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Elucidating the Ramularia eucalypti species complex

The genus Ramularia includes numerous phytopathogenic species, several of which are economically important. Ramularia eucalypti is currently the only species of this genus known to infect Eucalyptus by causing severe leaf-spotting symptoms on this host. However, several isolates identified as R. eucalypti based on morphology and on nrDNA sequence data of the ITS region have recently been isolated from other plant hosts, from environmental samples and also from human clinical specimens. Identification of closely related species based on morphology is often difficult and the ITS region has previously been shown to be unreliable for species level identification in several genera. In this study we aimed to resolve this species-complex by applying a polyphasic approach involving morphology, multi-gene phylogeny and matrix assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS). Six partial genes (ITS, ACT, TEF1-α, HIS3, GAPDH and RPB2) were amplified and sequenced for a total of 44 isolates representing R. eucalypti s.lat. and closely related species. A multi-gene Bayesian phylogenetic analysis and parsimony analysis were performed, and both the resulting trees showed significant support for separation of seven species in R. eucalypti s.lat., including two previously described (R. eucalypti and R. miae), four novel species here described (R. haroldporteri, R. glennii, R. mali and R. plurivora) and one undescribed Ramularia species (sterile). Additionally, Mycosphaerella nyssicola is newly combined in Ramularia as R. nyssicola. Main mass spectra (MSPs) of several R. eucalypti s.lat. strains were generated using MALDI-TOF MS and were compared through a Principal Component Analysis (PCA) dendogram. The PCA dendrogram supported three clades containing R. plurivora, R. glenni/R. mali and R. eucalypti/R. miae. Although the dendrogram separation of species differed from the phylogenetic analysis, the clinically relevant strains were successfully identified by MALDI-TOF MS.

Italy that exhibited severe leaf spotting symptoms (Crous et al. 2007). It is currently the only species of the genus known to infect Eucalyptus and Corymbia, since R. pitereka and aggregate species have been reassigned to Quambalaria (Quambalariaceae) (de Beer et al. 2006). Over the past few years several isolates have been collected and identified as R. eucalypti based on morphology and on sequence data of the ITS region of the nrDNA operon which has recently been adopted as the universal DNA barcode for fungi (Schoch et al. 2012). However, in several genera of phytopathogenic fungi, ITS phylogenies have often failed to separate closely related species, and a better resolution could only be achieved by using protein-coding loci (Lombard et al. 2010, Cabral et al. 2012, Crous et al. 2013, Quaedvlieg et al. 2013, Woudenberg et al. 2013).
In Italy, R. eucalypti has been reported as an emerging problem on pome fruit in cold storage where it causes lenticel rot in healthy fruits of apple (Malus domestica cv. Ambrosia) and pear (Pyrus communis cv. Conference) (Giordani et al. 2012). Investigations into the epidemiology showed that apple trees in the orchards had leaf spots caused by R. eucalypti, and symptomless fruits harvested from infected plants exhibited disease symptoms during the subsequent four months of cold storage (Gianetti et al. 2012).
In the Netherlands, R. eucalypti has been isolated not only from different plant hosts but has also been obtained from clinical specimens in different hospitals. This is the first time a Ramularia species is associated with human infection and little is known of its epidemiology. However, this is not the first time a plant pathogenic fungus has been reported to be able Elucidating the Ramularia eucalypti species complex S.I.R. Videira 1,3 , J.Z. Groenewald 1 , A. Kolecka 1 , L. van Haren 1 , T. Boekhout 1 , P.W. Crous 1,2,3 to infect human hosts (Mostert et al. 2006, van Baarlen et al. 2007, Phillips et al. 2013). The number of infections caused by filamentous fungi previously considered of low clinical relevance has increased in the past few years, especially among immunocompromised patients (Cassagne et al. 2011, Lu et al. 2013. Mycoses caused by hyaline, septate fungal hyphae fall under the medical term hyalohyphomycosis and some of the pathogens involved have been demonstrated to be resistant to certain antifungals (Tortorano et al. 2014). Therefore, a fast and accurate diagnosis is critical for patient management in order to determine appropriate treatment. The identification of microbial species is usually based on microscopy and biochemical methods that are time consuming and require high expertise. The DNA sequencing approach gives more reliable and faster results but still remains laborious. Recently, a technique known as Matrix-Assisted Laser Desorption Ionisation Time of Flight Mass Spectrometry (MALDI-TOF MS) has been revolutionising the clinical diagnostics field. This technique allows the identification of microorganisms by analysing their unique protein peak pattern and comparing it with a database of reference main mass spectra (MSPs). The peaks present in the MSP represent mostly ribosomal proteins but structural proteins, cold-shock proteins and others may also be detected. MALDI-TOF MS is a simple, fast and accurate procedure that has been validated in numerous laboratories for the identification of yeasts (Goyer et al. 2012, Kolecka et al. 2013) and bacteria (Seng et al. 2009) from clinical samples. Recently, an effort has been made towards the validation of standardised procedures for routine mould identification (Cassagne et al. 2011, Lau et al. 2013 and dermatophytes (L'Ollivier et al. 2013) from clinical samples and all reports showed that MALDI-TOF MS had a good discrimination power for species separation, effectively decreased the time of identification and improved its accuracy. Furthermore, MALDI-TOF MS has also been used in studies were it was used as a complementary tool for taxonomical discriminatory purposes: Degenkolb et al. (2008) used MALDI-TOF MS in a polyphasic approach to support the description of Trichoderma brevicompactum as a novel species and Brun et al. (2013) tested this technique to discriminate closely related species of Alternaria.
In this study we aimed to: i. resolve the R. eucalypti species-complex by applying a polyphasic approach involving morphology, multi-gene phylogeny and MALDI-TOF mass spectrometry; and ii. build an in-house library of MSPs of R. eucalypti s.lat. strains in order to evaluate the taxonomic resolution power of this technique for the identification of the set of clinical isolates within this species complex.

Fungal strains
The 44 isolates used in this study are maintained in the culture collection of the CBS-KNAW Fungal Biodiversity Centre (CBS), Utrecht, The Netherlands, and the working collection of Pedro Crous (CPC), housed at CBS (Table 1).

DNA extraction, amplification and sequencing
The fungal strains (

Phylogenetic analyses
Ramularia nyssicola (CBS 127665) has recently been revised and separated from R. endophylla (= Mycosphaerella punctiformis) (Minnis et al. 2011). Ramularia nyssicola is basal in the genus Ramularia (Videira, unpubl. data) and was therefore considered as an adequate outgroup for the R. eucalypti species complex. The generated sequences for each gene were aligned with MAFFT v. 6.864b (http://mafft.cbrc.jp/alignment/ server/index.html) according to the gene characteristics. The alignments were manually checked and improved where necessary using MEGA v. 5 (Tamura et al. 2011) and were concatenated with Mesquite v. 2.75 (Maddison & Maddison 2011). In order to check the stability of each species clade a neighbour-joining analysis using the HKY85 substitution model was applied to each gene partition individually using PAUP v. 4.0b10 (Swofford 2003) (data not shown). Alignment gaps were treated as missing data and all characters were unordered and of equal weight. When ties were encountered they were randomly broken. The robustness of the obtained trees was evaluated by 1 000 bootstrap replications (Hillis & Bull 1993).
Parsimony and Bayesian analyses were used to estimate phylogenetic relationships for the aligned combined dataset. Parsimony analyses were conducted with PAUP v. 4.0b10 (Swofford 2003). Alignment gaps were treated as fifth base and all characters were unordered and of equal weight. The robustness of the obtained trees was evaluated by 1 000 bootstrap replications (Hillis & Bull 1993).
MrModeltest v. 2.2 (Nylander 2004) was used to determine the best nucleotide substitution model settings for each data partition in order to perform a model-optimised Bayesian phylogenetic reconstruction using MrBayes v. 3.2.0 (Ronquist & Huelsenbeck 2003). The heating chain was set to 0.15 and the Markov Chain Monte Carlo (MCMC) analysis of four chains was started in parallel from a random tree topology and lasted until the average standard deviation of split frequencies reached a value of 0.01. Burn-in was set to 25 % after which the likelihood values were stationary. Trees were saved each 100 generations and the resulting phylogenetic tree was printed with Geneious v. 5.5.4 (Drummond et al. 2011). All new sequences generated in this study were deposited in NCBIs GenBank nucleotide database (www.ncbi.nlm.nih.gov) and the accession numbers of the sequences used for the phylogenetic analyses are detailed in Table 1. The alignment and phylogenetic tree were deposited in TreeBASE (www.TreeBASE.org).

Sample preparation
The cultures were prepared according to the method used in the protocol for the construction of the Filamentous fungi v. 1 Library (Bruker Daltonics, Germany) with a few modifications. Falcon tubes (15 mL) containing 7 mL of Saboraud dextrose broth (Difco, REF 238230) were inoculated with the isolates and incubated at 21 °C for 48-72 h on a tube rotator SB2 (Stuart). The tubes were centrifuged (1 min, 3 000 rpm) and 1.5 mL of the sediment was collected into 1.5 mL Eppendorf tubes. These were centrifuged (3 min, 14 000 rpm), the supernatant was removed and 1 mL of sterile Milli-Q water was added to the pellet followed by vortexing. This washing step was performed twice. The supernatant was removed and 1.2 mL of 70 % ethanol was added. The samples were stored up to 5 d at room temperature. The crude protein content was extracted using the Formic Acid/Ethanol sample preparation method (Bruker Daltonics, Germany) with a few modifications. The samples were centrifuged (3 min, 14 000 rpm), the supernatant was removed and the pellets were air-dried in a laminar flow cabinet for 30 min. The pellets were incubated for 10-20 min in 20-40 µL of 70 % formic acid (FA) (Sigma-Aldrich, Zwijndrecht, The Netherlands), followed by 10-20 min in 20-40 µL of 100 % acetonitrile (ACN) (Fluka) and were then centrifuged (2 min, 14 000 rpm). The supernatant, now containing the protein crude extract, was immediately used to generate mass spectra.

In-house library and identification
The in-house library of Ramularia comprises 22 reference MSPs, of which 21 were created from strains of R. eucalypti s.lat. and one from a strain of R. vizellae. The reference MSPs were generated with a MALDI Biotyper 3.0 Microflex LT (Bruker Daltonics, Germany) mass spectrometer. For each strain, 1 µL of protein crude extract was deposited on eight spots of a polished steel target plate (Bruker Daltonics, Germany), air-dried and covered with 1 µL of alpha-cyano-4-hydroxycinnamic acid (HCCA) matrix solution (Kolecka et al. 2013). Twenty-four spectra were acquired per isolate using FlexControl v. 2.4 (Bruker Daltonics, Germany). A minimum of 20 high quality spectra were selected with Flex analysis v. 3.3 (Bruker Daltonics, Germany) to create the respective reference MSP entry to be stored in the in-house library. Comparison of the MSPs was performed by Principal Component Analysis (PCA) (Shao et al. 2012) resulting on a distance score-oriented dendrogram (Fig. 1). The library was challenged with the identification of a set of four clinical isolates. The identification of each isolate was performed in duplicate using MALDI Biotyper 3.0 RTC application (Bruker Daltonics, Germany) with the standardised parameters recommended by the manufacturer for routine diagnostics in hospitals (Kolecka et al. 2013). In the automatic identification runs the clinical isolates were compared with reference MSPs selected simultaneously from the BDAL Bruker database (5627 MSPs), the Bruker Filamentous fungi v. 1 Library (365 MSPs) and the Ramularia in-house library (24 MSPs). Identification results were scored as log-values and, according to the manufacturer, classified as follows: secure genus and species identification (> 2.0), secure genus identification (1.7-2.0) and no reliable identification (< 1.7).

Taxonomy
The 33 isolates belonging to R. eucalypti s.lat. were in oculated on Synthetic Nutrient-poor Agar (SNA) (Crous et al. 2009c) and incubated at 21 °C for 7 d. Morphology of the strain CBS 118743 was also observed and described at 33 °C, because it showed morphological dimorphism at different temperatures. Observations of the conidiogenous structures were performed using a Nikon Eclipse 80i light microscope with differential interference contrast (DIC) illumination ( Fig. 4-9). Slides were prepared using the inclined coverslip method (Kawato & Shinobu 1959, Nugenta et al. 2006) and also with transparent adhesive tape (Titan Ultra Clear Tape, Conglom Inc., Toronto, Canada) (Bensch et al. 2012). Lactic acid (clear) was used as mounting medium for the measurements and Lactophenol cotton blue was used in some preparations to improve the contrast of the naturally hyaline structures. The terminology of morphological structures followed those used for description of Ramularia species by Crous et al. (2011). The recorded measurements represent the minimum value followed by the 95 % confidence interval of 30 individual measurements and the maximum value for both length and width. For colony macro-morphology the isolates were inoculated on Potato Dextrose Agar (PDA), Oatmeal Agar (OA) and Malt Extract Agar (MEA) (Crous et al. 2009c), and incubated in the dark at 25 °C. After 14 d, the colony diameter was measured and the colony colour was described according to the mycological colour charts of Rayner (1970  included in a growth study. The isolates were inoculated onto MEA plates in triplicate, and placed in a serial incubator, in the dark, at temperatures ranging from 6-36 °C, with 3 °C intervals, and also at 40 °C. Measurements of colony diameters were taken after 14 d (Fig. 2). Nomenclatural data was deposited in MycoBank (Crous et al. 2004a).

DNA amplification and phylogenetic analysis
New primers for the GAPDH, TEF-1α and RPB2 loci were designed based on a larger dataset of Ramularia and other cercosporoid genera (Videira, unpubl. data) that proved to be effective for species within the genus Ramularia. These primers were used when no amplification was obtained with the standard primers (Table 2).
In the phylogenetic analysis six of the 10 screened loci were used, namely ITS, RPB2, GAPDH, ACT, TEF-1α and HIS3. The LSU sequences obtained were nearly identical to one another and did not provide useful information to resolve the speciescomplex and were therefore not included in the subsequent phylogenetic analyses. The amplification of CAL and CHS-1 was not successful for all the isolates and the inclusion of missing data in the alignment would negatively influence the posterior probability and bootstrap support values. The amplification of bTUB often generated multiple PCR products and was only successful for a reduced number of isolates. Although these sequences were excluded from the phylogenetic analyses, they have been deposited in GenBank under accession numbers KJ504473 -KJ504495 (TUB), KJ504496 -KJ504529 (CAL), KJ504530-KJ504550 (CHS) and KJ504724-KJ504764 (LSU).
Neighbour-joining analysis using the HKY85 substitution model was applied to each data partition in order to check the stability and robustness of each species clade (data not shown). The ITS locus did not differentiate species well, supporting only R. eucalypti, R. miae, R. plurivora and Ramularia sp., while most of the isolates formed a basal polytomy. The tree based on the ACT gene had a better resolution by additionally segregating strains of R. glennii and R. haroldporteri. The HIS3 phylogeny resolved seven species but with very low bootstrap support values. The individual trees based on the RPB2, GAPDH and TEF1-α loci all supported seven species with high bootstrap support. These genes also suggested a split of R. glennii in two clades but with a low support value and with internal subclades that were not supported either by the geographical origin or by the morphological characteristics of the isolates.
The concatenated alignment contained 33 strains, including the outgroup sequence (R. nyssicola). A representative strain was selected from strains representing the same substrate and country and which shared identical sequences for all loci (Table 1). The final alignment contained a total of 2 651 characters divided in 6 partitions containing 665 (RPB2), 486 (ITS), 551 (GAPDH), 358 (HIS3), 177 (ACT), 389 (TEF-1α) characters, respectively. From the total alignment, 40 characters were excluded from the phylogenetic analysis: 25 characters were artificially introduced as spacers to separate the genes and 15 characters in the GAPDH locus (alignment positions 1216 -1230, see TreeBASE) represented a longer sequence in the outgroup compared to the ingroup sequences.
The results of the MrModelTest analyses indicated that the ITS partition had fixed (equal) base frequencies, whereas all the other partitions had dirichlet base frequencies. The optimised models for this dataset were K80+I+G for ITS and GTR+I+G for all the other partitions.
The Bayesian analysis generated 1 702 trees from which 424 trees were discarded (25 % burnin). The 50 % majority rule consensus tree ( The parsimony analysis generated the maximum limit of 1 000 equally most parsimonious trees and the bootstrap support values (right numbers) higher than 75 % are displayed (Fig. 3). The gaps in the alignment were treated as fifth base and from the analysed characters 1 670 were constant, 234 were variable and parsimony-uninformative and 707 were parsimonyinformative. A parsimony consensus tree was calculated from the equally most parsimonious trees and the branches present in the strict consensus tree are mapped with a thicker stroke on the Bayesian tree (Fig. 3).
Phylogenetic trees based on the combined dataset and generated with both parsimony and Bayesian analyses (Fig. 3) separated strains into seven well-supported species within this complex: R. eucalypti, R. glennii, R. haroldporteri, R. mali, R. miae, R. plurivora and Ramularia sp. Ramularia eucalypti is no longer the only species of the genus to be found on Eucalyptus with the addition of the newly described R. glennii. The clinical isolates do not cluster in the same clade as R. eucalypti, and are here described as R. glennii and R. plurivora. The species causing the apple and pear fruit damage in storage is a new species as well (R. mali) considering both the branch length and the posterior probability value separating it from the closest species (R. glennii). The clades of R. eucalypti, R. glennii and R. plurivora show some interspecific variability within the evaluated genes, but not strong enough to support further division into additional species.

MALDI-TOF MS
A total of 22 strains from Ramularia were used to create the Ramularia in-house library. Twenty-one strains belonged to R. eucalypti s.lat. and one strain of R. vizellae was used as a reference species outside the complex while still within the same genus. It was not always possible to obtain good quality MSPs for all strains (e.g. R. haroldporteri and Ramularia sp.) as the crude protein extraction performed with the current protocol was problematic for a few strains. For the PCA dendrogram 26 MSPs were used in total, including the 22 MSPs from the Ramularia in-house library and four Cladosporium strains from the Bruker Filamentous fungi v. 1 Library that were used as an outgroup (Fig. 1). The distance level presented on the dendrogram is a relative measure of the differences among the MSP peak patterns and three clades can be observed: R. plurivora, R. glennii /R. mali and R. eucalypti /R. miae. The PCA dendogram topology shows a broadly similar topology to the DNA phylogeny but it is unable to separate the species R. glennii from R. mali and R. eucalypti from R. miae, which are closely related. The MALDI-TOF MS identification results of the four clinical isolates confirmed their identity as R. plurivora and R. glenni, respectively, as secure genus and species identification was attained with log-score > 2.0. The identification results showed that the top ten identification hits per tested spot per isolate were matching only with MSPs of Ramularia species.

Ramularia haroldporteri
Culture characteristics -On MEA surface convex, strongly folded, smoke-grey, with undulate and concave margin, flat aerial mycelium, reverse greyish sepia, reaches 18 mm after 2 wk at 25 °C. On OA folded with undulate margins, smokegrey, flat aerial mycelium, reaches 15 mm after 2 wk at 25 °C. On PDA surface folded with undulate margins, smoke-grey, flat aerial mycelium, reverse olivaceous-grey, reaches 15 mm after 2 wk at 25 °C. Notes -Ramularia haroldporteri differs from R. miae by producing significantly shorter ramoconidia, intercalary and terminal conidia and by not producing exudate droplets on top of the mycelium. In the individual gene phylogenetic trees, all genes except the ITS separates R. haroldporteri from R. miae.   Videira & Crous,Fig. 7 Etymology. Named after its occurrence on apple (Malus).

Ramularia sp.
Culture characteristics -On MEA surface convex, folded, white with very few and small droplets of pale luteous exudates, margin undulate and feathery, reverse umber with ochreous margin, reaching 18 mm after 2 wk at 25 °C. On OA surface convex, white and feathery, margin undulate and without aerial mycelium, 2 mm hazel ring around the colony, reaching 20 mm after 2 wk at 25 °C. On PDA surface convex, white, margin slightly undulate and feathery, reverse dark mouse grey with pale luteus margin, reaching 18 mm after 2 wk at 25 °C; culture sterile. Specimen examined. swedeN, Uppland, Knovsta, isolated from Epilobium hirsutum L., 22 Sept. 1989, E. Gunnerbeck, CBS 114568. Notes -This strain was previously identified as R. epilobiana. The type specimen of R. epilobiana was described from Epilobium hirsutum in France, and no ex-type culture is available. The culture CBS 114568 was sterile and we were unable to compare its morphology with that of the type description. However, it is very doubtful that it represents the true R. epilobiana since all species of the R. eucalypti complex have catenate, narrow conidia, and R. epilobiana is characterised by having broadly ellipsoid-ovoid conidia that are formed singly. The DNA sequences obtained from this strain differ significantly from the sequences of the closest strain CBS 129581. Therefore, we rename it as 'Ramularia sp.' and retain it as a potential new species pending the collection of fresh material from the same host and country.

dISCuSSIon
Eucalyptus is one of the most important commercially afforested genera cultivated to meet the increasing global demand for wood and paper pulp. Over the years, more than 50 species of the family Mycosphaerellaceae have been described causing diseases on Eucalyptus trees (Quaedvlieg et al. 2014). However, since the introduction of molecular techniques, many wellestablished plant pathogens have been revealed to represent species complexes , Damm et al. 2012a, b, Weir et al. 2012. The pathogen R. eucalypti has certainly proved to be no exception.
Using a polyphasic approach involving morphology, multi-gene phylogeny and MALDI-TOF MS, a total of seven species were accepted within the complex: R. eucalypti, R. glennii, R. haroldporteri, R. mali, R. miae, R. plurivora and one undescribed Ramularia species. Species discrimination was mostly based on the multigene phylogeny since it clearly separated them into stable and strongly supported monophyletic clades while the morphological features and the MALDI-TOF MS PCA dendrogram did not consistently discriminate all species.
Within the clades of R. eucalypti and R. glennii, some phylogenetic structure was observed that was not resolved consistently in all gene trees (data not shown) and, in accordance with the Genealogical Concordance Phylogenetic Species Recognition (GCPSR) concept, the transition from concordance to conflict determined the limit of these species (Taylor et al. 2000). The isolates within these clades have been collected worldwide and the phylogenetic structure observed suggests that the isolates studied may represent populations in the process of divergence. It has been shown that Mycosphaerella populations can be carried within asymptomatic Eucalyptus trees transported and planted across the world and, given time, they have genetically diverged sometimes to the point of being recognised as distinct species .
The ITS barcode was not sufficient to achieve species level identification, just like previously reported among other cercosporoid genera, e.g. Cercospora  and Pseudocercospora (Crous et al. 2013). The need to use secondary barcodes to achieve species identification has been highlighted in several studies in recent years (e.g. Fitzpatrick et al. 2006, Aguileta et al. 2008. Secondary barcodes are usually protein-coding genes since their intron sequences introduce more variability that is valuable for species discrimination. From the five protein-coding genes that were used in this study, any of the partial genes TEF1-α, RPB2 or GAPDH could be used as a secondary barcode since they all delineate the seven recognised species. However, further studies are necessary to determine which of these loci would be more adequate to discriminate species within the genus Ramularia. Cultural morphological traits have been used in the past for species discrimination within species complexes in other genera, e.g. Cercospora apii s.lat. . The species in this study (Fig. 2), however, showed few morphological or cultural features that could be consistently and reliably used to identify them. All the strains used in this study were cultures that were deposited in the CBS or CPC fungal collections and no fresh material was collected. Therefore, any features that may exclusively develop in association with the original host or substrate have not been examined.
Although the genus Ramularia is currently accepted as a hostspecific genus this assumption has not been tested experimentally. In the present study, R. haroldporteri and R. mali have been isolated from a single host while R. eucalypti, R. glennii, R. miae and R. plurivora were isolated from multiple hosts, suggesting that both host-specific and plurivorous species may occur in this genus, even within the same species complex. Some species of the Mycosphaerellaceae are known to have the ability of colonising different hosts in order to disperse further in an attempt to find the host to which they are truly pathogenic . This ability makes it more difficult to determine whether they act as true pathogens, are opportunistic and take advantage of an already debilitated host, or if they are simply saprophytes.
The pathogen responsible for causing lenticel rot in fruits of apple (Malus malus cv. Ambrosia) and pear (Pyrus communis cv. Conference) in the Piemont Province in Italy (Gianetti et al. 2012, Giordani et al. 2012) is here newly described as R. mali. The apple tree orchards in Piemont are an important crop that in 2011 produced 140 000 t of fruit. Healthy apple fruits (Malus domestica cv. Ambrosia) collected from trees with leaf spots caused by R. mali in the orchards, exhibited disease symptoms during the subsequent months of cold storage (Gianetti et al. 2012). Artificial inoculations of healthy apple (Malus domestica cv. Ambrosia) with R. mali also caused the development of symptoms indicating that this is a true pathogen (Giordani et al. 2012). It is thought that the fungus was already present in the country and that the gradual abandonment of the use of broad-spectrum fungicides in the fruit sector allowed the emergence of this pathogen that had passed unnoticed until now. In 2013, in the Trentino Alto-Adige province in Italy, apples from a different cultivar (Malus domestica cv. Golden Delicious), also developed the lenticel rot in cold storage and the disease affected 50-60 % of the crop. In the same year and province, Malus domestica cv. Braeburn and Malus domestica cv. Rosy Glow were also affected. Molecular analysis of these isolates were identical to those of R. eucalypti (Crous et al. 2007) (100 % ITS, 99 -100 % LSU) deposited on GenBank. However, artificial inoculation of these isolates on ripe fruits of Malus domestica cv. Golden Delicious did not result in de development of disease symptoms (Lindner 2013). No isolates from this province were available in the present study. Since the ITS barcode is not sufficient for species identification, the mentioned pathogen can be R. eucalypti, R. mali, or a different species. If it is R. mali, it may be a mere opportunist on Malus domestica cv. Golden Delicious and only truly pathogenic to the Ambrosia cultivar. Information on the biology and behaviour of R. mali is still lacking and no preventive measures to control this fungus from spreading have been taken.
The newly described species R. glennii and R. plurivora include strains that were obtained not only from plants but also from human clinical specimens. This is the first time species of the genus Ramularia are reported in association with a human host and little is known about their pathogenicity. Some pathogens are able to infect hosts from different kingdoms (van Baarlen et al. 2007) and other plant pathogens have been reported capable of infecting humans (Mostert et al. 2006, Phillips et al. 2013). The fact that only a limited number of isolates was obtained and no previous report is known about Ramularia species infecting patients support the hypothesis that this is an opportunistic fungus. However, if potential host species are immunocompromised, opportunistic pathogens may turn into aggressive pathogens (van Baarlen et al. 2007). Furthermore, R. plurivora (strain CBS 118743) displayed morphological dimorphism (Fig. 9) and was able to grow at 40 °C (Fig. 2). These characteristics are similar to, for example, Talaromyces marneffei (syn. Penicillium marneffei, Eurotiomycetes) (Vanittanakom et al. 2006, Houbraken & Samson 2011, a human pathogen known to cause lethal systemic infections in immunocompromised patients. Therefore, further studies are needed to appraise the pathogenicity of R. plurivora in order to determine if measures for its rapid identification, containment and treatment should be taken.
MALDI-TOF MS has become a powerful tool in the clinical microbiology workflow for the identification of bacteria and yeasts (Bader 2013, Lau et al. 2013. The use of MALDI-TOF MS for routine filamentous fungal identification from clinical samples has only recently been standardised and validated for several species (Cassagne et al. 2011, Lau et al. 2013, L'Ollivier et al. 2013. Filamentous fungi present some challenges when compared to yeast and bacteria. They have thicker cell walls that make the protein extraction more difficult, the presence of cell wall pigments inhibit the ionisation process and sporebased protein extractions result in a low variability of mass spectra peaks (Bader 2013). The use of Sabouraud broth as culture media has been shown to inhibit pigmentation and spore production in most species thus improving the quality of the spectra. Furthermore, filamentous fungi have complex phylogenetic relationships that make their species boundaries more difficult to define. The need to use secondary barcodes to resolve species complexes also challenges the MALDI-TOF MS to perform identifications almost at the level of intraspecies subtyping (Degenkolb et al. 2008, Cassagne et al. 2011, Welker & Moore 2011, Bader 2013, Brun et al. 2013. The species in this complex are very closely related and the PCA dendrogram topology (Fig. 1) individualised only three clades containing R. plurivora, R. glennii /R. mali and R. eucalypti / R. miae. The dendrogram represents the relative similarity of the peak patterns and is based on a scoring algorithm that is influenced not only by the available number of MSPs that are representative of each species, but also by the intensity of the peaks. The first parameter can be improved by creating more MSPs from different strains of the same species. However, the second parameter can only be improved by preparing all the samples on the same day, using the same amount of protein and using the same settings on the machine, which is virtually impossible when building a large database. Furthermore, the protocol for the crude protein extraction recommended by the manufacturer still needs to be optimised, since it did not work for all strains in this study.
Nevertheless, the use of MALDI-TOF MS as an identification tool has still proven to be reliable not only in previous studies but also in this one. When the in-house Ramularia library was challenged with the identification of the clinical isolates of R. glenni and R. plurivora, a secure genus and species identification log-score (> 2.0) was attained.
In conclusion, the R. eucalypti species complex has been resolved with the circumscription of the R. eucalypti s.str. and the description of four new species. Ramularia eucalypti and R. glennii are the only species of this genus described so far from the economically important Eucalyptus hosts. Ramularia mali is an important pathogen of apple cv. Ambrosia and may become a serious pathogen on other apple cultivars. For the first time, two Ramularia species, R. glennii and R. plurivora have been reported from clinical specimens and R. plurivora has the potential of becoming an important human pathogen. The identification of the clinical isolates with MALDI-TOF MS was successful and their MSPs should be added to the commercially available Bruker database of MSPs (BDAL). This would promote a fast and accurate identification of these species in clinical laboratories and would contribute to further investigate the epidemiological relationship with the human host.