Evolutionary Phytopathology

Producing food sufficient in quality and quantity remains paramount for sustaining quality of life. Inadvertent introductions of phytopathogenic fungi have had dire consequences to nature and to cultivated crops on various continents in the past. The economic impact of such introductions can be seen in yield loss and in increased input costs for cultivation and disease control, as well as in social impact. To combat these diseases on an international scale, it is important to clarify whether the same species and genotypes occur in various countries, since each different species and genotype can be expected to have different patterns of attack, as well as different responses to fungicides and to climatological conditions. With such pathogens, it is also important to know what their host ranges and mating strategies are, and how this relates to different disease control mechanisms. The global movement of agricultural and forestry produce is inextricably cross-linked, and will continue to be so in future. Knowing which pathogens occur where and on what crops facilitates trade in agricultural produce. In this programme, we address these economically vital matters by investigating the speciation and host adaptation of various important phytopathogenic fungi.

Host specificity and speciation in Mycosphaerella
The genus Mycosphaerella and its associated anamorph genera represent more than 10.000 species, being associated with diseases on most genera of plants. Most species have been described on the assumption that they are highly host-specific. With the implementation of molecular phylogeny as the basis of modern taxonomy, host relationships and specificity can now be tested. A major aim of our research is to determine how exclusive the host-pathogen relationship of Mycosphaerella species is. Investigations based on genomic analysis are in progress on fungal species from a wide range of plant hosts. Mycosphaerella has been linked to numerous asexual reproductive states that may have evolved into exclusively asexual species. Such asexual forms were often difficult to trace to a sexual ancestor and were thus historically placed in separate genera. One such example is the genus Cercospora, which represents several thousand names, of which roughly a thousand can be recognised based on morphology. During the past evaluation period we have monographed the genus Cercospora, and are now in the process of studying its phylogeny and sexual behaviour. To this end we used the celery pathogen, Cercospora apii (with approx. 300 morphologically indistinguishable synonyms), and the sugarbeet pathogen, Cercospora beticola, as model to study variation and speciation within Cercospora. The genus Cercospora appears to be largely asexual: very few species have been reported to have Mycosphaerella states. To investigate this matter further, mating type primers were developed to screen populations of C. beticola, C. apii, C. zeae maydis (on maize), and two newly described species, C. apiicola (on celery), and C. zeina (on maize). The results of this screening indicate that some species are undergoing cryptic sex, and probably have functional Mycosphaerella teleomorphs that have yet to be found, while others appear to be truly asexual. A similar situation of cryptic sex was revealed when the Mycosphaerella complex of Pinus (Dothistroma red band needle disease) was investigated, as was “Cladosporium leaf spot”of tomato (Passalora fulva). A DNA phylogeny approach was used to investigate the evolution and inter-relationships of Mycosphaerella species causing defoliation and deformation of various hosts. These include species occurring on Pinus, Eucalyptus, Acacia (cultivated for timber, paper and pulp industries), Olea (olives), Protea (cut-flowers), and Musa (eating and cooking bananas). Several examples were found of Mycosphaerella species jumping between hosts (citrus, acacia, eucalypts, proteas, banana, etc.), and although this adaptation is not yet well understood, we will study it further in coming years using novel sequencing technologies.  We were also able to demonstrate the presence of several novel species, first reports and / or new hosts for these species. Numerous species of Mycosphaerella were found to be associated with the Sigatoka disease complex of banana. Several of these species appear to be confined to certain regions, while others were more global in distribution.

Mycosphaerella mating types and genomics
We found numerous species of Mycosphaerella to be associated with the Sigatoka disease complex of banana and the possibility of interaction and hybridisation among these species is being investigated. Analyses of the cloned mating type genes of M. fijiensis, M. musicola and M. eumusae indicated an extraordinary organization of the idiomorphs as well as the presence of Mycosphaerella unique genes, Functional analyses of these unique genes and characterization of the mating type loci of additional Mycosphaerella spp. is currently underway.  Also, the distribution of the mating type loci within populations is being determined. This is done to assess the occurrence of sexual reproduction, a factor controlling genetic recombination and genotypic diversity. Further mating type studies that have been completed concern the Cercospora apii complex, the Dothistroma needle blight pathogens (D. pini, D. septospora) of pines, and Passalora (Cladosporium) fulva.
Specific TaqMan probes have been developed in collaboration with Plant Research International (Wageningen University), which will facilitate the early detection and monitoring of the disease.
To aid our understanding of the pathology of the genus Mycosphaerella, two model species of Mycosphaerella, M. graminicola and M. fijiensis, were selected by the International Mycosphaerella Genomics Consortium, in which CBS participates, for complete genome sequencing. The genome sequences for both species are finished and publicly available. The genome sequence of M. graminicola is of extremely high quality and it is expected to be the first completely finished fungal genome (all chromosomes being covered from telomere to telomere and no gaps remaining). Surprisingly, the genome of M.fijensis is almost twice the size of M. graminicola  (~73MB vs ~41 MB) and contains an extremely high proportion of transposons. Our research group is actively involved in the ongoing annotation of both genomes. The differences observed between the genomes of the two Mycosphaerella species illustrates the high genetic variability and flexibility within this genus. Therefore, this project will be further coordinated with sequencing efforts planned for other Mycosphaerella species and relatives to greatly increase the power of future comparative genomics analyses.

Cladosporium and their Davidiella teleomorphs
The genus Davidiella was established for Mycosphaerella-like teleomorphs with Cladosporium anamorphs. Species of Cladosporium are common and widespread, and interact with humans in every phase of life, from producing allergens in the indoor environment, to causing fruit decay and plant disease, or being associated with human mycoses. Although Cladosporium is one of the largest and most heterogeneous genera of hyphomycetes (700 odd names), only a mere fraction of these species are known from culture, and few have been characterised based on molecular data.  By employing a multi-gene phylogenetic approach, integrated with anamorph and teleomorph morphology, Cladosporium could be circumscribed, and the family Davidiellaceae distinguished from the other genera and families in the Capnodiales. Two species complexes have thus far been elucidated, namely the C. sphaerospermum and the C. herbarum complex, both of which contained numerous undescribed species.  Future attention will now be directed towards the C. cladosporioides complex, which contains numerous undescribed species, several of which have Davidiella teleomorphs. In 2009 we envisage to publish a monograph of Cladosporium, in collaboration with Prof. dr U. Braun (Martin-Luther Univ., Halle, Germany), and Dr K. Schubert (Botanische Staatssammlung München, Germany).

Botryosphaeria canker pathogens
Botryosphaeria is a species-rich genus with a cosmopolitan distribution, commonly associated with dieback and cankers of woody plants. As many as 18 anamorph genera have been associated with Botryosphaeria, most of which have been reduced to synonymy under Diplodia (conidia mostly ovoid, pigmented, thick-walled), or Fusicoccum (conidia mostly fusoid, hyaline, thin-walled). However, there are numerous conidial anamorphs with morphological characteristics intermediate between Diplodia and Fusicoccum, and there are also several records of species outside the Botryosphaeriaceae that have anamorphs apparently typical of Botryosphaeria sensu stricto. Recent molecular studies have also linked Botryosphaeria to species with pigmented, septate ascospores and anamorphs in Dothiorella, or to species with hyaline ascospores and Fusicoccum anamorphs linked to Dichomera synanamorphs. By employing DNA sequence data for various loci, different lineages, representing 12 clades, could be resolved within the Botryosphaeriaceae. Two of these lineages clustered outside the molecularly reconceived Botryosphaeriaceae sensu stricto; both were groups with diplodia-like anamorphs occurring on maize. These phylogenetically disparate lineages are best accommodated in Stenocarpella (Diaporthales) and in an unresolved clade including species of Camarosporium / Microdiplodia. The ten lineages retained within the Botryosphaeriaceae sensu stricto represented different anamorph-teleomorph combinations, many of which are new to science. Further studies are underway to resolve the taxonomic status of many of these generic and species complexes occurring on different woody hosts. In 2009–2010 we envisage to publish a monograph on the species of Botryosphaeria known from culture in collaboration with Dr A.J.L. Phillips (Universidade Nova de Lisboa, Portugal), and Prof. dr M.J. Wingfield (FABI, Univ. of Pretoria, South Africa).

Petri disease and phaeohyphomycosis caused by species of Phaeoacremonium
Species of Phaeoacremonium are involved in Petri disease and esca of grapevines. Additionally, several species of Phaeoacremonium also cause phaeohyphomycosis in humans. During this study, Togninia (Calosphaeriales) was confirmed as teleomorph of Phaeoacremonium by means of morphology, sexual compatibility, and DNA phylogeny. Three species of Phaeoacremonium have been associated with phaeohyphomycosis. These are Pm. parasiticum, Pm. inflatipes and Pm. rubrigenum. Numerous unknown isolates resembling Phaeoacremonium spp. have in recent years been isolated from human patients, as well as from woody plants that appear to be the main environmental source of these fungi. New species were identified based on their cultural and morphological characters, and phylogenetic analyses of partial sequences of the actin, β-tubulin and calmodulin genes. A multiple-entry electronic key based on morphological, cultural and β-tubulin sequence data was developed to facilitate routine species identification. The genus Togninia was monographed along with its Phaeoacremonium anamorphs. Ten species of Togninia and 22 species of Phaeoacremonium were recognised. Phylogenies of the SSU and LSU rRNA genes were used to determine whether Togninia had more affinity with the Calosphaeriales or the Diaporthales. A rapid molecular identification method was developed for the 22 species of Phaeacremonium. It involved the use of 23 species-specific primers, including 20 primers targeting the β-tubulin gene and three targeting the actin gene. Furthermore, the multiple-entry electronic key was updated to include the new species of Phaeoacremonium. Separate dichotomous keys were provided for the identification of the Togninia and Phaeoacremonium species, and their mating strategies elucidated. Keys for the identification of phaeoacremonium-like fungi and the genera related to Togninia were also provided.

Hybridisation in Phytophthora and Pythium
Pythium and Phytophthora are two highly economically significant genera of fungus-like Oomycetes responsible for many types of crop disease and tree decline. The best known of the crop diseases is potato late blight (Phytophthora infestans), the cause of the Irish potato famine and a major agent of crop damage to this day. A study of the organization of the 5S rRNA gene family was performed for 87 species and varieties of Pythium. For the four different patterns of 5S organization that were found to occur within the genus, studies were conducted to determine how they arose and how evolutionarily stable they were. A number of Phytophthora strains were also included in the study as a reference outgroup giving insight into the ancestral organisation of the 5S gene family. The most parsimonious interpretation of the data would be that a contiguously linked arrangement of 5S sequences was the ancestral condition. A DNA array was developed as tool for the rapid identification and detection of Pythium species in pure culture, as well as in environmentally mixed samples. Oligonucleotides complementary to specific diagnostic regions of ribosomal internal transcribed spacers (ITS) were designed for more than 100 Pythium species and varieties as well as for groups of related species. Specificity was tested in hybridisation experiments with DNA from ex-type strains and other representative strains. BLAST analyses against Pythium DNA sequences available in GenBank were used to confirm that species-specific oligonucleotides were unique to all the available strains of each species. In a blind test with 50 additional unidentified Pythium isolates from soil, the array hybridisation patterns obtained were found to concur with isolate identifications obtained via morphological study and ITS sequences. In another blind test, total DNA of soil samples was amplified and hybridised on the array. Results were compared to the results of isolation by soil dilution plating and root baiting. Thirteen species were detected by the DNA array. These species corresponded with those obtained by isolation, though isolation also revealed the presence of one species that was not represented on the array. From these results it can be concluded that the DNA array is a reliable tool for identification and detection of the majority of Pythium species in environmental samples. Simultaneous detection and identification of multiple species of soil-borne pathogens such as Pythium will be a major step forward for epidemiological and ecological studies. Investigations of a number of atypical Phytophthora isolates initially identified as P. cactorum disclosed that these isolates were actually inter-species hybrids. Isozyme analysis demonstrated the presence of two alleles rather than the usual single allele for the dimeric malic enzyme (MDHP) in these isolates. One allele of the pair was typical for P. cactorum while the other was typical for P. hedraiandra. Sequencing of ribosomal ITS loci showed that this marker was heterogeneous in the atypical isolates, and that the sequences of P. cactorum and P. hedraiandra were both present. Phytophthora is diploid, and hybrids are expected to combine the genetic characters of both parents as is normally seen in plants and animals (but not most fungi). The mitochondrial genome, however, is inherited maternally and will be present in a single type derived from one of the parents. Indeed, the presumed hybrids were found to possess only one type of the mitochondrial CoxI gene, either that of the P. cactorum or that of P. hedraiandra. Two isolates showed deviating combinations of the characters mentioned above, suggesting that evolution by genome rearrangement had already taken place in some later-generation progeny of the hybrid lineages. The hybrid Phytophthora isolates were found on a variety of plant hosts in public parks all over the Netherlands, making it appear highly likely that they have also become established in natural ecosystems. Phytophthora hedraiandra is a species that has probably only recently been imported into the Netherlands via the use of Mediterranean Viburnum shrubs in gardening, while P. cactorum is a long-established native phytopathogen. The recent proliferation of hybrids between these species appears to fulfill a long-standing prediction that novel pathogenic Oomycetes would arise as world trade in plant products brought Oomycetes from around the world into interaction with one another. Several of the hosts infected by the hybrids are not known to be infected by either parent species. This suggests that such hybridisations arising from a breakdown in geographic barriers could cause the emergence of novel and unpredictable phytopathogen epidemiologies.