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Simplified DNA extraction protocol coupled with PCR and DNA
sequence analyses successfully reveal novel Meliolaceae species
Xiang-Yu Zeng1,2,3, Rajesh Jeewon4, Ting-Chi Wen1*, Sinang Hongsanan2,
Saranyaphat Boonmee2 & Kevin D. Hyde 2,3,5
1The Engineering Research Center of Southwest Bio-Pharmaceutical Resources, Ministry of
Education, Guizhou University, Guiyang 550025, China
2Center of Excellence in Fungal Research, Mae Fah Luang University, Chiang Rai 57100,
Thailand
3School of Science, Mae Fah Luang University, Chiang Rai 57100, Thailand
4Department of Health Sciences, Faculty of Science, University of Mauritius, Reduit, 80837,
Mauritius
5Key Laboratory for Plant Diversity and Biogeography of East Asia, Kunming Institute of Botany,
Chinese Academy of Sciences, 132 Lanhei Road, Kunming 650201, China
*email: tingchiwen@yahoo.com
Abstract
Meliolaceae is an obligate biotrophic fungal family which is almost impossible to culture in
artificial media. This has resulted in a lack of DNA sequence data in public databases to better
resolve species taxonomy. The main criterion for interspecific classification of species therefore
relied heavily on host association. In this study, collections from living leaves of Croton persimilis
and Tamarindus indica with black colonies in Mae Fah Luang University, Thailand yielded one
new species, Irenopsis crotonicola sp. nov and a new record of Meliola tamarindi. These taxa are
described herein and morphological comparisons are made with known species. To better classify
the putative novel species that cannot be cultivated, we first outline an effective protocol to extract
DNA from fruiting bodies and generate phylogenetic data. Results indicate that this direct DNA
extraction method is suitable to yield quality DNA sufficient for PCR amplifications of several
commonly used DNA regions in fungal systematics. The 28S rDNA phylogram generated
confirms the position of our taxa within Meliolaceae and indicate a close relationship of Irenopsis
crotonicola sp. nov to I. walsurae.
Introduction
DNA extraction and PCR amplification from many fungal specimens (fruiting bodies) which do
not grow in artificial media has always been a major impediment towards proper species
identification and investigation of their phylogenetic relationships. Recently there has been major
breakthrough and for some species, direct amplification following DNA sequencing have been
successful1–3. However, there is no single protocol for all fungal taxa, as there is a wide variety of
fruiting bodies.
Meliolaceae is a biotrophic fungal group that comprises eight genera and approximately 2400
species4. Species from this family can infect a broad range of hosts in 194 host families4. Members
of Meliolaceae are characterized by branched, dark brown, superficial mycelium with phialides
and two-celled hyphopodia, dark brown, superficial ascomata, with 2–4-spored asci, and 3–
4-septate, dark brown, cylindrical ascospores. They are believed to be highly host-specific and
numerous new species have been introduced based on host association5–7.
Attempts at culturing species of Meliolaceae have been conducted, but most have failed or
ended up with germinating ascospores with no further growth5,8,9. Goos10 isolated Meliola
palmicola from hyphal fragment using cornmeal agar. However, it only produced black hyphae
without setae or hyphopodia, thus, could not be identified as a Meliola.
This is a major problem with many Meliolaceae species and to date (Jan 2017), there are only
65 specimens for which DNA sequence data are available in GenBank1,3,6,7,11,12,13. Traditional
methods may have either overestimated or underestimated species diversity and therefore there is
a need to develop new and efficient protocols that are in line with our DNA based sequence
strategies for reliable classification14,15. Some DNA extraction protocols have been used before
(e.g. 1,3,7, 12), however, there has always been a major risk of PCR contamination or generating
unreliable DNA sequences if too many ascomata are used for starting material for molecular
studies.
In this study, we employ a new protocol based on a Direct PCR kit to unleash DNA from
fruiting bodies of Meliolaceae species in a very efficient and rapid manner. DNA obtained were
then amplified and sequenced. Two taxa are described herein based on morphological characters
and phylogenetic inference. A new species of Irenopsis as well as Meliola tamarindi are described
and compared with known species.
Results
Sequences of SSU (ca. 1020 bp), ITS1 (ca. 214 bp), 5.8S (159 bp), ITS2 (ca. 212 bp), LSU (ca.
860 bp), TEF (ca. 480 bp), β-tubulin (ca. 450 bp) and GPDH (ca. 977 bp) gene regions were
successfully obtained. Other results for different primer pairs tested are shown in Supplementary
Table S1. Novel DNA sequences generated are deposited to GenBank with accession number
listed in Supplementary Table S2.
Despite the fact that we obtained reliable sequences from the SSU, ITS, TEF, β-tubulin and GPDH
genes, phylogenetic analyses have not been performed due to lack of related sequence data from
databases. The dataset of LSU gene region analyses consisted of 824 characters after alignment,
including 436 constant characters, 66 parsimony-uninformative variable characters, and 322
parsimony-informative characters (TL=1417, CI=0.439, RI=0.683, RC=0.300, HI=0.561). 1008
thorough maximum likelihood tree searches were carried out with a final ML optimization
likelihood value of -7272.076232 for the best scoring tree. Bayesian analysis generated 2031 trees
and finally 1524 trees were used to calculate posterior probabilities. The phylogram inferred from
the LSU dataset of 40 ingroup taxa from the Meliolaceae is presented in Fig. 1.
Meliola tamarindi MFLU 14-0080, which is morphologically similar to M. tamarindi MFLU
14-0282 cluster together with 100% bootstrap support and 1.00 Bayesian Posterior Probabilities
and constitutes a strongly monophyletic lineage with Asteridiella obesa. Analyses place all
Irenopsis species into a monophyletic clade (100% and 98% BS and 1.00 BPP) and our new taxon.
I. crotonicola clusters with I. walsurae with high bootstrap support.
Description of Irenopsis crotonicola X.Y. Zeng, T.C. Wen & K.D. Hyde sp. nov. MycoBank:
MB 819817; Facesoffungi number: FoF 02895 (Fig 2, 3)
Etymology: Referring to the host Croton persimilis.
Holotype: MFLU 14-0078.
Beeli formula: [3401.4220]
Epiphytes on the surface of living leaves. Colonies epiphyllous, subdense to dense, confluent,
black. Hyphae superficial, brown, straight, radiating outwardly, branched, septate, darker at septa,
reticulate, without hyphal setae. Hyphopodia 12–22 × 10–14 μm (x
̅ = 15 × 12 μm, n=20), 2-celled,
brown, spathulate, form one near the septa, alternate, antrorse. Sexual morph: Ascomata up to
160 μm diam., superficial, subdense, globose to subglobose, thick-walled, with ostiole and
ascomata setae. Ascomata setae simple, straight, up to 100μm long, dark brown, rounded at the
apex. Peridium comprising two strata, outer strata of cells with dark brown walls of textura
angularis and hyaline inner strata. Hamathecium with evanescent paraphyses. Asci unitunicate, 2–
3-spored, ovoid at young state, with short pedicel, asci wall attenuated or broken when mature,
without a certain shape. Ascospores 38–42 × 15–17 μm (x
̅ = 39 × 16 µm, n = 20), 2–3-seriate,
cylindrical or oblong, hyaline at young state, becoming dark brown when mature, 4-septate,
constricted and darker at the septa, rounded at both ends, apical cell sometimes tapering at the tip,
smooth-walled. Asexual morph: Phialides 15–19 (–22) × 7–9 μm (x
̅ = 18 × 8 µm, n = 10),
ampulliform, forming two at top of hyphal cell separate from hyphopodia, opposite. Conidia
undetermined.
Material examined: THAILAND, Chiang Rai, Mae Fah Luang University, on living leaves of
Croton persimilis (Euphorbiaceae), 3 Feb. 2014, Xiang-Yu Zeng (MFLU 14-0078, holotype)
Notes: This new species was found on Croton persimilis and is recognized as a species of
Irenopsis based on its branched, dark brown, superficial hyphae with phialides and two celled
hyphopodia; dark brown, superficial ascomata with setae, and dark brown, 4-septate ascospores.
Irenopsis crotonis is the only Irenopsis species reported from Croton4, with hooked setae and
amphigenous, thin colonies. However, this new collection has larger ascomata and ascospores,
epiphyllous, straight setae and subdense colonies. Phylogenetic analyses indicate that the new
collection clusters within the clade that represents the genus Irenopsis and is closely related to I.
walsurae, but forms a distant clade (Fig 1). It is 92% (1417/1538 nucleotides, 26/1538 gaps)
similar to the ITS-LSU sequence of I. walsurae. Morphologically, the new collection differs in
having subdense colony and ascomata, shorter ascomata setae, and longer ascospores, instead of
scattered colony and ascomata, longer ascomata setae and shorter ascospores.
Description of Meliola tamarindi Syd. & P. Syd. 1912 (Fig 4)
Beeli formula: [3113.4332]
Epiphytes on the surface of living leaves. Colonies epiphyllous, scattered to dense,
sometimes confluent, black. Hyphae superficial, brown, substraight to undulate, radiating
outwardly, branched, septate, darker at septa, closely reticulate, with hyphal setae. Hyphal setae
simple, straight, up to 330μm long, dark brown, rounded at the apex. Hyphopodia 17–26 × 10–15
μm (x
̅ = 12 × 12 μm, n = 20), 2-celled, brown, spathulate, form one near the septa, alternate or
unilateral, antrorse. Sexual morph: Ascomata up to 250 μm, superficial, dense, grouped, globose
to subglobose, thick-walled, with ostiole, without perithecial setae. Peridium comprising two
strata, outer strata of cells with dark brown walls of textura angularis and hyaline inner strata.
Hamathecium with evanescent paraphyses. Asci unitunicate, 2–3-spored, ovoid to ellipsoid at
young state, with short pedicel, asci wall attenuated or broken when mature, without a certain
shape. Ascospores 38–50 × 18–22 μm (x
̅ = 45 × 20 µm, n = 20), 2–3-seriate, fusiform to ellipsoid,
hyaline at young state, becoming dark brown when mature, 2–4-septate, constricted and darker at
the septa, rounded at both ends, smooth-walled. Asexual morph: Phialides 15–20 × 8–9 μm (x
̅ =
17 × 8 µm, n = 5), ampulliform, forming two at top of hyphal cell separate from hyphopodia,
opposite or unilateral, not mixed with capitate hyphopodia. Conidia undetermined.
Material examined: THAILAND, Chiang Rai, Mae Fah Luang University, on living leaves of
Tamarindus indica (Fabaceae), 3 Feb. 2014, Xiang-Yu Zeng (MFLU 14-0080)
Notes: Meliola tamarindi was described by H. Sydow & P. Sydow in 191216. Our collection
is recognized as a species of Meliola based on branched, dark brown, superficial mycelium with
hyphal setae, phialides and two celled hyphopodia; dark brown, superficial ascomata without
ascomata setae and dark brown, 4-septate ascospores. Our collection is identical to the LSU
sequence data of M. tamarindi MFLU14-0282 with only 1 base different. However, it has larger
ascospores than in the protologue (38–50×18–22 μm versus 36–44×13–17 μm)16.
Discussion
Species of Meliolaceae are characterized by branched, dark brown, superficial mycelium with
phialides and two-celled hyphopodia, dark brown, superficial ascomata, with 2–4-spored asci, and
dark brown, cylindrical, 3–4-septate ascospores7. They are believed to be host-specific and
numerous new species were introduced based on host association. Irenopsis, with I. tortuosa F.
Stevens 1927 as type species, is a genus in the family Meliolaceae with 152 taxa4. This genus,
along with its close relatives, Meliola, are commonly reported as black mildews and infect a wide
variety of plants such as Fabaceae, Lamiaceae and Sapindaceae7. The main morphs characterizing
this genus include colonies that can be hypophyllous, amphigenous or epiphyllous; hyphae which
can be straight to flexuous and loosely to closely reticulate; hyphopodia which can be in different
arrangement (alternate or opposite); phialides which mostly opposite and mixed with hyphopodia;
ascomata mostly scattered, with simple, straight or curved ascomata setae, and ascospores which
are mostly 4-septate constricted and cylindrical7,17. The genus Irenopsis differs from any other
genera of Meliolaceae in having ascomata setae. There are about 150 species under this genus,
including the latest newly described species Irenopsis walsurae7. Lack of sequence data is always
a problem for this group and to date there are only DNA sequences from six different species (11
strains) of this genus in GenBank.
One new species of Meliolaceae, Irenopsis crotonicola, isolated from Croton persimilis, is
described and illustrated in this study. There is a report of Irenopsis crotonis, reported from the
host Croton sp. from Trinidad in 192618. While our new species has been collected from the same
host genus and share some morphological similarities with I. crotonis, it differs remarkably in
colony, setae, ascomata and ascospore characteristics. Irenopsis crotonicola is characterized by
epiphyllous, subdense to dense, confluent colonies, straight setae with subdense and larger
ascomata and ascospore dimensions (see Supplementary Table S3 for further details). DNA
sequence data for I. crotonis is not available for further comparison as well for Irenopsis
chrysophylli, which is a species described from a Sapotaceae host12. The latter was collected from
Panama and differs from our new taxon with phialides that are mixed with hyphopodia and longer
ascomata setae that are bent at the apex. Another species, Irenopsis vincensii collected from Hevea
brasiliensis is also distinct in terms of its amphigenous, scattered colonies, phialides that are
mixed with hyphopodia, setae that are bent at the apex, and larger ascomata. The results of our
DNA sequence analyses also display a phylogenetic distinction between Irenopsis vincensii and I.
crotonicola and strains of the former constitute a separate lineage with I. cornuta as a sister taxon
(Fig 1). Of particular interest in our phylogeny, we noted a close relationship (with high support)
between the new taxon and I. walsurae, which has been described from Walsura tubulata
(Meliaceae) and Chiang Mai, Thailand. We ruled out the possibility that our taxon can be I.
walsurae. In addition, I. walsurae differs from our new taxon by having scattered, discrete
colonies, longer ascomata setae and smaller ascospores. Upon further verification of DNA
sequences from the ITS and LSU regions, we noticed sufficient differences to warrant species
delineation as outlined by Jeewon & Hyde15. We believe that a close phylogenetic relationship
between the two taxa could be an artifact of taxon sampling given that there are insufficient DNA
sequence data available for this genus.
Development of rapid, reliable and effective methods to extract DNA and perform PCR for
further taxonomic work is of growing interest, as many undiscovered fungal species cannot be
cultured on artificial media for further molecular characterisation. In this paper, we have outlined
a reliable DNA extraction method which directly recovers target DNA from fruiting bodies. Other
researchers have also successfully isolated DNA from Meliolaceae species. For example, Saenz &
Tay lor1 used 500 μL 5% chelex (Bio-Rad, Richmond, CA) solution to obtain DNA from 5–20
ascomata by following a modified protocol developed by Walsh et al.19. The use of DNA
extraction kit has also been quite popular recently (e.g. 3,6,7,12), but most of them relied on more
than one chemical agent to extract DNA, which were rather time consuming, needed a high
number of fruiting bodies and PCR amplification success was rather low. In addition, it is noted
that many collections or specimens of this fungal group do not have enough clean ascomata, or
sometimes are not in good condition to allow further molecular studies. The protocol that we are
using in this paper needs only two to four ascomata, sometimes even one, as the starting material
and takes less than 30 mins for the whole process. We believe that this method is quite practical
and relies on minute samples as well which is quite important, especially if mycologists are
targeting to work on specimens and link current species to old names20. PCR amplifications under
standardized conditions from several commonly used genes in fungal systematics have been
attempted as well. As detailed in Supplementary Table S1, the results from the PCR are rather
promising with a wide range of genes and for those where PCR failed, there might be a need to
optimize PCR profiles. When compared with other DNA extraction methods used and the output
with PCR, we believe that the one described in this paper is quite rapid, convenient and cost
effective and will facilitate mycologists to better use DNA sequences from unculturable species or
old specimens for a wide range of applications including better species identification and
clarifying phylogenetic relationships.
Materials & Methods
Morphological studies. Fresh living leaves of Croton persimilis and Tamarindus indica with
black colonies were collected from Mae Fah Luang University, Thailand and returned to the
laboratory in paper envelopes. The samples were processed and examined following the methods
described by Taylor & Hyde21. Photographs of ascomata were taken under a compound
stereomicroscope (Zeiss Discovery.V8 with camera AxioCam ERc 5S). Sections were made using
a stereomicroscope (Motic) and mounted in water. Photomicrographs of fungal structures were
taken with a light microscope (Nikon Eclipse Ni–U) fitted with a digital camera (Canon
DS126311 EOS 600D) and a scanning electron microscope (SEM) (Hitachi S-3400N). The
holotype is deposited at Mae Fah Luang University (MFLU) Herbarium. MycoBank and
Facesoffungi numbers are registered22,23 and new species are justified based on recommendations
outlined by Jeewon & Hyde15.
DNA sequencing. Small pieces of leaves containing several clean ascomata, with as little other
fungi as possible, were checked under the stereomicroscope. Precautions were taken to avoid
picking any other associated materials that could lead to potential contamination. Accurate visual
examinations were performed to ensure that there was no mixture of other fungal colonies that
could contaminate our target sample. Examined leaf pieces were then cut and kept in Eppendorf
tubes with silica gel inside for later DNA extraction24. Total genomic DNA was extracted directly
from ascomata using the Lysis Buffer for Microorganism to Direct PCR (TaKaRa, 9164, China) as
follows: (1) Transfer 2‒4 clean ascomata (broken by a clean sterile razor blade while ensuring that
ascomata are of the same fungus) or small amount of clean mycelium from leaf surface into a
sterile Eppendorf tube using a sterilized needle. (2) Transfer 50µl of the lysis buffer to the
Eppendorf tube and vortex gently for 10 sec. (3) Incubate at 80℃ for 15 mins. (4) Centrifuge at
3500 rpm for 1 min. (5) Transfer the supernatant to another sterile and clean Eppendorf tube to be
used as the DNA template for later PCR amplification (the supernatant can be stored at -20℃ for
1 year).
PCR reactions were employed in 20 μL reaction mixture containing 10 μL 2×Bench Top Taq
Master Mix (Biomiga, AT1201, China), 7 μL ddH2O, 1 μL forward and reward primers (10μM/μL)
and 1 μL DNA template obtained from above. Amplification were performed in a T100TM Thermal
Cycler (BIO-RAD), which were programmed for 3-min denaturation at 95 °C followed by 34
cycles of 95 °C for 30 s, annealing for 30 s and extension at 72 °C, with a final 5-min elongation
step at 72 °C. Details of primers used in this study are listed in Supplementary Table S4. and
annealing temperature with extension time of different primer pairs are listed in Supplementary
Table S5. After amplification, the PCR fragments were electrophoresed in 1.5% agarose gels at
120 V for 15 min. Gels were checked under 254 nm UV in a Gel Doc™ XR+ System (BIO-RAD).
PCR products were sequenced by using appropriate PCR primers used in amplification reactions
by SinoGenoMax Co., Ltd, Beijing, China.
Phylogenetic analyses. Sequences generated from forward and reward primers were reassembled
with BioEdit v.7.2.525 to obtain consensus sequences. Consensus sequences were aligned with
sequences of Meliolales downloaded from GenBank by Mafft v7.18726 and then manually aligned
where necessary. Chaetosphaeria innumera was chosen as the outgroup taxon based on previous
studies7,27. Phylogenetic trees based on the LSU dataset were inferred from maximum parsimony,
maximum likelihood and Bayesian algorithms. Other details are as outlined by Jeewon et al.28,29
Daranagama et al.30 and Hyde et al.13. Maximum parsimony analysis was performed using PAUP
v4.0b1031. Trees were inferred using heuristic search option with 1000 random taxa addition.
Maxtrees were set up to 5000, branches of zero length were collapsed and all multiple
parsimonious trees were saved. Parsimony scores including tree length (TL), consistency index
(CI), retention index (RI) and homoplasy index (HI) were calculated for trees generated under
different optimality criteria. Clade stability was assessed using a bootstrap (BT) analysis with
1000 replicates, each with 100 replicates of random stepwise addition of taxa32. Maximum
likelihood analysis was performed at the CIPRES webportal33 using “RAxML-HPC v.8 on
XSEDE” tool34,35 and under a general time reversible model (GTR). A tree was obtained by
simultaneously running a fast bootstrap search of 1000 pseudoreplicates followed by a search for
the most likely tree. Bayesian analysis36 was performed in MrBayes v.3.2.637 based on the Akaike
Information Criteria (AIC) in MrModeltest 2.338. The Markov Chain Monte Carlo (MCMC)
algorithm of 6 chains started from a random tree topology with 2 parallel runs. The run was
stopped automatically when the average standard deviation of split frequencies falls below 0.01.
Trees were sampled every 100 generations and burn-in was set at 25% after which the likelihood
values were stationary. The remaining trees were used to calculate posterior probabilities (PP). All
trees were visualized in Fig Tree v1.4.039. Details of sequence data used are presented in
Supplementary Table S6.
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Acknowledgement
This study was supported by the Science and Technology Foundation of Guizhou Province (No.
[2012]3173). Rajesh Jeewon is grateful to Mae Fah Luang University for inviting him as a visiting
professor and a keynote speaker for the COEIC 2017 conference. Kevin D. Hyde thanks the
Chinese Academy of Sciences, project number 2013T2S0030, for the award of Visiting
Professorship for Senior International Scientists at Kunming Institute of Botany.
Author Contributions
X.Y.Z. and T.C.W. designed the study. X.Y.Z. conducted all the experiments. X.Y.Z., R.J. and S.H.
analysed the result. X.Y.Z., R.J., T.C.W., S.H., S.B. and K.D.H edited the manuscript. All authors
reviewed the manuscript and approved the manuscript for publication.
Competing financial interests: The authors declare no competing financial interests.
Figure 1. The phylogram shows the phylogenetic relationships within the order Meliolales based
on LSU sequence data. Maximum parsimony/likelihood bootstrap support values greater than 50%
and Bayesian posterior probabilities greater than 0.90 are shown in above and below. New data
generated in this study are in boldface, and branches with strong support are given in bold. The
tree is rooted with Chaetosphaeria innumera SMH 2748 (Chaetosphaeriaceae, Chaetosphaeriales).
Figure 2. Irenopsis crotonicola MFLU 14-0078. (a) Colony on surface of leaf; (b–c) Ascomata
on host substrate; (d) Hyphae with capitate hyphopodia; (e) Hypha with phialides; (f) Ascomata
setae; (g–n) Ascus from young state to mature state; (o) Ascospore.
Figure 3. Structure of Irenopsis crotonicola MFLU 14-0078 under SEM. (a) Host plant; (b)
Colony on surface of leaf; (c) Hyphae on surface of leaf; (d) Hypha with capitate hyphopodia and
phialides; (e) Immature ascoma; (f) Ascomata on host substrate; (g) Ascomata setae; (h) Ascus; (i–
j) Germinated ascospore on host substrate.
Figure 4. Meliola tamarindi MFLU 14-0080. (a) Host plant; (b–c) Ascomata on host substrate;
(d) Ascoma; (e) Ascomata under SEM; (f) Hyphopodia under SEM; (g) Phialides; (h). Hyphal
setae under SEM; (i). Peridium; (j). Peridium under SEM; (k). Ascospores under SEM; (l–o).
Ascus from young state to mature state; (p–q) Ascospore.
