Journal of Medical Microbiology Papers in Press. Published May 22, 2015 as doi:10.1099/jmm.0.000091
Journal of Medical Microbiology Differentiation of Clinically Relevant mucorales Rhizopus microsporus and R. arrhizus by Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry (MALDI-TOF MS) --Manuscript Draft-Manuscript Number:
JMM-D-15-00142R1
Full Title:
Differentiation of Clinically Relevant mucorales Rhizopus microsporus and R. arrhizus by Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry (MALDI-TOF MS)
Short Title:
MALDI-TOF MS of Rhizopus spp.
Article Type:
Standard
Section/Category:
Diagnostics, typing and identification
Corresponding Author:
Somayeh Dolatabadi Centraalbureau voor Schimmelcultures NETHERLANDS
First Author:
Somayeh Dolatabadi
Order of Authors:
Somayeh Dolatabadi Anna Kolecka Matthijs Versteeg Sybren G de Hoog Teun Boekhout
Abstract:
This study addresses the usefulness of Matrix-Assisted Laser Desorption IonizationTime of Flight Mass Spectrometry (MALDI-TOF MS) for reliable identification of the two most frequently occuring clinical species of Rhizopus, namely R. arrhizus with its two varieties arrhizus and delemar and R. microsporus. The test-set comprised 38 isolates of clinical and environmental origin previously identified by ITS sequencing of rDNA. Multi-locus sequence data targeting three gene markers (ITS, ACT, TEF) showed two monophylic clades for R. arrhizus and R. microsporus (bootstrap values of 99%). Cluster analysis confirmed the presence of two distinct clades within R. arrhizus representing its varieties arrhizus and delemar. The MALDI Biotyper 3.0 Microflex LT platform (Bruker Daltonics) was used to confirm the distinction between R. arrhizus and R. microsporus and the presence of two varieties within the R. arrhizus. An in-house database of thirty reference main spectra (MSPs) was initially tested for correctness using commercially available databases of Bruker Daltonics. By challenging the database with the same strains of which an inhouse database was created, automatic identification runs confirmed that MALDI-TOF MS is able to recognize the strains at the variety level. Based on the Principal Component Analysis (PCA), two MSP dendrograms were created and showed concordant with the multi-locus tree thus MALDI-TOF MS is a useful tool for diagnostics of mucoralean species.
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Journal of Medical Microbiology 01-05-2015
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Differentiation of Clinically Relevant mucorales Rhizopus microsporus and R. arrhizus by
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Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry (MALDI-
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TOF MS)
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Running title: MALDI-TOF MS of Rhizopus spp.
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Somayeh Dolatabadi1,2, Anna Kolecka1, Matthijs Versteeg1, Sybren G de Hoog1,2,3,4,5,6,7, Teun
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Boekhout1, 5
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CBS-KNAW Fungal Biodiversity Centre, Utrecht, The Netherlands,2Institute for Biodiversity
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and Ecosystem Dynamics, University of Amsterdam, Amsterdam, The Netherlands,3Peking
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University Health Science Center, Research Center for Medical Mycology, Beijing, China, 4Sun
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Yat-sen Hospital, Sun Yat-sen University, Guangzhou, China, 5Changzheng Hospital, Second
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Military Medical University, Shanghai, China, 6Basic Pathology Department, Federal University
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of Paraná State, Curitiba, Paraná, Brazil, 7King Abdulaziz University, Jeddah, Saudi Arabia
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Corresponding author: Teun Boekhout, CBS-KNAW Fungal Biodiversity Centre, Utrecht, The
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Netherlands, tel: +31302122600, fax: +31 3025122097, e-mail:
[email protected]
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1
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Abstract
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This study addresses the usefulness of Matrix-Assisted Laser Desorption Ionization–Time of
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Flight Mass Spectrometry (MALDI-TOF MS) for reliable identification of the two most
28
frequently occuring clinical species of Rhizopus, namely R. arrhizus with its two varieties
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arrhizus and delemar and R. microsporus. The test-set comprised 38 isolates of clinical and
30
environmental origin previously identified by ITS sequencing of rDNA. Multi-locus sequence
31
data targeting three gene markers (ITS, ACT, TEF) showed two monophylic clades for R.
32
arrhizus and R. microsporus (bootstrap values of 99%). Cluster analysis confirmed the presence
33
of two distinct clades within R. arrhizus representing its varieties arrhizus and delemar.
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The MALDI Biotyper 3.0 Microflex LT platform (Bruker Daltonics) was used to confirm the
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distinction between R. arrhizus and R. microsporus and the presence of two varieties within the
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R. arrhizus. An in-house database of thirty reference main spectra (MSPs) was initially tested for
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correctness using commercially available databases of Bruker Daltonics. By challenging the
38
database with the same strains of which an in-house database was created, automatic
39
identification runs confirmed that MALDI-TOF MS is able to recognize the strains at the variety
40
level. Based on the Principal Component Analysis (PCA), two MSP dendrograms were created
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and showed concordant with the multi-locus tree thus MALDI-TOF MS is a useful tool for
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diagnostics of mucoralean species.
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Keywords: MALDI-TOF MS; Rhizopus microsporus; Rhizopus arrhizus; Identification
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Introduction
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Rhizopus is a genus of Mucorales, family Rhizopodaceae which are commonly present in indoor
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environments where they occupy habitats in early stages of organic matter decay (Dolatabadi,
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2015). Several species are spoilage agents of fresh and manufactured food (Hesseltine, 1983;
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Jennessen et al., 2005), but they are also commonly applied in food fermentation of oriental
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foods, such as tempe, sufu, koji, and ragi (Hesseltine, 1983; Jennessen et al., 2005; Nout &
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Rombouts, 1990). Rhizopus species have also numerous biotechnological applications in the
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production of enzymes (e.g. lipases, amylases) and secondary metabolites (e.g. fumarate, malate
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and lactic acid) (Gosh & Ray, 2011; Abe et al., 2007). On the other hand they are spoilage
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organisms of fruits and vegetables.
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Rhizopus species comprised etiologic agents of mucormycosis, an acute fungal infection with
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high morbidity and mortality in patients with severe underlying metabolic or immune disorders,
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such as stem cell / solid organ transplants, diabetic mellitus, ketoacidosis, increased serum iron,
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neutropenia, hematologic malignancies, or birth prematurity (Skiada et al., 2011; Lanternier et
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al., 2012; Roilides et al., 2012; Chowdhary et al., 2014). The disease is angioinvasive, leading to
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thrombosis and extended necrosis, and is rapidly progressive with high rates of mortality
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(>50%), that approaching 100% among patients with disseminated disease or those with
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persistent neutropenia. Clinical manifestation of the disease occurs as rhinocerebral, pulmonary,
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gastrointestinal, renal or disseminated infection (Ibrahim et al., 2012). The number of cases is
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increasing especially in patients with impaired immunity due to e.g. organ transplantation.
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Rhizopus arrhizus (previously known as R. oryzae) is the prime species responsible for 70% of
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all cases of mucormycosis and 90% of rhinocerebral cases, while R. microsporus is the third
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most frequent opportunist in the Mucorales with 15%–25% of cases worldwide (Roden et al.,
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2005). Thus, the genus Rhizopus is the most important clinical entity involved in mucoralean
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infections (Skiada et al., 2011; Lanternier et al., 2012; Roilides et al., 2012; Walsh et al., 2012;
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Rammaert et al., 2012). Thus far, diagnostic methods have focused mostly on Lichtheimia and
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partly Mucor spp. which are the second etiological agents of mucormycosis in Europe and the
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U.S.A. (Skiada et al., 2011; Schrödl et al., 2011) and studies related to Rhizopus remained
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neglected.
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Treatment of mucormycosis is based on antifungal therapy combined with surgery, using
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amphotericin B as a prime drug. The choice of antifungal agents used, differs widely between 3
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various mucoralean fungi at species or even variety level; e. g amphotricin B shows different
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activities against R. microsporus and R. arrhizus thus confirming different susceptibility patterns
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between species or even varieties of mucoralean fungi (Chowdhary et al., 2014; Caramalho et
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al., 2015).
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Currently, reliable identification of Mucorales species remains challenging for many laboratories
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as it relies on microscopy and molecular methods. Reliable identification of clinical isolates at
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the species level is challenging and time consuming in many routine laboratories which makes
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the choice of optimal treatment difficult (Ibrahim et al., 2008; Lamaris et al., 2009; Lass-Flörl,
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2009). Culturing of mucoralean species from patient material may also be difficult because of
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lack of sporulation in tissue and coenocytic structure of the mycelium. Histopathological
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observation remains inconclusive for species or even generic identification, which is essential for
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optimal antifungal therapy selection (Griffin & Hanson, 2014). DNA-based methods, such as
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ITS sequencing are time-consuming and require several preparatory steps (e.g. DNA extraction,
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gene PCR amplification, and data analysis). Some agents of mucormycosis have a rather high
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intraspecific molecular variability, as the internal transcribed spacer (ITS) of the ribosomal DNA
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(rDNA) may vary up to 3.24%, which is in contrast to 1.96% as a maximum molecular
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variability in ascomycetous species (Nilsson et al., 2008) and this is also reflected in their
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diverse antifungal susceptibility even at variety level (e.g posaconazole MIC range) (Chowdhary
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et al., 2014). Machouart et al. (2001) introduced restricted fragment length polymorphism
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(RFLP) as a rapid method for the separation of the main species of Mucorales, but this technique
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requires availability of a set of specific primers. No serological indicator for mucormycosis is
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available at this moment (Griffin & Hanson, 2014). Considering the difficulties in early
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diagnostics, time needed for identification, the acute invasive infection type, and a steady
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increase in the number of cases, the availability of reproducible and rapid species recognition are
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essential to decrease mortality rates due to this infection.
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Here we present the application of Matrix-Assisted Laser Desorption Ionization-Time of Flight
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Mass Spectrometry (MALDI-TOF MS) for the identification of the most important clinical
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species of Rhizopus, viz. R. microsporus and R. arrhizus, with its two varieties var. arrhizus, var.
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delemar (Dolatabadi et al., 2014a). The data generated by multi-locus sequence typing (MLST)
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was compared with MALDI-TOF MS for correct species differentiation. The accuracy of
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MALDI-TOF MS results was compared with four other clinically relevant genera of Mucorales,
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including the type species of Rhizopus, R. stolonifer.
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Materials and methods
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Strains and cultivation
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Thirty nine reference and type strains from Fungal Biodiversity Centre (CBS-KNAW), Utrecht,
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the Netherlands were used (Table 1). These included 26 strains of R. arrhizus with 13 strains of
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var. arrhizus and 13 strains of var. delemar, and 13 strains of R. microsporus. For preservation,
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serial transfer and DNA isolation, strains were cultured on 5% malt extract agar (MEA, Oxoid,
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Basingstoke, U.K.) in 8 cm ø culture plates and incubated for 72 hours at 30 °C.
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Multi-locus sequencing
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DNA extraction and PCR conditions were followed according to methods described previously
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(Dolatabadi et al., 2014 b). Markers included the rDNA internal transcribed spacer (ITS) region,
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parts of the actin (ACT), and translation elongation factor 1-α (TEF) genes. Consensus sequences
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were constructed by means of SeqMan v. 9.0.4 (DNASTAR, WI, U.S.A.). Sequences of each
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marker were aligned in MEGA5 of the Lasergene software package (DNASTAR) using Clustal
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W. Concatenated sequences were imported to MEGA5 for the construction of a final MLST tree
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using Maximum Likelihood (ML). The optimal substitution model (T92 + G + I, Tamura 3) was
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selected by MEGA5. Robustness of tree topology was estimated by bootstrapping with 1000
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replicates. The final tree was drawn in Adobe Illustrator Artwork 15.1.
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MALDI-TOF MS
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Sample preparation
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A protocol of standardized liquid cultivation with constant rotation, developed for cultivation of
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filamentous fungi and used for the construction of the Filamentous Fungi v. 1 Library by Bruker
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Daltonics (Bremen, Germany) was followed. Falcon tubes (15 mL) containing 7 mL of
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Sabouraud dextrose broth (Difco, REF 238230) were inoculated and incubated on a tube rotator
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SB2 (Stuart) (20 r.p.m.) at room temperature for 24–72h followed by centrifugation (1 min, 3000
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r.p.m.). 1.5 mL of the fungal biomass sediment was transferred to Eppendorf tubes and 1 mL of
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sterile Milli-Q water was added to the pellet followed by vortexing. A washing step was 5
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performed three times. The final supernatant was removed and 1.2 mL of 70% ethanol was
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added.
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A crude protein extraction protocol using the Formic Acid / Ethanol sample preparation method
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(Bruker Daltonics, Germany) was followed (Marklein et al., 2009; Kolecka et al., 2013). The
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samples were centrifuged (3 min, 14000 r.p.m.), the supernatant was removed and the pellets
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were air-dried in a laminar flow cabinet for 30 min. The pellets were firstly dissolved in 25–40
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µL of 70% formic acid (FA) (Sigma-Aldrich, Zwijndrecht, Netherlands), depending on the pellet
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size, and after vortexing an equal volume of 100% acetonitrile (ACN) (Fluka) was added and
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mixed. Samples were then centrifuged (14000 r.p.m., 2 min) and supernatants were immediately
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used to generate reference mass spectra.
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MALDI-TOF MS in-house library and identification
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Mass spectra were generated with a MALDI Biotyper 3.0 Microflex LT platform (Bruker
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Daltonics, Germany). From 38 strains with 25 strains representing R. arrhizus and 13 R.
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microsporus an in-house database was created. For each strain, 1 μL of crude protein extract was
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deposited on eight spots of a 96-spot polished steel target plate (Bruker Daltonics), air-dried, and
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covered with 1 μl of HCCA matrix solution (Bruker Daltonics) (Kolecka et al., 2013). As a
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positive control and calibration reference, 1 μL of Bacterial Test Standard (BTS, Bruker
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Daltonics) was used. The Main Spectrum (MSP) was acquired from 24 individual spectra
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generated per isolate using the MALDI Biotyper automated Flex Control software v.3.0 (Bruker
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Daltonics). Minimum of 20 individual, high-quality spectra were selected with Flex Analysis
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software v. 3.3 (Bruker Daltonics) to create the respective MSP to be stored as an in-house
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database entry. Comparison of the MSPs was performed by Principal Component Analysis
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(PCA) resulting in a distance score oriented dendrogram (Fig. 1). The identification of the
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isolates, each represented by an MSP, was tested in silico using MALDI Biotyper 3.0 RTC
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software (Bruker Daltonics) with the two Bruker Daltonics commercial databases
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simultaneously, namely BDAL database (4110 MSPs) and Filamentous Fungi v. 1 Library and
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the in-house Rhizopus library.
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Results 6
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Multi-locus sequencing
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The set of 39 strains studied included the type strains of R. microsporus and the two varieties of
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R. arrhizus and their synonyms from clinical, environmental, and food-associated sources
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obtained from diverse geographical origins. Sequences were generated targeting three gene
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markers (ITS, ACT, TEF) and concatenated sequences were used to generate the tree. A
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Maximum Likelihood (ML) phylogenetic tree confirmed the correct concepts of the Rhizopus
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species and their separation in accordance with literature data (Fig. 1). The multi-locus tree
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showed separation òf two main clades, supported by bootstrap values of 99% for R. microsporus
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and R. arrhizus. In R. arrhizus separation of two varieties was observed but this was only
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supported by bootstrap values of 47% due to high sequence similarity between the varieties.
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Phylogenetic analysis proved monophyly for each species, which was concordant with data
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shown previously (Dolatabadi et al., 2014a, b) and was considered as proof of correct
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identification of strains to be used in the MALDI-TOF MS experiment. Sequences generated for
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the analysis are deposited in GenBank (Dolatabadi et al., 2014a, b).
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MALDI-TOF MS
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The MSPs were automatically classified and identified using MALDI Biotyper 3.0 software. The
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CBS in-house Rhizopus library was created with 38 MSPs out of 39 Rhizopus strains (Table 1).
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Strain CBS 264.28 was excluded from the further MALDI-TOF MS analyses as after two
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unsuccessful attempts, this isolate did not show sufficient growth in liquid media although robust
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growth on solid media was confirmed (Fig. 1).
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The mass spectra of the relevant type strains (i.e. CBS 699.68, CBS 120.12, and CBS 112.07 in
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Table 1) were unique for the two species and the two varieties enabling reliable separation (data
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not shown). Identification results with the tested set confirmed correct identity for all MSPs at
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species level and no false positive results were observed. Discrimination of Rhizopus isolates at
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species and variety levels was later confirmed as shown in the MSP dendrogram generated using
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the PCA application of MALDI Biotyper 3.0 software (Fig. 1).
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The results of the above set of 38 strains were supplemented with MSPs from the Filamentous
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Fungi v. 1 Library including the four mucoralean genera Rhizopus, Mucor, Rhizomucor and
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Lichtheimia to show unambiguous separation of all species (Fig. 2). Thirty five MSPs from these
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four clinically relevant genera were selected from the commercial Bruker Filamentous Fungi v. 1 7
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Library, viz. Rhizopus (16 MSPs), Mucor (6 MSPs), Rhizomucor (2 MSPs), and Lichtheimia (11
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MSPs) (Fig. 2). The topology of the extended PCA dendogram showed a clustering similar to the
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MLST phylogeny and supported the separation of the varieties arrhizus and delemar of R.
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arrhizus.
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Discussion
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MALDI-TOF MS has primarily been used for identification of bacteria and yeasts, and is
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increasingly applied in identification of filamentous fungi such as Fusarium, Aspergillus,
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Penicillium and Ramularia (Alanio et al., 2011; Bader et al., 2010; Hettick et al., 2008; De
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Carolis et al., 2012; Videira et al., 2015). In the basal lineages of the fungal kingdom, a
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comprehensive report on MALDI-TOF MS identification of Lichtheimia species has appeared
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(Schrödl et al., 2011). Other studies investigated more diverse sets of strains, but either the
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number of mucoralean fungi was limited or not the focus of the study and lacked comparison
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with the phylogeny of the fungi (De Carolis et al., 2012; Ranque et al., 2014; Becker et al.,
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2014).
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Rhizopus microsporus and R. arrhizus (including both varieties) do not show significant
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differences in their physiological profiles of growth temperature, nitrogen and carbon
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assimilation, and enzyme production (Dolatabadi et al., 2014 a, b). Two varieties of R. arrhizus
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however differ in the production of fumaric-malic (var. delemar) or lactic acid (var. arrhizus)
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(Abe et al., 2007). Size, shape, and ornamentation of spores that have been used in the past for
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species and variety distinction, remained inadequate for differentiation of taxa (Dolatabadi et al.,
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2014 a, b).
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Virulence of Rhizopus strains was shown to be independent from the source of isolation of the
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respective strains (Kaerger et al., 2015). Within Mucorales and between Rhizopus species,
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significant differences in antifungal susceptibility were observed (Vitale et al., 2012). Therefore,
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identification of clinical isolates at species level is helpful to decide on appropriate antifungal
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therapy based on known susceptibility profiles of the species. R. arrhizus has a high MIC range
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for AmB followed by R. microsporus spp. among other members of Mucorales which can be due
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to the ignorance of two varieties in past (Caramalho et al., 2015).
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Within both Rhizopus species studied, several varieties have been described in older literature,
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but recent molecular studies considered R. microsporus to be monophyletic and within R. 8
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arrhizus two varieties were maintained (Dolatabadi et al., 2014a, b). Three type strains of former
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varieties, i.e. varieties microsporus, azygosporus, and chinensis were included in the set of
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analyzed strains and were found randomly distributed within the R. microsporus cluster in the
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MALDI-TOF MS dendrogram and the multi-locus tree, thus confirming that they belong to one
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species (Fig. 1). Two strains of R. microsporus, CBS 700.68 and CBS 699.68, both isolated from
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soil, occurred at an external position in the multi-locus analysis (Dolatabadi et al., 2014b) and
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were also among the deviating strains in the MALDI-TOF MS dendrogram (Fig. 1). The two
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varieties of Rhizopus arrhizus can be identified by ITS sequence data (Dolatabadi et al., 2014a),
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having a maximum of 5 bp difference, and phenotypically they can be distinguished by either
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fumaric-malic acid or lactic acid production (Abe et al., 2007). Single-locus trees based on ITS,
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TEF and ACT were concordant (Dolatabadi et al., 2014b). The differences between the varieties
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were also observed with AFLP profiling (Dolatabadi et al., 2014b; Chowdhary et al., 2014) and
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with MALDI-TOF MS these two groups could be discerned and the result was concordant with
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the MLST tree (Fig. 1). These results show that MALDI-TOF MS may contribute to the
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clarifying of the taxonomy of these filamentous fungi.
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Considering the increasing number of fungal infections, MALDI-TOF MS provides
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fastidentification in the routine clinical laboratory. With most molecular methods interspecies
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distances in Mucorales tends to be quite high (3. 24%) compared to the observed intraspecific
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variation, leading to a considerable barcoding gap despite high intraspecific variability, which
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enhances reliable species differentiation (Walther et al., 2013). This is also expressed in the
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results obtained by MALDI-TOF MS and MLST analysis where overlap between species was
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neglectable.
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Compare to the molecular methods of identification which needs culturing, DNA extraction,
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PCR and sequencing; MALDI-TOF MS has a shorter period of extraction and PCR and
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sequencing steps are omitted. So the process can be easily shortened for two days.
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MALDI-TOF MS has been implemented as a rapid, simple, and cost-effective high-throughput
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proteomic technique that yielded correct identification at the species and variety levels for 38
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strains of Rhizopus. Addition of further clinical and environmental strains from four other genera
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from the Flamentous Fungi v. 1 Library showed that the identification potential of MALDI-TOF
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MS in Mucorales covers a wide range of infective mucoralean species. However, the availability
9
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of a reliable database based on a well-resolved taxonomy using molecular data is needed to allow
264
correct identification of isolates, as was considered exemplarily in our study.
265 266 267 268
Acknowledgments
269
This publication was made possible by NPRP grant 5-298-3-086 from the Qatar National
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Research Fund (a member of the Qatar Foundation) to Teun Boekhout. The statements herein are
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solely the responsibility of the authors. We want to thank Markus Kostrzewa from Bruker
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Daltonics GmbH, Breimen, Germany for his collaboration on MALDI-TOF MS.
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Transparency declaration
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The authors declare that they have no conflicts of interest.
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Fig. 1: Cluster analysis of 38 MALDI-TOF Main mass Spectra’s (MSPs) of strains belonging to
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Auth= authentic strain.
393
= R. microsporus,
= R. arrhizus var. arrhizus,
= R. arrhizus var. delemar
394 395
Fig. 2: Cluster analysis of MALDI-TOF Main mass Spectra’s (MSPs) of strains belonging to
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15
Table 1 Click here to download Table: Somay-list maldi.docx
CBS number
Current name
Country
Source
CBS 102277 CBS 112586 CBS 112588 CBS 124669 CBS 258.79 CBS 289.71
R. microsporus R. microsporus R. microsporus R. microsporus R. microsporus R. microsporus
NA Indonesia Indonesia Greece Sweden Italy
CBS 294.31 T CBS 338.62 CBS 357.93 T CBS 388.34 T CBS 608.81 CBS 699.68 NT CBS 700.68 CBS 102660 CBS 112.07 T CBS 118614 CBS 120591 CBS 120596 CBS 127.08 Auth? CBS 148.22 CBS 264.28 T CBS 286.55 CBS 387.34 T CBS 395.95 CBS 400.95 CBS 515.94 CBS 120.12 T CBS 126971 CBS 279.38 T CBS 295.31 Auth CBS 385.34 T CBS 386.34 T CBS 389.34
R. microsporus R. microsporus R. microsporus R. microsporus R. microsporus R. microsporus R. microsporus var. arrhizus var. arrhizus var. arrhizus var. arrhizus var. arrhizus var. arrhizus var. arrhizus var. arrhizus var. arrhizus var. arrhizus var. arrhizus var. arrhizus var. arrhizus var. delemar var. delemar var. delemar var. delemar var. delemar var. delemar var. delemar
France Indonesia Indonesia Japan Denmark Ukraine Georgia NA The Netherlands Turkey France France NA NA China NA Japan NA NA Singapore Japan NA India Germany Japan Japan Japan
Human, rhinocerebral Tempe Tempe Human, soft palate Saw mill dust Starch-containing material Cow foetus Tempe Tempe Ragi NA Soil Forest soil NA NA Palate Sinus NA NA NA Chinese yeast Rabbit brain NA NA NA Tempe NA NA NA Pig NA NA NA
Physiological group
AB AA AA AB AA AA AA AA AA AA AA AB AA DD DD DD DD DD DD DD
GenBank accession number ITS ACT TEF KC206521 KC206531 KC206532 KC206525 AY291263 KC206528
KC822407 KC822394 KC822398 KC822399 KC822409 KC822421
KC315413 KC315438 KC315425 KC315430 KC315417 KC315420
KC206522 AB097389 AB097392 KC206524 KC206527 AB097385 AB097386 KJ551384 AB097313 KJ551386 KJ551388 KJ551392 AB181304 KJ551403 KJ551404 JN206328 DQ641274 AY213684 AB097325 JN206325 AB181318 KJ551397 AB181319 AB181320 DQ641288 AB181322 AB181323
KC822420 AB512238 AB512242 KC822419 KC822408 AB512247 AB512248 KJ551423 AB281499 KJ551425 KJ551426 KJ551430 AB281501 KJ551440 AB281506 KJ551444 AB281516 KJ551452 KJ551464 KJ551458 AB281500 KJ551435 AB281509 AB281510 AB281514 AB281515 AB281517
KC315433 AF157288 KC315434 KC315423 KC315411 AB512270 AB512271 KJ551466 AB281528 KJ551468 KJ551469 KJ551472 AB281530 KJ551480 AB281535 KJ551482 AB281545 KJ551485 KJ551491 KJ551486 AB281529 KJ551502 AB281538 AB281539 AB281543 AB281544 AB281546
CBS 391.34 T CBS 393.34 T CBS 395.54 CBS 401.51 CBS 402.51 T CBS 406.51 T?
var. delemar var. delemar var. delemar var. delemar var. delemar var. delemar
Japan Japan Georgia Japan Japan Japan
NA NA Human, diabetic NA NA NA
DD DD DC DD DD DD
AB181325 AB181327 AB181317 DQ641297 AB181328 AB181330
AB281519 AB281521 AB281523 KJ551456 AB281524 AB281526
AB281548 AB281550 AB281552 KJ551505 AB281553 AB281555
Table 1: Isolates studied, with origin and substrates. NA = not available, CBS = Centraalbureau voor Schimmelcultures Fungal Biodiversity Centre, Utrecht, The Netherlands, T = type strain of (synonymized) taxonomic entity, Auth = authentic for a taxonomic entity. AA, AB: var. arrhizus strains (lactic acid producers) based on Abe et al. (2007). DD, DC: var. delemar strains (fumaric-malic acid producers) based on Abe et al. (2007). GenBank accession numbers are provided.
FIG. 1 Click here to download Figure: COMBINED TREES.pdf
CBS 387.34 T
CBS 402.51
CBS 286.55
CBS 385.34
37 CBS 120596
CBS 279.38
CBS 395.95
CBS 295.31
CBS 264.28 T
CBS 389.34
CBS 148.22
CBS 386.34
61
CBS 400.95 47
CBS 515.94
CBS 391.34
CBS 120591
CBS 120.12T
CBS 118614
CBS 393.34
CBS 102660 CBS 112.07 T 60 CBS 127.08 Auth?
CBS 402.51 T CBS 120.12 T CBS 279.38 T CBS 386.34 T CBS 126971 CBS 393.34 T
67
CBS 401.51 CBS 406.51 T? 99
CBS 295.31 Auth CBS 391.34 T
73
CBS 389.34 CBS 385.34 T CBS 395.54
CBS 608.81 CBS 102277 CBS 258.79 67
0.05 substitution / site
CBS 126971 CBS 406.51 CBS 401.51 CBS 387.34 CBS 127.08 CBS 515.94 CBS 400.95 CBS 120591 CBS 120596 CBS 112.07 CBS 118614 CBS 286.55 CBS 148.22 CBS 102660 CBS 395.95 CBS 700.68 CBS 699.68 CBS 102277
CBS 700.68
CBS 112586
CBS 699.68 NT
CBS 112588
CBS 338.62
CBS 289.71
CBS 357.93 T
CBS 294.31
CBS 294.31 T
CBS 258.79
CBS 388.34 T
CBS 608.81
CBS 112586
22
CBS 395.54
CBS 388.34
CBS 124669
CBS 338.62
CBS 112588
CBS 124669
CBS 289.71
CBS 357.93 0
100
200
300
Distace level
900
1000
Fig. 2 Click here to download Figure: fig 2. dendro rhizopus bruker method.pdf
R.stolonifer
R. microsporus
M. circinelloides
var. arrhizus
var. delemar
var. arrhizus Rh. pusillus
L. corymbifera
1000
900
800
700
600
500
400
300
200
100
0