple, equally parsimonious trees were saved. Other measures calculated included tree length, consistency index, retention index and rescaled consistency index (TL, CI, RI and RC, respectively). The robustness of the obtained trees was evaluated by 10000000 fast stepwise (ITS alignment) or 1000 bootstrap heuristic bootstrap replications (combined alignment). Sequences were deposited in GenBank (Table 1) and the alignments in TreeBASE (www.treebase.org).

Morphology

Growth rates and colony morphology were recorded from colonies grown on MEA plates after 30 d incubation in darkness at 24 ºC. Colony colours (surface and reverse) were assessed after growth on MEA and oatmeal agar (OA, Gams et al. 2007) using the colour charts of Rayner (1970). Microscopic observations were made from colonies cultivated on MEA and OA. Preparations were mounted in lactic acid and studied under a light microscope (× 1000 magnification). The 95 % confidence intervals were derived from 30 observations of spores formed on MEA or OA, with extremes given in parentheses. All cultures obtained in this study are maintained in the culture collection of the Centraalbureau voor Schimmelcultures (CBS) in Utrecht, the Netherlands or the working collections of Pedro Crous (CPC) or Mahdi Arzanlou (X, S numbers) at CBS (Table 1). Nomenclatural novelties and descriptions were deposited in MycoBank (www.MycoBank.org) (Crous et al. 2004a).

RESULTS

DNA phylogeny

Two alignments of DNA sequences were subjected to phylogenetic analyses. The first alignment consisted of ITS sequences generated in this study as well as sequences obtained from the NCBI GenBank nucleotide sequence database. The ITS alignment consisted of a total number of 113 sequences (including one outgroup); 508 characters including alignment gaps were

30 Gene genealogies of Mycosphaerella species on banana

Davidiella tassiana DQ289800 X1023 89 AY725537 Mycosphaerella communis AY725535 100 EF394854 Dissoconium australiensis X1021 Dissoconium musae 84 X1022 AF309625 S1024 Mycosphaerella lateralis AF309624 DQ267587 81 DQ302984 Mycosphaerella marksii DQ303075 Ps. epispermogoniana 100 DQ267588 AY424802 X398 X818 X819 Mycosphaerella musae X813 X879 65 AY923761 X814 EU041796 Ramichloridium biverticillatum 97 EU041801 Ramichloridium musae 86 DQ530216 Mycosphaerella pseudovespa EU041783 Periconiella velutina 100 74 DQ632677 100 X1084 Stenella queenslandica X55 100 X877, X878 X70, X745 Stenella musae X45, X47 EF535708 Stenellopsis liriopes 100 X1019 Stenella musicola X126 AY752146 X743 X742 Mycosphaerella citri AF181703 AF181704 DQ632684 X215 M. colombiensis X24 M. colombiensis 100 X53 M. thailandica X883 M. thailandica Mycosphaerella colombiensis / X22 M. thailandica 86 X882 M. thailandica Mycosphaerella thailandica AY752156 M. thailandica AF309612 M. colombiensis EF394849 M. thailandica CPC 12684 10 changes 100 CPC 12682 AY266162 Cercospora apii CPC 12683 AF297230 AY840519 Cercospora apii AY152590 Mycosphaerella laricina 100 AY752163 93 AY752162 Passalora sp. AY348311 X138 Passalora loranthi X28 DQ676520 Passalora sp. 89 AF362058 Mycosphaerella confusa X34 X884 Mycosphaerella mozambica EU042175 AY509744 Mycosphaerella aurantia AY626981 DQ267577 Mycosphaerella africana X847 97 X850 CIRAD 86 X92 Mycosphaerella fijiensis X84 AY266150, AY923765 95 X843 X104, X110 DQ289829 X988 Pseudocercospora assamensis X1083 Pseudocercospora sp. DQ885903 Passalora schizolobii DQ267602 Pseudocercospora paraguayensis AF309595 Pseudocercospora basiramifera X991 69 X992 Pseudocercospora indonesiana 67 X865 95 X870 AY923758 Mycosphaerella eumusae S1037G, S1037H 66 X208, X209 74 97 AY646484, AY646483 Mycosphaerella sp. X474, X475 Pseudocercospora longispora 98 X42, AY266148 X67 100 X602, X63 X589, X596 Mycosphaerella musicola X858, X860 X588, X857

Fig. 1. One of 11780 equally most parsimonious trees obtained from a heuristic search with simple taxon additions of the ITS sequence alignment. The scale bar shows ten changes, and bootstrap support values (65 % and higher) from 10000000 fast stepwise replicates are shown at the nodes. Thickened lines indicate the strict consensus branches. The tree was rooted to sequences of strain CPC 11600 (GenBank accession number DQ289800). M. = Mycosphaerella and Ps. = Pseudocercospora.

31 Chapter 2

Davidiella tassiana 100 X1023 Mycosphaerella communis 100 X1021 Dissoconium musae X1022 100 X34 Mycosphaerella mozambica 90 X884 100 X28 Passalora loranthi X138 X53 M. thailandica 100 X215 M. colombiensis M. thailandica / 65 X22 M. thailandica M. colombiensis X882 M. thailandica X883 M. thailandica 100 X879 X398 Mycosphaerella musae 67 X814 X813 X878 100 X47 X70 Stenella musae 100 65 X745 X877 X45 X847 100 X850 87 X92 Mycosphaerella fijiensis 10 changes X84 X110 65 X843 X104 100 X475 Pseudocercospora longispora 100 X474 100 X588 X602 X589 100 X596 X860 Mycosphaerella musicola X858 X857 X63 67 X67 83 X988 Pseudocercospora assamensis X991 Pseudocercospora indonesiana X865 X870 X866 100 X875 X867 S1037B Mycosphaerella eumusae 86 X869 X876 X873 83 S1030B S1037C

Fig. 2. One of eight equally most parsimonious trees obtained from a heuristic search with 100 random taxon additions of the combined (ITS, ACT, HIS, mtSSU) sequence alignment. The scale bar shows 10 changes, and bootstrap support values (65 % and higher) from 1000 replicates are shown at the nodes. Thickened lines indicate the strict consensus branches. The tree was rooted to sequences of Davidiella tassiana strain CPC 11600 (GenBank accession number DQ289800, DQ289867, EF679665, EU514455, respectively). M. = Mycosphaerella.

32 Gene genealogies of Mycosphaerella species on banana subjected to the analyses. Of these characters, 224 were parsimony-informative, 42 variable and parsimony-uninformative, and 242 were constant. Trees supporting the same clades were obtained irrespective of the analysis method used. The parsimony analysis yielded 11780 equally most parsimonious trees that mainly differed in the order of taxa at the terminal nodes; one of the trees is presented in Fig. 1 (TL = 861 steps; CI = 0.569; RI = 0.934; RC = 0.532). The sequence data in the second alignment were analysed as one combined set consisting of 1648 characters (incl. alignment gaps) (number of included characters: ITS: 509, ACT: 188, HIS: 375, mtSSU: 576). This second alignment included 54 sequences (including the outgroup) and of the 1648 characters 517 were parsimony-informative, 93 were variable and parsimony-uninformative, and 1038 were constant. Trees supporting the same clades were obtained irrespective of the analysis method used. The parsimony analysis yielded eight equally most parsimonious trees that mainly differed in the order of taxa at the terminal nodes; one of the trees is presented in Fig. 2 (TL = 1513 steps; CI = 0.654; RI = 0.901; RC = 0.589). Similar to the results obtained for the ITS alignment, the same lineages were found with the combined alignment. The ACT and HIS data were found to be more variable within species than the ITS and mtSSU data (data not shown for individual loci, variation within clades in Fig. 2). The phylogenetic results obtained are discussed where applicable in the descriptive notes below.

Taxonomy

The results of this study showed a rich diversity of Mycosphaerella spp. on Musa. Phylogenetic analyses revealed that more than 20 species of Mycosphaerella or its anamorphs occur on banana, including species known from hosts other than banana, namely Cercospora apii, Mycosphaerella citri, M. thailandica, M. communis, M. lateralis and Passalora loranthi (Fig. 1). Furthermore, eight species proved to be morphologically and phylogenetically distinct from the species presently known from banana. These new species are described below:

Cercospora apii Fresen, Beitr. Mykol. 3: 91. 1863. = Cercospora hayi Calp., Studies on the Sigatoka disease of bananas and its fungus pathogen, Atkins Garden and Research Laboratory, Cuba: 63. 1955.

Specimens examined: Cuba, Musa paradisiaca var. sapientum, 1955, L. Calpouzos, holotype FH, ex-type culture ATCC 12234. India, Bangladesh, Musa cv. Cavendish, Oct. 2005, I. Buddenhagen, CBS H-20035, culture CBS 119395.

Notes: In their treatment of the genus Cercospora, Crous & Braun (2003) considered C. hayi to be a synonym of the older name, Cercospora apii, which is known to have a wide host range. Based on a comparison of DNA sequence data with the ex-type strain of C. apii (GenBank AY840519; Groenewald et al. 2006), this synonymy appears to be correct.

33 Chapter 2

Fig. 3. Dissoconium musae (CBS 122453). A–D. Conidiophores with sympodially proliferating conidiogenous cells, which produce primary and secondary conidia in pairs. E–G. Primary conidia with truncate base. H–L. anastomoses between hyphae, primary and secondary conidia and primary conidia. Scale bar = 10 µm.

Dissoconium musae Arzanlou & Crous, sp. nov. MycoBank MB505972; Figs 3–4.

Dissoconio communi simile, sed coloniis in vitro tarde crescentibus (usque ad 10 mm diam. post 30 dies ad 24 °C in agaro maltoso).

Etymology: Named after its host plant, Musa.

In vitro on MEA: Mycelium submerged and superficial; submerged hyphae hyaline to subhyaline, thin-walled, smooth, forming a dense network with numerous anastomoses, 2–3 µm wide; aerial hyphae subhyaline, smooth, 2–3 µm wide. Conidiophores arising orthotropically from vegetative hyphae, often reduced to conidiogenous cells and continuous with supporting hyphae, thin- walled, smooth, pale brown, unbranched, straight, subulate to lageniform, tapering towards the apex, (10–)19–25(–53) × (2.5–)3–5 µm. Conidiogenous cells terminal, proliferating sympodially (but appearing as annellides under the light microscope), giving rise to a short conidium-bearing rachis, loci somewhat darkened and thickened. Conidia forming in sympodial order in pairs on

34 Gene genealogies of Mycosphaerella species on banana

Fig. 4. Dissoconium musae (CBS 122453). Scale bar = 10 µm. a conidiogenous cell; the primary conidium is two-celled, while the secondary conidium is aseptate; primary conidia pale olivaceous-brown, thin-walled, smooth, ellipsoidal to obclavate, 1-septate, apex obtuse, base obconically-truncate, (11–)22–26(–35) × (3–)4–5 µm, hilum unthickened; about 1 µm diam. Secondary conidia 1-celled, pale olivaceous-brown, pyriform to turbinate, 4–5 × 3–4 µm, base truncate, flat, unthickened, about 0.5 µm diam. Both conidial types are discharged forcibly in pairs and then anastomose on the agar surface. Anastomosis between primary conidia occurs as well and primary conidia may show multiple anastomoses. Primary conidia germinate from both ends and produce several conidiogenous cells and conidia (microcyclic conidiation). Germination of secondary conidia was not observed.

Cultural characteristics: Colonies on MEA slow-growing, reaching 10 mm diam after 30 d at 24 °C, erumpent, unevenly folded, with sparse aerial mycelium, colonies with granulate margin; surface hazel to isabelline in centre, and vinaceous-buff in outer region; brown-vinaceous in reverse. Colonies on OA reaching 25 mm diam after 30 d at 24 °C, effuse, with moderate aerial mycelium, later become powdery in centre, surface hazel; olivaceous in reverse.

Specimen examined: India, Tamil Nadu, Tiruchirapally, Musa cv. Nendran (Plantain) AAB, 2005, I. Buddenhagen, holotype CBS H-20036, culture ex-type X1021 = CBS 122453.

Notes: The genus Dissoconium is characterised by producing pairs of forcibly discharged primary and secondary conidia on sympodially proliferating conidiogeneous cells. Sympodial proliferation of the conidiogenous cells gives rise to a conidium-bearing rachis, which resembles that encountered in the genus Ramichloridium. The recent revision of the genus Ramichloridium and allied genera (Arzanlou et al. 2007b) revealed that R. apiculatum, the type species of the genus, is phylogenetically close to the species in the genus Dissoconium. However, Dissoconium is morphologically distinct from Ramichloridium by producing two types of forcibly discharged conidia. So far, seven species of Dissoconium have been described from different substrates (de Hoog et al. 1991, Jackson et al. 2004). Dissoconium musae is phylogenetically distinct from the other species of this genus, but morphologically similar to D. commune and D. dekkeri (teleomorph: Mycosphaerella lateralis), from which it differs based on its slower growth rate in culture.

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Mycosphaerella eumusae Crous & Mour., Sydowia 54: 36. 2002. Anamorph: Pseudocercospora eumusae Crous & Mour., Sydowia 54: 36. 2002.

Specimen examined: Réunion, on leaves of Musa sp., 2001, J. Carlier, PREM 57314 (holotype of teleomorph), PREM 57315 (holotype of anamorph), cultures ex-type (CIRAD 1156, 1157 = CPC 4579, 4580 = CBS 114824, CBS 114825).

Notes: Based on the DNA sequence data obtained in this study (Fig. 2), it appears that M. eumusae is heterogeneous as presently circumscribed. Further studies would be required to determine if the phylogenetic variation also correlates with differences in morphology.

Mycosphaerella fijiensis M. Morelet, Ann. Soc. Sci. Nat. Archéol. Toulon Var 21: 105. 1969. = Mycosphaerella fijiensis var. difformis J.L. Mulder & R.H. Stover, Trans. Brit. Mycol. Soc. 67: 82. 1976. Anamorph. Pseudocercospora fijiensis (M. Morelet) Deighton, Mycol. Pap. 140: 144. 1976. Basionym: Cercospora fijiensis M. Morelet, Ann. Soc. Sci. Nat. Archéol. Toulon Var 21: 105. 1969. ≡ Paracercospora fijiensis (M. Morelet) Deighton, Mycol. Pap. 144: 51. 1979. = Cercospora fijiensisvar. difformis J.L. Mulder & R.H. Stover, Trans. Brit. Mycol. Soc. 67: 82. 1976. ≡ Paracercospora fijiensis var. difformis (J.L. Mulder & R.H. Stover) Deighton, Mycol. Pap. 144: 52. 1979.

Specimens examined: Hawaii, on leaves of Musa sp., D.S. Meredith & J.S. Lawrence, holotype IMI 136696. Cameroon, date and collector unknown, epitype designated here CBS H-20037, culture ex-epitype CIRAD 86 = CBS 120258.

Note: The specimen and associated strain designated here as epitype, represent the strain that was selected by the Mycosphaerella consortium to obtain the full genome sequence of M. fijiensis (www.jgi.doe.gov/sequencing/why/CSP2006/mycosphaerella.html).

Mycosphaerella musae (Speg.) Syd. & P. Syd., Philipp. J. Sci. 8: 482. 1913. Basionym: Sphaerella musae Speg., Anal. Mus. Nac. Hist. Nat. Buenos Aires 19: 354. 1909. = Sphaerella musae Sacc., Atti Accad. Sci. Veneto-Trentino-Istriana, Ser. 3, 10: 67. 1917, homonym.

Specimen examined: Argentina, Jujuy, Orán, on leaves of Musa sapientum, Mar. 1905, holotype LPS, slide ex-type IMI 91165.

Notes: Mycosphaerella musae is reported to be the causal organism of Mycosphaerella speckle disease. However, as shown in the present study (Fig. 1), several distinct species appear to be able to induce these symptoms. Further collections would thus be required to recollect this species. All cultures examined in the present study were sterile.

Mycosphaerella musicola R. Leach ex J.L. Mulder, Trans. Brit. Mycol. Soc. 67: 77. 1976. Basionym: Mycosphaerella musicola R. Leach, Trop. Agric. 18: 92. 1941. (nom. nud.). Anamorph: Pseudocercospora musae (Zimm.) Deighton, Mycol. Pap. 140: 148. 1976.

36 Gene genealogies of Mycosphaerella species on banana

≡ Cercospora musae Zimm., Centralbl. Bakteriol. Parasitenk. 2. Abt. 8: 219. 1902. = Cercospora musae Massee, Bull. Misc. Inform. 28: 159. 1914.

Specimens examined: Jamaica, on leaves of Musa sapientum, Jan. 1959, R. Leach, holotype IMI 75804a. Cuba, on leaves of Musa sp., epitype designated here CBS H-20038, culture ex- epitype IMI 123823 = CBS 116634.

Mycosphaerella mozambica Arzanlou & Crous, sp. nov. MycoBank MB505973; Figs 5–6. Anamorph: ramichloridium-like.

Ascosporae rectae vel curvatae, fusoideo-ellipsoideae utrinque obtusae, ad septum medianum vix constrictae, (9–)10–11(–12) × 3–3.5(–4) µm.

Etymology: Named after the country of origin, Mozambique.

In vivo: Leaf spots amphigenous, irregular to sub-circular,1–7 mm diam, grey to pale brown on adaxial surface, grey on abaxial surface, with dark brown margins. Ascomata amphigenous, intermingled among those of M. musicola, dark brown, subepidermal, becoming erumpent, globose, 70–90 µm diam; wall consisting of 2–3 layers of medium brown textura angularis. Asci aparaphysate, fasciculate, bitunicate, subsessile, obovoid to broadly ellipsoid, straight to slightly curved, 8-spored, 28–35 × 7–9 µm. Ascospores bi- to tri-seriate, overlapping, hyaline, non-guttulate, thin-walled, straight to curved, fusoid-ellipsoidal with obtuse ends, widest in middle of apical cell, medianly 1-septate, not to slightly constricted at the septum, tapering towards both ends, but more prominently towards the lower end, (9–)10–11(–12) × 3–3.5(–4) µm; ascospores becoming distorted upon germination after 24 h on MEA, becoming constricted at the septum, 6–7 µm wide with irregular, wavy germ tubes, growing 90 ° to the long axis, and not arising from the polar ends of the spore.

In vitro on MEA: Mycelium submerged and superficial; submerged hyphae hyaline to sub-hyaline, thin-walled, smooth or slightly rough, 2–4 µm wide; aerial hyphae pale olivaceous, smooth or finely verruculose. Conidiophores arising from unbranched or loosely branched hyphae, occasionally reduced to conidiogenous cells or integrated, hyaline, subcylindrical, 2–2.5 µm wide and up to 35 µm long. Conidiogenous cells integrated, terminal, polyblastic, sympodial, loci aggregated, flat, not protuberant (not denticle-like), unthickened, but somewhat darkened. Conidia solitary, obovoid, ellipsoidal, obclavate 0(–1)-septate, hyaline, thin-walled, smooth, (5–)9–12(–22) × 2–2.5(–3) µm; hilum truncate, flat, broad, unthickened, slightly darkened, about 1 µm diam. Although rarely observed, older conidia can become elongated, obclavate, and up to 4-septate.

Cultural characteristics: Colonies on MEA reaching 45 mm diam after 30 d at 24 °C; erumpent, folded, with moderate velvety to hairy aerial mycelium, with smooth, entire margins; surface pale vinaceous to mouse-grey; brown-vinaceous in reverse. Colonies on OA reaching 51 mm diam after 30 d at 24 °C; effuse, with sparse aerial mycelium and entire edge; surface vinaceous- buff to vinaceous, and pale vinaceous in reverse.

Specimens examined: Mozambique, Chimoio, Bairro, on leaf of Musa cv. 2003, A. Viljoen, holotype CBS H-20039, culture ex-type X34 = CBS 122464; CBS H-20040, CBS H-20041, CBS H-20042.

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Fig. 5. Mycosphaerella mozambica (CBS 122464). A. Verruculose hyphae. B–E. Unbranched or loosely branched conidiophores with sympodially proliferating conidiogenous cells. F–G. Sympodially proliferating conidiogenous cells give rise to short conidium-bearing rachis. H. Conidia with truncate base. Scale bars = 10 µm.

Fig. 6. Mycosphaerella mozambica (CBS 122464). A. Ascus with biseriate ascospores. B. Ascospore germination pattern. C. Conidiophores with sympodially proliferating conidiogenous cells, which give rise to short conidium- bearing rachis. D. Conidia. Scale bar = 10 µm.

38 Gene genealogies of Mycosphaerella species on banana

Fig. 7. Pseudocercospora assamensis (CBS 122467). A. Conidiophore with sympodial and percurrent growth of conidiogenous cells. B–C. Conidia. Scale bar = 10 µm.

Notes: Sympodially proliferating conidiogenous cells are somewhat confusing with other morphologically similar genera such as Ramichloridium and Veronaea. The type species and most of the taxa referred to these genera are dematiaceous. The scars in Ramichloridium are subhyaline and slightly prominent. Veronaea has pigmented, truncate, flat loci and conidia with truncate bases. A recent revision of Ramichloridium and allied genera (Arzanlou et al. 2007b) revealed the type species of Ramichloridium, R. apiculatum, to be allied to the Dissoconium clade in the Capnodiales, while the type species of Veronaea, V. botryosa, resides in the Chaetothyiales. Mycosphaerella mozambica appeared to occur quite commonly on the banana samples investigated from Mozambique. Based on DNA sequence data, the ex-type strain appears similar to an isolate collected in Australia (CBS 121391 = X884). Unfortunately, however, the latter strain was sterile, so this could not be confirmed based on morphology.

Pseudocercospora assamensis Arzanlou & Crous, sp. nov. MycoBank MB505974; Figs 7–8.

Pseudocercosporae musae similis, sed conidiis longioribus et angustioribus, (30–)59–70(–83) × 2–3 µm.

Etymology: Named after the locality of origin, India, Assam.

In vitro on MEA: Mycelium submerged and superficial; submerged hyphae smooth, branched, septate, medium brown, 2.5–4 μm wide; aerial hyphae thin-walled, smooth, medium brown. Conidiophores solitary, arising from superficial hyphae, medium brown, thin-walled, smooth, unbranched or branched above, 0–1-septate, subcylindrical, straight, up to 20 µm long, 2–3 µm wide. Conidiogenous cells integrated, terminal, or conidiophores reduced to conidiogenous cells, subcylindrical, tapering to truncate or bluntly rounded apices, medium brown, smooth, proliferating sympodially; conidial scars inconspicuous. Conidia solitary, pale brown, smooth, subcylindrical, with truncate bases and, bluntly rounded apices, thin-walled with irregular swellings in older conidia, straight or curved, pluriseptate, (30–)59–70(–83) × 2–3 µm; hila about 1 µm wide, neither thickened nor darkened-refractive; microcyclic conidiation observed.

Cultural characteristics: Colonies on MEA reaching 47 mm diam after 30 d at 24 °C. Colonies elevated at the centre, with abundant aerial mycelium, and entire, smooth margin; surface pale mouse-grey to mouse-grey, olivaceous in reverse. Colonies on OA reaching 35 mm diam after 30 d at 24 °C; effuse, with moderate, velvety aerial mycelium, and entire, smooth margins; surface pale mouse-grey, and iron-grey in reverse.

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Fig. 8. Pseudocercospora assamensis (CBS 122467). Scale bar = 10 µm.

Specimen examined: India, Assam, Naojan, on leaf of Musa cv. Nanderan (Plantain), 2005, I. Buddenhagen, holotype CBS H-20044, culture ex-type X988 = CBS 122467.

Notes: Based on its characteristic conidial shape and dimensions, P. assamensis appears distinct from those species presently known from this host. Pseudocercospora musae conidia are shorter and above all wider (10–80 × 2–6 µm; Carlier et al. 2000) than in P. longispora. Pseudocercospora longispora has much longer and somewhat wider conidia.

Pseudocercospora indonesiana Arzanlou & Crous, sp. nov. MycoBank MB505975; Figs 9–10.

Pseudocercosporae longisporae similis, sed conidiis modice brunneis, hyphis tenuitunicatis, modice brunneis, non inflatis et non monilioidibus-muriformibus, coloniis in vitro celeriter crescentibus (usque ad 27 mm diam post 30 dies ad 24 °C in agaro maltoso).

Etymology: Named after its country of origin, Indonesia.

In vitro on MEA: Mycelium submerged and superficial; submerged hyphae thin-walled, smooth, branched, septate, medium brown, 2.5–4 μm wide; aerial hyphae, thin-walled, smooth, medium brown. Conidiophores solitary, arising from superficial hyphae, medium brown, smooth, unbranched, 0–2-septate, subcylindrical, straight, up to 30 µm long, 2–2.5 µm wide. Conidiogenous cells integrated, terminal, subcylindrical, tapering to truncate or bluntly rounded

40 Gene genealogies of Mycosphaerella species on banana

Fig. 9. Pseudocercospora indonesiana (CBS 122473). A–D. Conidia. E. Intercalary conidiogenous cell. Scale bar = 10 µm.

Fig. 10. Pseudocercospora indonesiana (CBS 122473). Scale bar = 10 µm. apices, medium brown, smooth, proliferating sympodially, frequently reduced to conidiogenous loci; conidial scars inconspicuous. Conidia solitary, pale brown, smooth, subcylindrical, bases truncate, apices bluntly rounded, thin-walled, straight or curved, guttulate, 3–7-septate, (40–) 78–95(–120) × 2–3 µm; hila unthickened, neither darkened nor refractive.

41 Chapter 2

Fig. 11. Pseudocercospora longispora (CBS 122469). A–E. Conidia. Scale bar = 10 µm.

Cultural characteristics: Colonies on MEA reaching 27 mm diam after 30 d at 24 °C. Colonies low convex, with abundant aerial mycelium, and entire, smooth margin; surface pale mouse- grey to mouse-grey; in reverse dark mouse-grey. Colonies on OA reaching 35 mm diam after 47 d at 24 °C; effuse, with moderate aerial mycelium, and entire, smooth margins; surface pale mouse-grey; in reverse olivaceous-black.

Specimen examined: Indonesia, Western Sumatra, Kumango, on leaf of Musa cv. Buai, 2004, I. Buddenhagen, holotype CBS H-20045, culture ex-type X992 = CBS 122473.

Notes: Pseudocercospora indonesiana is phylogenetically distinct from the other species of Pseudocercospora occurring on Musa. Morphologically it has longer conidia than P. musae (teleomorph M. musicola) and P. assamensis, though they are very similar to those of P. longispora; it can however, be distinguished from the latter by having medium brown conidia (those of P. longispora being pale brown), and its faster growth rate on MEA and OA.

Pseudocercospora longispora Arzanlou & Crous, sp. nov. MycoBank MB505976; Figs 11– 12.

Pseudocercosporae musae similis, sed conidiis longioribus, 82–120 × 2.5–4 µm.

Etymology: Named after its characteristically long conidia.

In vitro on OA: Mycelium submerged and superficial; submerged hyphae smooth, branched, septate, medium brown, thin-walled, 2–3 μm wide; aerial hyphae smooth, medium brown; hyphal cells become thick-walled, swollen, forming dark-brown monilioid, muriform cells, 5–17 × 7–12 µm. Conidiophores solitary, arising from superficial hyphae; conidiophores medium brown, smooth, unbranched or branched above, 0–2-septate, subcylindrical, straight, up to 30 µm long, 2–3 µm wide. Conidiogenous cells integrated, terminal, subcylindrical, tapering to truncate or bluntly rounded apices, medium brown, smooth, forming conidia by sympodial proliferation, rarely by means of percurrent proliferation; conidial scars inconspicuous. Conidia solitary, pale brown, thin-walled, smooth, cylindrical to subcylindrical, widest in the middle of conidium, tapering towards the apex, bases truncate, straight, multi-septate, 82–120 × 2.5–4 µm; hila about 1 µm diam, neither thickened nor darkened-refractive.

42 Gene genealogies of Mycosphaerella species on banana

Fig. 12. Pseudocercospora longispora (CBS 122469). Scale bar = 10 µm.

Cultural characteristics: Colonies reaching 15 mm diam after 30 d at 24 °C. Colonies erumpent, with moderate aerial mycelium, and entire, smooth edges; surface buff to rosy-buff, mouse-grey to dark grey; in reverse dark mouse-grey. Colonies on OA reaching 15 mm diam after 30 d at 24 °C, effuse, with abundant aerial mycelium, and entire, smooth margins; surface pale mouse- grey; in reverse dark mouse-grey.

Specimen examined: Malaysia, Felcra Plantation, Melaka, Musa cv. Pisang Byok AAA/AAB, July 1988, D.R. Jones, holotype CBS H-20043, culture ex-type X475 = CBS 122470.

Notes: Pseudocercospora longispora resembles P. musae (teleomorph Mycosphaerella musicola) in its colony morphology on MEA and OA. However, in P. musae conidia are much shorter (10–80 × 2–6 µm; Carlier et al. 2000) than in P. longispora.

Stenella musae Arzanlou & Crous, sp. nov. MycoBank MB505977; Figs 13–14A.

Conidiophora ex hyphis superficialibus oriunda, modice brunnea, tenuitunicata, verruculosa vel verrucosa, 0–3-septata, subcylindrica, recta vel geniculata-sinuosa, non ramosa, ad 30 µm longa et 2–2.5 µm lata. Cellulae conidiogenae integratae, terminales, interdum intercalares, modice brunneae, verruculosae, subcylindricae, apicem versus attenuatae, sympodiales, locis truncatis, subdenticulatis, 1–1.5 µm diam., inspissatis et fuscatis-refringentibus praeditae. Conidia solitaria, dilute brunnea, verruculosa, tenuitunicata, subcylindrica vel obclavata, recta vel curvata, 0–7-septata, (7–)27–40(–70) × 1.5–3 µm, hilo inspissato obscuriore refringente, 1–1.5 µm diam praedita.

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Fig. 13. Stenella musae (CBS 122477). A–D. Conidiophores with sympodially proliferating conidiogenous cells. E–F. Conidia. Scale bar = 10 µm.

Etymology: Named after its host, Musa.

In vitro on MEA: Mycelium submerged and superficial; submerged hyphae smooth to verrucose, thin-walled, subhyaline to medium brown, 2–3 µm wide, with thin septa; aerial hyphae coarsely verrucose, olivaceous-brown to medium brown, rather thick-walled, 2–2.5 µm wide, with thin septa. Conidiophores arising from superficial hyphae, medium brown, rather thick-walled, finely verrucose to verruculose, 0–3-septate, subcylindrical, straight to geniculate-sinuous, unbranched, up to 30 µm long, 2–2.5 µm wide. Conidiogenous cells integrated, terminal, sometimes intercalary, unbranched, medium brown, finely verruculose, subcylindrical, tapering towards flat-tipped, subdenticulate apical loci, 1–1.5 µm diam, proliferating sympodially; loci thickened, darkened, refractive. Conidia solitary, thin-walled, pale brown, finely verrucose, subcylindrical to obclavate, with subobtuse apex,and long obconically subtruncate to obconically subtruncate base, straight to curved, 0–7-septate, (7–)27–40(–70) × 1.5–3 µm; hilum thickened, darkened, refractive, 1–1.5 µm diam.

Cultural characteristics: Colonies on MEA reaching 30 mm diam after 30 d at 24 °C. Colonies erumpent, unevenly folded, with moderate aerial mycelium, and entire, smooth margin; surface pale mouse-grey to mouse-grey; in reverse dark mouse-grey. Colonies on OA reaching 48 mm diam after 30 d, at 24 °C; effuse, with moderate aerial mycelium, and entire margins; surface pale mouse-grey to mouse-grey, and dark mouse-grey in reverse.

Specimens examined: Tonga, ACIAR Plot, Tongatapu, Musa cv. TU8 AAAA, Mar. 1990, R.A. Fullerton, holotype CBS H-20047, culture ex-type X745 = CBS 122477. Windward Islands, St Lucia, on Musa cv., 2003, E. Reid, culture X47 = CBS 122476.

Notes: Stover (1994) discussed and illustrated a Stenella sp. from banana, and named it ‘Cercospora non-virulentum’, which was considered as a prevalent co-inhabitant with Black Leaf Streak and Sigatoka. Mycosphaerella musae is the causal agent of Mycosphaerella Speckle disease of banana (Carlier et al. 2000). A comparison made between strains isolated from Mycosphaerella Speckle disease symptoms (presumed M. musae), and ‘Cercospora non- virulentum’ isolates in culture, suggested that the two species are identical, both producing brown, verruculose conidia with thickened scars on agar medium (Stover 1994). An inoculation assay carried out by using a mixture of conidia and mycelium of ‘Cercospora non-virulentum’

44 Gene genealogies of Mycosphaerella species on banana

Fig. 14. A. Stenella musae (CBS 122477). B. Stenella queenslandica (CBS 122475). C. Stenella musicola (CBS 122479). Scale bar = 10 µm. on banana ‘Cavendish Valery’ leaves resulted in leaf spot symptoms after 70 d incubation, resembling those obtained using ascospores derived from ‘M. musae’ strains. Because ‘Cercospora non-virulentum’ was never validly published, it is difficult to make a comparison with Stenella musae. However, based on the description provided by Stover (1994), S. musae has shorter conidia (7–70 × 1.5–3 µm) than ‘Cercospora non-virulentum’ (55–200 × 2.6–3.2 µm). A further complication lies in the fact that several phylogenetically distinct species of Mycosphaerella have in the past been isolated from Mycosphaerella Speckle disease symptoms of banana. All the ‘M. musae’ isolates examined in this study were sterile, and thus could not be used for morphological comparison. Mycosphaerella musae was originally described from Musa sapientum leaves collected in Argentina. An examination of the type (IMI 91165) shows ascospores to be straight to slightly curved, fusoid-elllipsoidal with narrowly obtuse ends, being widest at the median septum (Fig. 15). Further collections would thus be required to clarify the identity of this species.

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Fig. 15. Ascospores of Mycosphaerella musae (IMI 91165). Scale bar = 10 µm.

Stenella musicola Arzanlou & Crous, sp. nov. MycoBank MB505978; Figs 14C, 16.

Stenellae musae similis, sed conidiophoris leviter longioribus et latioribus, (18–)30–36(–45) × (2–)2.5–3(–4)µm, conidiis saepe longioribus, (7–)37–57(–120) × 2–4 µm µm. A Stenella queenslandica conidiophoris 0–2-septatis et conidiis 2–4 µm latis differt.

Etymology: Named after its host, Musa.

In vitro on MEA: Mycelium submerged and superficial; submerged hyphae smooth to verrucose, thin-walled, subhyaline to olivaceous brown, 2–3 µm wide, with thin septa; aerial hyphae coarsely verrucose, olivaceous-brown, rather thick-walled, 2–2.5 µm wide, with thin septa. Conidiophores arising from superficial hyphae, pale brown, rather thick-walled, finely verruculous, 0–2-septate, occasionally continuous with supporting hyphae, subcylindrical, straight to geniculate-sinuous, unbranched, (18–)30–36(–45) × (2–)2.5–3(–4)µm. Conidiogenous cells integrated, terminal, sometimes intercalary, unbranched, pale brown, smooth or finely verruculose, cylindrical to subcylindrical, sometimes swollen at the apex, with flat-tipped apical loci, proliferating sympodially; 1–1.5 µm diam, loci thickened, darkened, refractive. Conidia solitary, rarely in unbranched chains, medium brown, thin-walled, finely verruculose subcylindrical to obclavate, with subobtuse apex, and long obconically subtruncate to obconically subtruncate base, straight to curved, 0–pluri-septate, (7–)37–57(–120) × 2–4 µm; hilum thickened, darkened, refractive, 1–1.5 µm wide.

Cultural characteristics: Colonies on MEA reaching 28 mm diam after 30 d at 24 °C; effuse, slightly raised at the centre, with moderate, velvety to hairy aerial mycelium; folded, with entire smooth margin; surface pale mouse-grey to mouse-grey; in reverse dark mouse-grey. Colonies on OA reaching 39 mm diam after 30 d at 24 °C; effuse, with moderate velvety to hairy aerial mycelium, and entire, smooth margins; surface pale mouse-grey to mouse-grey, and olivaceous in reverse.

Specimen examined: India, Tamil Nadu, Tiruchirapally, on leaf of Musa cv. Grand Nain AAA (Cav.), 2005, I. Buddenhagen, holotype CBS H-20046, culture ex-type X1019 = CBS 122479.

Notes: Stenella musicola morphologically also resembles S. citri-grisea (Teleomorph: Mycosphaerella citri), which is known from Citrus (Pretorius et al. 2003). It differs from the later species, however, based on its conidial dimensions. In S. musicola conidia range from (7–)37– 57(–120) × 2–4 µm, while in S. citri-grisea conidia are longer and narrower, namely 25–200 × 1.5–3 µm. The three new Stenella species on Musa spp. are morphologically very similar and only gradually differentiated in the size and septation of the conidiophores and conidia.

46 Gene genealogies of Mycosphaerella species on banana

Fig. 16. Stenella musicola (CBS 122479). A–E. Conidiophores with sympodially proliferating conidiogenous cells and darkened, thickened loci. F–G. Hyphal anastomoses. H–I. Conidia. Scale bar = 10 µm.

Stenella queenslandica Arzanlou & Crous, sp. nov. MycoBank MB505979; Figs 14B, 17.

Stenellae musae similis, sed conidiis longioribus, 51–83 × 2–2.5 µm. A Stenella musicola conidiophoris 1–4-septatis et conidiis saepe longioribus et angustioribus, 51–83 × 2–2.5 µm, differt.

Etymology: Named after Queensland, the state in Australia where this fungus was collected.

47 Chapter 2

Fig. 17. Stenella queenslandica (CBS 122475). A. Conidiophore with terminal conidiogenous cells. B–D. Conidia. Scale bar = 10 µm.

In vitro on MEA: Mycelium submerged and superficial; submerged hyphae smooth, thin-walled, subhyaline to olivaceous-brown, 2–3 µm wide, with thin septa; aerial hyphae coarsely verrucose, olivaceous-brown, rather thick-walled, 2–2.5 µm wide, with thin septa. Conidiophores arising from superficial hyphae, pale brown, thin-walled, finely verrucose, 1–4-septate, occasionally reduced to conidiogenous cells, subcylindrical, straight to geniculate-sinuous, unbranched, up to 40 µm long and 2–3 µm wide. Conidiogenous cells integrated, terminal, sometimes intercalary, unbranched, pale brown, smooth or finely verruculose, cylindrical, tapering to a bluntly rounded apex with flat-tipped apical loci that proliferate sympodially; loci thickened, darkened, refractive about 1 µm diam. Conidia solitary, medium brown, thin-walled, verruculose, subcylindrical to obclavate, with subobtuse to obtuse apex and long obconically subtruncate to obconically subtruncate base, straight to curved, 0–multi-septate, 51–83 × 2–2.5 µm; hilum thickened, darkened, refractive, 0.5–1 µm wide.

Cultural characteristics: Colonies on MEA reaching 24 mm diam after 30 d at 24 °C. Colonies effuse, slightly elevated at the centre with abundant aerial mycelium, and entire, smooth margins; surface mouse-grey to dark mouse-grey; dark mouse-grey in reverse. Colonies on OA reaching 41 mm diam after 30 d at 24 °C, colonies effuse, with moderate aerial mycelium, and entire, smooth margin; surface olivaceous-grey; iron-grey in reverse.

Specimen examined: Australia, Queensland, Mount Lewis, Mount Lewis Road, 16°34’47.2” S, 145°19’7” E, 538 m alt., on Musa banksii leaf, Aug. 2006, P.W. Crous, W. Gams & B. Summerell, holotype CBS H-20050, culture ex-type CBS 122475.

Notes: The ITS sequence of Stenella queenslandica is identical to that of Mycosphaerella obscuris (Burgess et al. 2007), a pathogen of Eucalyptus known from Vietnam and Indonesia. However, the latter fungus is a species of Teratosphaeria with a Readeriella anamorph (CBS 119973), which appears to be a synonym of T. suttonii (Crous & Wingfield 1997, Crouset al. 2007), and the deposited sequences (DQ632676, DQ632677) belong to another species.

DISCUSSION

The present study is the first multi-gene DNA phylogenetic study of a global set ofMycosphaerella isolates associated with the Sigatoka disease complex of banana. Considering that Sigatoka diseases are the economically most important diseases of banana and the main constraint for

48 Gene genealogies of Mycosphaerella species on banana

Fig. 18. A–C. Conidia in Pseudocercospora eumusae, P. fijiensis and P. musae, respectively. D–F. Ascospore germination pattern in M. eumusae, M. fijiensis and M. musicola. Scale bar = 10 µm. banana production worldwide (reviewed in Jones 2000), there was a huge paucity of knowledge relating to the identity of other Mycosphaerella species occurring on banana. Even though several species of Mycosphaerella have in the past been described from Musa, the majority has never been known from culture (Pont 1960, Stover 1963, 1969, 1977, 1980, 1994, Mulder & Stover 1976, Crous et al. 2003, Aptroot 2006, Arzanlou et al. 2007a). The integration of DNA analyses and morphology in the present study revealed more than 20 species of Mycosphaerella to occur on banana. Five of these species were shown to have wider host ranges than banana only, and we describe a further eight new species of Mycosphaerella from various Musa collections. The three primary agents of the Sigatoka disease complex, M. musicola, M. fijiensis and M. eumusae can be distinguished based on their conidial morphology and ascospore germination patterns (Jones 2000, Crous & Mourichon, 2002). Conidia of M. fijiensis are medium brown, and

49 Chapter 2

Fig. 19. Pseudocercospora fijiensis. A–C. Conidiophores with sympodially proliferating conidiogenous cells. D–F. Obclavate conidia with darkened hilum. Scale bar = 10 µm. have a characteristic thickening along the basal rim of the hilum, which is absent in M. musicola and M. eumusae; these two species have medium and pale brown conidia, respectively. Ascospores of M. fijiensis and M. musicola germinate from both polar ends, do not become distorted (4–5 µm wide), with a germ tube parallel to the long axis of the spore. However, in M. musicola a mucoid sheath surrounds the germinating ascospores, and the germ tubes are more irregular in width than in M. fijiensis. Ascospores of M. eumusae show some distortion upon germination (5–6 µm wide), and frequently germinate by means of 3–4 germ tubes, which grow parallel or lateral to the long axis of the spore (Figs 18–19). Thus, all of these species can be identified based on a combination of morphology and cultural characteristics, but proper identification remains problematic to the non-specialist. Hence the DNA barcodes generated in this study, along with the Taqman probes (Arzanlou et al. 2007a), are an alternative method of identification. Besides the three primary agents of the Sigatoka complex disease, which have Pseudocercospora anamorphs, three additional Pseudocercospora species were described from Musa in the present study. One of these, Pseudocercospora longispora, has in the past been confused with P. musae (teleomorph: M. musicola), and has been isolated from similar, Sigatoka disease lesions. Although these species can be distinguished based on differences in conidial size and shape, these characters overlap among the various Pseudocercospora species, making explicit identification solely possible by means of additional markers such as DNA sequence data (Fig. 2). Much confusion still surrounds the identity of M. musae. According to Stover (1994), M. musae is identical to a Stenella species called ‘Cercospora non-virulentum’. This species was considered as a prevalent co-inhabitant with black Sigatoka and yellow Sigatoka. A comparison made between isolates isolated from Mycosphaerella Speckle disease symptoms, revealed several phylogenetically distinct species to be associated with this disease. In the present study we treat four Stenella species, three of which proved to be new on banana. None of these three new species fit with the description provided for ‘Cercospora non-virulentum’ by Stover (1994), which appears to represent yet another undescribed species of Stenella. Further collections would thus be required to resolve the status of ‘Cercospora non-virulentum’ and M. musae. Data obtained in the present study revealed three species of Dissoconium on Musa, of which one is described as new. The recent revision of the genus Ramichloridium and allied genera (Arzanlou et al. 2007b), revealed that R. apiculatum, type species of Ramichloridium, has

50 Gene genealogies of Mycosphaerella species on banana phylogenetic affinity with the genusDissoconium . However, the latter genus is morphologically distinct from Ramichloridium by producing forcibly discharged pairs of primary and secondary conidia. Thus far seven species of Dissoconium have been described from different substrates, and as in the Pseudocercospora species occurring on Musa, identification is best achieved by means of molecular sequence data. It is interesting to note that up to six species have been reported during the course of the present study as occurring on hosts other than Musa. Although our present data suggest the causal agents of Sigatoka to be highly specific to banana, no information is presently available to elucidate the ecology and possible pathology of the wide host-range species, and inoculation studies would now be required to fully resolve their status as foliar pathogens of banana. The possibility exists that some of the species described here as new have been described previously on hosts other than banana. However, none of the sequences presently in GenBank, or in the MycoBank database, match any known comparable species. From the data presented in this study, it is clear that the Sigatoka disease complex is caused by a multitude of Mycosphaerella species. However, the exact contribution of each of these species to the disease complex remains unclear. The multi-locus DNA sequence data set established in this study can be used to develop species-specific molecular detection tools, which is a good alternative for traditional diagnostics. These tools can subsequently be implemented in disease management programmes.

ACKNOWLEDGEMENTS

The work of Mahdi Arzanlou was funded by the Ministry of Science, Research and Technology of Iran, which we gratefully acknowledge. Several colleagues from different countries provided material without which this work would not have been possible. Errol Reid from the Windward Islands, Yasmina Jaufeerally-Fakim from Mauritius, Gert Kema from Netherlands and Randy Christopher Ploetz from Florida, USA. We thank Marjan Vermaas for preparing the photographic plates, and Arien van Iperen for taking care of the cultures. Dr Lute-Harm Zwiers and Prof. dr P.J.G.M. de Wit are thanked for their critical review of this article, and dr Walter Gams for providing the Latin diagnoses.

REFERENCES

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51 Chapter 2

Carlier, J., Lebrun, M. H., Zapater, M. F., Dubois, C., & Mourichon, X. 1996. Genetic structure of the global population of banana black leaf streak fungus, Mycosphaerella fijiensis. Mol. Ecol. 5: 499–510. Carlier, J., Zapater, M. F., Lapeyre, F., Jones, D. R., & Mourichon, X. 2000. Septoria leaf spot of banana: a newly discovered disease caused by Mycosphaerella eumusae (anamorph Septoria eumusae). Phytopathology 90: 884–890. Cortinas, M. N., Crous, P. W., Wingfield, B. D., & Wingfield, M. J. 2006. Multi-gene phylogenies and phenotypic characters distinguish two species within the Colletogloeopsis zuluensis complex associated with Eucalyptus stem cankers. Stud. Mycol. 55: 133–146. Crous, P. W. 1998. Mycosphaerella spp. and their anamorphs associated with leaf spot diseases of Eucalyptus. Mycol. Mem. 21: 1–170. Crous, P. W., Aptroot, A., Kang, J. C., Braun, U., & Wingfield, M. J. 2000. The genus Mycosphaerella and its anamorphs. Stud. Mycol. 45: 107–121. Crous, P. W., & Braun, U. 2003. Mycosphaerella and its anamorphs. 1. Names published in Cercospora and Passalora. CBS Biodiv. Series 1: 1–571. Crous, P. W., Braun, U., & Groenewald, J. Z. 2007a. Mycosphaerella is polyphyletic. Stud. Mycol. 58: 1–32. Crous, P. W., Gams, W., Stalpers, J. A., Robert V., & Stegehuis, G. 2004a. MycoBank: an online initiative to launch mycology into the 21st century. Stud. Mycol. 50: 19–22. Crous, P. W., Groenewald, J. Z., Aptroot, A., Braun, U., Mourichon, X., & Carlier, J. 2003. Integrating morphological and molecular data sets on Mycosphaerella, with specific reference to species occurring on Musa. Pages 34–57 in: Proceedings of 2nd international workshop on Mycosphaerella leaf spot disease of bananas, Costa Rica. Jacome, L., Lepoivre, P., Marin, D., Ortiz, R., Romero, R., & Escalant, J. V. eds. 20–23 May 2002, San José, Costa Rica, INIBAP. Crous, P. W., Groenewald, J. Z., Pongpanich, K., Himaman, W., Arzanlou, M., & Wingfield, M. J. 2004b. Cryptic speciation and host specificity among Mycosphaerella spp. occurring on Australian Acacia species grown as exotics in the tropics. Stud. Mycol. 50: 457–469. Crous, P. W., Kang, J. C., & Braun, U. 2001. A phylogenetic redefinition of anamorph genera in Mycosphaerella based on ITS rDNA sequence and morphology. Mycologia 93: 1081–1101. Crous, P. W., & Mourichon, X. 2002. Mycosphaerella eumusae and its anamorph Pseudocercospora eumusae spp. nov.: causal agent of eumusae leaf spot disease of banana. Sydowia 54: 35–43. Crous, P. W., Summerell, B. A., Carnegie, A. J., Mohammed, C., Himaman, W., Groenewald, J. Z. 2007b. Foliicolous Mycosphaerella spp. and their anamorphs on Corymbia and Eucalyptus. Fungal Div. 26: 143–185. Crous, P. W., Verkley, G. J. M., & Groenewald, J. Z. 2006a. Eucalyptus microfungi known from culture. 1. Cladoriella and Fulvoflamma genera nova, with notes on some other poorly known taxa. Stud. Mycol. 55: 53–63. Crous, P. W., & Wingfield, M. J. 1997. Colletogloeopsis, a new coelomycete genus to accommodate anamorphs of Mycosphaerella occurring on Eucalyptus. Can. J. Botany 75: 667–674. Crous, P. W., Wingfield, M. J., Mansilla, J. P., Alfenas, A. C., & Groenewald, J. Z. 2006b. Phylogenetic reassessment of Mycosphaerella spp. and their anamorphs occurring on Eucalyptus. II. Stud. Mycol. 55: 99–131. Deighton, F. C. 1976. Studies on Cercospora and allied genera. VI. Pseudocercospora Speg., Pantospora Cif. and Cercoseptoria Petr. Mycol. Pap. 140: 1–168. Deighton, F. C. 1979. Studies on Cercospora and allied genera. VII. New species and redispositions. Mycol. Pap. 144: 1–56.

52 Gene genealogies of Mycosphaerella species on banana

Farr, D. F., Bills, G. F., Chamuris, G. P., & Rossman, A. Y. 1995. Fungi on plants and plant products in the United States. St Paul, MN, USA: APS Press. Gams, W., Verkley, G. J. M., & Crous, P. W. 2007. CBS course of mycology, 5th ed. Centraalbureau voor Schimmelcultures, Utrecht. Goodwin, S. B., Dunkle, D. L., & Zismann, V. L. 2001. Phylogenetic analysis of Cercospora and Mycosphaerella based on the internal transcribed spacer region of ribosomal DNA. Phytopathology 91: 648–658. Groenewald, M., Groenewald, J. Z., Braun, U., & Crous, P. W. 2006. Host range of Cercospora apii and C. beticola and description of C. apiicola, a novel species from celery. Mycologia 98: 275–285. Hayden, H. L., Carlier, J., & Aitken, E. A. B. 2003. Genetic structure of Mycosphaerella fijiensis populations from Australia, Papua New Guinea and the Pacific Islands. Plant Pathol. 52: 703–712. Hoog, G. S. de, Hijwegen T., & Batenburg-van der Vegte W. H. 1991. A new species of Dissoconium. Mycol. Res. 95: 679–682. Hunter G. C., Wingfield B. D., Crous P. W., & Wingfield M. J. 2006. A multi-gene phylogeny for species of Mycosphaerella occurring on Eucalyptus leaves. Stud. Mycol. 55. 147–161. Jackson, S. L., Maxwell, A., Neumeister-Kemp, H. G., Dell, B., & Hardy, G. E. StJ. 2004. Infection, hyperparasitism and conidiogenesis of Mycosphaerella lateralis on Eucalyptus globulus in Western Australia. Australas. J. Plant Pathol. 33: 49–53. Jones, D. R. 2000. Diseases of banana, abaca and enset. CAB International, Wallingford, Oxon, UK. Jones, D. R. 2003. The distribution and importance of the Mycosphaerella leaf spot diseases of banana. Pages 25–42 in: Proceedings of 2nd international workshop on Mycosphaerella leaf spot disease of bananas, Costa Rica. Jacome, L., Lepoivre, P., Marin, D., Ortiz, R., Romero, R., & Escalant, J. V. eds. 20–23 May 2002, San José, Costa Rica, INIBAP. Leach, R. 1941. Banana leaf spot Mycosphaerella musicola, the perfect stage of Cercospora musae Zimm. Trop. Agr. Trin.18: 91–95. Li, K. N., Rouse, D. I., & German, T. L. 1994. PCR primers that allow intergeneric differentiation of ascomycetes and their application to Verticillium spp. Appl. Environ. Microb. 60: 4324– 4331. Maxwell, A., Jackson, S. L., Dell, B., & Hardy, G. E. StJ. 2005. PCR-identification of Mycosphaerella species associated with leaf diseases of Eucalyptus. Mycol. Res. 109: 992– 1004. Morelet, M. 1969. Micromycètes du var et d’ailleurs (2 ème Note). Ann. Soc. Sci. Nat. Archéol. Toulin Var 21:104–106. Mourichon, X., & Fullerton, R. A. 1990. Geographical distribution of the two species Mycosphaerella musicola Leach (Cercospora musae) and M. fijiensis Morelet (C. fijiensis), respectively agents of Sigatoka disease and black leaf streak disease in bananas and plantains. Fruits 45: 213–218. Mulder, J. L., & Stover, R. H. 1976. Mycosphaerella species causing banana leaf spot. Trans. Brit. Mycol. Soc. 67: 77–82. Park, R. F., Keane, P. J., Wingfield, M. J., & Crous, P. W. 2000. Fungal diseases of eucalypt foliage. Pages 153–259 in: Diseases and pathogens of eucalypts. Keane, P. J., Kile, G. A., Podger, F. D., & Brown, B. N. eds. CSIRO Publishing, Collingwood, Australia. Pons, N. 1987. Notes on Mycosphaerella fijiensis var. difformis. Trans. Brit. Mycol. Soc. 89: 120–124. Pont, W. 1960. Three leaf speckle diseases of the banana in Queensland. Queensland J. Agri. Sci. 17: 271–309.

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Pretorius, M. C., Crous, P. W., Groenewald, J. Z., & Braun, U. 2003. Phylogeny of some cercosporoid fungi from Citrus. Sydowia 55: 286–305. Rayner, R. W. 1970. A mycological colour chart. CMI and British Mycological Society. Kew. Rhodes, P. L. 1964. A new banana disease in Fiji. Common Phytopath. News 10: 38–41. Rivas, G. G., Zapater, M. F., Abadie, C., & Carlier, J. 2004. Founder effects and stochastic dispersal at the continental scale of the fungal pathogen of bananas Mycosphaerella fijiensis. Mol. Ecol. 13: 471–482. Stover, R. H. 1963. Leaf spot of bananas caused by Mycosphaerella musicola: associated ascomycetous fungi. Can. J. Bot. 41: 1481–1485. Stover, R. H. 1969. The Mycosphaerella species associated with banana leaf spots. Trop. Agric. Trin. 46 325– 331. Stover, R. H. 1977. A non-virulent benomyl tolerant Cercospora from leaf spots caused by Mycosphaerella fijiensis var. difformis and M. musicola. Trans. Brit. Mycol. Soc. 69: 500– 502. Stover, R. H. 1980. Sigatoka leaf spots of banana and plantain. Plant Dis. 64: 750–755. Stover, R. H. 1994. Mycosphaerella musae and Cercospora non-virulentum from Sigatoka leaf spots are identical. Fruits 49: 187–190. Swofford, D. L. 2003. PAUP*. Phylogenetic Analysis Using Parsimony (*and other Methods). Version 4. Sinauer Associates, Sunderland, Massachusetts. Verkley, G. J. M., & Starink-Willemse, M. 2004. A phylogenetic study of some Septoria species pathogenic to Asteraceae based on ITS ribosomal DNA sequences. Mycol. Prog. 3: 315– 322. White, T. J., Bruns, T., Lee, S., & Taylor, J. 1990. Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. Pages 315–322 in: PCR Protocols: a guide to methods and applications. Innis, M. A., Gelfand, D. H., Sninsky, J. J., & White, T. J. eds. Academic Press, San Diego, California.

54 CHAPTER 3

Molecular diagnostics in the Sigatoka disease complex of banana

M. Arzanlou1,2, E.C.A. Abeln2,3, G.H.J. Kema4, C. Waalwijk4, J. Carlier5, I. de Vries4, M. Guzmán6, and P.W. Crous1,2

1Laboratory of Phytopathology, Wageningen University, Binnenhaven 5, 6709 PD Wageningen, The Netherlands; 2CBS Fungal Biodiversity Centre, Uppsalalaan 8, 3584 CT Utrecht, The Netherlands; 3TNO Quality of Life, Utrechtseweg 48, 3700 AJ Zeist, The Netherlands; 4Plant Research International BV, P.O. Box 16, 6700 AA Wageningen, The Netherlands; 5Centre de coopération internationale en recherche agronomique pour le développement (CIRAD), Montpellier, France; 6CORBANA S.A., San José, Costa Rica.

Phytopathology (2007) 97: 1112–1118.

55 Chapter 3

ABSTRACT

The Sigatoka disease complex of bananas involves three related ascomycetous fungi, viz. Mycosphaerella fijiensis, M. musicola and M. eumusae. The exact distribution of these three species, and their disease epidemiology remain unclear, since their symptoms and life cycles are rather similar. Disease diagnosis in the Mycosphaerella complex of banana is based on the presence of host symptoms and fungal fruiting structures, which hamper preventive management strategies. In the present study we have developed rapid and robust species specific molecular- based diagnostic tools for detection and quantification of M. fijiensis, M. musicola and M. eumusae. Conventional species-specific PCR primers were developed based on the actin gene that detected as little as 100, 1 and 10 pg/µl DNA from M. fijiensis, M. musicola and M. eumusae, respectively. Furthermore, TaqMan real-time quantitative PCR assays were developed based on the β-tubulin gene and detected quantities as low as 1 pg/µl DNA for each Mycosphaerella species from pure cultures and 1.6 pg/µl DNA/mg of dry leaf tissue for M. fijiensis that was validated using naturally infected banana leaves.

56 Molecular diagnostics in the Sigatoka disease complex of banana

INTRODUCTION

Banana and plantain (Musa spp.) are among the world’s most important staple food crops, ranking fourth after wheat, rice and corn, with exported bananas valued at 4.5 to 5 billion US$ per year during 1998–2000 (Ploetz 2000, Marin et al. 2003). However, bananas are susceptible to a variety of devastating diseases, including Fusarium wilt, caused by F. oxysporum f. sp. cubense, banana bunchy top virus, and Mycosphaerella associated leaf spot diseases. Although several species of Mycosphaerella have been described from banana (Crous et al. 2003, Jones 2003, Crous et al. 2004, Crous & Groenewald 2005), three species, namely M. fijiensis (causal agent of black Sigatoka disease), M. musicola (causal agent of yellow Sigatoka disease), and M. eumusae (causal agent of eumusae leaf spot disease), cause major economic losses (Ploetz 2000, Crous & Mourichon 2002). These diseases are serious threats for banana production worldwide as they reduce photosynthetic capacity of plants through necrotic leaf lesions, which result in reduced crop yield and fruit quality. Yellow Sigatoka was first described on banana in Java in 1902, and became a serious global disease epidemic during the next 40 years. The disease was especially severe in the Sigatoka Valley in Fiji, where bananas were intensively sprayed to control disease; it was also here that black Sigatoka disease first appeared in 1963 (Jones 2003). Black Sigatoka predominantly occurs in lowlands, but at altitudes of >1500 m in Colombia and Costa Rica both pathogens are equally severe (Marin et al. 2003). Mycosphaerella fijiensis is considered a quarantine organism in many banana-producing areas and continues to occupy new ecological niches, although it is presently still unknown from many countries in Latin America and the Caribbean region. More recently it has been reported from Trinidad (Fortune et al. 2005), and we have confirmed its presence in Grenada in the Caribbean region (Carlier & Arzanlou, unpublished data). Yellow Sigatoka is normally observed under cooler conditions than black Sigatoka, and ascospore and conidial germ tubes of M. musicola grow faster at cooler conditions than those of M. fijiensis (reviewed in Jones 2000, Marin et al. 2003). Yellow Sigatoka has mostly been replaced by black Sigatoka disease in Central America and the coastal areas of the Caribbean, but it remains a considerable problem in subtropical regions (Balint- Kurti et al. 2001, Jones 2003, Marin et al. 2003). Mycophaerella fijiensis is more aggressive than M. musicola, and causes in premature fruit ripening with losses of 33 to 76 % (Romero & Sutton 1997, Jones 2003). In the mid-1990’s a third Mycosphaerella species, M. eumusae, was recognized as a new constituent of the Sigatoka complex of banana (Carlier et al. 2000, Crous & Mourichon 2002). Presently M. eumusae is known from South-East Asia and parts of Africa, where it affects cultivars that are highly resistant to both M. musicola and M. fijiensis (Jones 2003). The chronology of disease records around the world suggests that South-East Asia is the centre of origin for all these three species (Jones 2003, Rivas et al. 2004). Asia is the centre of diversity of bananas as well, where earliest domestication of edible bananas took place and many wild banana cultivars still grow naturally in the forests (Jones 2003). Even though M. fijiensis has been present in South-East Asia for a long time, it has not become the dominant pathogen over M. musicola in this region. This might be due to competition among the three species or to the existence of different host populations in this region, which could lead to co-evolution of each species with different host populations in different areas (Carlier et al. 2000, Rivas et al. 2004). However, the exact distribution of the three species in this region remains unclear, since their symptoms are rather similar (Crous & Mourichon 2002, Jones 2003). A component of successful plant disease management is early detection of the pathogens that cause the Sigatoka disease complex, encompassing eradication, chemical and quarantine measurement strategies. Traditionally, diagnosis of the constituent species in the Sigatoka

57 Chapter 3 complex was based on disease symptoms, which have limited diagnostic value due to their similarity, and the long latent period that varies from 14 to 35 days, depending on weather conditions and cultivar (Balint-Kurti et al. 2001, Marin et al. 2003). Species of Mycosphaerella are distinguished by morphological characteristics that require expertise to distinguish, including ascospore morphology, germination patterns, and minute differences in the morphology of their anamorphs (Crous 1998, Crous et al. 2004). Furthermore, traditional diagnosis is time consuming, and inappropriate for eradication and quarantine management strategies. In recent years, conventional PCR-based techniques have emerged as robust tools for diagnosis and detection of phytopathogenic fungi, and have contributed greatly to plant disease management (Johanson & Jeger 1993, Johanson et al. 1994, Waalwijk et al. 2004, Lievens & Thomma 2005). However, post-amplification procedures and presence of chemical elements in plant extracts still prevent the large-scale application of diagnostic PCR in plant pathology. Moreover, in conventional PCR, the amplification efficiency is not consistent during all PCR cycles, and remains unreliable for quantitative analysis (Waalwijk et al. 2004, Lievens &

Table 1. Mycosphaerella strains and other fungi isolated from Musa included in this study. Species Isolate1 Geographical origin Mycosphaerella fijiensis X845; CBS 121360 Indonesia X848; CBS 121363 New Caledonia X842; CBS 121358 Costa Rica X841 Colombia X843 Honduras CIRAD 89 Gabon Mycosphaerella musicola X858; CBS 121372 Australia X862; CBS 121375 Cameroon X856; CBS 121370 Martinique X954 Costa Rica X62 Windward Islands CIRAD 318 Unknown Mycosphaerella eumusae X871 India X869; CBS 121380 Sri Lanka S1037D Mauritius S1037E Mauritius S1031B Mauritius CIRAD 670 Vietnam Mycosphaerella musae X38 Mozambique Mycosphaerella lateralis X1023 India Mycosphaerella thailandica X881; CBS 121388 Martinique Cordana musae CBS 151.34 Unknown Cordana johnstonii X1071 Indonesia Metulocladosporiella musae CBS 161.74 Honduras Metulocladosporiella musicola CBS 113862 Kenya 1CBS: Centraalbureau voor Schimmelcultures, Utrecht, The Netherlands; CIRAD; Centre de coopération internationale en recherche agronomique pour le développement (CIRAD), Montpellier, France; X, S: Culture collection of Mahdi Arzanlou, housed at CBS.

58 Molecular diagnostics in the Sigatoka disease complex of banana

Thomma 2005). Real-time PCR alleviates these difficulties as it combines thermal cycling with online fluorescent detection of amplificons. Unlike the end-point PCR, accumulation of amplicons is monitored during all PCR cycles. In real-time PCR reactions, the specific cycle number at which a statistically significant increase in fluorescence can be detected is defined as the threshold cycle (Ct). By definition, the tC value is inversely proportional to the log-value of the initial template DNA quantity. Therefore, real-time technology is widely used to detect and quantify DNA from plant pathogens (Waalwijk et al. 2004, Lievens & Thomma 2005). This technique enables DNA quantification at very low concentrations (pg/ml), and has been applied in quantitative detection of plant pathogens, biological control agents, plant-microbe interaction experiments (Vandermark & Barker 2003, Valsesia et al. 2005), and monitoring studies (Rohel et al. 2002, Hietala et al. 2003). The aim of the present study was to develop and optimise molecular-based detection and quantification tools for M. fijiensis, M. musicola and M. eumusae, the primary species of the Sigatoka complex of banana to further facilitate ecological and epidemiological studies of this diseases complex. A future goal would be to implement these tools in decision support systems that will be established for disease management.

MATERIALS AND METHODS

Genomic DNA for PCR analysis

The Mycosphaerella strains as well as fungal species commonly occurring on banana leaves that were used to test primer and probe specificity are listed in Table 1. Genomic DNA was extracted from axenic cultures grown on agar plates using the PureGene kit (Gentra Systems Inc., Minneapolis, MN, USA) whereas the DNeasy Plant Mini Kit (Qiagen, Germany) was used to isolate DNA from uninfected banana leaves as well as banana leaves naturally infected with M. fijiensis, M. musicola or M. eumusae. Genomic DNA was used to test for possible cross- reactions and to verify the specificity and efficacy of the real-time PCR primers and probes.

Primers for qualitative PCR

A generally applicable primer pair ACTF/ACTR (Table 2; expected amplicon size 820 bp), was designed and used to generate actin sequences from different Mycosphaerella species and other fungi commonly causing leaf spot on banana. Amplification reactions were performed with the ACTF/ACTR primer pair in a total reaction volume of 25 µl, which was composed of 1× PCR

Buffer (Applied Biosystems, Foster City, USA), 1.5 mM MgCl2, 60 µM dNTPs, 0.2 µM ACTF and ACTR primers, 1.5 U of Taq DNA polymerase (Roche Diagnostics, Indianapolis, USA) and 1 ng of genomic DNA. PCR cycle conditions were 5 min of 95 °C, followed by 36 cycles of 94 °C for 30 s, 60 °C for 30 s, 72 °C for 60 s, and a final elongation at 72 °C for 7 min. PCR amplicons were sequenced using Amersham Dye chemistry (Amersham Biosciences, USA). Primers for conventional PCR were designed based on sequence alignment of the actin gene for the 17 Mycosphaerella species associated with the Sigatoka leaf spot disease complex of banana; six known species (Table 1) and 11 undescribed Mycosphaerella species from banana (Chapter 2, this thesis). Species-specific primer combinations were designed with expected amplicon sizes of 500 bp from M. fijiensis (ACTR/MFactF), 200 bp from M. musicola (MMactF2/MMactRb) and 630 bp for M. eumusae (ACTF/MEactR).

59 Chapter 3

Table 2. Conventional and real-time PCR primers and probes designed and used in this study. Identifier Sequence (5’ → 3’) Target Gene Primers ACTF1 TCCAACCGTGAGAAGATGAC General Actin ACTR1 GCAATGATCTTGACCTTCAT General Actin MFactF CTCATGAAGATCTTGGCTGAG M. fijiensis Actin MMactF2 ACGGCCAGGTCATCACT M. musicola Actin MMactRb GCGCATGGAAACATGA M. musicola Actin MEactR GAGTGCGCATGCGAG M. eumusae Actin TMG31 CTTTCTGGCAGACCATCTCC General β-tubulin TMG41 AAGAGCTGACCGAAAGGAACC General β-tubulin MFBF CGACACAGCAAGAGCAGCTTC M. fijiensis β-tubulin MFBRtaq TTCGAAAGCCTTGGCACTTCAA M. fijiensis β-tubulin MMBF CACACATCAAGAGCAGCACAG M. musicola β-tubulin MMBRtaq TGGCACTTGGCGGAAGTTTG M. musicola β-tubulin MEBFtaq CACCTCAAGAGCAGGAGTGGAA M. eumusae β-tubulin MEBRtaq TTGGCAATTGGAGGTAGTTGTCC M. eumusae β-tubulin Probes MFBP CTGAGCACGACTGACCACAACGCA M. fijiensis β-tubulin FMEP CACGTCTGATCTCCAGCTCGAGCGCATG M. musicola & M. eumusae β-tubulin 1Resulted in amplified products for all taxa listed in Table 1.

Multiplex PCR

Specific primers for each species were used in combination with the β-tubulin gene primer set, TMG3F/TMG4 (Table 2), as an internal control to confirm that a fungus was the causal organism of the leaf spot. Specificity and cross reactions of these primer sets were verified on DNA extracted from uninfected or naturally infected banana leaves as well as from fungal species frequently occurring on banana (Table 1). The PCR amplification was performed under the same conditions as used with the ACTF/ACTR primer set.

Primer and probe design for quantitative PCR

Primer/probe sets for real-time PCR assays were designed based on partial sequences of the β-tubulin gene. Primer set TMG3/TMG4 (Table 2) was used to amplify part of the β-tubulin gene in the Mycosphaerella species listed in Table 1, and the 11 additional, though unnamed Mycosphaerella species associated with the Sigatoka disease complex (Chapter 2, this thesis). Primers and probes were designed manually and then evaluated with Beacon Designer v.4.01 software (Premier Biosoft International, http://www.premierbiosoft.com). One probe and primer combination was designed for M. fijiensis and a common probe was designed for M. musicola and M. eumusae in combination with selective primers for these two species (Table 2). Both probes were labeled with 6-carboxyfluorescein (FAM) at the 5’-end and drake deep quencher dye at the 3’-end (Eurogentec, Belgium). A potato leaf-roll virus (PLRV) probe was used as a positive internal control to discriminate between uninfected samples and false negatives due to possible PCR inhibitors (Waalwijk et al. 2004). Real-time PCR was performed using a MicroAmp Optical 96-well reaction plate and MicroAmp Optical Caps (Applied Biosystems, Foster City, USA). An ABI Prism 7700

60 Molecular diagnostics in the Sigatoka disease complex of banana

Sequence Detection System (Applied Biosystems) was used to perform the PCR and assess fluorescence. Each amplification reaction consisted of 1 ng of genomic DNA, 1× real-time

PCR buffer (Applied Biosystems), 5 mM MgCl2, 83 nM of the FAM-labeled TaqMan probe, 83 nM of the VIC-labeled PLRV probe, 1.5 U of Hot Gold star DNA polymerase (Eurogentec, Belgium) and 333 nM of forward and reverse primer for each target DNA, and the positive PLRV internal control in a reaction volume of 30 µl. In each assay, non-template DNA and uninfected banana DNA templates were run in parallel as negative controls. The thermocycling profile for conventional diagnostic PCR consisted of an initial incubation of 2 min at 50 °C followed by incubation of 10 min at 95 °C, and 40 cycles of 15 s at 95 °C and 1 min at 60 °C.

Standard curves

To calculate the amount of fungal biomass in field samples a standard regression curve was made using serial dilutions of pure genomic DNA of M. fijiensis, M. musicola and M. eumusae in distilled water (10000, 1000, 100, 10 and 1 pg/µl). Samples without DNA template and uninfected banana DNA template served as negative controls. Standard regression curves for each species were constructed by plotting the log of the known amount of DNA versus the Ct values measured by SDS 7700 software (Applied Biosystems). Serial dilutions were included in each new run, and used to calculate the amount of fungal biomass in naturally infected banana leaves.

Validation of TaqMan real-time assay

Infected plant material was used to test the sensitivity of TaqMan primers and probes, namely artificially inoculated banana leaves (detached leaf inoculation), and naturally infected field samples. For artificial inoculation, isolates of M. fijiensis, M. musicola and M. eumusae were individually or in a three-way mixture inoculated on to banana leaves of cv. ‘Grand Naine’ (AAA, Cavendish subgroup) using a detached leaf assay. These banana plants were initiated from tissue culture and grown in a glasshouse for five to seven months. Leaf pieces (6 × 6 cm) were cut from the youngest, fully mature leaf and placed in Petri dishes with the adaxial surface on the medium (0.4 % water agar and 50 ppm benzimidazole, Sigma). Mycosphaerella fijiensis isolate CIRAD 89, M. musicola isolate CIRAD 318 and M. eumusae isolate CIRAD 670 were cultured at 20 °C for 10 to 14 days under 60 µmoles m2/s of continuous and cool-white fluorescent light on modified V8-juice medium (100 ml of V8 juice, 0.2 g of CaCo3, 20 g agar per liter of medium, pH 6) to initiate conidiation. Water was used to dislodge conidia from the culture and the resulting suspension was adjusted to 3000 conidia/ml with 1000 conidia of each isolate/ml for the mixtures (Abadie et al. 2005). One ml of the suspension was used to inoculate each leaf fragment with an artist’s airbrush (Badger air-brush n°150-1-M). Two leaf pieces in two replicates were used for each treatment. Water-sprayed leaf fragments were used as controls. After inoculation, leaf fragments were incubated at 25 °C with a 12 h photoperiod (4000 lux) for symptom expression. For each treatment, two leaf pieces were collected at 10 and 30 days after inoculation and stored at -20 °C. Leaf pieces were lyophilized and ground to a fine powder, and 10 mg were used to extract DNA using the DNeasy Plant Mini Kit (Qiagen, Germany) according to the recommendations of the manufacturer. Each sample was analyzed in two replicates in a single run. To validate TaqMan real-time assay on field samples, we used naturally infected banana leaves with black Sigatoka symptoms. Samples (three to six leaf pieces with early and advanced symptoms) were collected from nine banana fields in Costa Rica at different altitudes

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(Table 3). Sample preparation and DNA extraction were performed as explained above for artificially inoculated leaves, and analyzed using the M. fijiensis (the major pathogen in the region) primer and probe set in three different runs. The data were analyzed using a t-student test to evaluate the statistical significance between treatments (95 % confidence interval, α = 0.05).

Reproducibility of real-time PCR assays

Different sources of variation that could influence real-time PCR efficiency were evaluated using banana leaves that were naturally-infected with M. fijiensis, M. musicola or M. eumusae. The identity of the Mycosphaerella species on these leaves was determined after isolation using morphological characters and was confirmed by sequencing the β-tubulin gene. Samples represented 10 cm2 leaf pieces, 10 of which were selected for each treatment. We determined (i) intra-assay accuracy, influenced by well-to-well differences in signal measurements, pipetting, and PCR efficiency; (ii) Inter-assay variability, that may be due to quantitative differences in reaction components among runs and (iii) inter-sample reproducibility, which could be affected by differences in sample selection, DNA extraction efficiency and/or amplification efficacy among different samples (Winton et al. 2002, Vandermark & Barker 2003). One sample of each Mycosphaerella species was analyzed in 12 replicates in a single assay, to evaluate the intra- assay accuracy. To evaluate the inter-assay variability, the same DNA samples were analyzed in five replicates over three separate assays. The third source of variation was assessed on DNA extracted from eight leaf samples for each of the three Mycosphaerella species.

M 1 2 3 4 5 6 7 8 9 10 11 500 bp 300 bp

A

200 bp B

630 bp

C

Fig. 1. Amplification results with species-specific primer sets in combination with a positive internal control (TMG3/TMG4) using template DNA from M. fijiensis (ACTR/MFactF) (A), M. musicola (MMactF2/MMactRb) (B), and M. eumusae (MEactR/ACTF) (C). Genomic DNA from pure cultures of M. fijiensis, M. musicola and M. eumusae (lanes 1, 4 and 7, respectively), and naturally-infected bananas with M. fijiensis (lanes 2–3), M. musicola (lanes 5–6), M. eumusae (lanes 8–9) and banana (lane 10); Lane 11 negative control (water and no template DNA). Lane labelled M contains DNA marker.

62 Molecular diagnostics in the Sigatoka disease complex of banana

Table 3. Source of field samples used for validation of real-time PCR assays developed in this study. Locality Altitude (meter) Cultivar Country La Rita 100 Cavendish Costa Rica Siquires 100 Gros Michel Costa Rica Catie 620 Cavendish Costa Rica Tres Equis 650 Gros Michel Costa Rica Santa Marta 700 Plantain Costa Rica Verbena 1110 Gros Michel Costa Rica La Victoria 1240 Plantain Costa Rica Cervantes 1340 Gros Michel Costa Rica San Luis 1360 Cavendish Costa Rica

RESULTS

Qualitative and quantitative PCR assays

Primer set TMG3/TMG4 amplified part of the β-tubulin gene from theMycosphaerella species listed in Table 1, and no amplicon was obtained when Musa DNA was used as template (data not shown). Sequence alignment of amplicons showed unique sites for each species, which enabled us to design species-specific primers and probes.

Primers for conventional PCR

An actin sequence from a M. graminicola EST (expressed sequence tag) data base (Kema et al. 2003) was used to design a generally applicable primer pair ACTF/ACTR that was used to generate actin sequences from six Mycosphaerella species known from banana (Table 1), and 11 additional unnamed Mycosphaerella species (Chapter 2, this thesis). This primer set resulted in an 820 bp amplicon. Alignment of these sequences enabled the design of three Mycosphaerella species-specific primer sets: ACTR/MFactF amplified a 500 bp DNA fragment from M. fijiensis; MMactF2/MmactRb amplified a 200 bp DNA fragment from M. musicola, and ACTF/MEactR amplified a 630 bp DNA fragment fromM. eumusae (Table 2, Fig. 1). Each PCR reaction was performed in combination with the β-tubulin primer set (TMG3F/TMG4), as a universal fungal internal control to check whether a fungal agent was responsible for the disease symptom (Fig. 1). The specific primer sets forM. fijiensis, M. musicola and M. eumusae were also tested against the naturally-infected banana leaves (field samples) and resulted in the expected diagnostic bands (Fig. 1). The sensitivity of the primer sets enabled reliable detection of M. fijiensis DNA as low as 100 pg, whereas for M. musicola and M. eumusae a higher sensitivity was achieved of 1 pg and 10 pg genomic DNA, respectively (Fig. 2).

Real-time PCR

In order to quantify the fungal biomass in positively diagnosed diseased leaves we developed real time probe and primer sets for the three Mycosphaerella species. The aforementioned β-tubulin primer set TMG3F/TMG4 did not enable the design of species-specific primers for conventional PCR assays but was successfully used to design a TaqMan real-time assay primer and probe as it amplified 330 bp fragments from the Mycosphaerella species listed in Table 1. The species-specific real-time PCR primers that we designed produced unique amplicons

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M 1 2 3 4 5 M 1 2 3 4 5 M 1 2 3 4 5

500 bp

A B C

Fig. 2. Sensitivity of the species-specific primer pairs. Lanes 1–5 contain 10-fold serial dilutions (1000, 100, 10, 1, 0.1 pg/µl DNA respectively) of M. fijiensis (A), M. musicola (B), and M. eumusae (C). Lane labelled M contains DNA marker.

35

30

25

CT value y = -3.63x + 33.95 2 20 R = 0.99 PCR efficiency value = 88.34% 15 0 1 2 3 4 5 M. fijiensis DNA(pg) log(10)

Fig. 3. Standard curve regression between the Ct values and the log of known amounts of M. fijiensis DNA. Such curves were developed using purified fungal DNA in each assay in order to estimate the amount of fungal DNA in naturally-infected leaves. The PCR efficiency value was calculated using the following formula: efficiency = -1+10(-1/slope). from target species; a 134 bp obtained from M. fijiensis using primer set (MFBF/MFBRtaq), primer set (MMBF/MMBRtaq) produced a 142 bp amplicon from M. musicola, and primer set (MEBFtaq/MEBRtaq) yielded a 134 bp amplicon from M. eumusae. In TaqMan real-time assays, the M. fijiensis specific primer set was used in combination with the MFBP probe, which is specific for this species. For M. musicola and M. eumusae the species-specific primer set for each species was used in combination with the common FMEP probe (Table 2). The fluorescent signals measured for non-target species and non-template DNA controls were all at the base line

(Ct = 40), whereas the fluorescent signals for the target species passed the baseline threshold at

Ct = 24 for M. fijiensis, at Ct = 22 for M. musicola, and at Ct = 24 for M. eumusae. The real-time PCR assay enabled the detection of 1 pg of DNA for each Mycosphaerella species. The standard curves were generated by plotting the Ct values against the log of the known amounts of serially diluted DNA in distilled water, which resulted in linear relationships for each Mycosphaerella species. For M. fijiensis R2 = 0.99, with a PCR efficiency value of 88.34 % (Fig. 3); for M. musicola R2 = 0.99, with a PCR efficiency value of 110 %, and for, M. eumusae R2 = 0.99, with a PCR efficiency value = 87 % (data not shown).

64 Molecular diagnostics in the Sigatoka disease complex of banana

100000,0 Early symptom 10000,0 Advanced symptom

1000,0

100,0 weight 10,0 Pg DNA/mg of dryPg DNA/mg of 1,0 100 100 620 650 700 1110 1240 1340 1360 Altitude Fig. 4. Validation of TaqMan real-time assay using DNAs extracted from naturally infected banana leaves with M. fijiensis. The DNA concentration was estimated based on serial dilutions of purified fungal DNA included in each experiment. Bars show the standard deviation values, each value is the mean of 3–6 independently sampled leaves.

Validation of TaqMan real-time assay

Each primer and probe set was used to amplify DNA from all target species in separate and mixed reactions without any undesired cross-reaction with non-target species (data not shown). Each primer and probe specifically detected the target species and was sensitive to detect the target species within 10 days post inoculation (dpi). However, quantitative analysis on the detached leaf material was not reliable, which was apparently mainly due to the artificial inoculation of detached leaves. The quantitative data obtained were inconsistent between replicates of each treatment. A considerable amount of variation was observed in the fungal biomass detected between different replicates of each treatment in mixed and separate inoculations. In order to validate the efficacy of primers and probes, we subsequently collected and analyzed naturally infected black Sigatoka field samples. The results obtained from the field sample analyses further confirmed the efficacy of primers and probes developed in this study. The M. fijiensis primer and probe set detected and quantified biomass as low as 1.6 pg of target DNA per milligram dry weight leaf tissue (Fig. 4). There was a significant difference (P = 0.056) in fungal biomass between early and advanced symptoms, which supports the sensitivity of the quantification tool (Fig. 4).

Reproducibility of real-time assays

Banana leaves naturally infected with M. fijiensis, M. musicola and M. eumusae were used to test the reproducibility of the TaqMan real-time assay. The intra-assay values revealed very low standard deviations among the repeats, e.g. the mean Ct value for M. fijiensis, M. musicola, and M. eumusae were 23 ± 0.77, 22.4 ± 0.31, and 24 ± 0.8, respectively (Fig. 5). The inter-assay experiments, testing the reproducibility within the same experiment in different runs, also produced consistent results with low standard deviations, e.g. the mean Ct value for M. fijiensis, M. musicola and M. eumusae were 22.7 ± 0.93, 22.5 ± 0.22, and 23 ± 0.7, respectively. As expected, standard deviations from the inter-sample reproducibility test were higher than the above mentioned assays, e.g. the mean Ct values for M. fijiensis and M. musicola were 21.5 ± 1.4, 23.5 ± 1.4, and 25 ± 1.7, respectively.

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35 M. fijiensis M. musicola 30 M. eumusae

25 Ct value Ct

20

15 Intra-assay Inter-assay Inter-sample Fig. 5. Assessment of inter-assay, intra-assay and inter-sample variability using DNAs extracted from banana leaves naturally infected with M. fijiensis, M. musicola and M. eumusae. Values are the average of replications (12 replicates in a single run for intra-assay; five replicates in three repeated experiments for inter-assay, and eight DNA samples in a single run for the inter-sample assay). Bars show the standard deviation values.

DISCUSSION

Understanding of the banana Sigatoka disease complex is a challenge for plant pathologists. Knowledge about the identity of the Mycosphaerella species and their distribution in banana producing areas as well as on specific host-pathogen interactions should enhance the ability of pathologists to understand the dynamics of these pathogens and thereby allow better management of this disease complex. Our primary aim was to develop a rapid and robust detection tool with the feasibility of wide application. Even though a PCR-based detection tool has previously been developed (Johanson & Jeger 1993, Johanson et al. 1994), those primers could only differentiate M. fijiensis from M. musicola. We, therefore, developed and optimised a qualitative and quantitative molecular diagnostics set for the three dominant Mycosphaerella species currently recognized in the Sigatoka complex on banana. We successfully developed species-specific primers for conventional diagnostic PCR assays for M fijiensis, M. musicola and M. eumusae that differentiate these species from each other, and from other fungal species commonly occurring on banana (Arzanlou, unpublished data), without any undesired cross-reaction. Selective primers for quantitative Taqman assays were designed based on sequence data of the β-tubulin gene that provided unique sites to design the selective primer and probe sets for Taqman real-time PCR with an expected amplicon size of ≤142 bp. These very short amplicons (≤142 bp) could not be used for conventional PCR as they cannot be visualized on agarose gels. These short amplicons are suitable for Taqman quantification assays, as amplification is monitored by the level of fluorescent signal. Each primer and probe set resulted in specific detection for the target species. Inclusion of sequence data from up to 11 unnamed Mycosphaerella species occurring on banana (Chapter 2, this thesis) in the alignment, ensured specificity of each primer and probe set designed for the quantification assay. The Sigatoka complex on banana used to be limited to M. musicola, M. fijiensis and more recently M. eumusae. Given the emergence of M. eumusae and the occurrence of many novel,

66 Molecular diagnostics in the Sigatoka disease complex of banana cryptic Mycosphaerella species on banana (Chapter 2, this thesis ), there is an urgent need for a robust, reliable and sensitive PCR primer set to detect and differentiate these commonly occurring three species from each other as well as from new additional Mycosphaerella species. Despite the fact that conventional PCR offers the advantage of being simple and relatively cheap per reaction, it is inappropriate for quantitative studies and time consuming in large- scale application. TaqMan real-time PCR assays, as developed in this study, facilitate the quantification of fungal DNA, even when present in very low amounts. Hence, TaqMan assays can support ecological and epidemiological studies, by quantifying the effect of agronomical measures such as fungicide applications, the efficacy of biological control agents, and host resistance on fungal biomass development (Rohel et al. 2002, Vandermark & Barker 2003, Heitala et al. 2003, Valsesia et al. 2005). The reproducibility of the real-time assays developed in this study was excellent. As expected, inter-sample variation affects the reproducibility of real-time assays, which can be due to differences in sample selection, DNA extraction, and PCR efficacy, emphasizing the importance of optimal sampling strategies (Valsesia et al. 2005). Biomass quantification in real-time assay is based on calibrated standard curves. These curves can be constructed by using serial dilutions of known amounts of fungal DNA in distilled water (Waalwijk et al. 2004), or in host DNA (Valsesia et al. 2005). The latter may be useful to compensate any possible effect of host DNA on the efficiency of the PCR reactions. As no such effect was observed, we used standard curves based on serial DNA dilutions of the target species in distilled water. Our assays showed that each primer and probe set could detect quantities as low as 1 pg/µl DNA for each Mycosphaerella species from pure culture. Our results confirmed each primer and probe as being specific, detecting the target species after 10 dpi without any undesired cross-reactivity in separate and mixed inoculations. However, quantification of fungal biomass using artificially inoculated banana leaves (detached leaf assay) was not reliable. The quantification results were inconsistent, as significant variation was observed between different replicates of each treatment. Nevertheless, the results obtained from analysing field samples showed a minimum detection level of 1.6 pg of DNA/mg of dry leaf tissue for M. fijiensis, which confirms the high sensitivity of the detection tool. Our data suggest that the detached leaf inoculation method is unsuitable for quantification studies. This might be due to the semi- biotrophic nature of Mycosphaerella pathogens of banana, which requires the host plant to be in optimal condition to facilitate disease development. The validation on field samples from Costa Rica was successful and further confirmed the presence ofM. fijiensis at higher altitudes, which indicates that M. fijiensis has the ability for adaptation to cooler conditions, suggesting that it might pose a potential threat for banana cultivation in subtropical countries. The molecular-based detection and quantification tools developed and optimized in this study are good starting points towards the better understanding of the Sigatoka disease complex of banana. The probe and primer sets we designed will facilitate further ecological and epidemiological studies on the Mycosphaerella pathogens of banana.

ACKNOWLEDGEMENTS

Mahdi Arzanlou was funded by the Ministry of Science, Research and Technology of Iran, which we gratefully acknowledge. Some of the infected banana materials used in this study was collected by dr I. Buddenhagen (University of California, Davis, USA), for which we are thankful. We also acknowledge the Dutch Mycosphaerella group for valuable discussions and support, and thank CORBANA S.A., San José, Costa Rica for advice, and scientific interaction.

67 Chapter 3

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Phylogenetic and morphotaxonomic revision of Ramichloridium and allied genera

M. Arzanlou1,2, J.Z. Groenewald1, W. Gams1, U. Braun3 , H.-D. Shin4 and P.W. Crous1,2

1CBS Fungal Biodiversity Centre, Uppsalalaan 8, 3584 CT Utrecht, The Netherlands; 2Laboratory of Phytopathology, Wageningen University, Binnenhaven 5, 6709 PD Wageningen, The Netherlands; 3Martin-Luther-Universität, Institut für Biologie, Geobotanik und Botanischer Garten, Neuwerk 21, D-06099 Halle (Saale), Germany; 4Division of Environmental Science & Ecological Engineering, Korea University, Seoul 146-701, Korea

Studies in Mycology (2007) 58: 57–93.

71 Chapter 4

ABSTRACT

The phylogeny of the genera Periconiella, Ramichloridium, Rhinocladiella and Veronaea was explored by means of partial sequences of the 28S (LSU) rRNA gene and the ITS region (ITS1, 5.8S rDNA and ITS2). Based on the LSU sequence data, ramichloridium-like species segregate into eight distinct clusters. These include the Capnodiales (Mycosphaerellaceae and Teratosphaeriaceae), the Chaetothyriales (), the , and five ascomycete clades with uncertain affinities. The type species of Ramichloridium, R. apiculatum, together with R. musae, R. biverticillatum, R. cerophilum, R. verrucosum, R. pini, and three new species isolated from Strelitzia, Musa and forest soil, respectively, reside in the Capnodiales clade. The human-pathogenic species R. mackenziei and R. basitonum, together with R. fasciculatum and R. anceps, cluster with Rhinocladiella (type species: Rh. atrovirens, Herpotrichiellaceae, Chaetothyriales), and are allocated to this genus. Veronaea botryosa, the type species of the genus Veronaea, also resides in the Chaetothyriales clade, whereas Veronaea simplex clusters as a sister taxon to the (Pleosporales), and is placed in a new genus, Veronaeopsis. Ramichloridium obovoideum clusters with Carpoligna pleurothecii (anamorph: Pleurothecium sp., Chaetosphaeriales), and a new combination is proposed in Pleurothecium. Other ramichloridium-like clades include R. subulatum and R. epichloës (incertae sedis, Sordariomycetes), for which a new genus, Radulidium is erected. Ramichloridium schulzeri and its varieties are placed in a new genus, Myrmecridium (incertae sedis, Sordariomycetes). The genus Pseudovirgaria (incertae sedis) is introduced to accommodate ramichloridium- like isolates occurring on various species of rust fungi. A veronaea-like isolate from Bertia moriformis with phylogenetic affinity to the Annulatascaceae (Sordariomycetidae) is placed in a new genus, Rhodoveronaea. Besides Ramichloridium, Periconiella is also polyphyletic. Thysanorea is introduced to accommodate Periconiella papuana (Herpotrichiellaceae), which is unrelated to the type species, P. velutina (Mycosphaerellaceae).

TAXONOMIC NOVELTIES

Myrmecridium Arzanlou, W. Gams & Crous, gen. nov., Myrmecridium flexuosum (de Hoog) Arzanlou, W. Gams & Crous, comb. et stat. nov., Myrmecridium schulzeri (Sacc.) Arzanlou, W. Gams & Crous var. schulzeri, comb. nov., Myrmecridium schulzeri var. tritici (M.B. Ellis) Arzanlou, W. Gams & Crous, comb. nov., Periconiella arcuata Arzanlou, S. Lee & Crous, sp. nov., Periconiella levispora Arzanlou, W. Gams & Crous, sp. nov., Pleurothecium obovoideum (Matsush.) Arzanlou & Crous, comb. nov., Pseudovirgaria H.-D. Shin, U. Braun, Arzanlou & Crous, gen. nov., Pseudovirgaria hyperparasitica H.-D. Shin, U. Braun, Arzanlou & Crous, sp. nov., Radulidium Arzanlou, W. Gams & Crous, gen. nov., Radulidium epichloës (Ellis & Dearn.) Arzanlou, W. Gams & Crous, comb. nov., Radulidium subulatum (de Hoog) Arzanlou, W. Gams & Crous, comb. nov., Ramichloridium australiense Arzanlou & Crous, sp. nov., Ramichloridium biverticillatum Arzanlou & Crous, nom. nov., Ramichloridium brasilianum Arzanlou & Crous, sp. nov., Ramichloridium strelitziae Arzanlou, W. Gams & Crous, sp. nov., Rhinocladiella basitona (de Hoog) Arzanlou & Crous, comb. nov., Rhinocladiella fasciculata (V. Rao & de Hoog) Arzanlou & Crous, comb. nov., Rhinocladiella mackenziei (C.K. Campb. & Al-Hedaithy) Arzanlou & Crous, comb. nov., Rhodoveronaea Arzanlou, W. Gams & Crous, gen. nov., Rhodoveronaea varioseptata Arzanlou, W. Gams & Crous, sp. nov., Thysanorea Arzanlou, W. Gams & Crous, gen. nov., Thysanorea papuana (Aptroot) Arzanlou, W. Gams & Crous, comb. nov., Veronaea japonica Arzanlou, W. Gams & Crous, sp. nov., Veronaeopsis Arzanlou & Crous, gen. nov., Veronaeopsis simplex (Papendorf) Arzanlou & Crous, comb.nov.

72 Ramichloridium and allied genera

Introduction

The anamorph genus Ramichloridium Stahel ex de Hoog 1977 presently accommodates a wide range of species with erect, dark, more or less differentiated, branched or unbranched conidiophores and predominantly aseptate conidia produced on a sympodially proliferating rachis (de Hoog 1977). This heterogeneous group of anamorphic fungi includes species with diverse life styles, viz. saprobes, human and plant pathogens, most of which were classified by Schol-Schwarz (1968) in Rhinocladiella Nannf. according to a very broad generic concept. Ramichloridium was originally erected by Stahel (1937) with R. musae Stahel as type species. However, because his publication lacked a Latin diagnosis, the genus was invalid. Stahel also invalidly described Chloridium musae Stahel for a fungus causing leaf spots (tropical speckle disease) on banana. Ellis (1976) validated Chloridium musae as Veronaea musae M.B. Ellis, and Ramichloridium musae as Periconiella musae Stahel ex M.B. Ellis. Periconiella Sacc. (1885) [type species P. velutina (G. Winter) Sacc.] differs from Veronaea Cif. & Montemart. chiefly based on its dark brown, apically branched conidiophores. However, de Hoog (1977) observed numerous specimens of V. musae to exhibit branched conidiophores in culture, as did Stahel (1937) for Ramichloridium musae. De Hoog (1977) subsequently re- introduced Ramichloridium, but typified it with R. apiculatum (J.H. Mill., Giddens & A.A. Foster) de Hoog. He regarded V. musae and P. musae to be conspecific, and applied the name R. musae (Stahel ex M.B. Ellis) de Hoog to both species, regarding Periconiella musae as basionym. The circumscription by de Hoog was based on their similar morphology and ecology. Central in his genus concept was the observed presence of more or less differentiated and pigmented conidiophores, with predominantly aseptate conidia produced on a sympodially proliferating rachis. De Hoog (1977) also used some ecological features as additional characters to discriminate Ramichloridium from other genera, noting, for instance, that species in Ramichloridium were non-pathogenic to humans (de Hoog 1977, Campbell & Al-Hedaithy 1993). This delimitation, however, was not commonly accepted (McGinnis & Schell 1980). De Hoog et al. (1983) further discussed the problematic separation of Ramichloridium from genera such as Rhinocladiella, Veronaea and Cladosporium Link. It was further noted that the main feature to distinguish Ramichloridium from Rhinocladiella, was the presence of exophiala-type budding cells in species of Rhinocladiella (de Hoog 1977, de Hoog et al. 1983, Veerkamp & Gams 1983). The separation of Veronaea from this complex is more problematic, as the circumscriptions provided by Ellis (1976) and Morgan-Jones (1979, 1982) overlap with that of Ramichloridium sensu de Hoog (1977). Cladosporium is more distinct, having very conspicuous, protuberant, darkened and thickened, coronate conidial scars, and catenate conidia (David 1997, Braun et al. 2003, Schubert et al. 2007). To date 26 species have been named in Ramichloridium; they not only differ in morphology, but also in life style. Ramichloridium mackenziei C.K. Campb. & Al-Hedaithy is a serious human pathogen, causing cerebral phaeohyphomycosis (Al-Hedaithy et al. 1988, Campbell & Al-Hedaithy 1993), whereas R. musae causes tropical speckle disease on members of the Musaceae (Stahel 1937, Jones 2000). Another plant-pathogenic species, R. pini de Hoog & Rahman, causes a needle disease on Pinus contorta (de Hoog et al. 1983). Other clinically relevant species of Ramichloridium are R. basitonum de Hoog and occasionally R. schulzeri (Sacc.) de Hoog, while the remaining species tend to be common soil saprobes. No teleomorph has thus far been linked to species of Ramichloridium. The main question that remains is whether shared morphology among the species in this genus reflects common ancestry (Seifert 1993, Untereiner & Naveau 1999). To delineate anamorphic genera adequately, morphology and conidial ontogeny alone are no longer satisfactory (Crous et al. 2006a, b),

73 Chapter 4 EU041848, EU041791 EU041847, EU041790 EU041846, EU041789 EU041845, EU041788 EU041844, EU041787 EU041843, EU041786 EU041842, EU041785 EU041824, EU041767 EU041823, EU041766 EU041822, EU041765 EU041841, EU041784 EU041840, EU041783 EU041839, EU041782 EU041838, EU041781 EU041837, EU041780 EU041836, EU041779 EU041835, EU041778 EU041834, EU041777 EU041833, EU041776 EU041832, EU041775 EU041831, EU041774 EU041830, EU041773 EU041829, EU041772 EU041828, EU041771 EU041827, EU041770 EU041826, EU041769 EU041825, EU041768 Genbank numbers ( L S U , ITS) U.S.A. Czech Republic Germany Czech Republic U.K. Guyana U.S.A. Korea Korea Korea Japan South Africa South South Africa South South Africa South Sri Lanka South Africa South South Africa South Switzerland Australia Netherlands South Africa South South Africa South Papua New Guinea Netherlands Germany Zaire Suriname Origin on Agrimonia pilosa Forest soil Lasioptera arundinis Incubator for cell cultures Phragmites australis Puccinia allii Poaceae Epichloë typhina On Pucciniastrum agrimoniae On Phragmidium sp. on Rubus coreanus On Phragmidium sp. on Rubus coreanus Pasania edulis Brabejum stellatifolium Brabejum stellatifolium Brabejum stellatifolium Turpinia pomifera Turpinia Ischyrolepsis subverticellata Ischyrolepsis Cannomois virgata Malus sylvestris — Triticum aestivum Triticum Wheat straw Wheat straw Soil Homo sapiens Soil Soil Soil Source 1 and similar genera used for DNA analysis and morphological studies. and similar genera used for DNA CBS 156.59*; ATCC 13211; IMI 13211; ATCC CBS 156.59*; 7991; 100716; JCM 6972; MUCL 15753; QM 7716 MUCL CBS 101010 CBS 912.96 CBS 405.76* CBS 287.84 CBS 115704 CBS 361.63*; MUCL 3124 CBS 361.63*; MUCL CBS 121739*; CPC 10753 CBS 121738; CPC 10704 CBS 121735; CPC 10702 CBS 209.95*; MFC 12477 CBS 101950; CPC 2264 CBS 101949; CPC 2263 CBS 101948*; CPC 2262 CBS 873.73* CBS 113477* CBS 114996 CBS 642.76 CBS 381.87 CBS 325.74; JCM 7234 CBS 305.73; JCM 6967 CBS 304.73 CBS 188.96 CBS 156.63 CBS 134.68; ATCC 16310 ATCC CBS 134.68; CBS 100.54; JCM 6974 Accession number CBS 398.76*; IMI 203547 sp. Table 1. Isolates of Ramichloridium Table Ramichloridium apiculatum Radulidium subulatum Radulidium Radulidium epichloës Pseudovirgaria hyperparasitica Pseudovirgaria Pleurothecium obovoideum Pleurothecium Periconiella velutina Periconiella levispora Periconiella arcuata Myrmecridium schulzeri Species Myrmecridium flexuosum

74 Ramichloridium and allied genera EU041873, EU041816 EU041872, EU041815 EU041871, EU041814 EU041870, EU041813 EU041869, EU041812 EU041868, EU041811 EU041867, EU041810 EU041866, EU041809 EU041865, EU041808 EU041864, EU041807 EU041863, EU041806 EU041862, EU041805 EU041861, EU041804 EU041860, EU041803 EU041859, EU041802 EU041858, EU041801 EU041857, EU041800 EU041856, EU041799 EU041855, EU041798 EU041854, EU041797 EU041853, EU041796 EU041852, EU041795 EU041851, EU041794 EU041850, EU041793 EU041849, EU041792 Genbank numbers ( L S U , ITS) Italy Australia Papua New Guinea Germany France Colombia United Arab Emirates United Israel Israel India Japan Canada France South Africa South U.K. Surinam — — Japan Brazil — Australia Pakistan South Africa South South Africa South Origin sp. sp. — Xanthorrhoea preissii Xanthorrhoea — Bertia moriformis Honey Soil Homo sapiens Homo sapiens Homo sapiens Decayed wood Homo sapiens Soil Fagus sylvatica Strelitzia Strelitzia Pinus contorta Musa sapientum Musa sapientum — Sasa sp. Forest soil Musa sapientum Musa banksii Soil Aloe Cucumis sativus Source 1 CBS 254.57*; IMI 070233; MUCL 9821 CBS 254.57*; IMI 070233; MUCL CBS 121.92 CBS 212.96* CBS 431.88* CBS 264.49; MUCL 9904 CBS 264.49; MUCL CBS 496.78*; IMI 287527 CBS 102590; NCPF 2853 CBS 368.92; UTMB 3170 CBS 367.92; NCPF 2738; UTMB 3169 CBS 132.86* CBS 101460*; IFM 47593 CBS 181.65*; ATCC 18655; DAOM ATCC CBS 181.65*; 8233; OAC 84422; IMI 134453; MUCL 10215 CBS 157.54; ATCC 15680; MUCL 15680; MUCL ATCC CBS 157.54; 15756 7992; MUCL 1081; MUCL CBS 121711 CBS 461.82*; MUCL 28942 CBS 461.82*; MUCL CBS 365.36*; JCM 6973; MUCL 9556 CBS 365.36*; JCM 6973; MUCL CBS 190.63; MUCL 9557 CBS 190.63; MUCL CBS 171.96 CBS 103.59* CBS 283.92* CBS 335.36 CBS 121710 CBS 400.76; IMI 088021 CBS 391.67; JCM 6966 Accession number CBS 390.67 sp. Table 1. (Continued). Table Veronaea botryosa Veronaea Thysanorea papuana Thysanorea Rhodoveronaea varioseptata Rhodoveronaea Rhinocladiella Rhinocladiella phaeophora Rhinocladiella fasciculata Rhinocladiella basitona Rhinocladiella anceps Ramichloridium strelitziae Ramichloridium pini Ramichloridium musae Ramichloridium indicum Ramichloridium cerophilum Ramichloridium brasilianum Ramichloridium biverticillatum Ramichloridium australiense Species

75 Chapter 4

and DNA data provide additional characters to help delineate species and genera (Taylor et al. 2000, Mostert et al. 2006, Zipfel et al. 2006). The aim of the present study was to integrate morphological and cultural features with DNA sequence data to resolve the species concepts and generic limits of the taxa currently placed EU041878, EU041821 EU041877, EU041820 EU041875, EU041818 EU041876, EU041819 EU041874, EU041817 Genbank numbers ( L S U , ITS) in Periconiella, Ramichloridium, Rhinocladiella and Veronaea, and to resolve the status of several new cultures that were isolated during the course of this study.

Materials and Methods — South Africa South Japan South Africa South India Origin

Isolates

Species names, substrates, geographical origins and GenBank accession numbers of the isolates included in this study are listed in Table 1. Fungal isolates are maintained in the culture collection of the Centraalbureau voor Schimmelcultures (CBS) in Utrecht, the Netherlands.

DNA extraction, amplification and sequence analysis Wine cellar Wine Acacia karroo On dead bamboo culm — Goat dung Source Genomic DNA was extracted from colonies grown on 2 % malt extract agar (MEA, Difco) (Gams et al. 2007) using the FastDNA kit (BIO101, Carlsbad, CA, U.S.A.). The primers ITS1 and ITS4 (White et al. 1990) were used

1 to amplify the internal transcribed spacer region (ITS) of the nuclear ribosomal RNA operon, including: the 3’ end of the 18S rRNA gene, the first internal transcribed spacer region (ITS1), the 5.8S rRNA gene, the second internal transcribed spacer region (ITS2) and the 5’ end of 28S CBS 146.36 CBS 588.66*; IMI 203547 CBS 776.83* CBS 268.75* Accession number CBS 350.65; IMI 115127; MUCL 7972 MUCL CBS 350.65; IMI 115127; rRNA gene. Part of the large subunit 28S rRNA (LSU) gene was amplified with primers LR0R (Rehner & Samuels 1994) and LR5 (Vilgalys & Hester 1990). The ITS region was sequenced only for those isolates for which these data were not available. The ITS analyses confirmed the proposed classification based on LSU analysis for each major clade and are not presented here in detail; but the sequences are deposited in ATCC: American Type Culture Collection, Virginia, U.S.A.; CBS: Centraalbureau Culture voor Virginia, Collection, Schimmelcultures, The Utrecht, Type American Netherlands; CPC: Culture collection of Pedro Crous, housed at ATCC: CBS; Table 1. (Continued). Table Zasmidium cellare Veronaeopsis simplex Veronaeopsis Veronaea japonica Veronaea Veronaea compacta Veronaea DAOM: Plant Research Institute, Department of Agriculture (Mycology), Ottawa, Canada; IFM: Research Center Japan; IMI: International Mycological Institute, for CABI-Bioscience, U.K.; JCM: Japan Collection of RIKEN Microorganism, Japan; Pathogenic BioResource MFC: Center, Matsushima Fungus Collection, Fungi and Microbial Toxicoses, Chiba University, Chiba, Kobe, Japan; MUCL: Mycotheque de Université l’ Catholique de Medical Louvain, Louvain-la-Neuve, Texas Belgium; of NCPF: University The UTMB: U.S.A.; National MA, Collection Army, of Pathogenic U.S. Fungi, Center, Holborn, Developement London, and Research U.K.; Quartermaster QM: OAC: Canada; Ont., Guelph, of University Genetics, and Botany of Department U.S.A. Texas, Branch, *Ex-type cultures. Species 1 GenBank where applicable. The PCR reaction

76 Ramichloridium and allied genera was performed in a mixture with 0.5 units Taq polymerase (Bioline, London, U.K.), 1× PCR buffer, 0.5 mM MgCl2, 0.2 mM of each dNTP, 5 pmol of each primer, approximately 10–15 ng of fungal genomic DNA, with the total volume adjusted to 25 µL with sterile water. Reactions were performed on a GeneAmp PCR System 9700 (Applied Biosystems, Foster City, CA) with cycling conditions consisting of 5 min at 96 °C for primary denaturation, followed by 36 cycles at 96 °C (30 s), 52 °C (30 s), and 72 °C (60 s), with a final 7 min extension step at 72 °C to complete the reaction. The amplicons were sequenced using BigDye Terminator v. 3.1 (Applied Biosystems, Foster City, CA) or DYEnamicET Terminator (Amersham Biosciences, Freiburg, Germany) Cycle Sequencing Kits and analysed on an ABI Prism 3700 (Applied Biosystems, Foster City, CA) under conditions recommended by the manufacturer. Newly generated sequences were subjected to a Blast search of the NCBI databases, sequences with high similarity were downloaded from GenBank and comparisons were made based on the alignment of the obtained sequences. Sequences from GenBank were also selected for similar taxa. The LSU tree was rooted using sequences of Athelia epiphylla Pers. and Paullicorticium ansatum Liberta as outgroups. Phylogenetic analysis was performed with PAUP (Phylogenetic Analysis Using Parsimony) v. 4.0b10 (Swofford 2003), using the neighbour-joining algorithm with the uncorrected (“p”), the Kimura 2-parameter and the HKY85 substitution models. Alignment gaps longer than 10 bases were coded as single events for the phylogenetic analyses; the remaining gaps were treated as missing data. Any ties were broken randomly when encountered. Phylogenetic relationships were also inferred with the parsimony algorithm using the heuristic search option with simple taxon additions and tree bisection and reconstruction (TBR) as the branch-swapping algorithm; alignment gaps were treated as a fifth character state and all characters were unordered and of equal weight. Branches of zero length were collapsed and all multiple, equally parsimonious trees were saved. Only the first 5000 equally most parsimonious trees were saved. Other measures calculated included tree length, consistency index, retention index and rescaled consistency index (TL, CI, RI and RC, respectively). The robustness of the obtained trees was evaluated by 1000 bootstrap replications. Bayesian analysis was performed following the methods of Crous et al. (2006c). The best nucleotide substitution model was determined using MrModeltest v. 2.2 (Nylander 2004). MrBa y e s v. 3.1.2 (Ronquist & Huelsenbeck 2003) was used to perform phylogenetic analyses, using a general time-reversible (GTR) substitution model with inverse gamma rates, dirichlet base frequencies and the temp value set to 0.5. New sequences were lodged with NCBI’s GenBank (Table 1) and the alignment and trees with TreeBASE (www. treebase.org).

Morphology

Cultural growth rates and morphology were recorded from colonies grown on MEA for 2 wk at 24 ºC in the dark, and colony colours were determined by reference to the colour charts of Rayner (1970). Microscopic observations were made from colonies cultivated on MEA and OA (oatmeal agar, Gams et al. 2007), using a slide culture technique. Slide cultures were set up in Petri dishes containing 2 mL of sterile water, into which a U-shaped glass rod was placed, extending above the water surface. A block of freshly growing fungal colony, approx. 1 cm square was placed onto a sterile microscope slide, covered with a somewhat larger, sterile glass cover slip, and incubated in the moist chamber. Fungal sporulation was monitored over time, and when optimal, images were captured by means of a Nikon camera system (Digital Sight DS- 5M, Nikon Corporation, Japan). Structures were mounted in lactic acid, and 30 measurements (× 1000 magnification) determined wherever possible, with the extremes of spore measurements given in parentheses.

77 Chapter 4

Athelia epiphylla AY586633 Paullicorticium ansatum AY586693 100 Conioscyphascus varius AY484512 Conioscypha lignicola AY484513 100 100 Carpoligna pleurothecii AF064645 Carpoligna pleurothecii AF064646 78 Carpoligna pleurothecii AY544685 98 74 Pleurothecium obovoideum CBS 209.95 Ascotaiwania hughesii AY316357 100 Ascotaiwania hughesii AY094189 Ophiostoma stenoceras DQ836904 Sordariomycetes 96 Magnaporthe grisea AB026819 100 Cryptadelphia polyseptata AY281102 Cryptadelphia groenendalensis AY281103 Rhodoveronaea varioseptata CBS 431.88 100 Annulatascus triseptatus AY780049 Annulatascus triseptatus AY346257 Thyridium vestitum AY544671 Myrmecridium schulzeri CBS 114996 Myrmecridium flexuosum CBS 398.76 95 Myrmecridium schulzeri CBS 188.96 Myrmecridium schulzeri CBS 381.87 Myrmecridium schulzeri CBS 305.73 89 Myrmecridium schulzeri CBS 304.73 Myrmecridium schulzeri CBS 100.54 Myrmecridium schulzeri CBS 642.76 Myrmecridium schulzeri CBS 134.68 Myrmecridium schulzeri CBS 156.63 100 88 Myrmecridium schulzeri CBS 325.74 Capronia pulcherrima AF050256 100 82 Exophiala dermatitidis AF050270 Capronia mansonii AY004338 Rhinocladiella sp. CBS 264.49 Rhinocladiella mackenziei CBS 368.92 100 Rhinocladiella mackenziei AF050288 Rhinocladiella mackenziei CBS 367.92 Rhinocladiella mackenziei CBS102590 Capronia coronata AF050242 Rhinocladiella fasciculata CBS 132.86 Rhinocladiella phaeophora CBS 496.78 Rhinocladiella anceps AF050284 Herpotrichiellaceae, 10 changes Rhinocladiella anceps CBS 181.65 Chaetothyriales, 100 Rhinocladiella anceps AF050285 Rhinocladiella anceps CBS 157.54 Chaetothyriomycetes Fonsecaea pedrosoi AF050276 86 Veronaea botryosa CBS 350.65 Veronaea botryosa CBS 121.92 Veronaea botryosa CBS 254.57 Thysanorea papuana CBS 212.96 Veronaea japonica CBS 776.83 Veronaea compacta CBS 268.75 Rhinocladiella basitona CBS 101460 80 Rhinocladiella atrovirens AF050289 60 Exophiala jeanselmei AF050271 96 Veronaeopsis simplex CBS 588.66 Repetophragma goidanichii DQ408574 pyrina EF114715 99 Metacoleroa dickiei DQ384100 Venturiaceae, Venturia chlorospora DQ384101 Pleosporales, Venturia inaequalis EF114713 Venturia hanliniana AF050290 Dothideomycetes Venturia asperata EF114711 54 Venturia carpophila AY849967

Fig. 1. One of 5000 equally most parsimonious trees obtained from a heuristic search with simple taxon additions of the LSU sequence alignment using PAUP v. 4.0b10. The scale bar shows 10 changes; bootstrap support values from 1000 replicates are shown at the nodes. Thickened lines indicate the strict consensus branches and ex-type sequences are printed in bold face. The tree was rooted to two sequences obtained from GenBank (Athelia epiphylla AY586633 and Paullicorticium ansatum AY586693).

78 Ramichloridium and allied genera

Radulidium sp. CBS 115704 100 Pseudovirgaria hyperparasitica CPC 10702 100 Pseudovirgaria hyperparasitica CPC 10753 90 Pseudovirgaria hyperparasitica CPC 10704 96 Radulidium subulatum CBS 287.84 Incertae sedis Radulidium subulatum CBS 912.96 99 Radulidium epichloës CBS 361.63 98 Radulidium epichloës AF050287 78 Radulidium subulatum CBS 405.76 64 Radulidium subulatum CBS 101010 71 Sporidesmium pachyanthicola DQ408557 99 Staninwardia suttonii DQ923535

Ramichloridium brasilianum CBS 283.92 Teratosphaeriaceae 100 Batcheloromyces proteae CBS 110696 54 Teratosphaeria alistairii DQ885901 Teratosphaeria toledana DQ246230 Readeriella considenianae DQ923527 77 Teratosphaeria molleriana EU019292 Teratosphaeria fibrillosa EU019282 67 Catenulostroma macowanii EU019254 58 Teratosphaeria suberosa DQ246235 100 Cibiessia dimorphospora EU019258 Teratosphaeria readeriellophora DQ246238 Xenomeris juniperi EF114709 Catenulostroma abietis EU019249 Devriesia staurophora DQ008150 100 Teratosphaeria parva DQ246240 Teratosphaeria parva DQ246243 87 Cladosporium cladosporioides EU019262 100 Cladosporium uredinicola EU019264 Cladosporium bruhnei EU019261 56 Cladosporium sphaerospermum EU019263 52 Ramichloridium indicum CBS 171.96 Dissoconium aciculare EU019266 Capnodiales 99 54 “Mycosphaerella” communis EU019267 99 “Mycosphaerella” lateralis EU019268 52 Ramichloridium apiculatum CBS 390.67 Ramichloridium apiculatum CBS 400.76 87 Ramichloridium apiculatum CBS 391.67 Ramichloridium apiculatum CBS 156.59 Ramichloridium pini CBS 461.82 , Dothideomycetes 100 Mycosphaerella endophytica DQ246252 Mycosphaerella endophytica DQ246255 Mycosphaerella gregaria DQ246251 100 Mycosphaerella graminicola EU019297 Septoria tritici EU019298

95 Cercosporella centaureicola EU019257 Mycosphaerellaceae 10 changes 99 Mycosphaerella punctiformis AY490776 56 Ramularia sp. EU019285 Ramularia miae DQ885902 55 Ramularia pratensis var. pratensis EU019284 Mycosphaerella walkeri DQ267574 Mycosphaerella parkii DQ246245 Mycosphaerella madeirae DQ204756 69 Periconiella levispora CBS 873.73 Mycosphaerella marksii DQ246249 Pseudocercospora epispermogonia DQ204758 80 Pseudocercospora epispermogonia DQ204757 Mycosphaerella intermedia DQ246248 98 Mycosphaerella marksii DQ246250 Periconiella arcuata CBS 113477 64 Rasutoria pseudotsugae EF114704 76 Rasutoria tsugae EF114705 Periconiella velutina CBS 101950 78 Periconiella velutina CBS 101948 94 Periconiella velutina CBS 101949 Ramichloridium biverticillatum CBS 335.36 Ramichloridium musae CBS 365.36 Ramichloridium musae CBS 190.63 56 Ramichloridium australiense CBS 121710 56 Ramichloridium strelitziae CBS 121711 77 Zasmidium cellare CBS 146.36 66 Ramichloridium cerophilum CBS 103.59 97 Ramichloridium cerophilum AF050286

Fig. 1. (Continued).

79 Chapter 4

Athelia epiphylla AY586633 Paullicorticium ansatum AY586693 Veronaeopsis simplex CBS 588.66 0.98 Repetophragma goidanichii DQ408574 Metacoleroa dickiei DQ384100 0.79 Venturia pyrina EF114715 Venturiaceae, 1.00 Venturia hanliniana AF050290 Pleosporales, Venturia inaequalis EF114713 0.84 Venturia chlorospora DQ384101 Dothideomycetes Venturia asperata EF114711 0.99 Venturia carpophila AY849967 Capronia pulcherrima AF050256 Capronia coronata AF050242 0.52 Rhinocladiella phaeophora CBS 496.78 Rhinocladiella fasciculata CBS 132.86 0.74 Exophiala dermatitidis AF050270 Capronia mansonii AY004338 1.00 Rhinocladiella mackenziei CBS 368.92 Rhinocladiella mackenziei AF050288 Rhinocladiella mackenziei CBS 367.92 1.00 Rhinocladiella mackenziei CBS 102590 Rhinocladiella anceps AF050284 Herpotrichiellaceae, 0.97 Rhinocladiella anceps CBS 181.65 1.00 Rhinocladiella anceps AF050285 Chaetothyriales, 0.80 Rhinocladiella anceps CBS 157.54 Chaetothyriomycetes 0.63 Rhinocladiella sp. CBS 264.49 Exophiala jeanselmei AF050271 0.99 Rhinocladiella atrovirens AF050289 0.50 Rhinocladiella basitona CBS 101460 Fonsecaea pedrosoi AF050276 0.68 Thysanorea papuana CBS 212.96 0.69 Veronaea japonica CBS 776.83 1.00 Veronaea compacta CBS 268.75 0.58 Veronaea botryosa CBS 350.65 Veronaea botryosa CBS 121.92 1.00 Veronaea botryosa CBS 254.57 1.00 Conioscyphascus varius AY484512 Conioscypha lignicola AY484513 1.00 Carpoligna pleurothecii AF064646 1.00 1.00 Carpoligna pleurothecii AF064645 Carpoligna pleurothecii AY544685 0.99 1.00 1.00 Pleurothecium obovoideum CBS 209.95 1.00 Ascotaiwania hughesii AY316357 Ascotaiwania hughesii AY094189

Magnaporthe grisea AB026819 Sordariomycetes 0.96 Ophiostoma stenoceras DQ836904 1.00 Cryptadelphia polyseptata AY281102 Cryptadelphia groenendalensis AY281103 0.99 Rhodoveronaea varioseptata CBS 431.88 0.84 Annulatascus triseptatus AY780049 0.91 1.00 Annulatascus triseptatus AY346257 Thyridium vestitum AY544671 0.1 expected changes per site Myrmecridium schulzeri CBS 114996 1.00 Myrmecridium schulzeri CBS 188.96 1.00 Myrmecridium schulzeri CBS 381.87 Myrmecridium schulzeri CBS 305.73 0.61 Myrmecridium schulzeri CBS 304.73 Myrmecridium flexuosum CBS 398.76 0.99 0.87 Myrmecridium schulzeri CBS 100.54 0.71 Myrmecridium schulzeri CBS 642.76 0.92 Myrmecridium schulzeri CBS 134.68 Myrmecridium schulzeri CBS 156.63 1.00 Myrmecridium schulzeri CBS 325.74

Fig. 2. Consensus phylogram (50 % majority rule) of 1500 trees resulting from a Bayesian analysis of the LSU sequence alignment using MrBa y e s v. 3.1.2. Bayesian posterior probabilities are indicated at the nodes. Ex-type sequences are printed in bold face. The tree was rooted to two sequences obtained from GenBank (Athelia epiphylla AY586633 and Paullicorticium ansatum AY586693).

80 Ramichloridium and allied genera

Pseudovirgaria hyperparasitica CPC 10702 0.99 Pseudovirgaria hyperparasitica CPC 10753 1.00 Pseudovirgaria hyperparasitica CPC 10704 Radulidium sp. CBS 115704 0.95 Radulidium subulatum CBS 287.84 0.86 Radulidium subulatum CBS 912.96 Incertae sedis Radulidium epichloës CBS 361.63 1.00 Radulidium epichloës AF050287 0.60 Radulidium subulatum CBS 405.76 0.57 Radulidium subulatum CBS 101010 0.91 Cladosporium cladosporioides EU019262 Cladosporium uredinicola EU019264 Davidiellaceae 1.00 Cladosporium bruhnei EU019261 0.64 0.81 Cladosporium sphaerospermum EU019263 1.00 Sporidesmium pachyanthicola DQ408557 Staninwardia suttonii DQ923535 0.61 1.00 Ramichloridium brasilianum CBS 283.92

1.00 Batcheloromyces proteae EU019247 Teratosphaeriaceae 0.96 Teratosphaeria alistairii DQ885901 0.99 Readeriella considenianae DQ923527 Teratosphaeria toledana DQ246230 1.00 Teratosphaeria molleriana EU019292 0.87 Teratosphaeria fibrillosa EU019282 Catenulostroma macowanii EU019254 Teratosphaeria suberosa DQ246235 Cibiessia dimorphospora EU019258 1.00 1.00 Teratosphaeria readeriellophora DQ246238 0.63 1.00 Teratosphaeria parva DQ246240 Teratosphaeria parva DQ246243 0.73 Devriesia staurophora DQ008150 0.72 Xenomeris juniperi EF114709 0.68 Catenulostroma abietis EU019249 0.95 Ramichloridium indicum CBS 171.96 1.00 Dissoconium aciculare EU019266 “Mycosphaerella” communis EU019267 Capnodiales 1.00 “Mycosphaerella” lateralis EU019268 Ramichloridium apiculatum CBS 390.67 Ramichloridium apiculatum CBS 400.76 0.1 expected changes per site 1.00 Ramichloridium apiculatum CBS 391.67 Ramichloridium apiculatum CBS 156.59

Mycosphaerella endophytica DQ246252 , 0.99 1.00 Mycosphaerella endophytica DQ246255 Dothideomycetes Mycosphaerella gregaria DQ246251 1.00 Cercosporella centaureicola EU019257 1.00 Mycosphaerella punctiformis AY490776 1.00 Ramularia sp. EU019285 0.64 Ramularia miae DQ885902 0.99 Ramularia pratensis var. pratensis EU019284 Mycosphaerellaceae 0.72 Mycosphaerella graminicola EU019297 1.00 Septoria tritici EU019298 Ramichloridium pini CBS 461.82 1.00 Mycosphaerella walkeri DQ267574 0.76 Mycosphaerella parkii DQ246245 Mycosphaerella madeirae DQ204756 0.60 0.95 Periconiella levispora CBS 873.73 0.97 Mycosphaerella marksii DQ246249 0.64 Pseudocercospora epispermogonia DQ204758 0.99 Pseudocercospora epispermogonia DQ204757 0.57 Mycosphaerella intermedia DQ246248 0.80 Mycosphaerella marksii DQ246250 Periconiella arcuata CBS 113477 0.64 Rasutoria pseudotsugae EF114704 1.00 Rasutoria tsugae EF114705 0.90 Periconiella velutina CBS 101950 0.95 Periconiella velutina CBS 101948 0.97 Periconiella velutina CBS 101949 Ramichloridium biverticillatum CBS 335.36 1.00 Ramichloridium musae CBS 365.36 0.94 Ramichloridium musae CBS 190.63 0.97 Ramichloridium australiense CBS 121710 0.96 Ramichloridium strelitziae CBS 121711 0.97 Zasmidium cellare CBS 146.36 0.97 Ramichloridium cerophilum CBS 103.59 0.96 Ramichloridium cerophilum AF050286 Fig. 2. (Continued).

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Results

Phylogeny

The manually adjusted alignment of the 28S rDNA data contained 137 sequences (including the two outgroups) and 995 characters including alignment gaps. Of the 748 characters used in the phylogenetic analysis, 373 were parsimony-informative, 61 were variable and parsimony- uninformative, and 314 were constant. Neighbour-joining analysis using the three substitution models on the LSU alignment yielded trees with similar topology and bootstrap values. Parsimony analysis of the alignment yielded 5000 equally most parsimonious trees, one of which is shown in Fig. 1 (TL = 2157, CI = 0.377, RI = 0.875, RC = 0.330). The Markov Chain Monte Carlo (MCMC) analysis of four chains started from a random tree topology and lasted 2000000 generations. Trees were saved each 1 000 generations, resulting in 2000 trees. Burn-in was set at 500000 generations after which the likelihood values were stationary, leaving 1500 trees from which the consensus tree (Fig. 2) and posterior probabilities (PP’s) were calculated. The average standard deviation of split frequencies was 0.043910 at the end of the run. Among the neighbour-joining, Bayesian and parsimony analyses, the trees differed in the hierarchical order of the main families and the support values (data not shown; e.g. the support within of the Capnodiales in Figs 1–2). The phylogenetic trees (Figs 1–2) show that the Ramichloridium species segregate into eight distinct clades, residing in the Capnodiales (Mycosphaerellaceae and Teratosphaeriaceae), the Chaetothyriales (Herpotrichiellaceae), the Pleosporales, and five other clades of which the relationships remain to be elucidated. The type species of Ramichloridium, R. apiculatum, together with R. musae, R. cerophilum (Tubaki) de Hoog, R. indicum (Subram.) de Hoog, R. pini and three new species respectively isolated from Musa banksii, Strelitzia nicolai, and forest soil, reside in different parts of the Capnodiales clade (all in the Mycosphaerellaceae, except for the species from forest soil which clusters in the Teratosphaeriaceae). The second clade (in the Chaetothyriomycetes clade), including the human-pathogenic species Rhinocladiella mackenziei and Rhinocladiella basitonum, together with Rhinocladiella fasciculatum V. Rao & de Hoog and Rhinocladiella anceps (Sacc. & Ellis) de Hoog, groups together with Rhinocladiella in the Herpotrichiellaceae. The third clade (in the Sordariomycetes clade) includes R. obovoideum (Matsush.) de Hoog, which in a Blast search was found to have affinity with Carpoligna pleurothecii F.A. Fernández & Huhndorf (Chaetosphaeriales). The fourth clade (in the Sordariomycetes clade) includes a veronaea-like isolate from Bertia moriformis, with phylogenetic affinity to the Annulatascaceae (Sordariomycetidae). The fifth clade (in the Sordariomycetes clade) includes R. schulzeri var. schulzeri and R. schulzeri var. flexuosum de Hoog, the closest relatives being Thyridium vestitum (Fr.) Fuckel in the Thyridiaceae and Magnaporthe grisea (T.T. Hebert) M.E. Barr in the Magnaporthaceae. The sixth clade (in the Incertae sedis clade) includes R. subulatum de Hoog, R. epichloës (Ellis & Dearn.) de Hoog and a species isolated from the Poaceae. Three ramichloridium-like isolates from Rubus coreanus and Agrimonia pilosa form another unique clade (in the Incertae sedis clade) with uncertain affinity. Veronaea simplex Papendorf clusters as sister taxon to the Venturiaceae representing the eighth clade (Dothideomycetes). The type species of Periconiella, P. velutina, clusters within the Mycosphaerellaceae (Capnodiales clade), whereas P. papuana Aptroot resides in the Herpotrichiellaceae (Chaetothyriales clade). Veronaea botryosa Cif. & Montemart., the type species of Veronaea, also resides in the Herpotrichiellaceae.

82 Ramichloridium and allied genera

Taxonomy

The species previously described in Ramichloridium share some morphological features, including erect, pigmented, more or less differentiated conidiophores, sympodially proliferating conidiogenous cells and predominantly aseptate conidia. Other than conidial morphology, features of the conidiogenous apparatus that appear to be more phylogenetically informative include pigmentation of vegetative hyphae, conidiophores and conidia, denticle density on the rachis, and structure of the scars. By integrating these data with the molecular data set, more natural genera are delineated, which are discussed below.

Key to ramichloridium-like genera

1. Conidiogenous cells integrated, terminal and lateral on creeping or ascending hyphae (differentiation between branched vegetativehyphae and conidiophores barely possible); conidiogenous loci bulging, more or less umbonate, apex rounded; occurring on rust pustules ...... Pseudovirgaria 1. Conidiogenous cells integrated in distinct conidiophores; conidiogenous loci non-umbonate (flat, not prominent; subcylindrical or conical denticles; or terminally flat-tipped; or thickened and darkened); rarely occurring on rust pustules, but if so, with a raduliform rachis and distinct denticles ...... 2

2. Conidia 0–2(–3)-septate, conidial base truncate, retaining a marginal frill after liberation [anamorphs of Sordariomycetes]...... Rhodoveronaea 2. Conidial base without marginal frill ...... 3

3. Conidiophores composed of a well-developed erect stalk and a terminal branched head .... 4 3. Conidiophores unbranched or, if branched, branches loose, irregular or dichotomous, but not distinctly separated into stalk and branched head ...... 5

4. Conidiophores dimorphic, either macronematous, dark brown with a dense apical cluster of branches or micronematous, undifferentiated, resembling vegetative hyphae; both kinds with a denticulate rachis; conidia predominantly 1-septate [anamorph of Chaetothyriales] ...... Thysanorea 4. Conidiophores monomorphic; branched head with fewer branches and looser; conidiogenous loci usually flat, non-prominent, less denticle-like; conidia aseptate to pluriseptate [anamorphs of Capnodiales] ...... Periconiella

5. Rachis with denticles 1–1.5 µm long, denticles almost cylindrical; conidia at least partly in short chains ...... Pleurothecium 5. Rachis with denticles less than 1 µm long, denticles not cylindrical or denticles lacking, rachis with flat, barely prominent scars...... 6

6. Conidia predominantly septate ...... 7 6. Conidia predominantly aseptate ...... 8

7. Conidiophores up to 200 µm long; rachis straight, not to slightly geniculate; conidiogenous loci more or less flat, barely prominent, unthickened, slightly darkened [anamorphs of Chaetothyriales, Herpotrichiellaceae]...... Veronaea

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7. Conidiophores up to 60 µm long; rachis distinctly geniculate; conidiogenous loci denticle- like, prominent, up to 0.5 µm high, slightly thickened and darkened [anamorph of Pleosporales, Venturiaceae] ...... Veronaeopsis

8. Vegetative mycelium entirely hyaline; rachis long, hyaline, with widely scattered pimple- shaped, terminally pointed, unpigmented denticles ...... Myrmecridium 8. Vegetative mycelium at least partly pigmented; conidiogenous loci distinct, non-denticulate, somewhat darkened-refractive, or denticles, if present, neither pimple-shaped nor pointed .... 9

9. Rachis distinctly raduliform, with distinct, prominent blunt denticles, 0.5–1 µm long; scars and hila unthickened, but pigmented...... Radulidium 9. Rachis not distinctly raduliform, at most subdenticulate; scars flat or only slightly prominent (subdenticulate), shorter ...... 10

10. Conidiophores usually poorly differentiated from the vegetative hyphae; conidial apparatus often loosely branched; exophiala-like budding cells usually present in culture [anamorphs of Chaetothyriales, Herpotrichiellaceae] ...... Rhinocladiella 10. Conidiophores usually well differentiated from the vegetative mycelium (macronematous), usually unbranched; without exophiala-like states [anamorphs of Capnodiales] ...... Ramichloridium

Capnodiales (Mycosphaerellaceae, Teratosphaeriaceae)

The type species of Ramichloridium, R. apiculatum, together with R. indicum cluster as a sister group to the Dissoconium de Hoog, Oorschot & Hijwegen clade in the Mycosphaerellaceae. Some other Ramichloridium species, including R. musae, R. biverticillatum Arzanlou & Crous, R. pini and R. cerophilum, are also allied with members of the Mycosphaerellaceae. Three additional new species are introduced for Ramichloridium isolates from Musa banksii, Strelitzia nicolai, and forest soil. Periconiella velutina, the type species of Periconiella, which also resides in the Mycosphaerellaceae, is morphologically sufficiently distinct to retain its generic status. Two new species of Periconiella are introduced for isolates obtained from Turpinia pomifera and Ischyrolepis subverticellata in South Africa. Zasmidium cellare (Pers.) Fr., the type species of Zasmidium (Pers.) Fr., is also shown to cluster within the Mycosphaerellaceae.

Periconiella Sacc., in Sacc. & Berlese, Atti Ist. Veneto Sci., Ser. 6, 3: 727. 1885.

In vitro: Colonies with entire margin; aerial mycelium rather compact, raised, velvety, olivaceous-grey; reverse olivaceous-black. Submerged hyphae verrucose, hyaline, thin-walled, 1–3 µm wide; aerial hyphae subhyaline, later becoming dark brown, thick-walled, smooth. Conidiophores arising vertically from creeping hyphae, straight or flexuose, up to 260 µm long, dark brown at the base, paler towards the apex, thick-walled; in the upper part bearing short branches. Conidiogenous cells terminally integrated, polyblastic, smooth or verrucose, subcylindrical, mostly not or barely geniculate-sinuous, variable in length, subhyaline, later becoming pale brown, fertile part as wide as the basal part, proliferating sympodially, sometimes becoming septate and forming a short, straight rachis with pigmented, slightly thickened and hardly prominent, more or less flat scars.Conidia solitary, occasionally in short chains, 0–multi- septate, subhyaline to rather pale olivaceous or olivaceous-brown, smooth to verrucose, globose, ellipsoidal to obovoid or obclavate, with a slightly darkened and thickened hilum; conidial secession schizolytic.

84 Ramichloridium and allied genera

Type species: P. velutina (G. Winter) Sacc., Miscell. mycol. 2: 17. 1884.

Notes: Periconiella is distinct from other ramichloridium-like genera by its conidiophores that are prominently branched in the upper part, and by its darkened, thickened conidial scars, that are more or less flat and non-prominent. Although conidiophores are also branched in the upper part in Thysanorea Arzanlou, W. Gams & Crous, the branching pattern in the latter genus is different from that of Periconiella. Thysanorea has a complex head consisting of up to six levels of branches, while in Periconiella the branching is limited, with mainly primary and secondary branches. Furthermore, Thysanorea is characterised by having dimorphic conidiophores and more or less prominent denticle-like conidiogenous loci.

Periconiella velutina (G. Winter) Sacc., Miscell. mycol. 2: 17. 1884. Fig. 3. Basionym: Periconia velutina G. Winter, Hedwigia 23: 174. 1884.

In vitro: Submerged hyphae verrucose, hyaline, thin-walled, 1–3 µm wide; aerial hyphae subhyaline, later becoming dark brown, thick-walled, smooth. Conidiophores arising vertically from creeping hyphae, straight or flexuose, up to 260 µm long, dark brown at the base, paler towards the apex, thick-walled; in the upper part bearing short branches, 10–35 µm long. Conidiogenous cells mostly terminally integrated, sometimes discrete, smooth or verrucose, cylindrical, variable in length, subhyaline, later becoming pale brown, fertile part as wide as the basal part, proliferating sympodially, sometimes becoming septate and forming a short, straight rachis with pigmented, slightly thickened and hardly prominent, more or less flat scars, less than 1 µm diam. Conidia 0(–1)-septate, subhyaline, thin-walled, verrucose or smooth, globose, ellipsoidal to obovoid, (7–)8–9(–11) × (2.5–)3(–4) µm, with a slightly darkened and thickened hilum, 1.5–2 µm diam.

Cultural characteristics: Colonies on MEA slow-growing, reaching 4 mm diam after 14 d at 24 °C, with entire margin; aerial mycelium rather compact, raised, velvety, olivaceous-grey; reverse olivaceous-black.

Specimens examined: South Africa, Cape Town, on Brabejum stellatifolium, P. MacOwan, herb. G. Winter (B), lectotype selected here; Cape Town, on leaves of Brabejum stellatifolium (= B. stellatum), P. Mac-Owan, PAD, F42165, F462166, isolectotypes; Stellenbosch, Jonkershoek Nature Reserve, on Brabejum stellatifolium, 21 Jan. 1999, J.E. Taylor, epitype designated here CBS H-15612, cultures ex-epitype CBS 101948–101950.

Periconiella arcuata Arzanlou, S. Lee & Crous, sp. nov. MycoBank MB504547. Figs 4, 7A.

Etymology: Named after its curved conidia.

Ab aliis speciebus Periconiellae conidiis obclavatis, rectis vel curvatis, (30–)53–61(–79) × (3–)5(–7) µm, distinguenda.

Submerged hyphae smooth, hyaline, thin-walled, 2 µm wide; aerial hyphae pale brown, smooth or verrucose, slightly narrower. Conidiophores arising vertically from creeping hyphae, straight or flexuose, up to 300 µm long, dark brown at the base, paler towards the apex, thick-walled; loosely branched in the upper part, bearing short branches. Conidiogenous cells integrated, cylindrical, variable in length, 20–50 µm long, subhyaline, later becoming pale brown, fertile part as wide as the basal part, proliferating sympodially, forming a geniculate conidium-bearing

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Fig. 3. Periconiella velutina (CBS 101948). A–B. Macronematous conidiophores with short branches in the upper part. C. Sympodially proliferating conidiogenous cell with darkened and slightly thickened scars. D. Conidia. Scale bar = 10 µm.

Fig. 4. Periconiella arcuata (CBS 113477). A–B. Sympodially proliferating conidiogenous cells with darkened, thickened and cone-shaped scars. C–E. Macronematous conidiophores with loose branches in the upper part. F–I. Conidia. Scale bar = 10 µm.

86 Ramichloridium and allied genera

Fig. 5. Periconiella levispora (CBS 873.73). A–C. Conidial apparatus at different stages of development, which gives rise to macronematous conidiophores with dense branches in the upper part. D. Sympodially proliferating conidiogenous cells with darkened and somewhat protruding scars. E–F. Conidia with truncate base and darkened hilum. Scale bar = 10 µm.

Fig. 6. A. Pseudovirgaria hyperparasitica (CBS 121739 = CPC 10753). B. Periconiella levispora (CBS 873.73). Scale bar = 10 µm.

87 Chapter 4 rachis with pigmented and thickened, prominent, cone-shaped scars, 1 µm diam. Conidia formed singly, obclavate, straight or mostly curved, 0(–4)-septate, coarsely verrucose, pale olive, thin- walled, tapering towards the apex, (30–)53–61(–79) × (3–)5(–7) µm, with a narrowly truncate base and a darkened, hardly thickened hilum, 2 µm diam; microcyclic conidiation observed in culture.

Cultural characteristics: Colonies on MEA reaching 12 mm diam after 14 d at 24 °C, with entire, smooth, sharp margin; mycelium compacted, becoming hairy, colonies up to 1 mm high; surface olivaceous to olivaceous-grey, reverse dark grey-olivaceous to olivaceous-black.

Specimen examined: South Africa, Western Cape Province, Kogelberg, on dead culms of Ischyrolepis subverticillata, May 2001, S. Lee, holotype CBS H-19927, culture ex-type CBS 113477.

Periconiella levispora Arzanlou, W. Gams & Crous, sp. nov. MycoBank MB504546. Figs 5–6B.

Etymology: (Latin) levis = smooth.

A simili Periconiella velutina conidiis levibus et maioribus, ad 23 μm longis distinguenda.

In vitro: Submerged hyphae smooth, hyaline, thin-walled, 2–2.5 µm wide; aerial hyphae subhyaline, later becoming dark brown, thick-walled, smooth. Conidiophores arising vertically from creeping aerial hyphae, dark brown at the base, paler towards the apex, thick-walled; in the upper part bearing several short branches, up to 120 µm long. Conidiogenous cells integrated, occasionally discrete, cylindrical, variable in length, 10–20 µm long, subhyaline, later becoming pale brown, fertile part as wide as the basal part, proliferating sympodially, forming a short rachis with pigmented and slightly thickened, somewhat protruding scars, less than 1 µm diam. Conidia solitary, 0(–2)-septate, smooth, pale olivaceous, cylindrical, ellipsoidal, pyriform to clavate, (7–)11–14(–23) × (3–)4–5(–6) µm, with a truncate base and a darkened, slightly thickened hilum, 2 µm diam.

Cultural characteristics: Colonies on MEA slow-growing, reaching 5 mm diam after 14 d at 24 °C, with entire margin; aerial mycelium compact, raised, velvety, olivaceous-grey; reverse olivaceous-black.

Specimen examined: Sri Lanka, Hakgala Botanic Gardens, on dead leaves of Turpinia pomifera, Jan. 1973, W. Gams, holotype CBS H-15611, culture ex-type CBS 873.73.

Ramichloridium Stahel ex de Hoog, Stud. Mycol. 15: 59. 1977.

In vitro: Colonies flat to raised, with entire margin; surface olivaceous-green to olivaceous- black. Mycelium consisting of submerged and aerial hyphae; submerged hyphae hyaline to subhyaline, thin-walled, aerial hyphae smooth or verrucose. Conidiophores straight, unbranched, rarely branched, thick-walled, dark brown (darker than the subtending hyphae), continuous or with several additional thin septa. Conidiogenous cells integrated, terminal, polyblastic, smooth, thick-walled, golden-brown, apical part subhyaline, with sympodial proliferation, straight or flexuose, geniculate or nodose, with conspicuous conidiogenous loci; scars crowded or scattered, unthickened, unpigmented to faintly pigmented, or slightly prominent denticles.

88 Ramichloridium and allied genera

Fig. 7. A. Periconiella arcuata (CBS 113477). B. Myrmecridium schulzeri (CBS 325.74). C. Thysanorea papuana (CBS 212.96). Scale bars = 10 µm.

Fig. 8. Ramichloridium apiculatum (CBS 156.59). A–C. Macronematous conidiophores with sympodially proliferating conidiogenous cells, which give rise to a conidium-bearing rachis with crowded and prominent scars. D. Conidia. Scale bar = 10 µm.

89 Chapter 4

Conidia solitary, 0–1-septate, subhyaline to pale brown, smooth to coarsely verrucose, rather thin-walled, obovate, obconical or globose to ellipsoidal, fusiform, with a somewhat prominent, slightly pigmented hilum; conidial secession schizolytic.

Type species: R. apiculatum (J.H. Mill., Giddens & A.A. Foster) de Hoog, Stud. Mycol. 15: 69. 1977.

Ramichloridium apiculatum (J.H. Mill., Giddens & A.A. Foster) de Hoog, Stud. Mycol. 15: 69. 1977. Fig. 8. Basionym: Chloridium apiculatum J.H. Mill., Giddens & A.A. Foster, Mycologia 49: 789. 1957. ≡ Veronaea apiculata (J.H. Mill., Giddens & A.A. Foster) M.B. Ellis, in Ellis, More Dematiaceous Hyphomycetes: 209. 1976. [non Rhinocladiella apiculata Matsush., in Matsushima, Icon. Microfung. Mats. lect.: 122. 1975]. = Rhinocladiella indica Agarwal, Lloydia 32: 388. 1969. [non Chloridium indicum Subram., Proc. Indian Acad. Sci., Sect. B, 42: 286. 1955].

In vitro: Submerged hyphae hyaline to subhyaline, thin-walled, 1–2.5 µm wide; aerial hyphae slightly darker, smooth-walled. Conidiophores generally arising at right angles from creeping aerial hyphae, straight, unbranched, thick-walled, dark brown, continuous or with 1–2(–3) additional thin septa, up to 100 µm long; intercalary cells 10–28 µm long. Conidiogenous cells integrated, terminal, smooth, thick-walled, golden-brown, straight, cylindrical, 25–37(– 47) × 2–3.5 µm; proliferating sympodially, resulting in a straight rachis with conspicuous conidiogenous loci; scars prominent, crowded, slightly pigmented, less than 1 µm diam. Conidia solitary, obovate to obconical, pale brown, finely verrucose, (3–)5–5.5(–7.5) × (2–)2.5–3(–4) µm, hilum conspicuous, slightly pigmented, about 1 µm diam.

Cultural characteristics: Colonies on MEA reaching 35 mm diam after 14 d at 24 °C; minimum temperature for growth above 6 °C, optimum 24 °C, maximum 30 °C. Colonies raised, velvety, dense, with entire margin; surface olivaceous-green, reverse olivaceous-black, often with a diffusing citron-yellow pigment.

Specimens examined: Pakistan, Lahore, from soil, A. Kamal, CBS 400.76 = IMI 088021. South Africa, from preserved Cucumis sativus in 8-oxyquinoline sulphate, M.C. Papendorf, CBS 390.67; Potchefstroom, from Aloe sp., M.C. Papendorf, CBS 391.67. USA, Georgia, isolated from forest soil, CBS 156.59 = ATCC 13211 = IMI 100716 = QM 7716, ex-type culture.

Ramichloridium australiense Arzanlou & Crous, sp. nov. MycoBank MB504548. Figs 9–10A.

Etymology: Named after its country of origin, Australia.

Ab aliis speciebus Ramichloridii conidiophoris ex hyphis verrucosis, crassitunicatis ortis distinguendum.

In vitro: Submerged hyphae hyaline, smooth, thin-walled, 1–2 µm wide; aerial hyphae pale brown, warted. Conidiophores arising vertically and clearly differentiated from creeping aerial hyphae, up to 400 µm tall, with several additional thin septa; intercalary cells, 8–40 × 2–5 µm, from the broadest

90 Ramichloridium and allied genera

Fig. 9. Ramichloridium australiense (CBS 121710). A–C. Macronematous conidiophores with thick-walled and warted subtending hyphae. D. Sympodially proliferating conidiogenous cell, which results in a short rachis with darkened and slightly thickened scars. E. Conidia. Scale bar = 10 µm.

Fig. 10. A. Ramichloridium australiense (CBS 121710). B. Ramichloridium brasilianum (CBS 283.92). C. Radulidium subulatum (CBS 405.76). D. Rhodoveronaea varioseptata (CBS 431.88). Scale bar = 10 µm.

91 Chapter 4

Fig. 11. Ramichloridium musae (CBS 365.36). A. Conidiophores with loose branches. B–D. Sympodially proliferating conidiogenous cells, resulting in a long conidium-bearing rachis. E. Rachis with hardly prominent, slightly darkened scars. F. Conidia. Scale bars = 10 µm.

Fig. 12. Ramichloridium biverticillatum (CBS 335.36). A–B. Profusely branched and biverticillate conidiophores. C. Sympodially proliferating conidiogenous cells, which give rise to a conidium-bearing rachis with crowded, slightly pigmented and thickened scars. D. Conidia. Scale bar = 10 µm. part at the base tapering towards the apex, subhyaline, later becoming pale brown and warted in the lower part. Subtending hyphae thick-walled, warted. Conidiogenous cells integrated, terminal, 10–18 µm long, proliferating sympodially, giving rise to a short rachis with conspicuous conidiogenous loci; scars slightly thickened and darkened, about 1 µm diam. Conidia solitary, aseptate, thin-walled, smooth, subhyaline, subcylindrical to obclavate, (10–)12–15(–23) × 2.5–3 µm, with a truncate base and a slightly darkened and thickened hilum,1.5–2 µm diam, rarely fusing at the basal part.

92 Ramichloridium and allied genera

Cultural characteristics: Colonies on MEA rather slow growing, reaching 8 mm diam after 14 d at 24 °C, with entire, smooth margin; mycelium flat, olivaceous-grey, becoming granular, with gelatinous droplets at the margin developing with aging; reverse pale olivaceous-grey.

Specimen examined: Australia, Queensland, Mount Lewis, Mount Lewis Road, 16°34’47.2” S, 145°19’7” E, 538 m alt., on Musa banksii leaf, Aug. 2006, P.W. Crous and B. Summerell, holotype CBS H-19928, culture ex-type CBS 121710.

Ramichloridium musae (Stahel ex M.B. Ellis) de Hoog, Stud. Mycol. 15: 62. 1977. Fig. 11. Basionym: Veronaea musae Stahel ex M.B. Ellis, in Ellis, More Dematiaceous Hyphomycetes: 209. 1976. ≡ Chloridium musae Stahel, Trop. Agric., Trinidad 14: 43. 1937 (nom. inval. Art. 36). Misapplied name: Chloridium indicum Subram., sensu Batista & Vital, Anais Soc. Biol. Pernambuco 15: 379. 1957.

In vitro: Submerged hyphae smooth, hyaline, thin-walled, 1–2 µm wide; aerial hyphae subhyaline, smooth. Conidiophores arising vertically and mostly sharply differentiated from creeping aerial hyphae, golden-brown; unbranched, rarely branched in the upper part, up to 250 µm tall, with up to 6 additional thin septa, cells 23–40 × 2–2.5 µm, basal cell occasionally inflated. Conidiogenous cells terminally integrated, cylindrical, variable in length, 10–40 µm long, golden-brown near the base, subhyaline to pale brown near the end, fertile part as wide as the basal part, later also becoming septate; rachis elongating sympodially, 2–2.5 µm wide, with hardly prominent, scattered, slightly pigmented scars, about 0.5 µm diam. Conidia solitary, aseptate, hyaline to subhyaline, ellipsoidal, (4–)7–8(–12) × 2–3 µm, smooth or verruculose, thin-walled, with slightly darkened hilum, about 1 µm diam.

Cultural characteristics: Colonies on MEA slow-growing, reaching 27 mm diam after 14 d at 24 °C, with entire, smooth, sharp margin; mycelium mostly submerged, some floccose to lanose aerial mycelium in the olivaceous-grey centre, becoming pale pinkish olivaceous towards the margin; reverse pale orange.

Specimens examined: Cameroon, from Musa sapientum, J.E. Heron, CBS 169.61 = ATCC 15681 = IMI 079492 = DAOM 84655 = MUCL 2689; from Musa sapientum, J. Brun, CBS 190.63 = MUCL 9557. Surinam, Paramaribo, from Musa sapientum leaf, G. Stahel, CBS 365.36 = JCM 6973 = MUCL 9556, ex-type strain of Chloridium musae; from Musa sapientum, G. Stahel, CBS 365.36; dried culture preserved as CBS H-19933.

Ramichloridium biverticillatum Arzanlou & Crous, nom. nov. MycoBank MB504549. Fig. 12. Basionym: Periconiella musae Stahel ex M.B. Ellis, Mycol. Pap. 111: 5. 1967. [non Ramichloridium musae (Stahel ex M.B. Ellis) de Hoog, 1977]. ≡ Ramichloridium musae Stahel, Trop. Agric., Trinidad 14: 43. 1937 (nom. inval. Art. 36). = Ramichloridium musae (Stahel ex M.B. Ellis) de Hoog, Stud. Mycol. 15: 62. 1977, sensu de Hoog, p.p.

Etymology: Named after its biverticillate conidiophores.

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In vitro: Submerged hyphae smooth, hyaline, thin-walled, 1–2 µm wide; aerial hyphae subhyaline, smooth, slightly darker. Conidiophores arising vertically from creeping aerial hyphae, pale brown, profusely branched, biverticillate, with up to three levels of main branches; branches tapering distally, 2–3 µm wide at the base, approx. 2 µm wide in the upper part, up to 250 µm long. Conidiogenous cells terminally integrated, cylindrical, variable in length, 15–50 µm long, rachis straight or geniculate, pale brown, as wide as the basal part; elongating sympodially, forming a rachis with crowded, slightly darkened and thickened minute scars, less than 0.5 µm wide. Conidia solitary, aseptate, hyaline to subhyaline, dacryoid to pyriform, (2–)3–4(–6) × (1.5–)2(–2.5) µm, smooth, thin-walled, with an inconspicuous hilum.

Cultural characteristics: Colonies on MEA slow-growing, reaching 16 mm diam after 14 d at 24 °C, with entire, smooth, sharp margin, rather compact, velvety; surface vinaceous-buff to olivaceous-buff; reverse buff.

Specimen examined: Surinam, from Musa sapientum, Aug. 1936, G. Stahel, CBS 335.36.

Notes: Ramichloridium biverticillatum is a new name based on Periconiella musae. The species is distinct from R. musae because of its profusely branched conidiophores, and conidia that are smaller (2–5 × 1.5–2.5 µm) than those of R. musae (5–11 × 2–3 µm).

Ramichloridium brasilianum Arzanlou & Crous, sp. nov. MycoBank MB504550. Figs 10B, 13.

Etymology: Named after its country of origin, Brazil.

A simili Ramichloridio cerophilo conidiis minoribus, ad 8 μm longis, et conidiis secundariis absentibus distinguendum.

In vitro: Submerged hyphae pale olivaceous, smooth or slightly rough, 1.5–2 µm wide; aerial hyphae olivaceous, smooth or rough, narrower and darker than the submerged hyphae. Conidiophores unbranched, arising vertically from creeping aerial hyphae, straight or flexuose, dark brown, with up to 10 additional septa, thick-walled, cylindrical, 2–2.5 µm wide and up to 70 µm long. Conidiogenous cells integrated, terminal, 10–30 µm long, proliferating sympodially, giving rise to a long, straight rachis with crowded, slightly darkened minute scars, about 0.5 µm diam. Conidia solitary, obovoid to fusiform with the widest part below the middle, thin-walled, verruculose, aseptate, pale brown, slightly rounded at the apex, truncate at the base, (4–)5–6(– 8.5) × 2–2.5(–3) µm, with a slightly thickened and darkened hilum, 1–1.5 µm diam.

Cultural characteristics: Colonies on MEA slow-growing, reaching 6 mm diam after 14 d at 24 °C, velvety to hairy, colonies with entire margin, surface dark olivaceous-grey; black gelatinous exudate droplets produced on OA. Specimen examined: Brazil, São Paulo, Peruibe, Jureia Ecological Reserve, forest soil, Jan. 1991, D. Attili, holotype CBS H-19929, culture ex-type CBS 283.92.

Ramichloridium cerophilum (Tubaki) de Hoog, Stud. Mycol. 15: 74. 1977. Fig. 14. Basionym: Acrotheca cerophila Tubaki, J. Hattori Bot. Lab. 20: 143. 1958. ≡ Cladosporium cerophilum (Tubaki) Matsush., in Matsushima, Icon. Microfung. Matsush. lect. (Kobe): 34. 1975.

94 Ramichloridium and allied genera

Fig. 13. Ramichloridium brasilianum (CBS 283.92). A–B. Macronematous conidiophores with sympodially proliferating conidiogenous cells, resulting in a conidium-bearing rachis. C. Rachis with crowded and slightly pigmented scars. D. Conidia. Scale bar = 10 µm.

Fig. 14. Ramichloridium cerophilum (CBS 103.59). A–C. Conidial apparatus at different stages of development, resulting in macronematous conidiophores and sympodially proliferating conidiogenous cells. D–E. Formation of secondary conidia. F. Conidia. Scale bar = 10 µm.

In vitro: Submerged hyphae pale olivaceous-brown, smooth or slightly rough, 1.5–3 µm wide; aerial hyphae olivaceous-brown, smooth or slightly rough, somewhat narrower and darker than the submerged hyphae. Conidiophores unbranched, arising vertically from creeping aerial hyphae, dark brown, thick-walled, smooth or verruculose, hardly tapering towards the apex, 2–3 µm wide, up to 50 µm long, with up to 3 additional septa. Conidiogenous cells integrated, terminal, proliferating sympodially, rachis short and straight, with crowded, prominent, pigmented unthickened scars, minute, approx. 0.5 µm diam. Conidia solitary, fusiform to clavate, thin-walled, smooth, 0(–1)-septate, subhyaline, (4–)6–7(–11) × (2–) 2.5(–3) µm, with a conspicuous hilum, about 0.5 µm diam, slightly raised with an inconspicuous marginal frill, somehow resembling those of Cladosporium. Conidia sometimes producing 1–3(–4) short secondary conidia.

Cultural characteristics: Colonies on MEA rather slow-growing, reaching 12 mm diam after 14 d at 24 °C, velvety to hairy, with entire margin; surface dark olivaceous-grey, with black gelatinous exudate droplets on OA.

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Fig. 15. Ramichloridium indicum (CBS 171.96). A–B. Macronematous conidiophores. C–E. Sympodially proliferating conidiogenous cells, resulting in a conidium-bearing rachis with pigmented and thickened scars. F. Conidia. Scale bar = 10 µm.

Specimen examined: Japan, isolated from Sasa sp., K. Tubaki, CBS 103.59, ex-type.

Notes: Phylogenetically, this species together with Ramichloridium apiculatum and R. musae cluster within the Mycosphaerellaceae clade. Ramichloridium cerophilum can be distinguished from its relatives by the production of secondary conidia and its distinct conidial hila.

Ramichloridium indicum (Subram.) de Hoog, Stud. Mycol. 15: 70. 1977. Fig. 15. Basionym: Chloridium indicum Subram., Proc. Indian Acad. Sci., Sect. B, 42: 286. 1955 [non Rhinocladiella indica Agarwal, Lloydia 32: 388. 1969]. ≡ Veronaea indica (Subram.) M.B. Ellis, in Ellis, More Dematiaceous Hyphomycetes: 209. 1976. = Veronaea verrucosa Geeson, Trans. Brit. Mycol. Soc. 64: 349. 1975.

In vitro: Submerged hyphae smooth, thin-walled, hyaline, 1–2.5 µm wide, with thin septa; aerial hyphae coarsely verrucose, olivaceous-green, rather thick-walled, 2–2.5 µm wide, with thin septa. Conidiophores arising vertically from creeping hyphae at right angles, straight, unbranched, thick-walled, smooth, dark brown, with up to 10 thin septa, up to 250 µm long, 2–4 µm wide, often with inflated basal cells. Conidiogenous cells terminally integrated, up to 165 µm long, smooth, dark brown, sympodially proliferating, rachis straight or flexuose, geniculate or nodose, subhyaline; scars thickened and darkened, clustered at nodes, approx. 0.5 µm diam. Microcyclic conidiation observed in culture. Conidia solitary, (0–)1-septate, not constricted at the septum, subhyaline to pale brown, smooth or coarsely verrucose, rather thin- walled, broadly ellipsoidal to globose, (5–)7–8(–10) × (4–)6–6.5(–9) µm, with truncate base; hilum conspicuous, slightly darkened, not thickened, about 1 µm diam.

Cultural characteristics: Colonies on MEA reaching 35 mm diam after 14 d at 24 °C. Colonies velvety, rather compact, slightly elevated, with entire, smooth, whitish margin, dark olivaceous- green in the central part.

Specimen examined: Living culture, Feb. 1996, L. Marvanová, CBS 171.96.

96 Ramichloridium and allied genera

Fig. 16. Ramichloridium strelitziae (CBS 121711). A–C. Conidial apparatus at different stages of development, resulting in macronematous conidiophores and sympodially proliferating conidiogenous cells. D–E. Rachis with crowded, slightly pigmented, thickened, circular scars. F. Conidia. Scale bars = 10 µm.

Ramichloridium pini de Hoog & Rahman, Trans. Brit. Mycol. Soc. 81: 485. 1983.

Specimen examined: U.K., Scotland, Old Aberdeen, branch of Pinus contorta (Pinaceae), 1982, M.A. Rahman, ex-type strain, CBS 461.82 = MUCL 28942.

Note: The culture examined (CBS 461.82) was sterile. For a full description see de Hoog et al. (1983).

Ramichloridium strelitziae Arzanlou, W. Gams & Crous, sp. nov. MycoBank MB504551. Figs 16–17A.

Etymology: Named after its host, Strelitzia.

Ab aliis speciebus Ramichloridii conidiophoris brevibus, ad 40 μm longis, et cicatricibus rotundis, paulo protrudentibus distinguendum.

In vitro: Submerged hyphae smooth, hyaline, thin-walled, 2–2.5 µm wide; aerial hyphae pale brown, verrucose. Conidiophores arising vertically from creeping aerial hyphae, clearly differentiated from the vegetative hyphae, subhyaline, later becoming pale brown, thick-walled, smooth or verruculose, with 1–3 additional septa; up to 40 µm long and 2 µm wide. Conidiogenous cells integrated, terminal, cylindrical, variable in length, 10–35 µm long, subhyaline, later turning pale brown, fertile part as wide as the basal part, proliferating sympodially, forming a straight rachis with slightly thickened and darkened, circular, somewhat protruding scars, approx. 0.5 µm diam. Conidia solitary, aseptate, smooth or verruculose, subhyaline, oblong, ellipsoidal to clavate, (3–)4–5(–5.5) × (1–)2(–2.5) µm, with truncate base and unthickened, non-pigmented hilum.

Cultural characteristics: Colonies on MEA slow-growing, reaching 5 mm diam after 14 d at 24 °C, with entire margin; aerial mycelium rather compact, raised, dense, olivaceous-grey; reverse olivaceous-black.

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Fig. 17. A. Ramichloridium strelitziae (CBS 121711). B. Veronaea japonica (CBS 776.83). C. Veronaeopsis simplex (CBS 588.66). Scale bar = 10 µm.

Specimen examined: South Africa, KwaZulu-Natal, Durban, near Réunion, on leaves of Strelitzia nicolai, 5 Feb. 2005, W. Gams & H. Glen, CBS-H 19776, holotype, culture ex-type CBS 121711.

Zasmidium Fr., Summa Veg. Scand. 2: 407. 1849.

In vitro: Submerged hyphae smooth, thin-walled, hyaline, with thin septa; aerial hyphae coarsely verrucose, olivaceous-green, thick-walled, with thin septa. Conidiophores not differentiated from vegetative hyphae, often reduced to conidiogenous cells. Conidiogenous cells integrated, predominantly terminal, sometimes lateral, arising from aerial hyphae, cylindrical, pale brown; polyblastic, proliferating sympodially producing crowded, conspicuously pigmented, almost flat, darkened, somewhat refractive scars. Conidia in short chains, cylindrical to fusiform, verrucose, obovate to obconical, pale brown, base truncate, with a conspicuous, slightly pigmented, thickened and refractive hilum. Primary conidia sometimes larger, subhyaline, verrucose or smooth-walled, 0–4-septate, variable in length, fusiform to cylindrical; conidial secession schizolytic.

Type species: Zasmidium cellare (Pers. : Fr.) Fr., Summa Veg. Scand. 2: 407. 1849.

98 Ramichloridium and allied genera

Fig. 18. Zasmidium cellare (CBS 146.36). A–D. Micronematous conidiophores with terminal, integrated conidiogenous cells. E. Conidiogenous cell with pigmented, thickened and refractive scars. F–G. Primary and secondary conidia. Scale bar = 10 µm.

Zasmidium cellare (Pers. : Fr.) Fr., Summa Veg. Scand. 2: 407. 1849. Fig. 18. Basionym: Racodium cellare Pers., Neues Mag. Bot. 1: 123. 1794. ≡ Antennaria cellaris (Pers. : Fr.) Fr., Syst. Mycol. 3: 229. 1829. ≡ Cladosporium cellare (Pers. : Fr.) Schanderl, Zentralbl. Bakteriol., 2. Abt., 94: 117. 1936. ≡ Rhinocladiella cellaris (Pers. : Fr.) M.B. Ellis, in Ellis, Dematiaceous Hyphomycetes: 248. 1971.

In vitro: Submerged hyphae smooth, thin-walled, hyaline, 2–3 µm wide, with thin septa; aerial hyphae coarsely verrucose, olivaceous-green, rather thick-walled, 2–2.5 µm wide, with thin septa. Conidiophores not differentiated from vegetative hyphae, often reduced to conidiogenous cells. Conidiogenous cells integrated, predominantly terminal, sometimes lateral, arising from aerial hyphae, cylindrical, 20–60 µm long and 2–2.5 µm wide, pale brown, proliferating sympodially producing crowded, conspicuously pigmented scars that are thickened and refractive, about 1 µm diam. Conidia cylindrical to fusiform, verrucose, obovate to obconical, pale brown, with truncate base, (6–)9–14(–27) × 2–2.5 µm, with a conspicuous, slightly pigmented, refractive hilum, approx. 1 µm diam. Primary conidia sometimes subhyaline, verrucose or smooth-walled, thin-walled, 0–1(–4)-septate, variable in length, fusiform to cylindrical.

Cultural characteristics: Colonies reaching 7 mm diam after 14 d at 24 °C. Colonies velvety, rather compact, slightly elevated with entire margin; surface dark olivaceous-green in the central part, margin smooth, whitish.

Specimen examined: Wall in wine cellar, Jun. 1936, H. Schanderl, ATCC 36951 = IFO 4862 = IMI 044943 = LCP 52.402 = LSHB BB274 = MUCL 10089 = CBS 146.36.

Notes: The name Racodium Fr., typified by Ra. rupestre Pers. : Fr., has been conserved over the older one by Persoon, with Ra. cellare as type species. De Hoog (1979) defended the use of Zasmidium in its place for the well-known wine-cellar fungus. Morphologically Zasmidium resembles Stenella Syd., and both reside in the Capnodiales, though the type of Stenella, S. araguata Syd., clusters in the Teratosphaeriaceae, and the type of Zasmidium, Z. cellare, in the Mycosphaerellaceae. When accepting anamorph genera as

99 Chapter 4 polyphyletic within an order, preference would be given to the well-known name Stenella over the less known Zasmidium, even though the latter name is older. Further studies are required, however, to clarify if all stenella-like taxa should be accommodated in a single genus, Stenella. If this is indeed the case, a new combination for Zasmidium cellare will be proposed in Stenella, and the latter genus will have to be conserved over Zasmidium.

Chaetothyriales (Herpotrichiellaceae)

The four “Ramichloridium” species residing in the Chaetothyriales clade do not differ sufficiently in morphology to separate them fromRhinocladiella (type Rh. atrovirens). Because of the pale brown conidiophores, conidiogenous cells with crowded, slightly prominent scars and the occasional presence of an Exophiala J.W. Carmich. synanamorph, Rhinocladiella is a suitable genus to accommodate them. These four species chiefly differ fromRamichloridium in the morphology of their conidial apparatus, which is clearly differentiated from the vegetative hyphae. The appropriate combinations are therefore introduced for Ramichloridium anceps, R. mackenziei, R. fasciculatum and R. basitonum. The genus Veronaea (type species: V. botryosa) also resides in the Chaetothyriales clade. Veronaea can be distinguished from Rhinocladiella by the absence of exophiala-type budding cells and its predominantly 1-septate conidia. Furthermore, the conidiogenous loci in Veronaea are rather flat, barely prominent.

Rhinocladiella Nannf., Svensk Skogsvårdsfören. Tidskr., Häfte 32: 461. 1934.

In vitro: Colonies dark olivaceous-brown, slow-growing, almost moist. Submerged hyphae hyaline to pale olivaceous, smooth; aerial hyphae, if present, more darkly pigmented. Exophiala-type budding cells usually present in culture. Conidial apparatus usually branched, olivaceous-brown, consisting of either slightly differentiated tips of ascending hyphae or septate, markedly differentiated conidiophores. Conidiogenous cells intercalary or terminal, polyblastic, cylindrical to acicular, with a sympodially proliferating, subdenticulate rachis; scars unthickened, non-pigmented to somewhat darkened-refractive. Conidia solitary, hyaline to subhyaline, aseptate, thin-walled, smooth, subglobose, with a slightly pigmented hilum; conidial secession schizolytic.

Type species: Rh. atrovirens Nannf., Svenska Skogsvårdsfören. Tidskr. 32: 461. 1934.

Rhinocladiella anceps (Sacc. & Ellis) S. Hughes, Can. J. Bot. 36: 801. 1958. Fig. 19. Basionym: Sporotrichum anceps Sacc. & Ellis, Michelia 2: 576. 1882. = Veronaea parvispora M.B. Ellis, in Ellis, More Dematiaceous Hyphomycetes: 210. 1976.

Misapplied name: Chloridium minus Corda sensu Mangenot, Rev. Mycol. (Paris) 18: 137. 1953.

In vitro: Submerged hyphae subhyaline, smooth, thick-walled, 2–2.5 µm wide; aerial hyphae pale brown. Swollen germinating cells often present on MEA, giving rise to an Exophiala synanamorph. Conidiophores slightly differentiated from vegetative hyphae, arising from prostrate aerial hyphae, consisting of either unbranched or loosely branched stalks, thick- walled, golden to dark-brown, up to 350 µm tall, which may have up to 15 thin, additional septa, intercalary cells 9–14 µm long. Conidiogenous cells terminal, rarely lateral, cylindrical,

100 Ramichloridium and allied genera

Fig. 19. Rhinocladiella anceps (CBS 181.65). A. Macronematous conidiophores. B–D. Conidial apparatus at different stages of development, resulting in semi-micronematous conidiophores and sympodially proliferating conidiogenous cells. E. Conidiogenous loci. F. Conidia. Scale bars = 10 µm. occasionally intercalary, variable in length, smooth, golden to dark brown at the base, paler toward the apex, later becoming inconspicuously septate, fertile part as wide as the basal part, 15–40 × 1.5–2 µm; with crowded, slightly prominent, unpigmented, conidium-bearing denticles, about 0.5 µm diam. Conidia solitary, subhyaline, thin-walled, smooth, subglobose to ellipsoidal, 2.5–4 × 2–2.5 µm, with a less conspicuous, slightly darkened hilum, less than 0.5 µm diam.

Cultural characteristics: Colonies on MEA reaching 6–12 mm diam after 14 d at 24 °C, with entire, smooth, sharp margin; mycelium powdery, becoming hairy at centre; olivaceous-green to brown, reverse dark-olivaceous.

Specimens examined: Canada, Ontario, Campbellville, from soil under Thuja plicata, Apr. 1965, G. L. Barron, CBS H-7715 (isoneotype); CBS H-7716 (isoneotype); CBS H-7717 (isoneotype); CBS H-7718 (isoneotype); CBS H-7719 (isoneotype), ex-type strain, CBS 181.65 = ATCC 18655 = DAOM 84422 = IMI 134453 = MUCL 8233 = OAC 10215. France, from stem of Fagus sylvatica, 1953, F. Mangenot, CBS 157.54 = ATCC 15680= MUCL 1081= MUCL 7992 = MUCL 15756.

Notes: Rhinocladiella anceps (conidia 2.5–4 µm long) resembles Rh. phaeophora Veerkamp & W. Gams (1983) (conidia 5.5–6 µm long), but has shorter conidia.

Rhinocladiella basitona (de Hoog) Arzanlou & Crous, comb. nov. MycoBank MB504552. Fig. 20. Basionym: Ramichloridium basitonum de Hoog, J. Clin. Microbiol. 41: 4774. 2003.

In vitro: Submerged hyphae hyaline, smooth, thin-walled, 2 µm wide; aerial hyphae rather thick-walled, pale brown. Conidiophores slightly differentiated from vegetative hyphae, profusely and mostly verticillately branched, straight or flexuose, pale-brown, 2–2.5 µm wide. Conidiogenous cells terminal, variable in length, 10–100 µm long, pale brown, straight or geniculate, proliferating sympodially, giving rise to a long, 2–2.5 µm wide rachis, with slightly

101 Chapter 4 prominent, truncate conidium-bearing denticles, slightly darkened. Conidia solitary, hyaline, thin-walled, smooth, pyriform to clavate, with a round apex, and slightly truncate base, (1–)3– 4(–5) × 1–2 µm, hilum conspicuous, slightly darkened and thickened, less than 0.5 µm diam.

Cultural characteristics: Colonies on MEA reaching 19 mm diam after 14 d at 24 °C, with entire, smooth, sharp margin; mycelium rather flat and slightly elevated in the centre, pale olivaceous-grey to olivaceous-grey; reverse olivaceous-black.

Specimen examined: Japan, Hamamatsu, from subcutaneous lesion with fistula on knee of 70- year-old male, Y. Suzuki, ex-type culture CBS 101460 = IFM 47593.

Rhinocladiella fasciculata (V. Rao & de Hoog) Arzanlou & Crous, comb. nov. MycoBank MB504553. Fig. 21. Basionym: Ramichloridium fasciculatum V. Rao & de Hoog, Stud. Mycol. 28: 39. 1986.

In vitro: Submerged hyphae subhyaline, smooth, thick-walled, 2–2.5 µm wide; aerial hyphae pale brown. Conidiophores arising vertically from ascending hyphae in loose fascicles, unbranched or loosely branched at acute angles, cylindrical, smooth, brown and thick-walled at the base, up to 220 µm long and 2–3 µm wide, with 0–5 thin additional septa. Conidiogenous cells terminal, cylindrical, 30–100 µm long, thin-walled, smooth, pale brown, fertile part as wide as the basal part, up to 2 µm wide, proliferating sympodially, giving rise to a rachis with hardly prominent, slightly pigmented, not thickened scars, less than 0.5 µm diam. Conidia solitary, smooth, thin- walled, subhyaline, ellipsoidal, (2.5–)4–5(–6) × 2–3 µm, with truncate, slightly pigmented hilum, about 0.5 µm diam. Synanamorph forming on torulose hyphae originating from giant cells; compact heads of densely branched hyphae forming thin-walled, lateral, subglobose cells, on which conidiogenous cells are formed; conidiogenous cells proliferating percurrently, giving rise to tubular annellated zones with inconspicuous annellations, up to 12 µm long, 1–1.5 µm wide. Conidia smooth, thin-walled, aseptate, subhyaline, globose, 2–2.5 µm diam.

Cultural characteristics: Colonies on MEA reaching 8 mm diam after 14 d at 24 °C, with entire, smooth, sharp margin; mycelium velvety, becoming farinose in the centre due to abundant sporulation, olivaceous-green to brown, reverse dark olivaceous. Blackish droplets often produced at the centre, which contain masses of Exophiala conidia.

Specimen examined: India, Karnataka, Thirathahalli, isolated by V. Rao from decayed wood, holotype CBS-H 3866, culture ex-type CBS 132.86.

Rhinocladiella mackenziei (C.K. Campb. & Al-Hedaithy) Arzanlou & Crous, comb. nov. MycoBank MB504554. Fig. 22. Basionym: Ramichloridium mackenziei C.K. Campb. & Al-Hedaithy, J. Med. Veterin. Mycol. 31: 330. 1993.

In vitro: Submerged hyphae subhyaline, smooth, thin-walled, 2–3 µm wide; aerial hyphae pale brown, slightly narrower. Conidiophores slightly or not differentiated from vegetative hyphae, arising laterally from aerial hyphae, with one or two additional septa, often reduced to a discrete or intercalary conidiogenous cell, pale-brown, 10–25 × 2.5–3.5 µm. Conidiogenous cells terminal or intercalary, variable in length, 5–15 µm long and 3–5 µm wide, occasionally slightly wider than the basal part, pale brown, rachis with slightly prominent, unpigmented, non- thickened scars, about 0.5 µm diam. Conidia golden-brown, thin-walled, smooth, ellipsoidal to obovate, subcylindical, (5–)8–9(–12) × (2–)3–3.5(–5) µm, with darkened, inconspicously thickened, protuberant or truncate hilum, less than 1 µm diam. 102 Ramichloridium and allied genera

Fig. 20. Rhinocladiella basitona (CBS 101460). A–B. Semi-micronematous conidiophores with verticillate branching pattern. C–D. Sympodially proliferating conidiogenous cells, giving rise to a long rachis with slightly prominent, truncate conidium-bearing denticles. E. Intercalary conidiogenous cell. F. Conidia. Scale bars = 10 µm.

Fig. 21. Rhinocladiella fasciculata (CBS 132.86). A. Conidiophores. B. Sympodially proliferating conidiogenous cells, which give rise to a long rachis with slightly prominent, unthickened scars. C. Conidia. D–E. Synanamorph consisting of conidiogenous cells with percurrent proliferation. F. Conidia. Scale bars = 10 µm.

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Cultural characteristics: Colonies on MEA reaching 5 mm diam after 14 d at 24 °C, with entire, smooth, sharp margin; mycelium densely lanose and elevated in the centre, olivaceous-green to brown; reverse dark olivaceous.

Specimens examined: Israel, Haifa, isolated from brain abscess, CBS 368.92 = UTMB 3170; human brain abscess, E. Lefler, CBS 367.92 = NCPF 2738 = UTMB 3169. Saudi Arabia, from phaeohyphomycosis of the brain, S.S.A. Al-Hedaithy, ex-type strain, CBS 650.93 = MUCL 40057 = NCPF 2808; from brain abscess, Pakistani male who travelled to Saudi Arabia, CBS 102592 = NCPF 7460. United Arab Emirates, from fatal brain abscess, CBS 102590 = NCPF 2853.

Notes: Morphologically Rhinocladiella mackenziei is somewhat similar to Pleurothecium obovoideum (Matsush.) Arzanlou & Crous, which was originally isolated from dead wood. However, P. obovoideum has distinct conidiophores, and the ascending hyphae are thick-walled, and the denticles cylindrical, up to 1.5 µm long. In contrast, Rh. mackenziei has only slightly prominent denticles. Rhinocladiella mackenziei is a member of the Chaetothyriales, while P. obovoideum clusters in the Chaetosphaeriales.

Thysanorea Arzanlou, W. Gams & Crous, gen. nov. MycoBank MB504555.

Etymology: (Greek) thysano = brush, referring to the brush-like branching pattern, suffix derived from Veronaea.

Veronaeae similis sed conidiophoris partim Periconiae similibus dense ramosis distinguenda.

In vitro: Submerged hyphae subhyaline, smooth, thin-walled; aerial hyphae pale brown, smooth or verrucose. Conidiophores dimorphic; micronematous conidiophores slightly differentiated from vegetative hyphae, branched or simple, multiseptate. Conidiogenous cells terminal, polyblastic, variable in length, smooth, golden- to dark brown at the base, paler towards the apex, later sometimes inconspicuously septate; fertile part often wider than the basal part, clavate to doliiform, with crowded, more or less prominent conidium-bearing denticles, unpigmented, but slightly thickened. Macronematous conidiophores consisting of well-differentiated, thick- walled, dark brown stalks; apically repeatedly densely branched, forming a complex head, each branchlet giving rise to a conidium-bearing denticulate rachis with slightly pigmented, thickened scars. Conidia of both kinds of conidiophore formed singly, smooth, pale brown, obovoidal to pyriform, (0–)1-septate, with a truncate base and darkened hilum; conidial secession schizolytic.

Type species: Thysanorea papuana (Aptroot) Arzanlou, W. Gams & Crous, comb. nov.

Thysanorea papuana (Aptroot) Arzanlou, W. Gams & Crous, comb. nov. MycoBank MB504556. Figs 7C, 23–24. Basionym: Periconiella papuana Aptroot, Nova Hedwigia 67: 491. 1998.

In vitro: Submerged hyphae subhyaline, smooth, thin-walled, 1.5–3 µm wide; aerial hyphae pale brown, smooth to verrucose, 1.5–2 µm wide. Conidiophores dimorphic; micronematous conidiophores slightly differentiated from vegetative hyphae, branched or simple, up to 6-septate. Conidiogenous cells terminal or intercalary, variable in length, 5–20 µm long, thin-walled, smooth, golden- to dark brown at the base, paler toward the apex, later sometimes becoming

104 Ramichloridium and allied genera

Fig. 22. Rhinocladiella mackenziei (CBS 368.92). A. Intercalary conidiogenous cell. B–E. Semi-micronematous conidiophores and sympodially proliferating conidiogenous cells, resulting in a rachis with slightly prominent, unthickened scars. F. Conidia. Scale bar = 10 µm.

Fig. 23. Thysanorea papuana (CBS 212.96). A. Intercalary conidiogenous cell. B–I. Semi-micronematous conidiophores and sympodially proliferating conidiogenous cells, resulting in a rachis with prominent conidium bearing denticles. J–K. Microcyclic conidiation observed in slide cultures. L. Conidia. Scale bar = 10 µm.

105 Chapter 4

Fig. 24. Thysanorea papuana (CBS 212.96), periconiella-like synanamorph. A. Macronematous conidiophores. B–C. Conidiophores with dense apical branches. D. Branches with different levels of branchlets. E–I. Conidiogenous cells at different stages of development; sympodially proliferating conidiogenous cells give rise to a denticulate rachis. J–K. Conidia. Scale bars = 10 µm. inconspicuously septate, fertile part wider than basal part, often clavate, with crowded, more or less prominent conidium-bearing denticles, about 1 µm diam, unpigmented but slightly thickened. Conidia solitary, subhyaline, thin-walled, smooth, cylindrical to pyriform, rounded at the apex and truncate at the base, pale brown, (0–)1-septate, (5–)7–8(–11) × (2–)3(–4) µm,

106 Ramichloridium and allied genera with a truncate base and darkened hilum, 1 µm diam. Macronematous conidiophores present in old cultures after 1 mo of incubation, consisting of well-differentiated, thick-walled, dark brown stalks, up to 220 µm long, (4–)5–6(–7) µm wide, with up to 15 additional septa, often with inflated basal cells; apically densely branched, forming a complex head, with up to five levels of branchlets, 20–50 µm long, each branchlet giving rise to a denticulate conidium- bearing rachis; scars slightly pigmented, thickened, about 1 µm diam. Conidia solitary, thin- walled, smooth, pale brown, obovoidal to pyriform, (0–)1-septate, (4–)5–6(–8) × (2–)3(–4) µm, with a truncate base and darkened hilum, 1–2 µm diam.

Cultural characteristics: Colonies on MEA reaching 10 mm diam after 14 d at 24 °C, with entire, sharp margin; mycelium velvety, elevated, with colonies up to 2 mm high, surface olivaceous- grey to iron-grey; reverse greenish black.

Specimen examined: Papua New Guinea, Madang Province, foothill of Finisterre range, 40.8 km along road Madang-Lae, alt. 200 m, isolated from unknown stipe, 2 Nov. 1995, A. Aptroot, holotype CBS-H 6351, culture ex-type CBS 212.96.

Veronaea Cif. & Montemart., Atti Ist. Bot. Lab. Crittog. Univ. Pavia, sér. 5, 15: 68. 1957.

In vitro: Colonies velvety, pale olivaceous-brown, moderately fast-growing. Submerged hyphae hyaline to pale olivaceous, smooth; aerial hyphae, more darkly pigmented. Exophiala- type budding cells absent in culture. Conidiophores erect, straight or flexuose, unbranched or occasionally loosely branched, sometimes geniculate, smooth-walled, pale to medium- or olivaceous-brown. Conidiogenous cells terminally integrated, polyblastic, occasionally intercalary, cylindrical, pale brown, later often becoming septate, fertile part subhyaline, often as wide as the basal part, rachis with crowded, flat to slightly prominent, faintly pigmented, unthickened scars. Conidia solitary, smooth, cylindrical to pyriform, rounded at the apex and truncate at the base, pale brown, 1(–2)-septate; conidial secession schizolytic.

Type species: Veronaea botryosa Cif. & Montemart., Atti Ist. Bot. Lab. Crittog. Univ. Pavia, sér. 5, 15: 68. 1957.

Veronaea botryosa Cif. & Montemart., Atti Ist. Bot. Lab. Crittog. Univ. Pavia, sér. 5, 15: 68. 1957. Fig. 25.

In vitro: Submerged hyphae hyaline to pale olivaceous, smooth; aerial hyphae more darkly pigmented. Conidiophores erect, straight or flexuose, unbranched or occasionally loosely branched, sometimes geniculate, smooth-walled, pale brown to olivaceous-brown, 2–3 µm wide and up to 200 µm long. Conidiogenous cells terminal, occasionally intercalary, cylindrical, 10– 100 µm long, pale brown, later often becoming septate, fertile part subhyaline, often as wide as the basal part, rachis with crowded, flat to slightly prominent, faintly pigmented, unthickened scars. Conidia solitary, smooth, cylindrical to pyriform, (3–)6.5–8.5(–12) × (1.5–)2–2.5(–3) µm, rounded at the apex and truncate at the base, pale brown, 1(–2)-septate, with a faintly darkened, unthickened hilum, about 0.5 µm diam.

Cultural characteristics: Colonies on MEA reaching 30 mm diam after 14 d at 24 °C, with entire, sharp margin; mycelium velvety, slightly elevated in the centre, surface olivaceous-grey to greyish-brown; reverse greenish black.

107 Chapter 4

Fig. 25. Veronaea botryosa (CBS 254.57). A–C. Semi-micronematous conidiophores and sympodially proliferating conidiogenous cells. D–E. Rachis with crowded and flat scars. F–G. Microcyclic conidiation. H. Conidia. Scale bars = 10 µm.

Fig. 26. Veronaea compacta (CBS 268.75). A–B. Semi-micronematous conidiophores and sympodially proliferating conidiogenous cells. C–D. Rachis with hardly prominent denticles. E. Conidia. Scale bar = 10 µm.

108 Ramichloridium and allied genera

Specimens examined: India, Ramgarh, about 38 km from Jaipur, isolated from goat dung, 1 Sep. 1963, B.C. Lodha, CBS 350.65 = IMI 115127 = MUCL 7972. Italy, Tuscany, Pisa, isolated from Sansa olive slag, 1954, O. Verona, ex-type strain, CBS 254.57 = IMI 070233 = MUCL 9821.

Veronaea compacta Papendorf, Bothalia 12: 119. 1976. Fig. 26.

In vitro: Submerged hyphae subhyaline, smooth, thin-walled, 1.5–3 µm wide; aerial hyphae rather thick-walled, pale brown. Conidiophores slightly differentiated from vegetative hyphae, lateral or occasionally terminal, often wider than the supporting hypha, up to 4 µm wide, unbranched or branched at acute angles, with 1–3 adititional septa, cells often inflated and flask- shaped, pale-brown, up to 60 µm long. Conidiogenous cells terminal, occasionally intercalary, variable in length, up to 10 µm long, pale brown, cylindrical to doliiform or flask-shaped, with hardly prominent denticles; scars flat, slightly pigmented, not thickened, about 0.5 µm diam. Conidia solitary, pale brown, smooth, thin-walled, ellipsoidal to ovoid, 0–1(–2)-septate, often constricted at the septa, (4–)6–7(–9) × 2–3 µm, with a round apex and truncate base; hilum prominent, slightly darkened, unthickened, about 0.5 µm diam.

Cultural characteristics: Colonies rather slow growing, reaching 15 mm diam on MEA after 14 d at 24 °C; surface velvety to lanose, slightly raised in the centre, pale grey to pale brownish grey; reverse dark grey.

Specimen examined: South Africa, soil, M.C. Papendorf, ex-type culture CBS 268.75.

Veronaea japonica Arzanlou, W. Gams & Crous, sp. nov. MycoBank MB504557. Figs 17B, 27.

Etymology: Named after the country of origin, Japan.

Veronaeae compactae similis, sed cellulis inflatis, aggregatis, crassitunicatis, fuscis in vitro formatis distinguenda.

In vitro: Submerged hyphae subhyaline, smooth, thin-walled, 1.5–3 µm wide; aerial hyphae slightly narrower, pale brown; hyphal cells later becoming swollen, thick-walled, dark brown, often aggregated. Conidiophores slightly differentiated from aerial vegetative hyphae, lateral, or terminal, often wider than the supporting hypha, 2–3 µm wide, up to 65 µm long, unbranched or occasionally branched, pale brown, thin-walled, smooth, with 1–3 additional septa. Conidiogenous cells terminal, occasionally intercalary, variable in length, up to 15 µm long, pale brown, cylindrical to clavate, with hardly prominent denticles; scars flat, slightly pigmented, not thickened, about 0.5 µm diam. Conidia solitary, pale brown, smooth, thin- walled, ellipsoidal to ovoid, (0–)1-septate, often constricted at the septum, (6–)7–8(–10) × 2–2.5(–4) µm, with a round apex and truncate base; hilum unthickened but slightly darkened, about 1 µm diam.

Cultural characteristics: Colonies rather slow growing, reaching 7.5 mm diam on MEA after 14 d at 24 °C; surface velvety to lanose, slightly raised in the centre, olivaceous-brown, with entire margin; reverse dark-olivaceous.

Specimen examined: Japan, Kyoto, Daitokuji Temple, Kyoto, inside dead bamboo culm, Dec. 1983, W. Gams, holotype CBS-H 3490, culture ex-type CBS 776.83.

109 Chapter 4

Fig. 27. Veronaea japonica (CBS 776.83). A. Intercalary conidiogenous cells. B–D. Semi-micronematous conidiophores and sympodially proliferating conidiogenous cells. E. Conidia. F. Thick-walled, dark brown hyphal cells. Scale bar = 10 µm.

Note: This species is morphologically similar to V. compacta (Papendorf 1976), but can be distinguished based on the presence of dark brown, swollen hyphal cells in culture, which are absent in V. compacta.

Pleurothecium obovoideum clade (Chaetosphaeriales)

Ramichloridium obovoideum was regarded as similar to “Ramichloridium” (Rhinocladiella) mackenziei by some authors, and subsequently reduced to synonymy (Ur-Rahman et al. 1988). However, R. obovoideum clusters with Carpoligna pleurothecii, the teleomorph of Pleuro- thecium Höhn. Because it is also morphologically similar to other species of Pleurothecium, we herewith combine it into that genus.

Pleurothecium obovoideum (Matsush.) Arzanlou & Crous, comb. nov. MycoBank MB504558. Fig. 28. Basionym: Rhinocladiella obovoidea Matsush., Icones Microfung. Mats. lect.: 123. 1975. ≡ Ramichloridium obovoideum (Matsush.) de Hoog, Stud. Mycol. 15: 73. 1977.

In vitro: Submerged hyphae smooth, hyaline, thin-walled, 1–2 µm wide; aerial hyphae hyaline to subhyaline, smooth. Conidiophores arising vertically from creeping hyphae, ascending hyphae thick-walled and dark brown; conidiophores 10–35 µm long, 1–2-septate, often reduced to a conidiogenous cell, unbranched, thick-walled, smooth, tapering towards the apex, pale brown. Conidiogenous cells integrated, cylindrical to ampulliform, 5–20 µm long, pale brown,

110 Ramichloridium and allied genera

Fig. 28. Pleurothecium obovoideum (CBS 209.95). A–C. Conidial apparatus consisting of conidiophores with sympodially proliferating conidiogenous cells as seen in slide cultures of ca. 14 d. D. Short chain of conidia. E–G. Sympodially proliferating conidiogenous cells, resulting in a short rachis with subcylindrical to cylindrical denticles. H. Conidia. Scale bar = 10 µm. elongating sympodially, with a short rachis giving rise to denticles, 1 µm long, slightly pigmented. Conidia aseptate, solitary or in short chains of up to 3, smooth, pale brown, ellipsoidal to obovate, (9–)11–12(–14.5) × (3–)4(–5) µm, smooth, thin-walled, with a more or less rounded apex, a truncate base and a slightly darkened, unthickened hilum, 1.5 µm diam.

Cultural characteristics: Colonies slow-growing, reaching 15 diam after 14 d at 24 °C, with entire, smooth margin; surface rather compact, mycelium mainly flat, submerged, some floccose to lanose aerial mycelium in the centre, buff; reverse honey.

Specimen examined: Japan, Kobe Municipal Arboretum, T. Matsushima, from dead leaf of Pasania edulis, CBS 209.95 = MFC 12477.

Incertae sedis (Sordariomycetes)

Ramichloridium schulzeri clade

Ramichloridium schulzeri, including its varieties, clusters near Thyridium Nitschke and the Magnaporthaceae, and is phylogenetically as well as morphologically distinct from the other genera in the Ramichloridium complex. To accommodate these taxa, a new genus is introduced below.

111 Chapter 4

Myrmecridium Arzanlou, W. Gams & Crous, gen. nov. MycoBank MB504559.

Etymology: (Greek) myrmekia = wart, referring to the wart-like denticles on the rachis, suffix -ridium from Chloridium.

Genus ab allis generibus Ramichloridii similibus rachide recta longa, subhyalina, denticulis distantibus, verruciformibus praedita distinguendum.

In vitro: Colonies moderately fast-growing, flat, with mainly submerged mycelium, and entire margin, later becoming powdery to velvety, pale orange to orange. Mycelium rather compact, mainly submerged, in the centre velvety with fertile bundles of hyphae. Conidiophores arising vertically and clearly distinct from creeping hyphae, unbranched, straight or flexuose, brown, thick-walled. Conidiogenous cells terminally integrated, polyblastic, cylindrical, straight or flexuose, pale brown, sometimes secondarily septate, fertile part subhyaline, as wide as the basal part, with scattered pimple-shaped, apically pointed, unpigmented, conidium-bearing denticles. Conidia solitary, subhyaline, smooth or finely verrucose, rather thin-walled, with a wing-like gelatinous sheath, obovoidal or fusiform, tapering towards a narrowly truncate base with a slightly prominent, unpigmented hilum; conidial secession schizolytic.

Type species: Myrmecridium schulzeri (Sacc.) Arzanlou, W. Gams & Crous, comb. nov.

Notes: Myrmecridium schulzeri was fully described as Acrotheca acuta Grove by Hughes (1951). The author discussed several genera, none of which is suitable for the present fungus for various reasons as analysed by de Hoog (1977). Only Gomphinaria Preuss is not yet sufficiently documented. Our examination of G. amoena Preuss (B!) showed that this is an entirely different fungus, of which no fresh material is available to ascertain its position. Myrmecridium can be distinguished from other ramichloridium-like fungi by having entirely hyaline vegetative hyphae, and widely scattered, pimple-shaped denticles on the long hyaline rachis. The conidial sheath is visible in lactic acid mounts with bright-field microscopy. The Myrmecridium clade consists of several subclusters, which are insufficiently resolved based on the ITS sequence data. However, two morphologically distinct varieties of Myrmecridium are treated here. The status of the other isolates in this clade will be dealt with in a future study incorporating more strains, and using a multi-gene phylogenetic approach.

Myrmecridium schulzeri (Sacc.) Arzanlou, W. Gams & Crous, comb. nov. MycoBank MB504560. var. schulzeri Figs 7B, 29. Basionym: Psilobotrys schulzeri Sacc., Hedwigia 23: 126. 1884. ≡ Chloridium schulzerii (Sacc.) Sacc., Syll. Fung. 4: 322. 1886. ≡ Rhinocladiella schulzeri (Sacc.) Matsush., Icon. Microfung. Mats. lect. (Kobe): 124. 1975. ≡ Ramichloridium schulzeri (Sacc.) de Hoog, Stud. Mycol. 15: 64. 1977 var. schulzeri. = Acrotheca acuta Grove, J. Bot., Lond. 54: 222. 1916. ≡ Pleurophragmium acutum (Grove) M.B. Ellis in Ellis, More Dematiaceous Hyphomycetes: 165. 1976. = Rhinotrichum multisporum Doguet, Rev. Mycol., Suppl. Colon. 17: 78. 1953 (nom. inval. Art. 36) [non Acrotheca multispora (Preuss) Sacc., Syll. Fung. 4: 277. 1886]. [non Acrothecium (?) multisporum G. Arnaud, Bull. Trimestriel Soc. Mycol. France 69: 288. 1953 (nom. inval. Art. 36)]. [non Acrothecium multisporum G. Arnaud sensu Tubaki, J. Hattori Bot. Lab. 20: 145. 1958].

112 Ramichloridium and allied genera

Fig. 29. Myrmecridium schulzeri (CBS 325.74). A. Macronematous conidiophores. B. Inflated basal cells visible in some conidiophores. C–E. Conidial apparatus at different stages of development, resulting in macronematous conidiophores and sympodially proliferating conidiogenous cells. F–G. Rachis with scattered, pimple-shaped denticles. H. Conidia. Scale bars: A =100 µm, B–H = 10 µm.

In vitro: Submerged hyphae hyaline, thin-walled, 1–2 µm wide; aerial hyphae, if present, pale olivaceous-brown. Conidiophores arising vertically from creeping aerial hyphae, unbranched, straight, reddish brown, thick-walled, septate, up to 250 µm tall, 2.5–3.5 µm wide, with 2–7 additional septa, basal cell often inflated, 3.5–5 µm wide. Conidiogenous cells integrated, cylindrical, variable in length, 15–110 µm long, subhyaline to pale brown, later becoming inconspicuously septate, fertile part subhyaline, as wide as the basal part, forming a straight rachis with scattered, pimple-shaped denticles less than 1 µm long and approx. 0.5 µm wide, apically pointed, unpigmented, slightly thickened scars. Conidia solitary, subhyaline, thin- walled, smooth or finely verrucose, surrounded by a wing-like, gelatinous conidial sheath, up to 0.5 µm thick, ellipsoid, obovoid or fusiform, (6–)9–10(–12) × 3–4 µm, tapering to a subtruncate base; hilum unpigmented, inconspicuous.

Cultural characteristics: Colonies reaching 29 mm diam after 14 d at 24 °C, pale orange to orange, with entire margin; mycelium flat, rather compact, later becoming farinose or powdery due to sporulation, which occurs in concentric zones when incubated on the laboratory bench.

Specimens examined: Germany, Kiel-Kitzeberg, from wheat-field soil, W. Gams, CBS 134.68 = ATCC 16310. The Netherlands, isolated from a man, bronchial secretion, A. Visser,

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CBS 156.63 = MUCL 1079; Lienden, isolated from Triticum aestivum root, C.L. de Graaff, CBS 325.74 = JCM 7234.

Myrmecridium schulzeri var. tritici (M.B. Ellis) Arzanlou, W. Gams & Crous, comb. nov. MycoBank MB504562. Basionym: Pleurophragmium tritici M.B. Ellis, in Ellis, More Dematiaceous Hyphomycetes: 165. 1976. ≡ Ramichloridium schulzeri var. tritici (M.B. Ellis) de Hoog, Stud. Mycol. 15: 68. 1977.

Specimen examined: Ireland, Dublin, on wheat stem, Oct. 1960, J.J. Brady, holotype IMI 83291.

Notes: No reliable living culture is available of this variety. Based on a re-examination of the type specimen in this study, the variety appears sufficiently distinct from Myrmecridium schulzeri var. schulzeri based on the frequent production of septate conidia.

Myrmecridium flexuosum (de Hoog) Arzanlou, W. Gams & Crous, comb. et stat. nov. MycoBank MB504563. Fig. 30. Basionym: Ramichloridium schulzeri var. flexuosum de Hoog, Stud. Mycol. 15: 67. 1977.

In vitro: Submerged hyphae hyaline, thin-walled, 1–2 µm wide. Conidiophores unbranched, flexuose, arising from creeping aerial hyphae, pale brown, up to 250 µm tall, 3–3.5 µm wide, thick-walled, smooth, with up to 24 thin septa, delimiting 8–12 µm long cells. Conidiogenous cells integrated, elongating sympodially, cylindrical, 20–150 µm long, flexuose, brown at the base, subhyaline in the upper part, later becoming inconspicuously septate; rachis slightly flexuose, subhyaline, as wide as the basal part, thick-walled near the base, hyaline and thin-walled in the apical part, with scattered pimple-shaped, unpigmented, approx. 0.5 µm long denticles. Conidia solitary, subhyaline, thin-walled, finely verrucose, with a wing-like gelatinous sheath, approx. 0.5 µm wide, ellipsoid to obovoid, (5–)6–7(–9) × 3–4 µm; hilum slightly prominent, unpigmented, approx. 0.5 µm diam.

Cultural characteristics: Colonies reaching 40 mm diam after 14 d at 24 °C; mycelium submerged, flat, smooth; centrally orange, later becoming powdery to velvety and greyish brown due to sporulation, with sharp, smooth, entire margin; reverse yellowish orange.

Specimen examined: Surinam, isolated from soil, J.H. van Emden, culture ex-type CBS 398.76 = JCM 6968.

Notes: This former variety is sufficiently distinguished from M. schulzeri s. str. by its flexuose conidiophores and conidia which lack an acuminate base, to be regarded as a separate species.

Ramichloridium torvi (Ellis & Everh.) de Hoog, Stud. Mycol. 15: 79. 1977. ≡ Ramularia torvi Ellis & Everh., Rep. Missouri Bot. Gard. 9: 119. 1898. ≡ Hansfordia torvi (Ellis & Everh.) Deighton & Piroz., Mycol. Pap. 101: 39. 1965. = Acladium biophilum Cif., Sydowia 10: 164. 1956. ≡ Hansfordia biophila (Cif.) M.B. Ellis, in Ellis, More Dematiaceous Hyphomycetes: 199. 1976.

Specimen: Jamaica, Port Marant, Dec. 1890, on leaves of Solanum torvum, holotype of Ramularia torvi (NY) (specimen not examined).

114 Ramichloridium and allied genera

Fig. 30. Myrmecridium flexuosum (CBS 398.76). A–C. Conidial apparatus at different stages of development, resulting in macronematous conidiophores with sympodially proliferating conidiogenous cells. D–H. Sympodially proliferating conidiogenous cells giving rise to a flexuose conidium-bearing rachis with pimple-shaped denticles. I. Conidia. Scale bar = 10 µm.

Notes: According to the description and illustration of R. torvi provided by de Hoog (1977), this appears to be an additional species of Myrmecridium. Although it is morphologically similar to M. flexuosumin having a flexuose rachis, it differs from the other species of the genus by having smooth, clavate conidia. Fresh collections and cultures would be required to resolve its status.

Pseudovirgaria H.-D. Shin, U. Braun, Arzanlou & Crous, gen. nov. MycoBank MB504564.

Etymology: Named after its morphological similarity to Virgaria.

Hyphomycetes. Uredinicola. Coloniae in vivo pallide vel modice brunneae, ferrugineae vel cinnamomeae, in vitro lentissime crescentes, murinae. Mycelium immersum et praecipue externum, ex hyphis ramosis et cellulis conidiogenis integratis compositum, conidiophoris ab hyphis vegetativis vix distinguendis. Hyphae ramosae, septatae, leves, tenuitunicatae, hyalinae vel pallide brunneae. Cellulae conidiogenae integratae in hyphis repentibus, terminales et intercalares, polyblasticae, sympodialiter proliferentes, subcylindricae vel geniculatae, cicatricibus conspicuis, solitariis vel numerosis, dispersis vel aggregatis, subdenticulatis, prominentibus, umbonatis vel apicem versus paulo attenuatis, non inspissatis, non vel parce fuscatis-refringentibus. Conidia solitaria, holoblastica, plus minusve obovoidea, recta vel leniter

115 Chapter 4 curvata, asymmetrica, continua, hyalina, subhyalina vel pallidissime olivaceo-brunnea, hilo subconspicuo vel conspicuo, truncato vel rotundato, non inspissato, non vel lenissime fuscato- refringente; secessio schizolytica.

Hyperparasitic on uredosori of rust fungi. Colonies in vivo pale to medium brown, rusty or cinnamom, in vitro slow-growing, pale to dark mouse-grey. Mycelium immersed and mainly aerial, composed of branched hyphae with integrated conidiogenous cells, differentiation between vegetative hyphae and conidiophores barely possible. Hyphae branched, septate, smooth, thin- walled, hyaline to pale brown. Conidiogenous cells similarly hyaline to pale brown, integrated in creeping threads (hyphae), terminal and intercalary, polyblastic, proliferation sympodial, rachis subcylindrical to geniculate, conidiogenous loci (scars) conspicuous, solitary to numerous, scattered to aggregated, subdenticulate, bulging out, umbonate or slightly attenuated towards a rounded apex, wall unthickened, not to slightly darkened-refractive. Conidia solitary, formation holoblastic, more or less obovoid, straight to somewhat curved, asymmetrical, aseptate, hyaline, subhyaline to very pale olivaceous-brown, with more or less conspicuous hilum, truncate to rounded, unthickened, not or slightly darkened-refractive; conidial secession schizolytic.

Type species: Pseudovirgaria hyperparasitica H.-D. Shin, U. Braun, Arzanlou & Crous, sp.nov.

Notes: Other ramichloridium-like isolates from various rust species form another unique clade, sister to Radulidium subulatum (de Hoog) Arzanlou, W. Gams & Crous and Ra. epichloës (Ellis & Dearn.) Arzanlou, W. Gams & Crous in the Sordariomycetidae. Although Pseudovirgaria is morphologically similar to Virgaria Nees, it has hyaline to pale brown hyphae, conidia and conidiogenous cells. The conidiogenous cells are integrated in creeping threads (hyphae), terminal and intercalary, and the proliferation is distinctly sympodial. The subdenticulate conidiogenous loci are scattered, solitary, at small shoulders of geniculate conidiogenous cells, caused by sympodial proliferation, or aggregated, forming slight swellings of the rachis, i.e., a typical raduliform rachis as in Virgaria is lacking. Furthermore, the conidiogenous loci of Pseudovirgaria are bulging, convex, slightly attenuated towards the rouded apex, in contrast to more cylindrical denticles in Virgaria (Ellis 1971). The scar type of Pseudovirgaria is peculiar due to its convex, papilla-like shape and reminiscent of conidiogenous loci in plant- pathogenic genera like Neoovularia U. Braun and Pseudodidymaria U. Braun (Braun 1998). The superficially similar genus Veronaea is quite distinct from Pseudovirgaria by having erect conidiophores with a typical rachis and crowded conidiogenous loci which are flat or only slightly prominent and darkened. Pseudovirgaria is characterised by its mycelium which is composed of branched hyphae with integrated, terminal and intercalary conidiogenous cells. A differentiation between branched hyphae and “branched conidiophores” is difficult and barely possible. It remains unclear if the “creeping threads” and terminal branches of hyphae are to be interpreted as “creeping conidiophores”. In any case, the mycelium forms complex fertile branched hyphal structures in which individual conidiophores are barely discernable. These structures and difficulties in discerning individual conidiophores remind one of some species of Pseudocercospora Speg. and other cercosporoid genera with abundant superficial mycelium in vivo.

Pseudovirgaria hyperparasitica H.-D. Shin, U. Braun, Arzanlou & Crous, sp. nov. MycoBank MB504565. Figs 6A, 31.

Etymology: Named after its hyperparasitic habit on rust fungi.

116 Ramichloridium and allied genera

Fig. 31. Pseudovirgaria hyperparasitica (CBS 121739). A–D. Conidial apparatus at different stages of development; conidiogenous cells with geniculate proliferation. E. Conidia. Scale bar = 10 µm.

Hyphae 1.5–4 µm latae, tenuitunicatae, ≤ 0.5 µm crassae. Cellulae conidiogenae 15–50 × 2–5 µm, tenuitunicatae (≤ 0.5 µm), cicatricibus (0.5–)1.0(–1.5) µm diam, 0.5–1 µm altis. Conidia saepe obovoidea, interdum subclavata, 10–20 × 5–9 µm, apice rotundato vel paulo attenuato, basi truncata vel rotundata, hilo ca 1 µm diam.

In vivo: Colonies on rust sori, thin to moderately thick, loose, cobwebby, to dense, tomentose, pale to medium brown, rusty or cinnamon. Mycelium partly immersed in the sori, but mainly superficial, composed of a system of branched hyphae with integrated conidiogenous cells (fertile threads), distinction between conidiophores and vegetative hyphae difficult and barely possible. Hyphae 1.5–4 µm wide, hyaline, subhyaline to pale yellowish, greenish or very pale olivaceous, light brownish in mass, thin-walled (≤ 0.5 µm), smooth, pluriseptate, occasionally slightly constricted at the septa. Conidiogenous cells integrated in creeping fertile threads, terminal or intercalary, 15–50 µm long, 2–5 µm wide, subcylindrical to geniculate, subhyaline to very pale brownish, wall thin, ≤ 0.5 µm, smooth, proliferation sympodial, with a single to usually several conidiogenous loci per cell, often crowded, causing slight swellings, up to 6 µm wide, subdenticulate loci, formed by the slightly bulging wall, convex, slightly narrowed towards the rounded apex, (0.5–)1.0(–1.5) µm diam and 0.5–1 µm high, wall of the loci unthickened, not or slightly darkened-refractive, in surface view visible as minute circle (only rim visible and dark). Conidia solitary, obovoid, often slightly curved with ± unequal sides, 10–20 × 5–9 µm, aseptate, subhyaline, pale yellowish greenish to very pale olivaceous, wall ≤ 0.5 µm thick, smooth, apex slightly attenuated to usually broadly rounded, base rounded to somewhat attenuated towards a more or less conspicuous hilum, (0.5–)1(–1.5) µm diam, convex to truncate, unthickened, not to slightly darkened-refractive.

In vitro: Submerged hyphae hyaline to subhyaline, smooth; aerial hyphae smooth, subhyaline, up to 4 µm wide. Conidiogenous cells arising imperceptably from aerial vegetative hyphae, terminal, occasionally intercalary, holoblastic, proliferating sympodially in a geniculate pattern, with more or less long intervals between groups of scars; loci slightly darkened, unthickened, approx. 0.5 µm diam. Conidia hyaline to subhyaline, aseptate, ovoid, often somewhat curved, (10–)13–15(–17) × (5–)6–7(–8) µm, with truncate base and acutely rounded apex; hila unthickened, slightly darkened-refractive.

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Cultural characteristics: Colonies on MEA rather slow-growing, reaching 11 mm diam after 14 d at 24 °C, pale to dark mouse-grey, velvety, compacted, with colonies being up to 1 mm high.

Specimens examined: Korea, Seoul, on uredosori of Frommeëlla sp., on Duchesnea chrysantha, 17 Sep. 2003, H.-D. Shin, paratype, 4/10, CPC 10702–10703 = CBS 121735–121736, HAL 2053 F; Chunchon, on Phragmidium griseum on Rubus crataegifolius, 20 Jul. 2004, H.-D. Shin, paratype, 2/8, HAL 2057 F; Suwon, on Phragmidium pauciloculare on Rubus parvifolius, 14 Oct. 2003, H.-D. Shin, paratype, 23/10, HAL 2055 F; Hongchon, on Phragmidium rosae- multiflorae on Rosa multiflora, 11 Aug. 2004, H.-D. Shin, paratype, 23/8, HAL 2056 F; Yangpyong, on Phragmidium sp. on Rubus coreanus, 30 Sep. 2003, H.-D. Shin, paratype, 11/10-1, CPC 10704–10705 = CBS 121737–121738, HAL 2052 F, and the same locality, 23 Jul. 2004, HAL 2058 F; Chunchon, on Pucciniastrum agrimoniae on Agrimonia pilosa, 7 Oct. 2002, H.-D. Shin, holotype, HAL 2054 F, culture ex-type CPC 10753–10755 = CBS 121739– 121741.

Radulidium subulatum and Ra. epichloës clade

Ramichloridium subulatum and R. epichloës form a distinct, well-supported clade with uncertain affinity. This clade is morphologically distinct and a new genus is introduced below to accommodate it.

Radulidium Arzanlou, W. Gams & Crous, gen. nov. MycoBank MB504566.

Etymology: Latin radula = A flexible tongue-like organ in gastropods, referring to the radula- like denticles on the rachis.

Genus ab aliis generibus Ramichloridii similibus denticulis densissimis, prominentibus, hebetibus in rachide e cellula conidiogena aculeata orta distinguendum.

Type species: Radulidium subulatum (de Hoog) Arzanlou, W. Gams & Crous, comb. nov.

In vitro: Colonies fast-growing, velvety, floccose near the margin, centrally with fertile hyphal bundles up to 10 mm high, about 2 mm diam, with entire but vague margin; mycelium whitish, later becoming greyish brown. Submerged hyphae smooth, thin-walled. Conidiophores usually reduced to polyblastic conidiogenous cells arising from undifferentiated or slightly differentiated aerial hyphae, terminally integrated or lateral, rarely a branched conidiophore present, smooth, slightly thick-walled, pale brown, cylindrical to acicular, widest at the base and tapering towards the apex; apical part forming a pale brown, generally straight rachis, with crowded, prominent, blunt denticles, suggesting a gastropod radula; denticles 0.5–1 µm long, apically pale brown. Conidia solitary, subhyaline, thin- or slightly thick-walled, smooth or verrucose, obovoidal, fusiform to subcylindrical, base subtruncate and with a slightly prominent, conspicuously pigmented hilum; conidial secession schizolytic.

Notes: Radulidium can be distinguished from other ramichloridium-like fungi by its slightly differentiated conidiophores and prominent, blunt, very dense conidium-bearing denticles. Although the Radulidium clade consists of several subclusters that correlate with differences in morphology, the ITS sequence data appear insufficient to resolve this species complex. Therefore, only two species of Radulidium with clear morphological and molecular differences are treated here. The phylogenetic situation of other taxa in this clade will be treated in a further study employing a multi-gene approach.

118 Ramichloridium and allied genera

Fig. 32. Radulidium subulatum (CBS 405.76). A–B. Macronematous conidiophores with sympodially proliferating conidiogenous cells, resulting in a conidium-bearing rachis. C–D. Rachis with crowded, blunt conidium-bearing denticles. E. Conidia. Scale bar = 10 µm.

Radulidium subulatum (de Hoog) Arzanlou, W. Gams & Crous, comb. nov. MycoBank MB504567. Figs 10C, 32. Basionym: Ramichloridium subulatum de Hoog, Stud. Mycol. 15: 83. 1977. Misapplied name: Rhinocladiella elatior Mangenot sensu dal Vesco & B. Peyronel, Allionia 14: 38. 1968.

In vitro: Submerged hyphae hyaline, thin-walled, 1–2.5 µm wide; aerial hyphae brownish. Conidiogenous cells arising laterally from vegetative hyphae, pale brown, smooth, thick-walled, sometimes without a basal septum, cylindrical to aculeate, tapering gradually towards the apex, widest at the base, 25–40 × 2–3 µm; proliferating sympodially, forming a pale brown rachis, with densely crowded, prominent, blunt conidium-bearing denticles, with pale brown apex. Conidia solitary, subhyaline, thin-walled, smooth, ellipsoidal to almost clavate, 5–7 × 1.5–2 µm, with a slightly pigmented, non-refractive hilum, about 1 µm diam.

Cultural characteristics: Colonies on MEA rather fast growing, reaching 50 mm diam after 14 d at 24 °C, with entire but vague margin, velvety, floccose near the margin, centrally with fertile hyphal bundles up to 10 mm high, about 2 mm diam; mycelium whitish, later becoming greyish brown; reverse grey, zonate.

Specimens examined: Czech Republic, on Phragmites australis, A. Samšiňáková, ex-type culture CBS 405.76; Opatovicky pond, from Lasioptera arundinis (gall midge) mycangia on Phragmites australis, M. Skuhravá, CBS 101010.

Radulidium epichloës (Ellis & Dearn.) Arzanlou, W. Gams & Crous, comb. nov. MycoBank MB504568. Fig. 33. Basionym: Botrytis epichloës Ellis & Dearn., Can. Record Sci. 9: 272. 1893. ≡ Ramichloridium epichloës (Ellis & Dearn.) de Hoog, Stud. Mycol. 15: 81. 1977.

In vitro: Submerged hyphae hyaline, thin-walled, 1–2.5 µm wide; aerial hyphae somewhat darker. Conidiogenous cells arising laterally or terminally from undifferentiated or slightly differentiated aerial hyphae, occasionally acutely branched in the lower part, smooth, thick-

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Fig. 33. Radulidium epichloës (CBS 361.63). A–C. Conidial apparatus at different stages of development, resulting in semi-micronematous conidiophores and sympodially proliferating conidiogenous cells. D. Rachis with crowded, blunt conidium-bearing denticles. E–F. Conidiophores with acute branches in the lower part. G. Conidia. Scale bar = 10 µm. walled, pale brown, more or less cylindrical, later with thin septa, 25–47 µm long; proliferating sympodially, forming a rather short, pale brown, straight or somewhat geniculate rachis, with crowded, prominent, blunt denticles with pale brown apex. Conidia solitary, subhyaline, rather thin-walled, verruculose, obovoidal to fusiform, (4.5–)7–8(–11) × 2–3 µm, with a pigmented hilum, 1–1.5 µm diam.

Cultural characteristics: Colonies reaching 45 mm diam after 14 d at 24 °C, with smooth, rather vague, entire margin; velvety, centrally floccose and elevated up to 2 mm high; surface mycelium whitish, later becoming greyish brown; reverse pale ochraceous.

Specimen examined: U.S.A., Cranberry Lake, Michigan, isolated from Epichloë typhina on Glyceria striata, G.L. Hennebert, CBS 361.63 = MUCL 3124; specimen in MUCL designated here as epitype.

120 Ramichloridium and allied genera

Veronaea-like clade, allied to the Annulatascaceae

A veronaea-like isolate from Bertia moriformis clusters near the Annulatascaceae, and is morphologically distinct from other known anamorph genera in the Ramichloridium complex, and therefore a new genus is introduced to accommodate it.

Rhodoveronaea Arzanlou, W. Gams & Crous, gen. nov. MycoBank MB504569.

Etymology: (Greek) rhodon = the rose, referring to the red-brown conidiophores, suffix -veronaea from Veronaea.

Genus ab aliis generibus Ramichloridii similibus basi condiorum late truncata et marginata distinguenda.

In vitro: Colonies slow-growing, velvety, floccose; surface olivaceous-grey to dark olivaceous- green; reverse olivaceous-black. Hyphae smooth, thin-walled, pale olivaceous. Conidiophores arising vertically from creeping hyphae, straight or flexuose, simple, thick-walled, red-brown, with inflated basal cell. Conidiogenous cells terminally integrated, polyblastic, sympodial, smooth, thick-walled, pale brown, rachis straight, occasionally geniculate, with crowded, slightly prominent conidium-bearing denticles; denticles flat-tipped, slightly pigmented.Conidia solitary, pale brown, thin- or slightly thick-walled, smooth, ellipsoidal to obovoidal, 0–multi- septate, with a protruding base and a marginal basal frill; conidial secession schizolytic.

Type species: Rhodoveronaea varioseptata Arzanlou, W. Gams & Crous, sp. nov.

Notes: Rhodoveronaea differs from other ramichloridium-like fungi by the presence of a basal, marginal conidial frill, and variably septate conidia.

Rhodoveronaea varioseptata Arzanlou, W. Gams & Crous, sp. nov. MycoBank MB504570. Figs 10D, 34.

Etymology: Named for its variably septate conidia.

Hyphae 2–3 µm latae. Conidiophora ad 125 µm longa et 3–5 µm lata. Cellulae conidiogenae 30–70 µm longae et 3–5 µm latae. Conidia 0–2(–3)-septata, (8–)11–13(–15) × (2–)3–4(–6) µm.

In vitro: Submerged hyphae smooth, thin-walled, pale olivaceous, 2–3 µm wide; aerial hyphae smooth, brownish and slightly narrower. Conidiophores arising vertically from creeping hyphae, straight or flexuose, simple, smooth, thick-walled, red-brown, up to 125 µm long, 3–5 µm wide, often with inflated basal cell. Conidiogenous cells terminally integrated, smooth, thick-walled, pale brown at the base, paler towards the apex, straight, variable in length, 30–70 µm long and 3–5 µm wide, rachis straight, occasionally geniculate; slightly prominent conidium-bearing denticles, crowded, with slightly pigmented apex, about 1 µm diam. Conidia solitary, pale brown, thin- or slightly thick-walled, smooth, ellipsoid to obovoid, 0–2(–3)-septate, (8–)11– 13(–15) × (2–)3–4(–6) µm with a protruding base, 1.5 µm wide, and marginal frill. Cultural characteristics: Colonies reaching 12 mm diam after 14 d at 24 °C, velvety, floccose; surface olivaceous-grey to dark olivaceous-green; reverse olivaceous-black.

Specimen examined: Germany, Eifel, Berndorf, on Bertia moriformis, Sep. 1987, W. Gams, holotype CBS-H 19932, culture ex-type CBS 431.88.

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Fig. 34. Rhodoveronaea varioseptata (CBS 431.88). A–D. Macronematous conidiophores with sympodially proliferating conidiogenous cells, resulting in conidium bearing rachis with slightly prominent conidium-bearing denticles. E–F. Conidia with minute marginal frill. Scale bar = 10 µm. Venturiaceae (Pleosporales)

The ex-type strain of Veronaea simplex (Papendorf 1969) did not cluster with the genus Veronaea (Herpotrichiellaceae), but is allied to the Venturiaceae. Veronaea simplex is distinct from species of Bonord. by having a well-developed rachis with densely aggregated scars. A new genus is thus introduced to accommodate this taxon.

Veronaeopsis Arzanlou & Crous, gen. nov. MycoBank MB504571.

Etymology: The suffix -opsis refers to its similarity with Veronaea.

Genus Veronaeae simile sed conidiophoris brevioribus (ad 60 μm longis) et rachide dense denticulata distinguendum.

In vitro: Colonies moderately fast-growing; surface velvety, floccose, greyish sepia to hazel, with smooth margin; reverse mouse-grey to dark mouse-grey. Conidiophores arising vertically from aerial hyphae, lateral or intercalary, simple or branched, occasionally reduced to conidiogenous cells, pale brown. Conidiogenous cells terminally integrated on simple or branched conidiophores, polyblastic, smooth, thin-walled, pale brown; rachis commonly straight, geniculate, with densely crowded, prominent denticles, and slightly pigmented scars. Conidia solitary, subhyaline to pale brown, thin- or slightly thick-walled, smooth, oblong- ellipsoidal to subcylindrical, (0–)1-septate, with a slightly darkened, thickened, hilum; conidial secession schizolytic.

Type species: Veronaeopsis simplex (Papendorf) Arzanlou & Crous, comb. nov.

Veronaeopsis simplex (Papendorf) Arzanlou & Crous, comb. nov. MycoBank MB504572. Figs 17C, 35.

122 Ramichloridium and allied genera

Fig. 35. Veronaeopsis simplex (CBS 588.66). A–C. Conidial apparatus at different stages of development, resulting in semi-micronematous conidiophores and sympodially proliferating conidiogenous cells. D–E. Rachis with crowded, prominent denticles. F. Intercalary conidiogenous cells. G. Conidia. Scale bar = 10 µm.

Basionym: Veronaea simplex Papendorf, Trans. Brit. Mycol. Soc. 52: 486. 1969.

In vitro: Submerged hyphae smooth, thin-walled, pale brown; aerial hyphae aggregated in bundles. Conidiophores arising vertically from aerial hyphae, lateral or intercalary, simple or branched, occasionally reduced to conidiogenous cells, pale brown, rather short, up to 60 µm long, 1.5–2 µm wide. Conidiogenous cells terminally integrated in the conidiophores, smooth, thin-walled, pale brown, variable in length, 5–25 µm long, rachis generally straight or irregularly geniculate, with crowded, prominent denticles, about 0.5 µm long, flat-tipped, with slightly pigmented apex. Conidia solitary, subhyaline to pale brown, thin- or slightly thick-walled, smooth, oblong-ellipsoidal to subcylindrical, (0–)1-septate, slightly constricted at the septum, (6–)10–12(–15) × (2–)2.5–3(–4) µm; hilum slightly darkened and thickened, not refractive, about 1 µm diam.

Cultural characteristics: Colonies reaching 25 mm diam after 14 d at 24 °C; surface velvety, floccose, greyish sepia to hazel, with smooth margin; reverse mouse-grey to dark mouse-grey.

Specimen examined: South Africa, Potchefstroom, on leaf litter of Acacia karroo, 1966, M.C. Papendorf, holotype, CBS H-7810; culture ex-type CBS 588.66 = IMI 203547.

Notes: The presence of 1-septate conidia in Veronaeopsis overlaps with Veronaea. However, Veronaeopsis differs from Veronaea based on its conidiophore and conidiogenous cell morphology. Veronaea has much longer, macronematous conidiophores than Veronaeopsis. Furthermore, Veronaea has a more or less straight rachis, whereas in Veronaeopsis the rachis is often geniculate. The conidiogenous loci in Veronaea are less prominent, i.e., less denticle-like.

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Discussion

The present study was initiated chiefly to clarify the status ofRamichloridium musae, the causal organism of tropical speckle disease of banana (Jones 2000). Much confusion surrounded this name in the past, relating, respectively, to its validation, species and generic status. As was revealed in the present study, however, two species are involved in banana speckle disease, namely R. musae and R. biverticillatum. Even more surprising was the fact that Ramichloridium comprises anamorphs of Mycosphaerella Johanson (Mycosphaerellaceae), though no teleomorphs have thus far been conclusively linked to any species of Ramichloridium. By investigating the Ramichloridium generic complex as outlined by de Hoog (1977), another genus associated with leaf spots, namely Periconiella, was also shown to represent an anamorph of Mycosphaerella. Although no teleomorph connections have been proven for ramichloridium- like taxa, de Hoog et al. (1983) refer to the type specimen of Wentiomyces javanicus Koord. (Pseudoperisporiaceae), on the type specimen of which (PC) some ramichloridium-like conidiophores were seen. Without fresh material and no anamorph-teleomorph connection proven in culture, however, this matter cannot be investigated further. It is interesting to note, however, that Wentiomyces Koord. shows a strong resemblance to Mycosphaerella, except for the external perithecial appendages. The genus Mycosphaerella is presently one of the largest genera of ascomycetes, containing close to 3000 names (Aptroot 2006), to which approximately 30 anamorph genera have already been linked (Crous et al. 2006a, b, 2007). By adding two additional anamorph genera, the Mycosphaerella complex appears to be expanding even further, though some taxa have been shown to reside in other families in the Capnodiales, such as Davidiella Crous & U. Braun (Davidiellaceae) and Teratosphaeria (Teratosphaeriaceae) (Braun et al. 2003, Crous et al. 2007, Schubert et al. 2007). Another family, which proved to accommodate several ramichloridium-like taxa, is the Herpotrichiellaceae (Chaetothyriales). Members of the Chaetothyriales are regularly encountered as causal agents of human mycoses (Haase et al. 1999, de Hoog et al. 2003), whereas species of the Capnodiales are common plant pathogens, or chiefly associated with plants. Species in the Chaetothyriales have consistently melanized thalli, which is a factor enabling them to invade humans, and cause a wide diversity of mycoses, such as chromoblastomycosis, mycetoma, brain infection and subcutaneous phaeohyphomycosis (de Hoog et al. 2003). The only known teleomorph connection in this genus is Capronia Sacc. (Untereiner & Naveau 1999). Rhinocladiella and Veronaea were in the past frequently confused with the genus Ramichloridium. However, Rhinocladiella, as well as Veronaea and Thysanorea, were shown to cluster in the Chaetothyriales, while Ramichloridium clusters in the Capnodiales. Rhinocladiella mackenziei, which causes severe cerebral phaeohyphomycosis in humans (Sutton et al. 1998), has in the past been confused with Pleurothecium obovoideum (Ur-Rahman et al. 1988). Data presented here reveal, however, that although morphologically similar, these species are phylogenetically separate, with P. obovoideum belonging to the Sordariales, where it clusters with sexual species of Carpoligna F.A. Fernández & Huhndorf that have Pleurothecium anamorphs (Fernández et al. 1999). In addition to the genera clustering in the Capnodiales and Chaetothyriales, several ramichloridium-like genera are newly introduced to accommodate species that cluster elsewhere in the ascomycetes, namely Pseudovirgaria, Radulidium and Myrmecridium, Veronaeopsis, and Rhodoveronaea. Although the ecological role of these taxa is much less known than that of taxa in the Capnodiales and Chaetothyriales, some exhibit an interesting ecology. For instance, the fungicolous habit of Pseudovirgaria, as well as some species in Radulidium, which are found on various rust species, suggests that these genera should be screened further to establish if

124 Ramichloridium and allied genera they have any potential biocontrol properties. Furthermore, these two genera share a common ancestor, and further work is required to determine whether speciation was shaped by co- evolution with the rusts. A further species of “Veronaea” that might belong to Pseudovirgaria is Veronaea harunganae (Hansf.) M.B. Ellis, which is known to occur on Hemileia harunganae Cummins on Harungana in Tanzania and Uganda (Ellis 1976). The latter species, however, is presently not known from culture, and needs to be recollected to facilitate further study. The genera distinguished here represent homogeneous clades in the phylogenetic analysis. Only the species of Rhinocladiella are dispersed among others morphologically classified in Exophiala or other genera. By integrating the phylogenetic data generated here with the various morphological data sets, we were able to resolve eight clades for taxa formerly regarded as representative of the Ramichloridium complex. According to the phylogeny inferred from 28S rDNA sequence data, the genera Ramichloridium and Periconiella were heterogeneous, requiring the introduction of several novel genera. Although the present 11 odd genera can still be distinguished based on their morphology, it is unlikely that morphological identifications without the supplement of molecular data would in the future be able to accurately identify all the novel isolates that undoubtably await description. The integration of morphology with phylogenetic data not only helps to resolve generic affinities, but it also assists in discriminating between the various cryptic species that surround many of these well-known names that are presently freely used in the literature. To that end it is interesting to note that for the majority of the taxa studied here, the ITS domain (Table 1) provided good species resolution. However, more genes will have to be screened in future studies aimed at characterising some of the species complexes where the ITS domain provided insufficient phylogenetic signal (data not shown) to resolve all of the observed morphological species.

ACKNOWLEDGEMENTS

The work of Mahdi Arzanlou was funded by the Ministry of Science, Research and Technology of Iran, which we gratefully acknowledge. Several colleagues from different countries provided material without which this work would not have been possible. We thank Marjan Vermaas for preparing the photographic plates, and Arien van Iperen for taking care of the cultures.

REFERENCES

Al-Hedaithy, S. S. A., Jamjoom, Z. A. B., & Saeed, E. S. 1988. Cerebral phaeohyphomycosis caused by Fonsecaea pedrosoi in Saudi-Arabia. Acta Pathol. Microbiol. Scand. 96 (Suppl.) 3: 94–100. Aptroot, A. 2006. Mycosphaerella and its anamorphs: 2. Conspectus of Mycosphaerella. CBS Biodiv. Series 5: 1–231. Braun, U. 1998. A monograph of Cercosporella, Ramularia and allied genera (phytopathogenic hypomycetes). Vol. 2. IHW-Verlag, Eching. Braun, U., Crous, P. W., Dugan, F., Groenewald, J. Z., & Hoog, G. S. de 2003. Phylogeny and taxonomy of Cladosporium-like hyphomycetes, including Davidiella gen. nov., the teleomorph of Cladosporium s. str. Mycol. Prog. 2: 3–18. Campbell, C. K., & Al-Hedaithy, S. S. A. 1993. Phaeohyphomycosis of the brain caused by Ramichloridium mackenziei sp. nov. in middle eastern countries. J. Med. Vet. Mycol. 31: 325–332.

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Crous, P. W., Groenewald, J. Z., Groenewald, M., Caldwell, P., Braun, U., & Harrington, T. C. 2006a. Species of Cercospora associated with grey leaf spot of maize. Stud. Mycol. 55: 189–197. Crous, P. W., Groenewald, J. Z., Risède, J-M., Simoneau, P., & Hyde, K. D. 2006b. Calonectria species and their Cylindrocladium anamorphs: species with clavate vesicles. Stud. Mycol. 55: 213–226. Crous, P. W., Slippers, B., Wingfield, M. J., Rheeder, J., Marasas, W. F. O., Phillips, A. J. L., Alves, A., Burgess, T., Barber, P., & Groenewald, J. Z. 2006c. Phylogenetic lineages in the Botryosphaeriaceae. Stud. Mycol. 55: 235–253. Crous, P. W., Summerell, B. A., Carnegie, A. J, Mohammed, C., Himaman, W. & Groenewald, J. Z. 2007. Foliicolous Mycosphaerella spp. and their anamorphs on Corymbia and Eucalyptus. Fungal Div. 26: 143–185. David, J. C. 1997. A contribution to the systematics of Cladosporium. Revision of the fungi previously referred to . Mycol. Pap. 172: 1–157. Ellis, M. B. 1971. Dematiaceous Hyphomycetes. Commonwealth Mycological Institute, Kew. Ellis, M. B. 1976. More Dematiaceous Hyphomycetes. Commonwealth Mycological Institute, Kew. Fernández, F. A., Lutzoni, F. M., & Huhndorf, S. M. 1999. Teleomorph-anamorph connections: the new pyrenomycetous genus Carpoligna and its Pleurothecium anamorph. Mycologia 91: 251–262. Gams, W., Verkley, G. J. M., & Crous, P. W. eds. 2007. CBS Course of Mycology, 5th ed. Centraalbureau voor Schimmelcultures, Utrecht. Haase, G., Sonntag, L., Melzer-Krick, B., & Hoog, G. S. de. 1999. Phylogenetic inference by SSU-gene analysis of members of the Herpotrichiellaceae with special reference to human pathogenic species. Stud. Mycol. 47: 80–97. Hoog, G. S. de. 1977. Rhinocladiella and allied genera. Stud. Mycol. 15: 1–140. Hoog, G. S. de. 1979. Nomenclatural notes on some black yeast-like hyphomycetes. Taxon 28: 347–348. Hoog, G. S. de, Rahman, M. A., & Boekhout, T. 1983. Ramichloridium, Veronaea and Stenella: generic delimitation, new combinations and two new species. Trans. Brit. Mycol. Soc. 81: 485–490. Hoog, G. S. de, Vicente, V., Caligiorne, R. B., Kantarcioglu, S., Tintelnot, K. A., Gerrits van den Ende, A. H. G., & Haase, G. 2003. Species diversity and polymorphism in the Exophiala spinifera clade containing opportunistic black yeast-like fungi. J. Clin. Microbiol. 41: 4767– 4778. Hughes, S. J. 1951. Studies on Microfungi. 5. Acrotheca. Mycol. Pap. 38: 1–8. Jones, D. R. 2000. Tropical speckle. Pages 116–120 in: Disease of banana, abaca and enset. Jones D. R., ed. CAB International, Wallingford, Oxon, UK. McGinnis, M. R., & Schell, W. A. 1980. The genus Fonsecaea and its relationship to the genera Cladosporium, Phialophora, Ramichloridium, and Rhinocladiella. Pan American Health Organisation Scientific Publication 396: 215–234. Morgan-Jones, G. 1979. Notes on hyphomycetes. 28. Veronaea bambusae sp. nov. Mycotaxon 8: 149–151. Morgan-Jones, G. 1982. Notes on hyphomycetes. 40. New species of Codinaea and Veronaea. Mycotaxon 14: 175–180. Mostert, L., Groenewald, J. Z., Summerbell, R. C., Gams, W., & Crous, P. W. 2006. Taxonomy and pathology of Togninia (Diaporthales) and its Phaeoacremonium anamorphs. Stud. Mycol. 54: 1–113.

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Nylander, J. A. A. 2004. MrModeltest v2.2. Program distributed by the author. Evolutionary Biology Centre, Uppsala University. Papendorf, M. C. 1969. New South African soil fungi. Trans. Brit. Mycol. Soc. 52: 483–489. Papendorf, M. C. 1976. Notes on Veronaea including V. compacta sp. nov. Bothalia 12: 119– 121. Rayner, R. W. 1970. A mycological colour chart. CMI and British Mycological Society. Kew. Rehner, S. A., Samuels, G. J. 1994. Taxonomy and phylogeny of Gliocladium analysed from nuclear large subunit ribosomal DNA sequences. Mycol. Res. 98: 625–634. Ronquist, F., & Huelsenbeck, J. P. 2003. MrBayes 3: Bayesian phylogenetic inference under mixed models. Bioinformatics 19: 1572–1574. Saccardo, P. A., & Berlese, A. N. 1885. Miscellanea mycologica. Ser. II. Atti dell’Istituto Veneto di Scienze, Lettere ed Arti Ser. 6 3: 711–742. Schol-Schwarz, M. B. 1968. Rhinocladiella, its synonym Fonsecaea and its relation to Phialophora. A. van Leeuw. J. Microb. 34: 119–152. Schubert, K., Groenewald, J. Z., Braun, U., Dijksterhuis, J., Starink, M., Hill, C. F., Zalar, P., Hoog, G. S.de, & Crous, P. W. 2007. Biodiversity in the complex (Davidiellaceae, Capnodiales), with standardisation of methods for Cladosporium taxonomy and diagnostics. Stud. Mycol. 58: 105–156 Seifert, K. A. 1993. Integrating anamorphic fungi into the fungal system. Pages 79–85 in: The fungal holomorph: mitotic, meiotic and pleomorphic speciation in fungal systematics. Reynolds D. R., & Taylor, J. W. eds. International Mycological Institute, Egham. Stahel, G. 1937. The banana leaf speckle in Surinam caused by Chloridium musae nov. spec. and another related banana disease. Trop. Agr. 14: 42–44. Sutton, D. A., Slifkin, M., Yakulis, R., & Rinaldi, M. G. 1998. U.S. case report of cerebral phaeohyphomycosis caused by Ramichloridium obovoideum (R. mackenziei): Criteria for identification, therapy, and review of other known dematiaceous neurotropic taxa. J. Clin. Microbiol. 36: 708–715. Swofford, D. L. 2003. PAUP*. Phylogenetic Analysis Using Parsimony (*and other Methods). Version 4. Sinauer Associates, Sunderland, Massachusetts. Taylor, J. W., Jacobson, D. J., Kroken, S., Kasuga, T., Geiser, D. M., Hibbett, D. S., & Fisher, M. C. 2000. Phylogenetic species recognition and species concepts in fungi. Fungal Genet. Biol. 31: 21–32. Untereiner, W. A., & Naveau, F. A. 1999. Molecular systematics of the Herpotrichiellaceae with an assessment of the phylogenetic positions of Exophiala dermatitidis and Phialophora americana. Mycologia 91: 67–83. Ur-Rahman, N., Mahgoub, E., & Chagla, A. H. 1988. Fatal brain abscesses caused by Ramichloridium obovoideum – report of three cases. Acta Neurochir. 93: 92–95. Veerkamp, J., & Gams, W. 1983. Los hongos de Colombia – VIII. Some new species of soil fungi from Colombia. Caldasia 13: 709–717. Vilgalys, R., & Hester, M. 1990. Rapid genetic identification and mapping of enzymatically amplified ribosomal DNA from several Cryptococcus species. J. Bacteriol. 172: 4238– 4246. White, T. J., Bruns, T., Lee, S., & Taylor, J. 1990. Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. Pages 315–322 in: PCR Protocols: a guide to methods and applications. Innis, M. A., Gelfand, D. H., Sninsky, J. J., & White, T. J. eds. Academic Press, San Diego, California. Zipfel, R. D., Beer, W. de, Jacobs, K., Wingfield, B. D., & Wingfield, M. J. 2006. Multi-gene phylogenies define Ceratocystiopsis and Grosmannia distinct from Ophiostoma. Stud. Mycol. 55: 75–97.

127 128 CHAPTER 5

Evolution of heterothallism in three major Mycosphaerella species associated with the Sigatoka disease complex of banana

M. Arzanlou1,2, P. W. Crous1,2, and L.-H. Zwiers1

1CBS Fungal Biodiversity Centre, Uppsalalaan 8, 3584 CT Utrecht, The Netherlands; 2Laboratory of Phytopathology, Wageningen University, Binnenhaven 5, 6709 PD Wageningen, The Netherlands

To be submitted

129 Chapter 5

ABSTRACT

In the present study we characterised mating type loci of the three primary agents of Sigatoka disease on banana viz., Mycosphaerella fijiensis, M. musicola and M. eumusae, using comparative genomics and bioinformatics. We extended this analysis to resolve the evolutionary history of the mating type loci in these three closely related species. Our analyses revealed that the idiomorphs are characterised by an expansion in size and the presence of two additional Mycosphaerella-specific genes (ORF1 and ORF2). Analysis of the proteins encoded by these ORFs suggests a recent shared sexual history of the three fungal pathogens. Moreover, analysis of the M. fijiensis genome sequence revealed the presence of an additional mating type-like locus containing a fusion between a Mat1-1 and a Mat1-2 gene. This fused mating type gene (Mat1/2) is not physically linked to the idiomorph and is also present in M. musicola and M. eumusae. This unique mating type-like locus is suggested to be a remnant of a homothallic ancestral state of Mycosphaerella species

130 Evolution of heterothallism in causal agents of the Sigatoka disease complex

INTRODUCTION

The most serious and economically important leaf spot diseases of bananas are caused by different species of Mycosphaerella. The Sigatoka leaf spot disease complex of bananas involves three related ascomycetous fungi; Mycosphaerella fijiensis (anamorph Pseudocercospora fijiensis) causing the black Sigatoka disease, M. musicola (anamorph Pseudocercospora musae) responsible for yellow Sigatoka disease and M. eumusae (anamorph Pseudocercospora eumusae), causing eumusae leaf spot disease (Crous & Mourichon 2002, Jones 2003, Arzanlou et al. 2007). Of these, M. fijiensis is presently regarded as the most important fungal pathogen causing premature ripening of the fruits and yield losses up to 50 %. Due to the clonal nature of the commercial banana cultivars and the difficulties encountered when breeding for disease-resistant cultivars, disease management relies heavily on the use of fungicides (Marin et al. 2003). The chronology of the disease record around the world suggests that South-East Asia is the centre of origin for all three pathogens as well as for their host genus Musa (Jones 2003, Rivas et al. 2004). Furthermore, a recent study on the phylogeny of Mycosphaerella species occurring on banana indicated that 20 species of Mycosphaerella or its anamorphs could occur on banana, several of which are able to co-infect a single leaf or even lesion. Moreover, a phylogeny inferred from the combined DNA sequence data set of four genes revealed that the three pathogens represent a monophyletic clade and share common ancestry (Chapter 2, this thesis). Sexual reproduction plays a major role in population diversity and disease epidemiology. In the euascomycetes, sexual development is controlled by a single mating type locus (MAT). In all heterothallic filamentous ascomycetes studied to date, the mating type locus contains one of two forms of dissimilar sequences (known as idiomorph) occupying the same chromosomal position in their genome (Metzenberg & Glass 1990). In contrast, homothallic species contain both mating type alleles in a single genome. By convention, mating type idiomorphs of complementary isolates are termed Mat1-1 and Mat1-2 (Turgeon & Yoder 2000). Since the first characterisation of the mating type locus of the ascomyceteous yeast Saccharomyces cerevisiae in 1981, the deployment of PCR-based techniques and the increasing availability of genome sequences have resulted in the isolation and characterisation of mating-type genes of a steadily increasing number of fungi (Astell et al. 1981, Arie et al. 1997, 2000, Waalwijk et al. 2002, Barve et al. 2003, Bennett et al. 2003, Goodwin et al. 2003, Inderbitzin et al. 2005, Debuchy & Turgeon 2006, Groenewald et al. 2006, 2007, Conde-Ferráez et al. 2007, Stergiopoulos et al. 2007). These studies show that mating type genes are commonly present, even in presumed asexual species, indicating that lack of (obvious) sexual recombination is not due to the absence of basal elements controlling the sexual reproductive machinery (Sharon et al. 1996). The presence of mating type genes in asexual species further supports the view that the asexual life style arose from sexual progenitors. The simplest euascomycetous idiomorphs can be found in heterothallic members of the Dothideomycetes, to which also the genus Mycosphaerella belongs. These idiomorphs are characterised by the presence of a single open reading frame (ORF) encoding a protein with an alpha domain for Mat1-1, and a single ORF encoding a MAT protein with a highly-mobility group (HMG) domain for Mat1-2, with corresponding genes labeled Mat1-1-1 and Mat1-2-1, respectively (Turgeon et al. 2000). Mat1-1-1 and Mat1-2-1 encode proteins, which act as global transcription factors controlling a signal transduction pathway involved in mating identity and development of the sexual cycle (Coppin et al. 1997, Wirsel et al. 1998, Nolting & Pöggeler 2006). Homothallic members of the Dothideomycetes carry both mating type genes in their

131 Chapter 5 haploid genome, either linked or unlinked. Structurally, the Mat genes of homothallic species can be complete, partial or even chimaeric (Yun et al. 1999). The close co-occurrence of multiple species of Mycosphaerella both in time and space on a single host can lead to close physical interactions and potential exchange of genetic material through interspecies mating, hybridisations or anastomosis. This could ultimately result in the origin of new species with altered virulence patterns or host specificity, and thus could be an example of microevolution or sympatric speciation. Analysing the structure of the mating type genes of the Mycosphaerella species occurring on banana might help us to understand the evolutionary history of these species on the one hand, and the evolutionary history of sexual reproduction of fungi on the other. The mating type idiomorph of M. fijiensis has recently been characterised (Conde-Ferráez et al. 2007). With this study, we aim to (i) isolate and characterise the mating type loci of the other two primary agents of the Sigatoka disease complex, namely M. musicola and M. eumusae, and (ii) use the obtained data to deduce the evolutionary history of sexual reproduction for all three species.

MATERIALS AND METHODS

Isolates

A list of the Mycosphaerella strains isolated from banana and used in this study is provided in Table 1. Isolates were maintained in a glycerol solution at -80 °C, and all are deposited at the Centraalbureau voor Schimmelcultures (CBS), Utrecht, Netherlands.

Isolation and characterisation of mating type loci of Mycosphaerella species

Previously published degenerate primers (Groenewald et al. 2006) were used to amplify a conserved region within Mat1-1-1 and Mat1-2-1, respectively, from different Mycosphaerella species occurring on banana. Amplification reactions were performed in a total reaction volume of 12.5 µl, containing 1× PCR Buffer (Bioline, London, UK), 1.5 mM MgCl2, 48 µM dNTPs, 8 pmol of each degenerate primer, 0.7 U of Taq DNA polymerase (Bioline, London, UK) and

Table 1. List of the isolates used in this study. Species Accession number1 Mating identity Origin Mycosphaerella eumusae CIRAD 487; CBS 121382 Mat1-1 Thailand CIRAD 670; CBS 111438 Mat1-1 Vietnam CIRAD 554; CBS 121379 Mat1-2 Sri Lanka CIRAD 485; CBS 121381 Mat1-2 Thailand Mycosphaerella fijiensis CIRAD 86; CBS 121380 Mat1-1 Cameroon CIRAD 251; CBS 121358 Mat1-2 Costa Rica Mycosphaerella musicola UQ433; CBS 121372 Mat1-1 Australia UQ2003; CBS 121374 Mat1-1 Australia CIRAD 90; CBS 121368 Mat1-2 Colombia CIRAD 102; CBS 121370 Mat1-2 Martinique 1CBS: Centraalbureau voor Schimmelcultures, Utrecht, The Netherlands; CIRAD: Centre de coopération internationale en recherche agronomique pour le développement (CIRAD), Montpellier, France; UQ: University of Queensland, Australia.

132 Evolution of heterothallism in causal agents of the Sigatoka disease complex

1–10 ng of genomic DNA. PCR amplifications were performed in a GeneAmp PCR System 9600 (Applied Biosystems, Foster City, CA). DNA was initially denatured for 5 min at 94 °C, followed by 15 cycles at 94 °C for 20 s, 52 °C for 20 s, 72 °C for 50 s, followed by 25 cycles at 94 °C for 20 s, 50 °C for 20 s, 72 °C for 50 s, and with a final elongation step at 72 °C for 7 min. PCR amplicons were sequenced using a DYEnamic ET Terminator Cycle Sequencing kit (Amersham Biosciences, Roosendal, The Netherlands) on an ABI Prism 3700 DNA Sequencer (Perkin-Elmer, Foster City, CA) using the same primers as used for PCR. Sequence fragments were assembled using SeqMan (Lasergene package, DNAstar, Madison, WI). Contigs obtained were then analysed using the basic local alignment search tools (BLAST) at NCBI (http://www. ncbi.nlm.nih.gov) (Altschul et al. 1997). Nested primer sets were designed based upon contigs corresponding to Mat1-1-1 and Mat 1-2-1 sequences. These primer sets were used in combination with primers provided with the DNA Walking SpeedUp kit (Seegene Inc., Rockville, USA) to amplify fragments adjacent to the initially cloned fragments. Amplified fragments were purified using the GFX PCR DNA and Gel Band Purification Kit (GE Healthcare, Piscataway, USA), and cloned in a pGEM-T Vector System I (Promega Madison, WI, USA). The identity of the cloned fragments was confirmed by sequencing, and subsequent genome walking steps were performed to obtain the complete idiomorph.

DNA and amino acid sequence comparisons and bioinformatics

Sequence data obtained from genome walking were assembled and edited using SeqMan and EditSeq programmes from the Lasergene package (DNAstar, Madison, WI). Consensus sequence files were exported to the Vector NTI v.10.1 software package (Invitrogen, USA) for further analysis and the creation of graphical maps. Sequence data were analysed using the basic local alignment search tools (BLAST) at NCBI (http://www.ncbi.nlm.nih.gov) (Altschul et al. 1997). Open reading frames (ORFs) and intron positions were predicted by comparing the sequence data with known MAT sequences from other filamentous fungi as well as by means of the FGENESH gene prediction module with the Stagonospora nodorum dataset as reference from the MOLQUEST software package (Soft berry Inc., NY, USA available at http://www. softberry.com/berry.phtml) (Salamov & Solovyev 2000). Deduced proteins were analysed for the presence of potential motifs using the MEME system (http://meme.sdsc.edu/meme/meme. html) (Bailey et al. 2006). Phylogenetic analyses were carried out based on amino acid sequence alignment data from the ORFs detected in mating type idiomorphs using CLUSTALW (http:// www.ebi.ac.uk) and a neighbour-joining phylogenetic tree was generated from the alignment using the Phylip software package (http://evolution.genetics.washington.edu/phylip.html).

DNA and RNA manipulations

Basic DNA and RNA manipulations were performed based on standard procedures (Sambrook et al. 1989). Escherichia coli strain JM109 (Promega) was used for propagation of constructs. Genomic DNA was extracted from axenic cultures grown on agar plates using a commercial DNA isolation kit (MoBio laboratories, Carlsbad, CA, USA), according to the vendor’s instructions. Total RNA was isolated using the Trizole® reagent (Life Technologies) from M. fijiensis isolates grown for 7 d in liquid potato dextrose broth. Isolates CIRAD 86 (= CBS 120258) and CIRAD 251 (= CBS 121358) were used for RNA isolation and subsequent RT-PCR. RT-PCR was performed on cDNA made using the Superscript™ III First-Strand Synthesis System for RT-PCR (Invitrogen) according to the manufacturer’s instructions. Primers used for RT-PCR were designed to distinguish between genomic DNA and cDNA (Table 2).

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Table 2. Primers used for RT-PCR, with expected amplicon size from genomic DNA and cDNA of the target genes. Primer combination (5’ → 3’) Target gene Expected size Expected size genomic DNA (bps) cDNA (bps) Mat1F: CATGAGCACGCTGCAGCAAG Mat1-1-1 702 547 Mat1R: GTAGCAGTGGTTGACCAGGTCAT

Mat2F: GGCGCTCCGGCAAATCTTC Mat1-2-1 720 616 Mat2R: CTTCTCGGATGGCTTGCGTG

ORF1F: CTATCCAGCAAGGCCCAG ORF1 441 382 ORF1R: TTCTGCTGCATCTCCTCCA

ORF2F: CTCACGCATGACACCTCCGA ORF2 733 683 ORF2R: GCGRTTCTGCGTAGTCACATC

Table 3. Predicted gene and intron size of the Mat1-1-1 and Mat1-2-1 genes in Mycosphaerella fijiensis (corrected values shown), M. musicola and M. eumusae and number of amino acids present in the encoded proteins. Commas separate the intron sizes where more than one intron is present. Mat1-1-1 Mat1-2-1 Species Gene size Intron size (bps) # amino acids Gene size Intron size (bps) Amino acids M. eumusae 1317 51, 50, 49 388 1376 54, 50 423 M. musicola 1322 51, 55, 49 388 1340 54, 50 411 M. fijiensis 1322 51, 55, 49 388 1406 54, 50 433

RESULTS

Cloning and characterisation of mating type loci

PCR amplification of genomic DNA from the M. musicola and M. eumusae mating type genes yielded either a ~800-bp fragment from Mat1-1 isolates or a ~250-bp fragment from Mat1-2 isolates. Sequence data from these fragments revealed substantial homology on nucleotide level with the mating type genes of other Dothidiomycetes deposited in GenBank. Several subsequent chromosome-walking steps were performed in both downstream and upstream directions to obtain the full sequence of the mating type genes as well as the whole idiomorph. The predicted gene structures of the cloned Mat1-1-1 and Mat1-2-1 genes of M. eumusae and M. musicola were compared to the published gene structures of the M. fijiensis Mat1-1-1 and Mat1-2-1 genes (Conde-Ferráez et al. 2007). The predicted Mat1-1-1 gene of the three pathogens is remarkably similar; all three encode a protein of 388 amino acids and in all three species the ORF is interrupted by three introns of almost identical sizes (Table 3). The first two introns are located in the α-domain encoding areas of Mat1-1-1 and at exactly the same positions as described for other Mat1-1-1 genes. The presence and location of the third intron downstream of the α-domain was exactly matching with the position of the third intron recently described for the Mat1-1-1 gene of several Cercospora species and (Groenewald et al. 2006, Stergiopoulos et al. 2007). Blastp analyses of the predicted Mat1-1-1 of M. eumusae and M. musicola showed that both proteins exhibited highest similarity to Mat1-1-1 of M. fijiensis

134 Evolution of heterothallism in causal agents of the Sigatoka disease complex

Fig. 1. Graphical representation of the pairwise alignment of cloned mating type loci of Mycosphaerella fijiensis (A), M. musicola (B), and M. eumusae (C). White boxes indicate the dissimilar sequences, black lines mark the position of the homologous areas (>90 % identity on nucleotide level) indicated as darkened boxes. Positions of the annotated genes are also indicated. with 94 % and 93 % identity, respectively. Second best hits were obtained with Mat1-1-1 of Mycosphaerella pini and P. fulva (70 % identity). The published size of the M. fijiensis Mat1-2-1 (440 amino acids) as well as the sizes of the published introns (33 and 50 nucleotides) present within the Mat1-2-1 deviated from the situation in M. eumusae and M. musicola. Therefore the M. fijiensis Mat1-2-1 was re-examined using the same prediction tools as used for M. eumusae and M. musicola, which revealed that the published intron boundaries for M. fijiensis are unlikely, and consequently the corrected Mat1-2-1 gene structures of the three banana pathogens are highly conserved (Table 3). The predicted proteins vary between 411 and 433 amino acids in length but the sizes and positions of the two predicted introns are identical. The positions of the predicted introns correspond to the positions of these introns in other Mycosphaerella species. Blastp analysis showed that the Mat1-2-1 protein of M. eumusae and M. musicola have highest similarity to the M. fijiensis Mat1-2-1 (89 % and 86 % identity, respectively). Second best hits were obtained with the Mat1-2-1 proteins of several Cercospora species (~60 % identity).

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Fig. 2. Organisation of mating type loci of Mycosphaerella graminicola, M. fijiensis, M. musicola, and M. eumusae. Dissimilar sequences (idiomorphs) are indicated by a white box, and identical sequences by a black box.

Approximately 11.5 kb of M. musicola genomic DNA from Mat1-1 and Mat1-2 isolates were obtained by genome walking and subsequently sequenced. Blast2 analyses of these sequences identified a 3.3 kb region of highly similar sequences (over 97 % identity on nucleotide level). Blast analyses of this 3.3 kb sequence revealed the presence of a DNA lyase gene which is also found upstream of the mating type loci in other ascomycetes. The M. musicola DNA lyase gene has the highest homology with M. fijiensis both on nucleotide (76 % identity over 1083 nucleotides) and protein level (67 % identity over 312 amino acids). Besides the DNA lyase gene, no other genes could be identified upstream of the mating type loci. Between the Mat1-1 and Mat1-2 isolates 7.4 kb of Mat1-1 and 8.4 kb of Mat1-2 were dissimilar, and therefore defined to be part of the idiomorph (Fig. 1). Approximately 10 kb of genomic DNA from Mat1-1 and Mat1-2 isolates of M. eumusae were sequenced. Blast analyses of these 10 kb sequences from Mat1-1 and Mat1-2 idiomorphs could not identify any of the genes flanking the idiomorphs in the published genomes of M. graminicola and M. fijiensis, indicating that these dissimilar sequences still belong to mating type idiomorphs in M. eumusae. Also, an attempt to bridge the M. eumusae idiomorph to the flanking regions by long range PCR between the idiomorph sequence and the DNA lyase gene, located upstream of the idiomorph in M. fijiensis, M graminicola and M. musicola was not successful. These results show that the mating type loci of M. musicola and M. eumusae have been expanded compared to the idiomorphs of other related species (Fig. 2).

136 Evolution of heterothallism in causal agents of the Sigatoka disease complex

A pairwise alignment of the Mat1-1 and Mat1-2 idiomorphs of M. musicola and M. eumusae using blast2 revealed the presence of two inverted regions with high levels of identity (Fig. 1). Such inversions have also been described to be present within the mating type idiomorphs of M. fijiensis (Conde-Ferráez et al. 2007). These inverted regions present within the M. musicola idiomorphs are very homologous to the inverted regions present within the M. fijiensis idiomorphs. The same pairwise alignment of the Mat1-1 and Mat1-2 idiomorphs of M. eumusae revealed a more complicated pattern including two inverted regions. One of these inversions shared high similarity with one of the inversions observed in M. musicola and M. fijiensis, whereas the other (a ~1.3 kb inversion) was restricted to M. eumusae. Adjacent to this large inversion, a homologous stretch of ~0.3 kb was observed which consisted of a duplication of sequences found within this inversion (Fig. 1).

The mating type loci of heterothallic members of the Sigatoka disease complex contain additional genes

The expanded idiomorphs of M. musicola and M. eumusae were analysed for the presence of additional genes by BlastX analysis and the FGENESH gene prediction module. Special attention was paid to the inverted regions found to be present within these incomplete idiomorphs. This analysis was also performed on the inverted regions observed upstream and downstream of both the Mat1-1-1 and Mat1-2-1 of M. fijiensis. The analysis of the M. fijiensis Mat1-1 idiomorph predicted the presence of two putative genes, designated here as ORF1 and ORF2, respectively. ORF1 is located on the same strand downstream of Mat1-1-1, whereas ORF2 is present in a head to tail orientation upstream of the Mat1-1-1 gene. Putative genes homologous to ORF1 and ORF2 were also discovered within the M. fijiensis Mat1-2 idiomorph again in an inverted order; ORF1 is located upstream of the Mat1-2-1 and ORF2 downstream of Mat1-2-1 (Fig. 2). Subsequent analysis of the cloned mating type loci of M. musicola, M. eumusae and the published idiomorphs of M. graminicola and S. passerinii indicated the presence of these new genes in all of these species, although not in all mating types (Fig. 2, Table 4). The idiomorphs of M. graminicola and S. passerinii are very similar, with ORF2 lacking in both Mat1-2 idiomorphs and ORF1 located outside the idiomorphs. Furthermore, ORF2 has not been

Table 4. Comparison of the gene organisation of the newly identified ORF1 and ORF2 in both idiomorphs of Mycosphaerella graminicola, M. fijiensis, M. eumusae and M. musicola. ORF1 ORF2 Species Gene size Intron size # Amino Gene size Intron size # Amino (bps) (bps) acids (bps) (bps) acids M. eumusae Mat1-1 791 59 244 1135 49 361 M. eumusae Mat1-2 818 142, 31 214 _ _ _

M. fijiensis Mat1-1 791 59 243 1136 50 361 M. fijiensis Mat1-2 818 59 252 1315 50 , 77 395

M. graminicola Mat1-1 901 53, 53 264 1226 56, 49, 57 354 M. graminicola Mat1-2 910 53, 53 267 _ _ _

M. musicola Mat1-1 791 59 243 1135 49, 102 327 M. musicola Mat1-2 818 141 224 1145 164 326

137 Chapter 5 identified in the expanded mating type locus of Mat1-2 isolates ofM. eumusae, but its position is occupied by a gene encoding a protein with a dynactin-p62 domain (e-value: 1e-104 based upon BlastP analysis of the deduced protein sequence). Additional genes identified in the mating type loci of M. eumusae include a gene with homology to a hypothetical protein of Magnaporthe grisea (e-value: 1e-24) found within the Mat1-1 idiomorph, and within the Mat1-2 idiomorph a gene resembling a histone H1-like protein (e-value: 4e-6) and a gene with similarity to a hypothetical gene from Aspergillus terreus (e-value: 4e-32). Besides the ORF1 and ORF2, no additional genes could be identified within the M. musicola idiomorphs (Fig. 2). A directed BlastX search using the sequences of both ORF1 and ORF2 of M. fijiensis against the NCBI databases identified only a single sequence with homology to each ORF. The single hit with ORF2 (e-value: 5e-21 based upon BlastX analysis) as well as the single hit with ORF1 (e-value: 6e-7, based upon BlastX analyses) correspond to recently published unknown genes (GenBank accession numbers DQ659350 & DQ659351, respectively) found within the idiomorph of Passalora fulva, a species that also belongs to the Mycosphaerellaceae (Stergiopoulos et al. 2007). Prediction of potential motifs in the deduced proteins encoded by ORF1 and ORF2 from M. graminicola, M. fijiensis, M eumusae and M. musicola suggests the presence of three blocks of conserved amino acids (Fig. 3). These putative conserved domains do not correspond to any known protein motif. Not all predicted motifs are present in all proteins. For instance, motif 2 and motif 3 are lacking in ORF1 of the M. eumusae and the M. musicola Mat1-2 isolate, whereas the highly conserved motif 2 of ORF2 is absent from M. graminicola. A phylogenetic comparison of the deduced proteins encoded by the ORF1 gene reveals that the interspecies homology between the three banana pathogens was higher than the intraspecies homology. Thus, the ORF1 from a M. fijiensis Mat1-1 isolate has higher homology to ORF1 from a M. eumusae (or M. musicola ) Mat1-1 isolate than to ORF1 from a M. fijiensis Mat1-2 isolate. The same phenomenon is observed for ORF2. This is in contrast to the situation in M. graminicola and S. passerinii where the intraspecies homology is the highest (Fig. 4).

Expression study

RT-PCR experiments were performed on M. fijiensis isolates of opposite mating type to answer the question whether the additional ORFs found within the idiomorphs of the heterothallic Mycosphaerella species are expressed and thus encode functional genes. No fragments indicative for expression of Mat1-1-1 or Mat1-2-1 were observed. However, RT-PCR performed on RNA isolated from the Mat1-2 isolate CIRAD 251 yielded a fragment corresponding to the expected size of an expressed ORF1. Similarly, a fragment with the expected size of an expressed ORF2 was detected in RNA isolated from the Mat1-1 isolate CIRAD 86 (Fig. 5). These fragments were cloned and sequence analysis confirmed their proper identity as well as the predicted intron boundaries. The RT-PCR experiment on fungal material grown in liquid was repeated twice, and in both experiments the expression of ORF1 was restricted to the Mat1-2 isolate, and expression of ORF2 was limited to the Mat1-1 isolate.

Heterothallic members of the Sigatoka disease complex contain an additional fused mating-type gene

The RT-PCR experiments did not reveal any expression of the M. fijiensis mating type genes Mat1-1-1 and Mat1-2-1; but these experiments did yield a surprising result. Negative control

138 Evolution of heterothallism in causal agents of the Sigatoka disease complex

Fig. 3. Schematic representation of the distribution of predicted protein motifs and the alignment of the predicted motifs of ORF1 (A) and ORF2 (B) from Mat1-1 and Mat1-2 isolates from Mycosphaerella eumusae, M. fijiensis, M. musicola and M. graminicola. Numbers and blocks indicate motifs.

139 Chapter 5

Fig. 4. Unrooted phylogenetic tree based upon a neighbour-joining analysis of the predicted amino acid sequences of ORF1 from both idiomorphs of Mycosphaerella eumusae, M. fijiensis, M. musicola and M. graminicola. reactions performed on genomic DNA of CIRAD 96 (Mat1-1) using Mat1-2-1 specific primers sometimes yielded a fragment. As the opposite phenomenon, amplification of a fragment using Mat1-1-1 specific primers from a Mat1-2 isolate, also irregularly occurred it was decided to study this in more detail. A BLAST analysis against the genome sequence of the recently released M. fijiensis isolate CIRAD 86 (http://genome.jgi-psf.org/Mycfi1/Mycfi1.home.html) was performed using the partial Mat1-2-1 sequence corresponding to the area amplified by theMat1-2-1 specific primers Mat2-F and Mat2-R. Surprisingly, this Mat1-2 specific sequence gave a significant hit with scaffold 15 of the genome sequence (>90 % identity in 336 bps). Therefore, the genome sequence surrounding this area on scaffold 15 was analysed in more detail. This analysis revealed the presence of a genomic region sharing extensive homology (>90 % identity on nucleotide level) to parts of both the Mat1-1-1 and Mat1-2-1 gene of M. fijiensis(Fig. 6). This partial Mat1/Mat2- fusion locus contains areas highly similar to parts of the 3’end of the Mat1-1-1 gene that are located amid areas highly similar to the 5’end of the Mat1-2-1 gene (Fig. 6). These homologous areas do not include areas that encode either the Mat1-1 specific alpha-domain or the Mat1-2 specific HMG-box. However, according to the FGENESH gene prediction programme, the

140 Evolution of heterothallism in causal agents of the Sigatoka disease complex

Fig. 5. RT-PCR analysis of ORF1 and ORF2 expression in the Mycosphaerella fijiensis Mat1-1 isolate CIRAD 86 and the M. fijiensis Mat1-2 isolate CIRAD 251. PCR with primer combinations ORF1F/ORF1R (lanes 1–4) and ORF2F/ORF2R (lanes 5–8) was performed on cDNA derived from CIRAD 251 (lanes 1 and 5), cDNA derived from CIRAD 86 (lanes 2 and 6), genomic DNA from CIRAD 251 (lanes 3 and 7) and on genomic DNA isolated from CIRAD 86 (lanes 4 and 8). Lane labelled M contains DNA marker.

Fig. 6. Schematic overview of the Mycosphaerella fijiensis Mat1-1/Mat1-2-fusion locus. The putative gene model as predicted by the FGENESH programme is indicated as a thick arrow, the predicted introns (1 and 2) as black horizontal lines, the areas with highest homology to Mat1-1-1 and Mat1-2-1 are marked in black and grey blocks, respectively, and the positions and names of the primers used are marked with small arrows.

Mat1-1/Mat1-2-fusion locus putatively encodes a gene of 1118 bps long interrupted by two introns of 172 and 160 bps, respectively. The observed similarity between the predicted gene and the Mat1-1-1 and Mat1-2-1 does extend beyond the predicted gene (Fig. 6). To answer the question whether the presence of this locus was restricted to Mat1-1 isolates of M. fijiensis or whether it would also be present in M. fijiensis Mat1-2 isolates, primers were

141 Chapter 5

Fig. 7. Representation of the areas sharing >90 % nucleotide identity between the Mycosphaerella fijiensis (A), M. eumusae (B), and M. musicola (C), Mat1-1-1, Mat1-2-1 mating type genes and their respective Mat1-1/Mat1-2-fusion locus. Black lines mark the position of the homologous areas indicated as darkened boxes. designed based upon the sequence of the predicted Mat1-1-Mat1-2-fusion locus (Fig. 6). Furthermore, these primers were used on Mat1-1 and Mat1-2 strains from the other two main constituents of the Sigatoka disease complex, M. musicola and M. eumusae. PCR reactions under conditions of relative low stringency (50 °C annealing) lead to successful amplification of fragments from both Mat1-1 and Mat1-2 isolates from all three banana pathogens. Using the primer Fus1 (5’-ATGGCTACTCAGGTCACTGC-3’) in combination with the primer Fus2 (5’-GAATGGCATAGGCTCGACAG-3’) a 969 bp fragment of M. eumusae, and a 993 bp fragment from M. musicola could be obtained. This primer combination yielded a fragment of 1004 bps from M. fijiensis. Sequence analysis of the cloned fragments confirmed the presence of the Mat1-1/Mat1-2-fusion locus in isolates of both mating-types of all three pathogens. The three fused loci shared ~86 % identity on nucleotide level which is only slightly lower than

142 Evolution of heterothallism in causal agents of the Sigatoka disease complex the interspecies identity of Mat1-1-1 and Mat1-2-1 genes of the three species (>90 % identity on nucleotide level). All Mat1-1/Mat1-2-fusion loci of the three heterothallic Mycosphaerella species exhibited the same organisation with Mat1-1-1 sequences being in between Mat1-2-1 sequences (Fig. 7). Finally, the M. fijiensis genome sequence surrounding the fusion locus was analysed in more detail. In total, approximately 60 kb of genomic sequence was annotated and examined for genes with a potential role in sex or mating (Fig. 8). Nearly 20 kb upstream of the Mat1-1/Mat1-2-fusion locus, an ORF was identified with high homology to the Mei2 protein of Schizosaccharomyces pombe (e-value: 7e-33 based upon BlastX analysis), a protein shown to be essential for meiosis in S. pombe (Watanabe et al. 1988). No other obvious mating / sex related candidates could be discriminated in this 60 kb genomic region. As the genome sequence of the related M. graminicola has already been released, it was examined to determine whether the gene organisation was conserved between the two genomes. The Mat1-1/Mat1-2-fusion locus was not found within the M. graminicola genome, but almost all of the genes found surrounding the M. fijiensis Mat1-1/Mat1-2-fusion locus were identified on a single scaffold (scaffold 6). Only the α-rhamnosidase was not located on the same scaffold, but on scaffold 3. However, this region on scaffold 6 was expanded over 17 times in M. graminicola, as compared to M. fijiensis (~60 kb vs ~1070 kb). Despite the large expansion in M. graminicola, still areas in which the order and grouping of the genes was conserved were observed (Fig. 8).

DISCUSSION

The simplest idiomorphs described to date belong to the Dothidiomycetes (Debuchy & Turgeon 2006). Since the initial characterisation of the idiomorphs of Cochliobolus heterosporus (Turgeon et al. 1993) mating type loci were characterised for many other species in this group of fungi. For all of these species a single ORF per idiomorph was characterised. This is in contrast to the situation in Sordariomycetes and Leotiomycetes where the mating type loci can contain additional genes besides the Mat1-1-1 and Mat1-2-1 genes. However, our results clearly show that the mating type loci of the three main constituents of the Sigatoka disease and other Mycosphaerella species are deviating from this “typical” Dothidiomycetous pattern. First, the idiomorphs show an expansion in size. The genomic walking strategy has not yet resulted in the amplification of the downstream flanking region of theM. musicola mating type loci and neither was it successful in amplifying the up- and downstream flanking regions of the M. eumusae mating type loci. Thus, the size of the M. fijiensis idiomorphs are 4.2 kb and 4.7 kb (Conde-Ferráez et al. 2007), whereas, the size of the M. musicola idiomorphs is over 8 kb and that of the M. eumusae mating type loci is at least 10 kb (Fig. 2). Second, in contrast to the other Dothidiomycetes, the Mycosphaerella idiomorphs contain additional genes (ORF1 and ORF2) and in an extraordinary organisation (Fig. 2). Homologs of ORF2 were also identified to be present in the published Mat1-1 idiomorphs of M. graminicola and S. passerinii, previously thought to adhere to the one gene per idiomorph rule (Goodwin et al. 2003, Waalwijk et al. 2002). Moreover, in these species a homolog of ORF1 was found downstream of both the Mat1-1 and Mat1-2 idiomorph.

Expansion of the mating type locus

The pattern of an increased mating type locus concomitant with the incorporation of flanking regions and genes inside the mating type loci has been described for Coccidioides immitis,

143 Chapter 5 -fusion locus and its comparison to the to comparison its and locus -fusion Mat1-1/Mat1-2 strain CIRAD 86 surrounding the surrounding 86 CIRAD strain fijiensis Mycosphaerella M. graminicola strain IPO323 genome sequence. Annotated region of 60 kb of the genomic sequence of sequence genomic the of kb 60 of region Annotated 8. F ig.

144 Evolution of heterothallism in causal agents of the Sigatoka disease complex

C. posadasii and most strikingly Cryptococcus neoformans (Lengeler et al. 2002, Fraser et al. 2007). In C. neoformans the idiomorph is largely expanded (>100 kb) and contains extensive rearrangements of genes between the mating types. For instance, the C. neoformans RP041α and RP041a genes are highly similar (97 %) but are organised in opposite directions. This is strikingly similar to the observed similarity and inversion of ORF1 and ORF2 in the mating type loci of M. eumusae, M. fijiensis and M. musicola. The expansion of mating type loci by the incorporation of adjacent genes could represent a step in the evolution towards sex chromosomes. The observed structural differences between the two mating type loci caused by gene rearrangements (inversions) would suppress recombination by an improper alignment of these chromosomal areas during meiosis (Lengeler et al. 2002, Fraser & Heitman 2004). If this hypothesis is true the mating type loci of the different heterothallic Mycosphaerella species described in this paper represent four different stages of such a development towards a tentative sex chromosome. In M. graminicola and S. passerinii only ORF2 is incorporated inside the idiomorph, whereas ORF1 is still located outside. In M. fijiensis both genes have been integrated inside the idiomorph, and these new “mating type genes” have been inverted. And finally, in M. musicola and M. eumusae, the idiomorphs represent a next step with the incorporation of more genes and additional internal rearrangements (duplications and inversions).

Presence and expression of ORF1 and ORF2

The newly identified ORF1 and ORF2 are seemingly unique for the genus Mycosphaerella. Blast analyses at NCBI indicated that a homolog could only be found in P. fulva, which also belongs to the Mycosphaerellaceae. A re-examination of the published mating type locus of S. passerinii also revealed the presence of these ORFs in an identical organisation as found in M. graminicola. Furthermore, additional studies aimed at the characterisation of mating type loci of the homothallic M. musae revealed the presence of ORF2 in close association with a mating type gene (data to be published elsewhere). Analysis of the proteins encoded by ORF1 and ORF2 indicated the presence of several conserved protein motifs with no known homologs present in the databases. These motifs can be used to design specific primers that can be used to test whether these genes are indeed specific for the Mycosphaerellaceae and thus potentially can yield a Mycosphaerella-specific barcode. The performed RT-PCR experiments showed that under in vitro growth conditions both ORF1 and ORF2 of M. fijiensis are expressed and thus are no pseudo-genes. A striking feature is the observed mating type dependent expression of these genes; ORF1 is solely expressed in the Mat1-2 isolate, and ORF2 in the Mat1-1 isolate. This mating-type-dependent expression is seemingly mating or sex independent as it was observed under conditions considered to be non conducive for mating. It would be interesting to see if and how the expression of ORF1 and ORF2 is regulated during mating, but several attempts to cross complementary isolates in vitro proved unsuccessful. The mating-type dependent expression could well be explained by the genomic organisation within the idiomorphs. In the Mat1-1 idiomorph, ORF2 is located upstream of the Mat1-1-1 gene on the opposite DNA strand in a head-to-head orientation. In the Mat1-2 idiomorph the position upstream of the Mat1-2-1 gene is occupied by ORF1. These results suggest that the intergenic region upstream of the mating type genes is determining the expression of ORF1 and ORF2. Thus, potentially the mating-type gene and ORF1 / ORF2 share the same (bidirectional) promoter. Considering the lack of Mat1-1-1 and Mat1-2-1 expression it can not be ruled out that expression of ORF1 or ORF2 is blocking the expression of Mat1-2-1 and Mat1-1-1, respectively. However, to address this properly promoter studies need to be performed. Finally, it is not clear whether the location of ORF1 and ORF2 near the

145 Chapter 5 mating type genes is meaningful in relation to a function in sexual development or mating. Currently, experiments are underway to generate ORF1 / ORF2 knock-out mutants to assess their function. An interesting observation was the interspecies homology of both ORF1 and ORF2 being higher between the three examined banana pathogens than the intraspecies homology. This result suggests a recent common evolutionary history for the Mat1-1 locus of M. eumusae, M. fijiensis and M. musicola, independent of the Mat1-2 locus (Fig. 4).

Heterothallic members of the Sigatoka disease complex contain an additional fused mating-type gene

The amplification of fragments from a M. fijiensis Mat1-1 isolate using Mat1-2-1 specific primers as well as from a M. fijiensis Mat1-2 isolate using Mat1-1-1-specific primers were unexpected and intriguing. Therefore, these amplicons were studied in more detail. This revealed the presence of a locus containing Mat1-1-1 sequences dispersed amid Mat1-2-1 sequences. Even more surprising was the finding that homologs of this locus were also found in M. eumusae and M. musicola. Furthermore, preliminary results of PCR amplification with a specific primer set for the fused mating-type gene revealed that this gene is also present in the other closely related Pseudocercospora spp. from banana, viz. P. longispora, P. indonesiana and P. assamensis, which based on organismal gene phylogeny display the same pattern of evolutionary history (Chapter 2, this thesis). However, attempts to amplify the fused mating- type gene from genomic DNA isolated from other Mycosphaerella species occurring on banana, e.g., the homothallic species M. musae and M. thailandica and the heterothallic M. citri (data not shown) as well as an in silico analysis of the publicly available genome of the related M. graminicola were unsuccessful. These results suggest that the presence of this locus is restricted to the monophyletic clade, containing these three major banana pathogens and the three novel Pseudocercospora spp. from banana. The FGENESH programme did predict the possibility that the Mat1-1/Mat1-2-fusion region contains a gene. Whether this model reflects reality is unknown as expression data are lacking. However, if the model is true, it will probably not function as a mating type gene as the encoded protein does not contain an α-domain nor a HMG-box domain. Overall this partial Mat1-1/Mat1-2-fusion locus is reminiscent of the mating locus structure of a homothallic species (Debuchy & Turgeon 2006), suggesting that this locus could be the remnant of a relatively recent homothallic ancestor. The consistent presence of Mat1-1-1 sequences amid Mat1-2-1 sequences and the conservation of the organisation of the Mat1-1/Mat1-2-fusion loci amongst the three examined species are also suggestive in this respect. To elucidate the potential history of the Mat1-1/Mat1-2-fusion locus as a homothallic mating-type locus the genomic surroundings of the Mat1-1/Mat1-2-fusion locus were analysed for the presence of genes potentially involved in sex and mating and a genomic comparison between M. fijiensis and M. graminicola was performed. The presence of a Mei2 homolog within 20 kb of the Mat1-1/Mat1-2-fusion locus was suggestive in this respect. Mei2 of S. pombe encodes an RNA recognition motif (RRM) protein which is essential for the initiation of meiosis (Watanabe et al.1988, Harigaya &Yamamoto 2007). Other potentially interesting genes found, were a Sec15 homolog and a BAR-domain encoding gene. Both genes can be involved in membrane dynamics, e.g. endocytosis and vesicular trafficking and thus could function in plasmogamy. The genomic comparison between M. fijiensis and M. graminicola revealed an expansion of this region in the M. graminicola genome, but also a conserved grouping for some of the genes present in the analysed region. These results could indicate that the ordering in this region has been maintained in the M. fijiensis genome as a consequence of a putative function as

146 Evolution of heterothallism in causal agents of the Sigatoka disease complex homothallic mating type locus in the recent evolutionary history of this species. It would be interesting to examine whether the same holds true for the genomic sequences surrounding the Mat1-1/Mat1-2-fusion locus in M. eumusae and M. musicola. The expansion in the M. graminicola genome would then imply that no selection for proper maintenance has occurred and consequently the region has been modified due to multiple insertions and rearrangements. Finally, this hypothesis of maintenance of the integrity of this genomic area in M. fijiensis is strengthened by the observation that the M. fijiensis genome is approximately 1.7 times larger than that of M. graminicola (73.4 Mb vs 41.2 Mb) indicating that overall there seems to be no restraint in genome expansion within M. fijiensis. Altogether these data suggest a complicated evolutionary history for M. eumusae, M. fijiensis and M. musicola. Basically, the data presented both in this chapter and in Chapter 2 suggest a stepwise evolution of these species. The identified Mat1-1/Mat1-2-fusion locus suggests that these three species share a common homothallic ancestor. This homothallic ancestor somehow became heterothallic but maintained a relic of the homothallic mating-type locus. The high interspecies homology of the identified Mycosphaerella-specific ORF1 and ORF2 suggests an active genetic / sexual exchange between these heterothallic successor species, originating from the homothallic ancestor, resulting in the gradual split into M. fijiensis and later into M. eumusae and M. musicola, which were stabilised by a constant increase in idiomorph size and genomic rearrangements. The high similarity between the Mat1-1-1, Mat1-2-1, ORF1 and ORF2 genes and the Mat1-1/Mat1-2-fusion loci among the different species could be explained by assuming that these events have occurred relatively recent and / or over a relatively short time span. All these data point to a frequent and relatively easy exchange of genetic material amongst Mycosphaerella species. Therefore, we postulate that the frequently observed co-existence of Mycosphaerella species on the same host and even the same lesion can be both the cause and the consequence of the development of new species with potentially altered host specificities or virulence.

ACKNOWLEDGEMENTS

Mahdi Arzanlou was funded by the Ministry of Science, Research and Technology of Iran, which we gratefully acknowledge. Several colleagues provided valuable technical and scientific help during this work, which we acknowledge. Dr Marizeth Groenewald, dr Edwin Abeln and Laura Conde-Ferráez are thanked for technical help and scientific discussions. We thank dr Ewald Groenewald, dr Gert Kema and Prof. dr Pierre de Wit for their critical review of this article. We also acknowledge the Dutch Mycosphaerella group for valuable discussions and support.

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148 Evolution of heterothallism in causal agents of the Sigatoka disease complex

pathogens Histoplasma capsulatum, Coccidioides immitis, and Coccidioides posadasii. Eukaryot. Cell 6: 622–629. Goodwin, S. B., Waalwijk, C., Kema, G. H. J., Cavaletto, J. R., & Zhang, G. 2003. Cloning and analysis of the mating-type idiomorphs from the barley pathogen Septoria passerinii. Mol. Genet. Genomics 269: 1–12. Groenewald, M., Barnes, I., Bradshaw, R. E., Brown, A., Dale, A., Groenewald, J. Z., Lewis, K. J., Wingfield, B. D., Wingfield, M. J., & Crous, P. W. 2007. Characterisation and worldwide distribution of the mating type genes in the Dothistroma needle blight pathogens. Phytopathology 97: 825–834. Groenewald, M., Groenewald, J. Z., Harrington, T. C., Abeln, E. C. A., & Crous P. W. 2006. Mating type gene analysis in apparently asexual Cercospora species is suggestive of cryptic sex. Fungal Genet. Biol. 43: 813–825. Harigaya, Y., & Yamamoto, M. 2007. Molecular mechanisms underlying the mitosis-meiosis decision. Chromosome Res. 15: 523–537. Inderbitzin, P., Harkness, J., Turgeon, B. G., & Berbee, M. 2005. Lateral transfer of mating system in Stemphylium. Proc. Natl. Acad. Sci. USA 102: 11390–11395. Jones, D. R. 2003. The distribution and importance of the Mycosphaerella leaf spot diseases of banana. Pages 25–42 in: Mycosphaerella leaf spot diseases of bananas: present status and outlook. Proceedings of 2nd international workshop on Mycosphaerella leaf spot disease of bananas, Costa Rica. Jacome, L., Lepoivre, P., Marin, D., Ortiz, R., Romero, R., & Escalant, J. V. eds. 20–23 May 2002, San José, Costa Rica, INIBAP. Lengeler, K. B., Fox, D. S., Fraser, J. A., Allen, A., Forrester, K., Dietrich, F. S., & Heitman, J. 2002. Mating-type locus of Cryptococcus neoformans: A step in the evolution of sex chromosomes. Eukaryot. Cell 1: 704–718. Marin, D. H., Romero, R. A., Guzmán, M., & Sutton, T. B. 2003. Black Sigatoka: an increasing threat to banana cultivation. Plant Dis. 87: 208–222. Maxwell, A., Dell, B., Neumeister-Kemp, H. G., & Hardy, G. E. StJ. 2003. Mycosphaerella species associated with Eucalyptus in south-western Australia: New species, new records and a key. Mycol. Res. 107: 351–359. Metzenberg, R. L., & Glass, N. L. 1990. Mating type and mating strategies in Neurospora. BioEssays 12: 53–59. Nolting N., & Pöggeler, S. 2006. A MADS box protein interacts with a mating-type protein and is required for fruiting body development in the homothallic ascomycete Sordaria macrospora. Eukaryot. Cell 5: 1043–1056. Rivas, G. G., Zapater, M. F., Abadie, C., & Carlier, J. 2004. Founder effects and stochastic dispersal at the continental scale of the fungal pathogen of bananas Mycosphaerella fijiensis. Mol. Ecol. 13: 471–482. Salamov, A., & Solovyev, V. 2000. Ab initio gene finding inDrosophila genomic DNA. Genome Res. 10: 516–522. Sambrook, J., Fritsch, E. F., & Maniatis, T. 1989. Molecular cloning: a laboratory manual, 2nd ed. Cold Spring Harbor, N.Y., Cold Spring Harbor Laboratory. Sharon, A., Yamaguchi, K., Christiansen, S. K., Horwitz, B. A., Yoder, O. C., & Turgeon, B. G. 1996. An asexual fungus has the potential for sexual development. Mol. Gen. Genet. 251: 60–68. Stergiopoulos, I., Groenewald, M., Staats, M., Lindhout, P., Crous, P. W., & Wit, P. J. de 2007. Mating-type genes and the genetic structure of a world-wide collection of the tomato pathogen Cladosporium fulvum. Fungal Genet. Biol. 44: 415–29. Turgeon, B. G. 1998. Application of mating type gene technology to problems in fungal biology. Annu. Rev. Phytopathol. 36: 115–137.

149 Chapter 5

Turgeon, B. G., Bohlmann, H., Ciuffetti, L. M., Christiansen, S. K., Yang, G., Schafer, W., & Yoder, O. C. 1993. Cloning and analysis of the mating-type genes from Cochliobolus heterostrophus. Mol. Gen. Genet. 238: 270–284. Turgeon, B. G., & Yoder, O. C. 2000. Proposed nomenclature for mating type genes of filamentous ascomycetes. Fungal Genet. Biol. 31: 1–5. Waalwijk, C., Mendes, O., Verstappen, E. C. P., Waard, M. A. de, & Kema, G. H. J. 2002. Isolation and characterisation of the mating-type idiomorphs from the wheat septoria leaf blotch fungus Mycosphaerella graminicola. Fungal Genet. Biol. 35: 277–286. Watanabe, Y., Lino, Y., Furuhata, K., Shimoda, C., & Yamamoto, M. 1988. The S. pombe mei2 gene encoding a crucial molecule for commitment to meiosis is under the regulation of cAMP. EMBO J. 7: 761–767. Wirsel, S., Horwitz, B., Yamaguchi, K., Yoder, O. C., & Turgeon, B. G. 1998. Single mating type- specific genes and their 3′ UTRs control mating and fertility inCochliobolus heterostrophus. Mol. Gen. Genet. 259: 272–281. Yun, S. H., Berbee, M. L., Yoder, O. C., & Turgeon, B. G. 1999. Evolution of the fungal self- fertile reproductive life style from self-sterile ancestors. Proc. NATL. Acad. Sci. USA 96: 5592–5597.

150 CHAPTER 6

GENERAL DISCUSSION

151 Chapter 6

INTRODUCTION

Fungi are heterotrophic organisms that live as saprophytes decomposing dead host substrate, as symbionts in close association with their hosts from which both benefit or as pathogens, damaging or even killing their hosts. Pathogens can spread and cause epidemics that are causes of biological, social and economic tragedies (Agrios 1997). Plant disease management is one of the major aims of plant pathologists, who employ different strategies to achieve this goal. A thorough knowledge of the identity of the causal organism is a basic requirement for sustainable disease management. The overall aim of this research project was to obtain a better understanding of the Sigatoka disease complex of banana by studying (i) diversity and phylogeny of associated Mycosphaerella species, (ii) species detection and quantification, and (iii) mating behaviour and the evolutionary plasticity of the mating type locus within this genus.

The Sigatoka disease complex

The first objective of this research was to study the biodiversity and phylogenetic relationships among the Mycosphaerella species occurring on banana (Chapter 2). Species from this genus constitute one of the major components of the disease triangle and a thorough knowledge of the identity of the causal agents is required for successful plant disease management. Three major species of Mycosphaerella, namely M. fijiensis (causal agent of black Sigatoka disease), M. musicola (causal agent of yellow Sigatoka disease), and M. eumusae (causal agent of eumusae leaf spot disease), are known to cause major economic losses on banana. Besides these three major species, several other Mycosphaerella species have been described from banana, most of which are not known from culture. Traditionally, Mycosphaerella systematics relied mainly upon host plant association and morphological characters of anamorphs and teleomorphs (Crous 1998, Arzanlou et al. 2007a), criteria which were repeatedly shown to be unreliable (Crous et al. 2004, Crous & Groenewald 2005). Due to the co-occurrence of multiple morphologically similar species on the same leaf or even the same lesion, accurate identification solely based on morphology is impossible. Therefore, I applied a polyphasic approach on a global collection of isolates from banana incorporating multi-gene nucleotide sequence data, morphology, and cultural characteristics, to study the diversity and phylogenetic relationship among the Mycosphaerella species associated with banana leaf spot diseases. The phylogeny inferred from the ITS region and the combined data set containing partial gene sequences of the actin gene, the small subunit mitochondrial ribosomal DNA and the histone H3 gene revealed a rich diversity of Mycosphaerella species on Musa. Integration of molecular and morphological data sets confirmed the occurrence of more than 20 species ofMycosphaerella (incl. anamorphs) on banana. I re-confirmed the presence of previously described species such asCercospora apii, M. citri and M. thailandica, and also identifiedMusa as a novel host for Mycosphaerella communis, M. lateralis and Passalora loranthi. Moreover, eight entirely new species were identified and described, namely Dissoconium musae, Mycosphaerella mozambica, Pseudocercospora assamensis, P. indonesiana, P. longispora, Stenella musae, S. musicola, and S. queenslandica. The identification of 20 species of Mycosphaerella on cultivated Musa is resembling the situation reported from other Mycosphaerella host plants, such as Eucalyptus where more than 80 species of Mycosphaerella can infect species in this host genus (Crous et al. 2006). Thus, it can be predicted that even more species might be present on Musa. Extensive sampling from wild Musa species especially in South-East Asia, the centre of origin for Musa, may lead to the discovery of additional novel Mycosphaerella species. Host sharing among the Mycosphaerella species is quite striking, as in the past, host association represented a major criterion used in Mycosphaerella systematics. The main question regarding

152 General discussion

Common ancestor

Step 1

Ancestor

Step 2

M. fijiensis

M. musicola M. eumusae

Fig. 1. The three major Mycosphaerella species on Musa have evolved in two evolutionary steps, step 1: M. fijiensis and the ancestor of M. musicola and M. eumusae, and step 2: M. musicola and M. eumusae. plant-associated Mycosphaerella species on Musa is the pathological relevance and degree of host specificity. The host range of the three main species, namely M. fijiensis, M. musicola and M. eumusae, is currently believed to be limited to Musa, whereas species like M. citri, M. thailandica and M. colombiensis have been reported from multiple hosts. Mycosphaerella citri is a major pathogen on Citrus, but it has also been reported from banana, Acacia and Eucalyptus in South-East Asia (Crous et al. 2004, Chapter 2). The pathological relevance of M. citri on banana and the other hosts remains to be studied. Regardless of pathological relevance, the fact that Mycosphaerella species co-occur on a single host and even in a single lesion, as well as their ability to jump between hosts (Crous & Groenewald 2005), may ease inter-species exchange of genetic material. It is likely that different means of interaction and genetic material exchanges take place among the species colonising a single lesion (Chapter 2). This could ultimately result in the origin of new species with altered virulence patterns or host specificity. My results further revealed that the three primary agents of the Sigatoka disease complex, which all have a Pseudocercospora anamorph, together with three novel Pseudocercospora species, represent a monophyletic clade within all data sets (Chapter 2). However, on other hosts such as Eucalyptus, Pseudocercospora spp. were shown to be polyphyletic (Hunter et al. 2006). This might indicate that Pseudocercospora species on banana share a common ancestry, which concurs with the results obtained from the mating type locus (Chapter 5). Even though the disease report chronology described M. musicola as the first pathogen on banana and M. fijiensis, M. eumusae and P. longispora as subsequent pathogens of this host, it is unclear whether these species were co-existing, whether the presence of these species was unnoticed or whether these species evolved recently. However, between the three species clear differences in virulence exist correlating with the timing of their (economical) appearance. Mycosphaerella fijiensis appears to be more virulent than M. musicola but M. eumusae appears to be the most virulent species, affecting cultivars that are resistant to both M. musicola and M. fijiensis (Jones 2003). From the multi-gene sequence data generated here as well as from the mating type locus studies (Chapter 2 and 5), it can be concluded that the three major species have evolved in two

153 Chapter 6 evolutionary steps from a common ancestor (Fig. 1). During the first step the common ancestor split M. fijiensis from the ancestor of M. musicola and M. eumusae. Subsequently, M. musicola and M. eumusae have evolved as two separate lineages from the latter ancestor. The highest level of genetic diversity for M. fijiensis and M. musicola populations has been reported from South-East Asia (Carlier et al.1994, 1996, Hayden et al. 2003, 2005, Rivas et al. 2004) and currently M. eumusae is only found in South-East Asia and parts of Africa (Carlier et al. 2000, Crous & Mourichon 2002, Jones 2003, Arzanlou et al. 2007a). South-East Asia is the centre of origin for the genus Musa, where the earliest domestication of edible bananas took place and still many wild species of the genus grow in forests (Rivas et al. 2004). Considering the presumed host specificity of these three species, it is conceivable that these species have co-evolved with their Musa host. This hypothesis could be tested by extensive sampling from other genera in Musaceae and thus establish whether these three species occur on different hosts in this family or are restricted to Musa. The co-evolution of a Mycosphaerella species with its host is not without precedent. The co-evolution of M. graminicola, causal agent of septoria leaf blotch disease, with wheat has been suggested from phylogeographical and population studies indicating that the centre of origin of M. graminicola resides in the Middle East coinciding with the earliest centres of wheat domestication and cultivation (Banke et al. 2004, Stukenbrock et al. 2007). From the data presented in this thesis, it is clear that the Sigatoka disease complex is caused by a multitude of Mycosphaerella species. However, the exact contribution of each of these species to the disease complex remains unclear.

Species identification and detection

In Chapter 3, qualitative and quantitative molecular diagnostics primer sets for the three major contributors of the Sigatoka disease complex, M. fijiensis, M. musicola and M. eumusae, were designed and tested. Morphology-based identification of causal agents of a disease or disease complex requires trained staff and is time-consuming, and is a limitation for eradication and quarantine management strategies. Molecular markers can provide an additional tool for rapid identification of known fungal species, even in the absence of visible disease symptoms. Based on the β-tubulin gene, TaqMan real-time quantitative PCR assays were developed capable of detecting quantities as low as 1 pg/µl DNA for each Mycosphaerella species from pure cultures and 1.6 pg/µl DNA/mg of dry leaf tissue for M. fijiensis using naturally infected banana leaves. Based on the actin gene conventional species-specific PCR primers were developed that could be used to detect as little as 100, 1 and 10 pg/µl DNA from M. fijiensis, M. musicola and M. eumusae, respectively. These molecular-based tests can be implemented in a plant disease management system, even before the disease symptoms manifest. The molecular-based detection and quantification tools developed and optimised in this study can act as good starting points towards an increased understanding of the Sigatoka disease complex of banana by facilitating further ecological and epidemiological studies on the Mycosphaerella pathogens of banana. In addition, equipment for performing at least the conventional amplifications are becoming increasingly more affordable and can be used routinely by staff with minimal training.

Polyphyly in Ramichloridium

Another aim of the present study was to clarify the confusion about the taxonomic position of Ramichloridium musae, causal agent of tropical speckle disease of banana. In order to determine the phylogenetic affinity of ramichloridium-like anamorphs to Mycosphaerella, I

154 General discussion studied their phylogenetic position in the fungal kingdom (Chapter 4). Many fungal species, including species in the genus ‘Ramichloridium’ are only known from their asexual states and lack teleomorph connections, and hence the taxonomic value of morphological traits for (presumed) exclusively anamorphic fungi is questionable as it frequently does not result in a natural grouping. It is a major difficulty to establish natural taxa in fungi due to their pleomorphic nature, which means that a given fungal species may display different morphological forms. The phylogeny inferred from 28S nrDNA sequence data revealed the genus Ramichloridium to be heterogeneous (Chapter 4). The phylogenetic analysis places Ramichloridium species in five different orders within , with the type species residing in the Capnodiales. Furthermore, Periconiella was shown to reside in Mycosphaerella; Rhinocladiella and Veronaea in the Chaetothyriales (Chapter 4). With this study, the phylogenetic affinities of two additional anamorph genera were established with the Capnodiales, to which the genus Mycosphaerella belongs. It is quite surprising that more than 30 morphologically distinguishable anamorph genera have been linked to Mycosphaerella (Crous et al. 2007a). Although these anamorph genera are of great help to phytomycologists and plant pathologists who need to identify these species based on morphology, their intrinsic taxonomic value is part of an ongoing debate. It has recently been shown that morphologically similar anamorph genera have evolved several times within different families and even within orders (Crous et al. 2007a). This can be explained by the assumption that phylogenetically distinct groups of fungi have evolved similar traits as result of adaptation to similar environments or ecological niches (convergence) or that they share a common ancestor (divergence). The lack of morphological differences and the absence of associated teleomorphs make it impossible to recognise such taxa without employing DNA sequence data. In an attempt to formulate a system that also has practical value to users, anamorph genera have been accepted as plastic entities that could be used as loose nouns in fungal orders (Arzanlou et al. 2007b, Crous et al. 2007a, b).

Mating type genes in Mycosphaerella

The final aim of the present study was to analyse the evolutionary history of sexual reproduction among the three major Mycosphaerella pathogens of banana. The evolutionary history of sexual reproduction is of common interest for biologists as it gives us insight in the potential of organisms to cross and hybridise and gives an outlook on speciation. Analyses of mating type loci (Chapter 5) revealed extraordinary expansions and rearrangements in the mating type loci of the three major constituents of the Sigatoka disease complex. The expansion of mating type locus has not been observed in any other member of the Dothideomycetes, to which the genus Mycosphaerella belongs. Analysis of mating type loci for different species in this class has shown the idiomorph length to vary from ~2 to ~5 kb (Waalwijk et al. 2002, Barve et al. 2003, Bennett et al. 2003, Goodwin et al. 2003, Inderbitzin et al. 2005, Debuchy & Turgeon 2006, Stergiopoulos et al. 2007). Mycosphaerella fijiensis has the largest idiomorph (4.7 kb) from all species in the genus Mycosphaerella with an already characterised mating type idiomorph (Waalwijk et al. 2002, Goodwin et al. 2003, Conde-Ferráez et al. 2007). Analyses of mating type loci of the other two Mycosphaerella species on banana showed further expansion of the mating type loci. The size of the M. musicola idiomorph is over 8 kb and the expansion seems to be even greater in M. eumusae with at least 10 kb of the idiomorph sequenced. Possibly the large idiomorphs are footprints of speciation events. The increase of the idiomorph and concomitant genomic reorganisations within the idiomorphs might help in stabilising the new species by reducing the chances of sexual recombination. Comparisons of the mating type genes of M. musicola and M. eumusae showed good homology to those of M. fijiensis. Mat1-1-1 and Mat1-2-1 genes showed over 90 % identity on

155 Chapter 6 nucleotide level among these three species and the intron / exon boundaries were conserved. Further analysis of the cloned mating type loci from M. eumusae and M. musicola and published sequences for M. fijiensis, M. graminicola and Septoria passerinii predicted the presence of two putative genes (ORF1 and ORF2) in both opposite mating types. The gene organisation and predicted proteins encoded by these ORFs were highly similar among the three major banana pathogens but differed from those of M. graminicola and S. passerinii (Chapter 5). The phylogeny inferred from amino acid sequences of these ORFs revealed higher inter-species homology than intra-species homology among the three major banana pathogens. Again, this was in contrast to M. graminicola and S. passerinii where a higher intraspecies homology was observed. To our knowledge, the presence of these ORFs is restricted to Mycosphaerellaceae, and hence analyses of these ORFs in the other three closely related Pseudocercospora spp. (described in Chapter 2) may provide more insight to the evolutionary history of these ORFs. The expression studies carried out in this research showed that these ORFs are true genes, which are expressed under normal vegetative growth conditions (non-mating conditions); however, the expression was mating type-related, e.g. ORF1 is solely expressed in the Mat1-2 isolate, and ORF2 in the Mat1-1 isolate. Further functional analyses are needed to determine the role of these ORFs in sexual reproduction of these fungi. The discovery of an additional fused mating locus containing Mat1-1-1 sequences dispersed amid Mat1-2-1 sequences in the three major components of the Sigatoka disease complex was very remarkable. This Mat1-1/Mat1-2- fusion locus does not include areas that encode either the Mat1-1-specific alpha-domain or the Mat1-2-specific HMG-box, and is seemingly restricted to the three banana pathogens. Potentially, the other three novel Pseudocercospora spp. (Chapter 2) also carry these homologs. Preliminary results of PCR amplification with a specific primer set for the fused mating type gene suggests that this is indeed the case. The high homology and conserved organisation of the fused mating type loci between the three heterothallic Mycosphaerella species, as well as the good synteny between the genes in the flanking regions of the Mat1-1/Mat1-2-fusion locus and the M. graminicola mating type locus, might give an indication for a homothallic ancestor for these three species. Furthermore, molecular phylogenies based on ITS and 28S nrDNA sequences indicate that homothallic species such as M. marksii are basal in the Mycosphaerellaceae (Crous & Groenewald, unpublished data). However, further genomic analyses of the synteny of the genes flanking the mating type loci, and their chromosomal position, in more Mycosphaerella species are required to provide an acceptable hypothesis of the evolution of heterothallism in the genus. In fungi, a heterothallic reproduction strategy is considered to be ancestral and a homothallic strategy descendant. However, recent studies on the evolution of mating types in Aspergilli are suggesting that homothallism is ancestral to heterothallism in these fungi (Galagan et al. 2005, Paoletti et al. 2005, Scazzocchio 2006). Scazzocchio (2006) predicted a hypothetical homothallic ancestor with closely linked mating type genes, which could generate other homothallic species (by reciprocal translocation) and heterothallic species (by loss of one of the mating types). Species in the genus Mycosphaerella display both homothallic and heterothallic mating strategies, but a large number of species in this genus are only known from their asexual cycle and no Mycosphaerella state has yet been discovered. Therefore, the mating strategy of Mycosphaerella species and the presence or absence of the Mat1-1/Mat1-2-fusion locus should be mapped onto a phylogeny of the genus after the genetic basis of the mating strategy is confirmed. This could also yield answers to the question of which mating strategy is ancestral in the genus. The presence of the fused mating type locus is an indication for a homothallic ancestor. However, the higher interspecies homology of the newly identified ORFs is suggestive for a heterothallic ancestor. This can be explained by assuming two evolutionary events. First

156 General discussion

Common homothallic ancestor

Heterothallic ancestor

M. fijiensis M. musicola M. eumusae

Fig. 2. Schematic diagram for presumed evolution of MAT loci in three major Mycosphaerella species on Musa; these three species are suggested to have evolved from a heterothallic ancestor, which itself has evolved from a homothallic ancestor. from a common homothallic ancestor, the ancestor of three species has evolved to a possibly heterothallic species, with this heterothallic ancestor giving rise to the three species (Fig. 2). Further indication for homothallism being ancestral in this genus, can be drawn from the mating type locus structure in M. citri (data to be published elsewhere). Analyses of this mating type locus shows pockets of identical sequences between the Mat-1 and Mat-2 idiomorph, which is suggestive of a homothallic ancestor for this species. To prove this hypothesis, mating type loci and flanking sequences should be analysed for homothallic species in this genus. If the length of the idiomorph is taken as measure of evolutionary age (with shorter being older), the expansion of mating type loci is in agreement with the phylogeny inferred from the combined data set of four genes (Chapter 2), which showed these three species being closely related and evolved from a common ancestor in two evolutionary steps. The data further indicates the expansion event being recent, the same for the evolution of M. musicola and M. eumusae from the second common ancestor. Disease chronology records are also correlated with the expansion in mating type loci; M. eumusae is the most recently occurring species among the three major Mycosphaerella species on banana and hence, has the largest idiomorph. If this hypothesis is true, the three novel Pseudocercospora spp., viz. P. longispora, P. indonesiana and P. assamensis, which display the same evolutionary history, should have the same expansion pattern in their mating type region. The three major Mycosphaerella species on banana have subsequently emerged during the last century. It is doubtful that these species appeared as the consequence of rapid evolutionary processes (in ~100 years). It is more conceivable that differences in cultural practices (selection of host-cultivars, frequency and type of fungicide treatments), and environmental factors (growth of banana at different altitudes, variation in weather patterns) have favoured the appearance and development / increase of virulence of previously hardly pathogenic or even non-pathogenic fungal species. Currently, the genomes of several Dothideomycetes species, including two Mycosphaerella species (M. graminicola and M. fijiensis) are being sequenced and further genomic analysis of genome sequence data will shed more light on the evolution of mating type loci in this class of fungi.

157 Chapter 6

Concluding remarks

The research presented in this thesis clarified various taxonomic aspects of the genus Mycosphaerella in general and Mycosphaerella species pathogenic on banana in particular, with an emphasis on biodiversity and phylogeny. The phylogenetic affinity of asexual genera such as Ramichloridium, Periconiella, Veronaea and Rhinocladiella to Mycosphaerella was investigated. Ramichloridium and Periconiella were shown to have a phylogenetic affinity with Mycosphaerella. A multi-locus DNA sequence data set was established to study the biodiversity and phylogenetic relationships among the Mycosphaerella species constituting the Sigatoka disease complex of banana, which revealed the occurrence of more than 20 Mycosphaerella species on (cultivated) Musa, of which eight species were described as new. Molecular tools for detection and quantification were developed for the major constituents of the Sigatoka disease complex, M. fijiensis, M. musicola and M. eumusae. Furthermore, the mating type loci and evolutionary history of sexual reproduction of these three major Mycosphaerella species on banana were studied. With insight gained from our study on the mating type loci, it is likely that mating type genes in these three pathogens have evolved in two evolutionary steps, the primary ancestor being homothallic and the secondary ancestor possibly heterothallic. The results presented here open a window of opportunity for classical plant pathology – inoculation experiments are needed to determine the pathogenicity of and control measures for the novel species described in this dissertation –, development of molecular identification tools for the novel species, characterisation of mating loci of related species and further expression studies of the mating type genes and the ORFs described in Chapter 5.

REFERENCES

Agrios, G. N. 1997. Plant Pathology, 4th ed. Academic Press, San Francisco, California, USA. Arzanlou, M., Abeln, E. C. A., Kema, G. H. J., Waalwijk, C., Carlier, J., Vries, I. de, Guzmán, M., & Crous, P. W. 2007a. Molecular diagnostics for the Sigatoka disease complex of banana. Phytopathology 97: 1112–1118. Arzanlou, M., Groenewald, J. Z., Gams, W., Braun, U., Shin, H. -D., & Crous, P. W. 2007b. Phylogenetic and morphotaxonomic revision of Ramichloridium and allied genera. Stud. Mycol. 58: 57–93. Banke, S., Peschon, A., & McDonald, B. A. 2004. Phylogenetic analysis of globally distributed Mycosphaerella graminicola populations based on three DNA sequence loci. Fungal Genet. Biol. 41: 221–238. Barve, M. P., Arie, T., Salimath, S. S., Muehlbauer, F. M., & Peever, E. 2003. Cloning and characterisation of the mating type (MAT) locus from Ascochyta rabiei (Teleomorph: Didymella rabiei) and a MAT phylogeny of legume-associated Ascochyta spp. Fungal Genet. Biol. 39: 151–167. Bennett, R. S., Yun, S.-H., Lee, T. Y., Turgeon, B. G., Arseniuk, E., Cunfer, B. M., & Bergstrom, G. C. 2003. Identity and conservation of mating type genes in geographically diverse isolates of Phaeosphaeria nodorum. Fungal Genet. Biol. 40: 25–37. Carlier, J., Lebrun, M. H., Zapater, M. F., Dubois, C., & Mourichon, X. 1996. Genetic structure of the global population of banana black leaf streak fungus, Mycosphaerella fijiensis. Mol. Ecol. 5: 499–510. Carlier, J., Mourichon, X., Gonzalez-de-Leon, D., Zapater, M. F., & Lebrun, M. H. 1994. DNA restriction fragment length polymorphisms in Mycosphaerella species that cause banana leaf spot diseases. Phytopathology 84: 751–6.

158 General discussion

Carlier, J., Zapater, M. F., Lapeyre, F., Jones, D. R., & Mourichon, X. 2000. Septoria leaf spot of banana: a newly discovered disease caused by Mycosphaerella eumusae (anamorph Septoria eumusae). Phytopathology 90: 884–890. Conde-Ferráez, L., Waalwijk, C., Canto-Canché, B. B., Kema, G. H. J., Crous, P. W., James, A. C., & Abeln, E. C. A. 2007. Isolation and characterisation of the mating type locus of Mycosphaerella fijiensis, the causal agent of black leaf streak disease of banana. Mol. Plant Pathol. 8: 111–120. Crous, P. W. 1998. Mycosphaerella species and their anamorphs associated with leaf spot diseases of Eucalyptus. Mycol. Mem. 21: 1–170. Crous, P. W., Braun, U., & Groenewald, J. Z. 2007a. Mycosphaerella is polyphyletic. Stud. Mycol. 58: 1–32. Crous, P. W., & Groenewald, J. Z. 2005. Hosts, species and genotypes: opinions versus data. Australas. Plant Path. 34: 463–470. Crous, P. W., Groenewald, J. Z., Pongpanich, K., Himaman, W., Arzanlou, M., & Wingfield, M. J. 2004. Cryptic speciation and host-specificity amongMycosphaerella species occurring on Australian Acacia species grown as exotics in the tropics. Stud. Mycol. 50: 457–469. Crous, P. W., & Mourichon, X. 2002. Mycosphaerella eumusae and its anamorph Pseudo- cercospora eumusae spp. nov.: causal agent of eumusae leaf spot disease of banana. Sydowia 54: 35–43. Crous, P. W., Schubert, K., Braun, U., Hoog, G. S. de, Hocking, A. D., Shin, H.-D., & Groenewald, J. Z. 2007b. Opportunistic, human-pathogenic species in the Herpotrichiellaceae are phenotypically similar to saprobic or phytopathogenic species in the Venturiaceae. Stud. Mycol. 58: 185–217. Crous, P. W., Wingfield, M. J., Mansilla, J. P., Alfenas, A. C., & Groenewald, J. Z. 2006. Phylogenetic reassessment of Mycosphaerella species and their anamorphs occurring on Eucalyptus. II. Stud. Mycol. 55: 99–131. Debuchy, R., & Turgeon, B. G. 2006. Mating-type structure, evolution and function in euascomycetes. Pages 293–323 in: The mycota I. growth, differentiation and sexuality. Kües, U., & Fischer, R. eds. Berlin, Heidelberg. Galagan, J. E., Calvo, S. E., Cuomo, C., Ma, L. J., Wortman, J. R., Batzoglou, S., Lee, S. I., Basturkmen, M., Spevak, C. C., Clutterbuck, J., Kapitonov, V., Jurka, J., Scazzocchio, C., Farman, M., Butler, J., Purcell, S., Harris, S., Braus, G. H., Draht, O., Busch, S., D‘Enfert, C., Bouchier, C., Goldman, G. H., Bell-Pedersen, D., Griffiths-Jones, S., Doonan, J. H., Yu, J., Vienken, K., Pain, A., Freitag, M., Selker, E. U., Archer, D. B., Penalva, M. A., Oakley, B. R., Momany, M., Tanaka, T., Kumagai, T., Asai, K., Machida, M., Nierman, W. C., Denning, D. W., Caddick, M., Hynes, M., Paoletti, M., Fischer, R., Miller, B., Dyer, P., Sachs, M. S., Osmani, S. A., & Birren, B. W. 2005. Sequencing of Aspergillus nidulans and comparative analysis with A. fumigatus and A. oryzae. Nature 438: 1105–1115. Goodwin, S. B., Waalwijk, C., Kema, G. H. J., Cavaletto, J. R., & Zhang, G. 2003. Cloning and analysis of the mating-type idiomorphs from the barley pathogen Septoria passerinii. Mol. Genet. Genomics 269: 1–12. Hayden, H. L., Carlier, J., & Aitken, E. A. B. 2003. Genetic structure of Mycosphaerella fijiensis populations from Australia, Papua New Guinea and the Pacific Islands. Plant Pathol. 52: 703–712. Hayden, H. L., Carlier, J., & Aitken, E. A. B. 2005. The genetic structure of Australian popula- tions of Mycosphaerella musicola suggests restricted gene flow at the continental scale. Phytopathology 95: 489–498. Hunter, G. C., Wingfield, B. D., Crous, P. W., & Wingfield, M. J. 2006. A multigene phylogeny for species of Mycosphaerella occurring on Eucalyptus leaves. Stud. Mycol. 55: 147–161.

159 Chapter 6

Inderbitzin, P., Harkness, J., Turgeon, B. G., & Berbee, M. 2005. Lateral transfer of mating system in Stemphylium. Proc. Natl. Acad. Sci. USA, 102: 11390–11395. Jones, R. D. 2003. The distribution and importance of the Mycosphaerella leaf spot diseases of banana. Pages 25-42 in: Proceedings of 2nd international workshop on Mycosphaerella leaf spot disease of bananas, Costa Rica. Paoletti, M., Rydholm, C., Schwier, E. U., Anderson, M. J., Szakacs, G., Lutzoni, F., Debeaupuis, J. P., Latgé, J. P., Denning, D. W., & Dyer, P. S. 2005. Evidence for sexuality in the opportunistic fungal pathogen Aspergillus fumigatus. Curr. Biol. 15: 1242–1248. Rivas, G. G., Zapater, M. F., Abadie, C., & Carlier, J. 2004. Founder effects and stochastic dispersal at the continental scale of the fungal pathogen of bananas Mycosphaerella fijiensis. Mol. Ecol. 13: 471–482. Scazzocchio, C. 2006. Aspergillus genomes: secret sex and the secrets of sex. Trends Genet. 22: 521–525. Stergiopoulos, I., Groenewald, M., Staats, M., Lindhout, P., Crous, P. W., & Wit, P. J. de. 2007. Mating-type genes and the genetic structure of a world-wide collection of the tomato pathogen Cladosporium fulvum. Fungal Genet. Biol. 44: 415–429. Stukenbrock, E. H., Banke, S., Javan-Nikkhah, M., & McDonald, B. A. 2007. Origin and domestication of the fungal wheat pathogen Mycosphaerella graminicola via sympatric speciation. Mol. Biol. Evol. 24: 398–411. Waalwijk, C., Mendes, O., Verstappen, E. C. P., Waard, M. A. de, & Kema, G. H. J. 2002. Isolation and characterization of the mating-type idiomorphs from the wheat septoria leaf blotch fungus Mycosphaerella graminicola. Fungal Genet. Biol. 35: 277–286.

160 APPENDIX

Summary in English

Samenvatting (Summary in Dutch)

AcknowledgementS

About the Author

List of Publications

Education certificate of the EPS Graduate School

161 Appendix

SUMMARY

The genus Mycosphaerella is phylogenetically heterogeneous, and has been linked to more than 30 anamorphic genera. Plant pathogenic species of Mycosphaerella are among the most common and destructive plant pathogens, causing considerable economic losses on a wide range of hosts by invading leaf and stem tissue, and distorting the host plant physiology. The Sigatoka leaf spot disease complex of bananas involves three related ascomycetous fungi: Mycosphaerella fijiensis, M. musicola and M. eumusae. Besides these three primary agents of the Sigatoka disease complex, several additional species of Mycosphaerella (or their anamorphs) have been reported from Musa. This thesis provides insight into various taxonomic aspects of the genus Mycosphaerella in general and Mycosphaerella species pathogenic on banana in particular, with an emphasis on biodiversity, phylogeny and evolutionary plasticity of mating type loci. Chapter 1 gives an introduction to the genus Mycosphaerella, with specific reference to the Sigatoka disease complex of banana, and further provides a brief review on sexual reproduction in ascomycetous fungi and Mycosphaerella in particular. Chapter 2 describes the biodiversity and phylogenetic relationships among the Mycosphaerella species constituting the Sigatoka disease complex of banana. From the data presented in this chapter, it is clear that the Sigatoka disease complex is caused by a multitude of Mycosphaerella species. More than 20 species of Mycosphaerella or associated anamorphs were identified on banana. Eight out of these twenty species were described for the first time, and five species were previously described to occur on other host crops. Chapter 3 describes the development and use of molecular tools to detect and quantify the major constituents of the Sigatoka disease complex (M. fijiensis, M. musicola and M. eumusae) in planta. In Chapter 4 I clarified the taxonomic position of Ramichloridium musae, the casual agent of tropical speckle disease of banana, and morphologically similar genera such as Periconiella, Veronaea and Rhinocladiella to Mycosphaerella. The phylogeny inferred from 28S nrDNA sequence data revealed the genus Ramichloridium to be heterogeneous. The phylogenetic analyses place Ramichloridium species in five different orders within the Ascomycota, with the type species residing in the Capnodiales. Furthermore, Periconiella was shown to reside in Mycosphaerella; Rhinocladiella and Veronaea in the Chaetothyriales. In Chapter 5 I characterised the mating type loci of the primary agents of the Sigatoka disease complex of banana by the application of a chromosome-walking strategy, genomic analyses and bioinformatics. The data revealed that the mating type loci of these three heterothallic Mycosphaerella species are characterised by an expansion in size and the presence of a number of new genes which are presumably unique to members of Mycosphaerella. These analyses were extended to study the evolutionary history of the mating type loci in M. fijiensis, M. musicola and M. eumusae. Finally, the results of this thesis are discussed with a broader outlook in Chapter 7. Various taxonomic aspects of the genus Mycosphaerella in general and Mycosphaerella species pathogenic on banana in particular are addressed, with emphasis on biodiversity and phylogeny. Furthermore, the evolutionary history of sexual reproduction of the three major Mycosphaerella species on banana is discussed. Altogether, the data presented in this thesis suggest a two step evolutionary history for the causal agents of the Sigatoka disease complex of banana.

162 Summary / Samenvatting

SAMENVATTING

Het geslacht Mycosphaerella is fylogenetisch zeer heterogeen en is gekoppeld aan meer dan dertig anamorfe (asexuele) geslachten. Mycosphaerella soorten behoren tot de meest algemene en meest destructieve plant pathogene schimmels. Deze schimmels kunnen op een grote verscheidenheid aan waardplanten aanzienlijke economische schade veroorzaken, als gevolg van de door de infectie veroorzaakte verstoring van de waardplant fysiologie. Het Sigatoka bladvlekken ziekte complex op banaan omvat drie tot Mycosphaerella behorende verwante schimmels: Mycosphaerella fijiensis, M. musicola en M. eumusae. Behalve van de drie primaire veroorzakers van het Sigatoka bladvlekken ziekte complex is het van verscheidene andere Mycosphaerella soorten bekend dat zij kunnen voorkomen op Musa. Dit proefschrift, met de nadruk op biodiversiteit, fylogenie en de evolutionaire plasticiteit van parings-type loci (mating type loci), geeft inzicht in verschillende taxonomische aspecten van het geslacht Mycosphaerella in het algemeen, en Mycosphaerella soorten welke pathogeen zijn op banaan in het bijzonder. In Hoofdstuk 1 wordt een inleiding tot het geslacht Mycosphaerella gegeven, met nadruk op het Sigatoka ziekte complex op banaan. Verder bevat dit hoofdstuk een kort overzicht van de sexuele reproductie in ascomyceten met de nadruk op schimmels behorend tot de Mycosphaerella. In Hoofdstuk 2 wordt de biodiversiteit en de fylogenetische verwantschap van de Mycosphaerella soorten behorend tot het Sigatoka ziekte complex van banaan beschreven. De gegevens gepresenteerd in dit hoofdstuk tonen aan dat het Sigatoka ziekte complex veroorzaakt wordt door een breed scala aan Mycosphaerella soorten. Meer dan twintig Mycosphaerella soorten (of geassocieerde anamorfs) kunnen worden geïdentificeerd op banaan. Acht van deze twintig soorten zijn nooit eerder beschreven en vijf soorten zijn bekend van een andere waardplant dan banaan. De ontwikkeling en de toepassing van een moleculaire test om de belangrijkste veroorzakers van het Sigatoka ziekte complex (M. fijiensis, M. musicola en M. eumusae) te kunnen detecteren en kwantificeren wordt inHoofdstuk 3 beschreven. De taxonomische positie van Ramichloridium musae, de veroorzaker van de tropische vlekziekte van banaan, en morfologisch vergelijkbare geslachten zoals Periconiella, Veronaea en Rhinocladiella in relatie tot Mycosphaerella is verhelderd in Hoofdstuk 4. De fylogenie afgeleid van de 28S nrDNA sequenties geeft aan dat Ramichloridium heterogeen is. Deze fylogenetische analyses geven aan dat Ramichloridium binnen vijf orders te plaatsen is en dat de type-soort blijkt te behoren tot de Capnodiales. Verder blijkt Periconiella te plaatsen in Mycosphaerella terwijl Rhinocladiella en Veronaea tot de Chaetothyriales behoren. In Hoofdstuk 5 worden de parings-type loci van de belangrijkste veroorzakers van het Sigatoka ziekte complex gekarakteriseerd en geanalyseerd door toepassing van een combinatie van chromosoom-walking, genoom vergelijking en bioinformatica. De gegevens laten zien dat de parings-type loci van deze drie heterothallische Mycosphaerella soorten gekenmerkt worden door een expansie van de grootte, en door de aanwezigheid van een aantal genen die waarschijnlijk uniek zijn voor het geslacht Mycosphaerella. De analyses van de paringstype loci worden in dit hoofdstuk ook gebruikt om een idee te krijgen van de evolutionaire geschiedenis van de paringstype loci in M. fijiensis, M. musicola en M. eumusae. Tenslotte, worden in Hoofdstuk 7 de resultaten van dit proefschrift bediscussieerd. Verscheidene taxonomische aspecten, met de nadruk op biodiversiteit en fylogenie, van het geslacht Mycosphaerella en de Mycosphaerella soorten pathogeen op banaan worden aan de orde gesteld. Verder wordt de evolutionaire geschiedenis van de sexuele voortplanting van de drie belangrijkste Mycosphaerella ziekteverwekkers op banaan besproken. De data gepresenteerd in dit proefschrift duiden op een evolutionaire ontstaansgeschiedenis voor deze ziekteverwekkers in twee stappen.

163 Appendix

ACKNOWLEDGEMENTS

This brings me back to June 2003, when I found myself at CBS. Indeed, it seems like yesterday when I came to the Netherlands and started a new life here. During the last five years, I did not only strengthen my scientific background, I also learned a lot about life and people from all over the world. The work presented in this book would never have been completed without the help and support of many people, which means that I have to think carefully not to miss anyone who helped me in so many different ways and made my stay in the Netherlands happy and joyful. First and foremost, I am grateful to God for everything in my life, whatever I have is from him. I also would like to thank the Ministry of Science, Research, and Technology of Iran, and the Plant Protection Department of Tabriz University (Iran) from where I was awarded a scholarship, for financial support. I am deeply indebted to my promoter prof. dr Pedro Crous for giving me the opportunity to do my PhD in his research group at CBS. This gave me an excellent opportunity to ‘feel the fungi’. Dear Pedro, your motivating ideas, suggestions and great guidance directed my work. I will never forget your personal support as well, especially during the period I was ill. You provided an inspiring and encouraging atmosphere during my PhD. I was always impressed by your enthusiasm and encouragements. Your office was always open for me to discuss both scientific and personal issues. Finally, I would like to thank you for all the time you spent on revising my manuscripts, despite the fact that you were very busy. I would like to express my gratitude to prof. dr Pierre de Wit, my second promoter. Dear Pierre, thank you for your great suggestions and advice throughout my PhD and for your critical review of my manuscripts and valuable comments. I am grateful to dr Edwin Abeln, my former co-promoter, who unfortunately left our research group late 2005. Dear Edwin you were the first one with whom I worked in the lab; I learned a lot from you in the lab and during our scientific discussions. I would like to express my thanks to dr Ewald Groenewald, my daily supervisor, who helped me from the very beginning of my study until the end. Dear Ewald, I really enjoyed working with you, thank you so much for everything, your teaching, your patience, your flexibility. I will never forget the time you gave me a ride to Schiphol when I was on the verge of missing the flight to Australia for IMC8, and the same when I went to hand in my thesis for examination. I would also like to thank you for your critical review of my manuscripts and my thesis as well. I really appreciate your friendship. I would like to acknowledge dr Lute-harm Zwiers, who joined our group in the middle of 2006 and replaced Edwin in my committee. Dear Lute, you introduced me to the world of genomics, I enjoyed working with you and all the discussions we had over science, specially mating type genes, and all the funny results we had!! We also travelled together to America for a conference; I really enjoyed your company, you are a wonderful person. I would also like to express my special thanks to prof. dr Walter Gams, whom I met for first time in Karaj, Iran, in 2001, when he was attending an Asian Mycology Congress. Dear Walter, thank you and Sophia, your wife, for all the warm receptions and hospitalities at your house in Baarn (Netherlands) and Bomarzo (Italy). I also would like to thank you for your critical review of my manuscripts. My special thanks goes to dr Gert Kema, my co-promoter, from whom I learned scientific goal orientation, inspiration and optimism. Dear Gert thank you for the valuable comments and suggestions you had on my work and manuscripts.

164 Acknowledgements

I also would like to thank dr Cees Waalwijk, my external adviser, for his valuable comments and suggestions on my work. Dear Cees, for part of my project we worked together at Plant Research International; I really enjoyed the work and discussions we had over the science. I was the third PhD student who joined the Evolutionary Phytopathology Group at CBS. I shared an office with dr Lizel Mostert, a former PhD student in our group. Dear Lizel, I learned a lot from you and enjoyed all the conversations we had over science, life, and religion. Our friendship will last forever. Dr Marizeth Groenewald, also a former PhD student in our group and one of my paranymphs. Dear Marizeth, thank you very much for accepting to be my paranymph, I appreciate all your help in the lab and discussions we had over science. I am grateful to the members of our research group, Evolutionary Phytopathology, for all the support, help and discussions we had during lab meetings; I really enjoyed working with all of you. Maikel Aveskamp, fellow PhD student, with whom I shared an office. Dear Maikel thanks for accepting to be one of my paranymphs. You are an excellent office mate, especially for those who are in last stages of their PhD and need silence for writing! By having you in the office, I did not need internet to translate Dutch texts. I would like to thank Arien van Iperen and Mieke Starink-Willemse for helping me with cultures and in the molecular lab, and organising social events in our research group. My thanks goes to the other (ex-)members of group: dr Arthur de Cock, dr Gavin Hunter, dr Hans-Josef Schroers, Henk Brouwer, Hans de Gruyter, Joyce Woudenberg and two interns, Olaf Daviena and Lizette Haazen, who were involved in my project. We had many guests in our group from all over the world; this brought me an opportunity to learn more about different cultures and life in general: dr Arantxa Avila de la Calle, Francesca Mela, dr Conrad Schoch, dr Ulrike Damm, dr Konstanze Schubert, Ana Cabral, Lorenzo Lombard, Ratchadawan (Joy) Cheewangkoon, dr Lam Duong Minh, dr Elena Turco, dr Uwe Simon and finally Salwa Essakhi, who was a great support for me for the period I was ill. I enjoyed the time I spent with all of you and, of course, the excursions we had together. I performed part of my project at the Molecular Phytopathology Cluster of Plant Research International, Wageningen. I warmly appreciate the assistance of Ineke de Vries in the lab and dr Theo van de Lee, dr Sarah Ware, dr Rahim Mehrabi and dr Ramin Roohparvar, Odette Mendes and the other staff members and PhD students for your excellent support and discussions we had over science and life. I enjoyed working with all of you. I was not the only Iranian at PRI, I was at the same department where dr Rahim Mehrabi, dr Hossein Jafari, and Reza Aghnoum were doing their PhD research; I enjoyed our coffee breaks and all the discussions we had on various subjects. In addition to the other members mentioned earlier, I would like to thank also these members of Dutch Mycosphaerella Group, dr Manoel Souza, Sarrah Ben M´Barak, Mahmod Tabib Ghaffary, dr Ioannis Stergiopoulos, Caucasella Díaz Trujillo, for all the discussions we had during our monthly meetings. The staff members and PhD students from the Phytopathology Department of Wageningen University, I thank you all for the support and discussions we had at Friday morning lab meetings. All the staff members of CBS have been very helpful and kind to me, I would like to thank you all for your great support and help during my study, which provided me a pleasant stay in the Netherlands. I will not be able to list all the names here, but would like to highlight some: Susan Maas from the personnel office, for arranging my visa and all the procedures with Dutch immigration, Tineke van den Berg-Visser and Manon Verweij for all their support with administrative part, Trix Merkx, Yda Vlug, Janny Holtman, and the other staff members of the culture collection department for providing cultures, the media preparation team for providing media and reagents, the IT team for solving the computer problems, the facility and financial services team for taking care of orders, facilities and mail and the cleaning team for providing a clean working environment.

165 Appendix

In addition to those mentioned before, I would like to thank the scientific staff members of CBS, PhD students, postdocs and guests researchers: Ferry, Jamal, Montarop, Hesti, Richard, Hamid, Javad, Shuwen, Kenneth, Marjan, Benedetta, Jingsi, Marcela, Jos, Bart, Bert, Kasper, Kittipan and Collin for your support and valuable discussions we had during Monday morning CBS seminars and during my study. Someone once said: ‘Where you have friends, you have family’. This is especially true for me; having so many Iranian friends here, I rarely felt homesick: Mohammad, Zohre, Sharam, Hadi, Hamid, Javad, Hossein, Esmaeil, Nasrin, Hasan, Mahnaz, Ramin, Mehdi, Sadegh, Saeed, Morteza, Kaka, Afsaneh, Yusef, Reza, Akbar, Ali and their families, where applicable. I would like to thank you all for all your emotional support and invitations. My special thanks goes to Marjan Vermaas for making excellent photo plates and the very nice design for the cover of my thesis. Dear Marjan, you did a great job! Thank you again. I would like to express my special thanks to Manon Verweij for doing such a nice lay-out for my thesis. Dear Manon, you did such an excellent job in a very short time! Thank you very much. Finally, I would like to express my special appreciation to my family. My dear father, you have always been the source of inspiration in my life, I am very happy that I could fulfil your desires. My dear mother, you are the source of love in my life. I promised you to finish my study in four years when I left you in June 2003. I am sorry for the 1-year delay I had, I hope you will forgive me as you always do. Words cannot express the appreciation I have for the two of you. I thank God that you both are in good health; I will come back to you soon. My brothers, Mohammad and Mohsen, you have both been a great support for me in my life. Being the youngest member of our family, I did not need to think more about the sorrows and troubles of life. You have both been taking care of everything, without your encouragements and support I would never have completed this work. I know that words cannot express the depth of gratitude that I feel for you, but I would like to thank you from the bottom of my heart.

It is always difficult to mention everybody, so I would like to thank all who helped me by their direct or indirect support and made me feel at home during this period of my life.

Mahdi Arzanlou 9th of April, 2008 CBS, Utrecht

166 About the author

ABOUT THE AUTHOR

Mahdi Arzanlou was born in 1975 in Firouragh, Khoy, located in northwest of Iran. After completing high school with majors in biological sciences in 1993, he was accepted in the Plant Protection Department of Tabriz University (East Azarbaijan Province, Iran) and obtained a BSc degree in plant protection in 1997. He continued his studies at Tehran University (Capital of Iran) and completed his MSc in plant pathology in 2000. In 2002 he was awarded scholarships from the Ministry of Science, Research and Technology of Iran (MSRT) and the Plant Protection Department of Tabriz University, to pursue a PhD study abroad. In June 2003, he started his PhD project in Evolutionary Phytopathology Group at CBS Fungal Biodiversity Centre, Utrecht, the Netherlands with enrolment at Wageningen University. He conducted his PhD study on phylogeny, detection, and mating behaviour of Mycosphaerella spp. occurring on banana. As of this date, he will return to Iran and will continue his scientific career as an assistant professor in the Plant Protection Department of Tabriz University.

167 Appendix

LIST OF PUBLICATIONS

Scientific Publications

Arzanlou M, Kema GHJ, Waalwijk C, Carlier J, Vries I de, Guzmán M, Vargas MA, Helder J, Crous PW (2008). Molecular diagnostics for the Sigatoka disease complex and Radopholus similis in banana. In proceedings of ISHS/Promusa Symposium, September 10–14, 2007, Greenway Woods Resort, White River, South Africa (in press). Arzanlou M, Groenewald JZ, Fullerton RA, Abeln ECA, Carlier J, Zapater M-F, Buddenhagen IW, Viljoen A, Crous PW (2008). Multiple gene genealogies and phenotypic characters differentiate several novel species of Mycosphaerella and related anamorphs on banana. Persoonia 20: 19–37. Arzanlou M, Abeln ECA, Kema GHJ, Waalwijk C, Carlier J, Vries I de, Guzmán M, Crous PW (2007). Molecular Diagnostics for the Sigatoka Disease Complex of Banana. Phytopathology 97: 1112–1118. Arzanlou M, Groenewald JZ, Gams W, Braun U, Shin HD, Crous PW (2007). Phylogenetic and morphotaxonomic revision of Ramichloridium and allied genera. Studies in Mycology 58: 57–93. Crous PW, Groenewald JZ, Pongpanich K, Himaman W, Arzanlou M, Wingfield MJ (2004). Cryptic speciation and host specificity among Mycosphaerella spp. occurring on Australian Acacia species grown as exotics in the tropics. Studies in Mycology 50: 457–469. Arzanlou M, Hedjaroude GH, Okhovat M, Sharifi-Tehrani A, Arjmand, MN (2002). Identification and pathogenicity ofFusarium spp. associated with sugar beet root rot in Karaj region of Iran. Sugar beet Scientific and Research Journal of Iran 16: 62–73. Arzanlou M, Hedjaroude GH, Okhovat M (2000). First report for occurrence of teleomorph of Rhizoctonia solani (AG-4) sugar beet isolate in Iran. Iranian Journal of Phytopathology 35: 179–180.

Popular Papers

Arzanlou M, Crous PW (2008). Devriesia strelitziae Arzanlou & Crous, sp. nov. Fungal Planet 22. Arzanlou M, Crous PW (2006). Phaeosphaeriopsis musae M. Arzanlou & Crous, sp. nov. Fungal Planet 6. Arzanlou M, Crous PW (2006). Strelitziana M. Arzanlou & Crous, gen. nov. Fungal Planet 8.

Oral and Poster Presentations

Arzanlou M, Crous PW, Zwiers L-H (2008). Genomic comparisons of mating type loci of Mycosphaerella spp belonging to the Sigatoka disease complex of banana. 9th European Conference on Fungal Genetics, April 5-8, Edinburgh, Scotland (Poster presentation). Arzanlou M (2007). The Mycosphaerella complex on banana. Mini-symposium Embrapa-PRI- CBS projects on Mycosphaerella/banana, October 16, Wageningen, the Netherlands (Oral presentation). Arzanlou M, Abeln ECA, Kema GHJ, Waalwijk C, Carlier J, Vries I de, Guzmán, Crous PW (2007). Molecular diagnostics in the Sigatoka disease complex of banana and for Radopholus similis. ISHS/Promusa Symposium, September 10–14, Greenway Woods Resort, White River, South Africa (Oral presentation by C. Waalwijk)

168 List of publications

Arzanlou M, Zwiers L-H, Crous PW (2007). The evolution of mating type idiomorphs in Mycosphaerella. 24th Fungal Genetics Conference, March 20–25, Asilomar, USA (Poster presentation). Arzanlou M, Groenewald JZ, Crous PW (2006). A phylogenetic approach to accommodate Ramichloridium orphans. 8th International Mycology Conference, August 20–25, Cairns, Australia (Oral presentation). Arzanlou M, Abeln ECA, Kema GHJ, Waalwijk C, Carlier J, Crous PW (2006). Molecular diagnostics in the Sigatoka disease complex of banana. 8th International Mycology Conference, August 20–25, Cairns, Australia (Poster presentation). Arzanlou M, Daviena O, Abeln ECA, Groenewald JZ, Crous PW (2004). ITS phylogeny reveals cryptic species within the Mycosphaerella leaf spot complex occurring on banana. CBS Centenary, May 12–14, Amsterdam, the Netherlands (Poster presentation). Arzanlou M, Hedjaroude GH, Okhovat M (2000). Identification and pathogenicity ofFusarium spp. associated with sugar beet root rot in Karaj region of Iran. 14th Iranian Plant Protection Congress, August 4–7, Isfahan, Iran (Poster Presentation). Arzanlou M, Hedjaroude GH, Okhovat M (2000). Identification and pathogenicity of fungal agents associated with sugar beet root rot in Karaj region of Iran. 14th Iranian Plant Protection Congress, August 4–7, Isfahan, Iran (Poster Presentation).

169 Appendix

Education Statement of the Graduate School Experimental Plant Sciences

Issued to: Arzanlou, Mahdi Date: 27 February 2008 Group: Evolutionary Phytopathology, CBS - KNAW & Phytopathology, Wageningen University

1) Start-up phase date Ź First presentation of your project Genetics and pathogenicity of Mycosphaerella species causing leaf spot disease on banana Nov 2003 Ź Writing or rewriting a project proposal Ź Writing a review or book chapter Molecular diagnostics for the Sigatoka disease complex and Radopholus similis in banana Sep 2007 Ź MSc courses Ź Laboratory use of isotopes Subtotal Start-up Phase 4.5 credits*

2) Scientific Exposure date Ź EPS PhD student days EPS PhD student day, Vrije Universiteit Amsterdam 03 Jun 2004 EPS PhD student day, Radboud University 02 Jun 2005 EPS PhD student day, Wageningen University 19 Sep 2006 Ź EPS theme symposia EPS theme 4 symposium ' Genome plasticity' 12 Dec 2003 Ź NWO Lunteren days and other National Platforms Annual PhD Student Day Graduate School Biodiversity 2004-2006 Ź Seminars (series), workshops and symposia Symposium 'Evolutionary Consequences of Life without Sex' 24 Nov 2003 CBS Symposium 'Fungal Phylogenomics' 11-12 May 2004 CBS Centenary 13-14 May 2004 Phytopathology day Jan 2004 Tracks of Evolution mini-symposium (2 seminars) 29 Oct 2004 Symposium in systems biology in honour of Prof. Pierre de Wit 04 Nov 2004 Mini-symposium Embrapa-PRI-CBS projects on Mycosphaerella/banana, The Netherlands 2007 Oct 2007 Current themes in ecology (Biological invasion) 02 Nov 2007 Ź Seminar plus Ź International symposia and congresses 8th international mycology congress, Australia 2006 20-25 Aug 2006 24th fungal genetics, USA 2007 20-25 Mar 2007 Ź Presentations CBS centenary poster presentation May 2004 IMC8 oral presentation Aug 2006 IMC8 poster presentation Aug 2006 Mini-symposium Embrapa-PRI-CBS projects on Mycosphaerella/banana presentation Oct 2007 9th European Fungal Genetics Conference poster presentation Apr 2008 Ź IAB Interview 19 Sep 2006 Ź Excursion Subtotal Scientific Exposure 12.8 credits*

3) In-Depth Studies date Ź EPS courses or other PhD courses Functional genomics: theory and hands-on data analysis 25-28 Aug 2003 Molecular phylogenies: reconstruction and interpretation 04-07 Nov 2003 Introduction to fungal Biodiversity 02-13 Feb 2004 Advanced topics in Phylogeny re-construction Feb 2005 Real-time PCR course Jun 2005 Ź Journal club Ź Individual research training Subtotal In-Depth Studies 7.7 credits*

4) Personal development date Ź Skill training courses Writing English for publication 2004-2005 Goal oriented working and planning Jan 2005 Techniques for writing and presenting a scientific paper Feb 2006 Ź Organisation of PhD students day, course or conference Ź Membership of Board, Committee or PhD council Subtotal Personal Development 3.9 credits*

TOTAL NUMBER OF CREDIT POINTS* 28,9 * A credit represents a normative study load of 28 hours of study

170 This research was financially supported by the Ministry of Science, Research and Technology (MSRT) of Iran.

Layout and design: Manon Verweij and Marjan Vermaas.

Front and back cover: Banana leaves showing Sigatoka disease complex symptoms. Photograph by Gert Kema.

Front cover insets: Top and middle: banana leaves showing Sigatoka disease complex symptoms (photographs by Gert Kema and the author). Bottom: light microscopy photograph of Pseudocercospora musae (teleomorph: Mycosphaerella musicola) conidia and conidiophores (photograph by the author).

Printed at: CPI Wöhrmann Print Service

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