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Phytotaxa 587 (3): 251–268
https://www.mapress.com/pt/
Copyright © 2023 Magnolia Press Article PHYTOTAXA
ISSN 1179-3155 (print edition)
ISSN 1179-3163 (online edition)
Accepted by Sajeewa Maharachchikumbura: 22 Feb. 2023; published: 16 Mar. 2023
https://doi.org/10.11646/phytotaxa.587.3.4
251
Three interesting fungi from American bullfrog larvae (Rana catesbeiana) in
Yunnan, China
ERFU YANG1,2,6, WENHUA LU1,7, SAOWALUCK TIBPROMMA1,8*, DONGQIN DAI1,9, YING GAO4,10,
ITTHAYAKORN PROMPUTTHA3,11 & SAMANTHA C. KARUNARATHNA1,5,12*
1 Center for Yunnan Plateau Biological Resources Protection and Utilization, College of Biological Resource and Food Engineering,
Qujing Normal University, Qujing, Yunnan 655011, People’s Republic of China
2 Master of Science Program in Applied Microbiology (International Program), Faculty of Science, Chiang Mai University, Chiang Mai
50200, Thailand
3 Department of Biology, Faculty of Science, Chiang Mai University, Chiang Mai, Thailand
4 Center of Excellence in Fungal Research, Mae Fah Luang University, Chiang Rai 57100, Thailand
5 National Institute of Fundamental Studies (NIFS), Sri Lanka
6
�
erfu20170431@gmail.com; https://orcid.org/0000-0003-2385-6402
7
�
reuven0319@gmail.com; https://orcid.org/0000-0001-7283-7596
8
�
saowaluckfai@gmail.com; https://orcid.org/0000-0002-4706-6547
9
�
cicidaidongqin@gmail.com; https://orcid.org/0000-0001-8935-8807
10
�
gaoying@mail.kib.ac.cn; https://orcid.org/0000-0001-8671-1978
11
�
itthayakorn.p@cmu.ac.th; https://orcid.org/0000-0003-3376-4376
12
�
samanthakarunarathna@gmail.com; https://orcid.org/0000-0001-7080-0781
* Corresponding authors:
�
saowaluckfai@gmail.com,
�
samanthakarunarathna@gmail.com
Abstract
American bullfrog (Rana catesbeiana) is an alien invasive species in southwest China native to the central and eastern United
States and southeastern Canada. After the 19th century, they extensively appear in aquaculture and natural environments
worldwide as a delicious food but also creating a serious threat to the survival and development of native species. In the
early rainy season, dead American bullfrog larvae floating on the water of unnamed ponds in Qujing Normal University,
Yunnan Province, China were collected and brought to the mycology laboratory, and three interesting fungal strains were
isolated from their intestinal contents. Phylogenetic analyses were carried out on the resultant isolates based on multiple
gene sequences (ITS, LSU, rpb2, tub2, tef1-α), and results confirmed that the three strains belong to three species, namely;
Boothiella tetraspora, Sordaria macrospora and Trichoderma virens. The morphological characteristics were also used to
describe the fungal taxa. Photographic plates, descriptions, and phylogenetic trees that show the placements of the fungal
species are reported herein.
Keywords: Amphibian, Boothiella, Microfungi, Sporulation, Sordaria, Trichoderma
Introduction
Rana catesbeiana (= Lithobates catesbeianus (Shaw 1802)), also known as American bullfrog, is native to the central
and eastern United States and southeastern Canada. Still, since the end of the 19th century, it has been introduced
throughout the world (Mazzoni 2000). American bullfrog was introduced into China around 1959 for aquaculture and
aquarium trades (Han et al. 1991). As they can adapt to a wide variety of environmental conditions and reproduce
rapidly, currently, invasive bullfrog species have successfully established their populations in many provinces from
eastern to western China (Wu et al. 2004, Liu et al. 2015). American bullfrogs require nonephemeral aquatic habitats,
prefer inhabiting open, permanent waters like lakes and ponds, breed from early spring through late summer, and
larvae are very large and typically require two to three years before metamorphosis (Shirose & Brooks 1995, Wang &
Li 2009). The American bullfrog is an opportunistic generalist predator, consumes a large number and variety of prey
species, and insects usually numerically dominant (Silva et al. 2009). They eat social wasps and odonates (damselflies
and dragonflies), and also hunt vertebrates viz; birds, reptiles, fish, mammals and frogs (Leivas et al. 2012, Jancowski
YANG ET AL.
252 • Phytotaxa 587 (3) © 2023 Magnolia Press
& Orchard 2013). The American bullfrog is now considered as one of the most ecologically destructive invasive
alien vertebrate species in the world. They result in population declines and local extinctions of native amphibians
through direct predation, interference competition, and disease transmission (Kats & Ferrer 2003, Garner et al. 2006).
Similarly, the field surveys concluded have significant negative correlations between American bullfrogs and native
amphibian populations (Adams 1999, Wang et al. 2009).
Chytrid fungus Batrachochytrium dendrobatidis is well studied as the major pathogenic fungus of amphibian
species implicated in global amphibian declines and numerous species extinctions (Garner et al. 2005), and more than
600 amphibian species were infected globally (Olson et al. 2013). The American bullfrog is also the reservoir of the
pathogenic chytrid fungus (Batrachochytrium dendrobatidis), capable of transmitting infections to native hosts (Miaud
et al. 2016). However, other fungi associated with the American bullfrog are rarely reported.
In this study, we report three interesting fungal species from dead American bullfrog larvae based on morphological
characteristics and multilocus phylogenetic analyses. Full descriptions, color photoplates of macro- and micro-
morphological characteristics, and phylogenetic trees to show the placements of three fungi are provided.
Materials and methods
Sampling and fungal isolation
The dead American bullfrog larvae were found floating in the water of a pond at Qujing Normal University, Qujing,
China (rainwater) (GPS: 103°44’35”E, 25°30’46”N, 1856.6 m) (Figure 1). The tadpole specimens were collected
and taken to the mycology laboratory of the university for further analyses. Cui et al. (2022) was followed for the
intestinal fungi isolation with slight modifications. Initially, the dead tadpole bodies were washed with sterilized water
and saline solution, soaked in 75% ethanol for 3 mins, dried with sterilized tissue paper, transferred to 3% sodium
hypochlorite solution for 3 mins, followed by rinsing with sterilized water, and at last dried with sterilized tissue paper.
The intestinal contents were obtained by a sterilized scalpel and sterilized glass rod and transferred to potato dextrose
agar (PDA). All the above steps were done in the UV-sterilized laminar flow. The PDA plates were incubated at 27 °C
for one week. A sterile needle was used to pick the mycelium from one-week-old culture and placed it on a new PDA
plate and repeated 3 to 5 times until getting a pure culture. Culture characteristics of fungi on PDA were observed after
one month. The dry cultures were deposited in the Kunming Institute of Botany Academia Sinica (HKAS), Kunming,
China, and living cultures were deposited at the Kunming Institute of Botany Culture Collection (KUNCC).
FIGURE 1. a The pond in Qujing Normal University. b A dead American bullfrog larvae floating on pond. c, d The dead larvae. e The
dead larvae in sterilized water.
THREE INTERESTING FUNGI FROM AMERICAN Phytotaxa 587 (3) © 2023 Magnolia Press • 253
Microscopic examination
The isolates were sporulated in PDA plates within one month at 27 °C. The observation and making slides were
carried out under an Olympus SZ61 (Japan). The morphology of colonies in PDA was photographed by a camera of a
Huawei P40 mobile phone (Huawei Inc., Shenzhen, China). The micro-morphological characteristics were observed
and captured by a digital camera (Canon EOS 600D, Canon Inc., Japan) mounted on a compound microscope (Nikon
ECLIPSE Ni, Nikon., Japan). Measurements of the microstructures were taken by the Tarosoft (R) Image Frame Work
program, and photo plates of the microstructures were edited using Adobe Photoshop CS3 Extended v. 10.0 (Adobe®,
San Jose, CA).
DNA extraction, PCR amplification and sequencing
Mycelia from pure cultures (one-month-old) were scraped using sterilized needles and transferred in 1.5 ml centrifugal
tubes. Genomic DNA extraction followed the instruction book of the manufacturer of Biospin Fungus Genomic DNA
Extraction Kit-BSC14S1 (BioFlux®, P.R. China). Extracted DNA were kept at -20 °C for long-term storage, while a
part of the DNA was stored at 4 °C for instant polymerase chain reaction (PCR). The PCR reaction mixture contained
8.5 µl of double-distilled water (ddH2O), 12.5 µl of 2×Power Taq PCR MasterMix (mixture of EasyTaqTM DNA
Polymerase, dNTPs, and optimized buffer, Beijing Bio Teke Corporation (Bio Teke), PR China), 2 µl DNA template,
and 2 µl of each primer (×10 pmol) (Yang et al. 2022). The PCR primers ITS4/ITS5 and LR0R/LR5 were used to
amplify portions of the internal transcribed spacer (ITS) and nuclear ribosomal large subunit (LSU) (Vilgalys & Hester
1990, White et al. 1990); the 5F/7cR were used to amplify portions of the partial RNA polymerase II subunit (rpb2)
(Liu et al. 1999); the T1/T22 were used to amplify portions of the beta-tubulin (tub2) (O’Donnell & Cigelnik 1997);
and the 728F/ALLErew were used to amplify portions of the partial translation elongation factor 1-alpha (tef1-α)
(Jaklitsch et al. 2005). The conditions for PCR of ITS, LSU and tef1-α genes consisted of an initial denaturation step
of 2 min at 95 °C, followed by 35 cycles of 30 s at 95 °C, 50 s at 55 °C and 1 min at 72 °C, and final denaturation step
of 10 min at 72 °C. The conditions for PCR of rpb2 genes consisted of an initial denaturation step of 2 min at 95 °C,
followed by 40 cycles of 30 s at 95 °C, 45 s at 57 °C and 1 min at 72 °C, and a final denaturation step of 10 min at 72
°C. The conditions for PCR of tub2 genes constituted of an initial denaturation step of 2 min at 95 °C, followed by 35
cycles of 30 s at 95 °C, 50 s at 52 °C and 1 min at 72 °C, and a final denaturation step of 10 min at 72 °C. The PCR
products were sent to Beijing Bio Teke Corporation for purification and sequencing.
Phylogenetic analyses
The reverse and forward sequences were assembled using the Geneious (Restricted) 9.1.2 (https://www.geneious.com).
They were subjected to BLASTn searches in the GenBank (www http://blast.ncbi.nlm.nih.gov/) to screen their most
probable closely related taxa, and the spreadsheet of accession numbers were formed (Table 1). Sequence alignments
were run on the website server version of MAFFT (www.ebi.ac.uk/Tools/mafft) (Katoh & Standley 2013) and edited
manually in BioEdit 7.2.3 (Hall 1999) whenever necessary. Uninformative gaps and ambiguous regions were removed
by trimAL v1.2 (http://trimal.cgenomics.org), and sequences were manually combined in BioEdit. Fasta files were
converted to PHYLIP (for ML) and NEXUS (for BI) format in Alignment Transformation Environment (ALTER) online
program (Glez-Peña et al. 2010). Maximum likelihood analyses (ML) were done on the CIPRES Science Gateway
v.3.3 (http:// www.phylo.org/portal2, Miller et al. 2010), selecting RAxML-HPC2 on XSEDE (8.2.12) (Stamatakis
2014) with the GTRGAMMA substitution model with 1000 bootstrap iterations. Bayesian analyses were conducted
with MrBayes v. 3.1.2 (Ronquist et al. 2012, Stamatakis 2014) in the CIPRES Science Gateway platform to evaluate
posterior probabilities (PP) (Zhaxybayeva & Gogarten 2002) using Markov Chain Monte Carlo sampling (MCMC).
The GTR+I+G evolution model was also applied in the BI analyses. Bayesian analyses of six simultaneous Markov
chains were run for 2,000,000 generations, and trees were sampled and printed to output at every 100th generation
(resulting in 20,000 total trees). Phylogenetic trees were visualized using FigTree v1.4.0 (Rambaut 2022), and were
edited by Microsoft PowerPoint. The reliable bootstrap support values of ML and BI were inserted above nodes.
YANG ET AL.
254 • Phytotaxa 587 (3) © 2023 Magnolia Press
TABE 1. Isolated taxa of Sordariaceae and Trichoderma used in this study and their GenBank accession numbers. The
newly generated sequences are in red, type strains are in bold, and “/” represents the sequence is unavailable.
Species Strain
GenBank accession numbers
ITS LSU tub2 tef-α rpb2
Boothiella tetraspora KUNCC22-12510 OP740944 OP740949 OP752164 / OP752161
Boothiella tetraspora CBS 334.67 MK926876 MH870684 MK926976 / MK876838
Boothiella tetraspora CBS 887.97 MK926875 / MK926975 / MK926975
Chaetomium globosum CBS 160.62 KT214565 KT214596 KT214742 / KT214666
Chaetomium microthecia LC4685 KP336786 KP336835 KP336884 / KT149506
Sordaria alcina CBS 109460 AY681198 AY681164 AY681232 / /
Sordaria arctica CBS 143.68 MH859096 MH870809 AY681209 / /
Sordaria brevicollis FGSC 1904 / FR774288 FR774338 / /
Sordaria clematidis MFLU 16-2138 NR_170818 MT214612 / MT394668 MT394717
Sordaria conoidea CBS 563.72 AY681179 AY681145 AY681213 / /
Sordaria equicola CBS 146992 NR_173047 MZ064492 MZ078267 / MZ078202
Sordaria fimicola CBS 508.50 AY681188 AY681160 AY681228 / /
Sordaria goundaensis var. latispora CBS 565.72 MH872279 MH872279 / / /
Sordaria humana CBS 248.89 MH862170 MH873860 / / /
Sordaria humana CBS 416.82 MH861509 MH873255 / / /
Sordaria islandica CBS 512.77 MH861097 MH872859 / / /
Sordaria lappae CBS 154.97 MH862634 MH874251 AY681205 / /
Sordaria mabokeensis CBS 564.72 MH860573 MH860573 / / /
Sordaria mabokeensis CBS 566.72 MH860575 MH860575 / / /
Sordaria macrospora NNIBRFG31693 OM758272 OM758252 / / /
Sordaria macrospora CBS 346.62 MH858175 MH869769 / / /
Sordaria macrospora FGSC 4818 / FR774290 FR774340 FR774389 /
Sordaria macrospora KUNCC22-12509 OP740943 OP740948 OP752163 / /
Sordaria nodulifera NBRC 32551 LC146761 LC146761 / / /
Sordaria prolifica CBS 567.72 AY681174 AY681140 AY681208 / /
Sordaria sclerogenia FGSC 2741 / FR774291 FR774341 / /
Sordaria sibutii HGUP190055 MZ724885 MZ724979 AY681214 / /
Sordaria sibutii CBS 768.96 AY681180 AY681146 / / /
Sordaria superba CBS 784.96 AY681173 AY681139 AY681207 / /
Sordaria superba P3 EU551188 / / / /
Sordaria superba IHEM 20009 OW986110 / / / /
Sordaria tamaensis NBRC 32552 LC146762 LC146762 / / /
Sordaria tomentoalba CBS 260.78 AY681195 FR774292 FR774342 FR774391 /
Sordaria tomentoalba CBS 569.72 MH860578 MH872281 / / /
......continued on the next page
THREE INTERESTING FUNGI FROM AMERICAN Phytotaxa 587 (3) © 2023 Magnolia Press • 255
TABLE 1 (Contniued)
Species Strain
GenBank accession numbers
ITS LSU tub2 tef-α rpb2
Trichoderma afroharzianum SZMC 12432 MZ754498 / / MZ773449 MZ773428
Trichoderma atroviride CBS 693.94 MH862501 / / KJ786838 KP009060
Trichoderma atroviride TRS638 KP009323 / / KP008946 KP009020
Trichoderma crassum TRS113 KP009300 / / KP008865 KP009102
Trichoderma crassum TAMA 0238 AB856632 / / AB856704 AB856775
Trichoderma harzianum T3 MT929295 / / KX632592 MG917685
Trichoderma harzianum T2 MW199106 / / KX632591 KX632534
Trichoderma neocrassum VSL178 KC020487 / / MH746776 AF545543
Trichoderma neocrassum VSL190 KC020488 / / / AF545542
Trichoderma virens LZ012 ON566042 / / ON010788 OM967095
Trichoderma virens TA2-2 MW325928 / / MW325733 MW325755
Trichoderma virens TRB2-17 MW325926 / / MW325732 MW325753
Trichoderma virens LZ002 ON566034 / / ON010784 OM967091
Trichoderma virens LZ051 MZ068176 / / OM967101 OL957049
Trichoderma virens ATCC 9645 KU729029 KU729113 / KU933428 KU933456
Trichoderma virens KUNCC22-12508 OP740942 OP740947 / OP752162 OP752160
Protocrea illinoensis TFC 96-98 NR_119701 / / EU703905 EU703952
Protocrea farinosa CPK 3144 EU703917 / / EU703894 EU703938
Results
Phylogenetic analyses
Analyses 1: The concatenated ITS, LSU and tub2 sequence dataset consisted of 34 strains of representative taxa in
Sordaria and Boothiella with Chaetomium globosum (CBS 160.62) and C. microthecia (LC4685) as the outgroup taxa
(Figure 2). The concatenated sequence matrix comprises 1930 characters, including gaps (830 characters for LSU,
601 characters for ITS, 499 characters for tub2). Phylogenetic investigation based on maximum likelihood analysis
conducted the best RAxML tree with a final likelihood value of −5309.079016, presented in Figure 2. The matrix had
349 distinct alignment patterns, with 19.74% undetermined characters or gaps. Estimated base frequencies were as
follows: A =0.241637, C = 0.252277, G = 0.280552, T = 0.225534; substitution rates AC = 1.418855, AG = 3.309772,
AT = 1.912284, CG = 1.496664, CT = 10.036836, GT = 1.000000; gamma distribution shape parameter α = 0.563213.
The final average standard deviation of split frequencies at the end of total MCMC generations was calculated as
0.009412 in BI analysis. Our two strains, the first strain (KUNCC22-12510) basal clades to Boothiella tetraspora
strains with high statistical supports (97% ML/ 1.00 BYPP, Figure 2) and another strain (KUNCC22-12509) grouped
with Sordaria macrospora, S. superba, S. humana, S. tamaensis and S. nodulifera stains as basal clades (Figure 2).
From this complex group, most of the species have ITS, and LSU genes (Table 1), and show a relatively high similarity
among each species, whereas Sordaria superba (CBS 784.96), Sordria macrospora (FGSC 4818) and our isolate
(KUNCC22-12509) are the only species that have tub2 gene. In addition, after comparing the morphological details of
those species, coupled with BLASTn results, our isolate (KUNCC22-12509) was identified as a new record of Sordria
macrospora.
YANG ET AL.
256 • Phytotaxa 587 (3) © 2023 Magnolia Press
FIGURE 2. Phylogram of Sordariaceae were constructed based on combined ITS, LSU and tub2 sequences. Related sequences were
taken from Phukhamsakda et al. (2020) and Huang et al. (2021). The tree is rooted with Chaetomium globosum (CBS 160.62) and C.
microthecia (LC4685). The BI and ML bootstrap support values equal to or above 0.90 BYPP and 60 % are shown at the first and second
positions above the nodes. Type strains are in bold, and newly generated strains are in red.
Analyses 2: The concatenated ITS, rpb2 and tef1-α sequence dataset consisted of 16 strains of Trichoderma
with Protocrea farinosa (CPK 3144) and P. illinoensis (TFC 96-98) as the outgroup taxa. The concatenated sequence
matrix comprises 2361 characters, including gaps (526 characters for ITS, 918 characters for rpb2, 917 characters
for tef1-α). Phylogenetic investigation based on maximum likelihood analysis conducted the best RAxML tree with
a final likelihood value of −7114.648023, presented in Figure 3. The matrix had 251 distinct alignment patterns, with
2.98% undetermined characters or gaps. Estimated base frequencies were as follows: A = 0.253910, C = 0.258671, G
= 0.259372, T = 0.228047; substitution rates AC = 1.700109, AG = 6.575298, AT = 1.926380, CG = 1.253106, CT =
13.844508, GT = 1.000000; gamma distribution shape parameter α = 0.492218. The final average standard deviation of
THREE INTERESTING FUNGI FROM AMERICAN Phytotaxa 587 (3) © 2023 Magnolia Press • 257
split frequencies at the end of total MCMC generations was calculated as 0.009306 in BI analysis. Multi-gene analyses
showed that our strain is Trichoderma virens (KUNCC22-12508), grouped within T. virens strains with good bootstrap
support values (Figure 3).
FIGURE 3. Phylogram of Trichoderma virens and closely related species constructed based on combined ITS, tef1-α and rpb2 sequences.
The tree is rooted with Protocrea farinosa (CPK 3144) and P. illinoensis (TFC 96-98). The BI and ML bootstrap support values above 0.95
BYPP and 60 % are shown at the first and second positions above the nodes. The newly generated strain is in red.
Taxonomy
Sordariaceae G. Winter, Rabenh. Krypt.-Fl.: 162 (1885)
Boothiella Lodhi & Mirza, Mycologia 54: 217 (1962)
Index Fungorum number: IF 627
Type species: Boothiella tetraspora Lodhi & Mirza, Mycologia 54: 217 (1962)
Notes: Boothiella was a monotypic genus and established by the type species B. tetraspora, which was isolated
from soil in Lahore City, Pakistan. Boothiella is similar to Thielavia but differs by having a colourless ascomatal wall
(Lodhi & Mirza 1962). Eriksson et al. (2004) and Kirk et al. (2008) proposed Boothiella should be accommodated in
the family Sordariaceae, and this phylogenetic placement was confirmed by other researchers later (Vu et al. 2019,
Wang et al. 2019). The sexual morph was described by having a superficial to immersed, globose to subglobose,
cleistothecial ascomata, solitary to aggregated, non-ostiolate, hyaline ascomatal walled, clavate to cylindrical asci
with four-spored, short pedicellate, uniseriate, evanescent. Ascospore ellipsoidal to broad ovoid, hyaline to yellow
to olivaceous brown with the maturity, one-celled, with apical germ pores, however, no asexual morphs have been
reported (Huang et al. 2021). Thielaviella humicola and Thielavia tetraspora were recently re-identified as Boothiella
YANG ET AL.
258 • Phytotaxa 587 (3) © 2023 Magnolia Press
tetraspora based on evidences of morphology and phylogeny (Wang et al. 2019). This genus has a single species (B.
tetraspora), and most collections were isolated from soil (Wang et al. 2019).
Boothiella tetraspora Lodhi & J.H. Mirza, Mycologia 54(2): 217 (1962) (Figure 4)
Index Fungorum number: IF 327083
FIGURE 4. Boothiella tetraspora (HKAS 125767). a Colony on PDA for one month. b, c Close-up of ascomata. d Hyphae. e, f Ascomata.
g–k Asci. l–o Ascospores. Scale bars: f = 300 μm; e = 200 μm; g–k = 50 μm; d, m–o = 20 μm; i = 10 μm.
THREE INTERESTING FUNGI FROM AMERICAN Phytotaxa 587 (3) © 2023 Magnolia Press • 259
Isolated from intestinal contents of dead American bullfrog larvae. Sexual morph on PDA: Mycelium 3–5 μm wide,
hyaline to yellow pale, thick-walled, branched, septate, with granules. Ascomata 260–370 × 240–330 μm (x =245 ×
285 μm, n=10), globose to subglobose, solitary or gregarious, superficial to immersed on PDA, brown to dark brown or
black, membranaceous, glabrous, without ostiolate. Ascomatal wall composed of membranaceous, subhyaline to pale
brown cells of textura angularis. Hamathecium not observed. Asci 80–100 × 15–20 μm (x =87.5 × 15.5 μm, n=20),
unitunicate, four-spored, clavate to broadly cylindrical, apically round, short pedicellate, furcate pedicel, evanescent.
Ascospores 20–30 × 10–20 μm (x = 24 × 14.5 μm, n=20), ovoid to ellipsoidal, hyaline to yellowish brown to dark
brown, aseptate, rough to verruculose, sometimes visible a germ pore at one or each end, without sheath. Asexual
morph: Undetermined.
Culture characteristics: Colonies growing on PDA reach 30–40 mm in diameter after one week at 27 °C, forming
the brown to dark brown fruiting bodies within one month in PDA. Obverse: flat, velvety, hairy, pale brown, entire
edge. Reverse: yellowish brown. Without pigments produced in PDA.
Known substratum: Soil (Lodhi & Mirza 1962, Wang et al. 2019); Sand (Wang et al. 2019); Intestinal contents
of dead American bullfrog larvae (this study).
Known Distribution: Pakistan (Lodhi & Mirza 1962); Spain, India (Wang et al. 2019), China (This study).
Material examined: China, Yunnan Province, Qujing Normal University, intestinal contents of dead American
bullfrog larvae, GPS: 103°44’35”E, 25°30’46”N, 1856.6 m, Wen-hua Lu, ER4, (Herb. HKAS 125767), living culture
KUNCC22-12510.
Notes: Based on detailed descriptions of Boothiella tetraspora, our isolate KUNCC22-12510 fits with previous
strains in morphology, forming brown to dark brown ascomata, with globose to subglobose, superficial to immersed,
non-ostiolate, additionally, both strains almost have the same size of asci and ascospores, with four-spored asci, ovoid to
ellipsoidal conidia (Lodhi & Mirza 1962, Wang et al. 2019). Phylogenetically, our isolate KUNCC22-12510 clustered
with Boothiella tetraspora (CBS 334.67 and CBS 887.97) with reliable statistical supports (99%ML/1 BYPP, Figure 2).
The BLASTn results of ITS, LSU, tub2, and rpb2 region demonstrate 99–100% similarity with Boothiella tetraspora
strains (CBS 334.67 and CBS 887.97). Therefore, our isolate KUNCC22-12510 is identified as B. tetraspora, a new
host and country record based on morphological features combined with phylogeny evidence.
Sordaria Ces. & De Not., Commentario della Società Crittogamologica Italiana 1 (4): 225 (1863)
Index Fungorum number: IF 5061
Type species: Sordaria fimicola (Roberge ex Desm.) Ces. & De Not., Commentario della Società Crittogamologica
Italiana 1 (4): 226 (1863)
Notes: Sordaria was introduced with the type species S. fimicola, and 264 records are listed in Index Fungorum
(2023), but only 23 species have available sequence data (Huang et al. 2021). The sexual morph of Sordaria was
characterized by having sub-immersed to superficial, perithecioid, coriaceous ascomata, contain with cylindrical
paraphyses, oblong, upright to slightly curved asci, with a lobate pedicel and distinct apical ring, with eight-spored,
uniseriate, ellipsoidal to ovoid, verruculose ascospore, immature ascospore enclosed in a hyaline gelatinous sheath,
however, sometimes thick and conspicuous to even difficult to detect (Ivanová et al. 2015; Phukhamsakda et al. 2020).
Species of Sordaria are commonly found in dung, soil, and seed pods (Furtado 1969; Watanabe 1989; Mungai et al.
2012). Notably, the filamentous ascomycete Sordaria macrospora was considered a model organism to study the
molecular mechanisms that regulate fruiting bodies’ development (Teichert et al. 2020).
Sordaria macrospora Auersw., Fungi Europaei exsiccati, Klotzschii herbarii vivi mycologici continuatio. Editio nov.
Series secunda. Cent. 10: no. 954 (1866) (Figure 5)
Index Fungorum number: IF 237763
Isolated from intestinal contents of dead American bullfrog larvae. Sexual morph on PDA: Ascomata 340–420 ×
270–380 μm (x = 330 × 380 μm, n=20), semi-immersed to superficial, perithecial, solitary or gregarious, ampulliform
or pyriform, globose, coriaceous, brown to black, ostiolate. Neck 140–180 × 85–120 μm (x = 160 × 99 μm, n=10)
central, oblong, straight to bent, extended, blunt ends. Peridium consist of dark-brown, thick-walled cells of textura
angularis. Hamathecium 6–15 wide (x = 9.5 μm, n=20), numerous, wide, branched, septate, markedly constricted at
the septa, hyaline, paraphyses. Asci 130–180 × 17–22 μm (x = 155×19 μm, n=20), unitunicate, eight-spored, slenderly
cylindrical, with a lobate pedicel and a prominent J- ring. Ascospores 20–30 × 15–20 μm (x =25.5 × 15.5 μm, n=20),
one-celled, ellipsoidal to ovoid, uniseriate, hyaline to golden to brown to black when mature, guttulate, with some
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260 • Phytotaxa 587 (3) © 2023 Magnolia Press
prominent oil droplets, with a thin gelatinous sheath, acute to round ends, with a basal germ pore. Asexual morph:
Undetermined.
Culture characteristics: Colonies growing on PDA reach around 60 mm in diameter after one week at 27 °C,
forming dark brown fruiting bodies within one month in PDA. Obverse: flat, white, entire edge, peripheral fertile.
Reverse: pale brown. Without pigments produced in PDA.
Known substratum: Pinus sylvestris, Fagus sylvatica (Petrini & Fisher 1988); Salix fragilis, Quercus robur
(Petrini & Fisher 1990); Olea europaea (Fisher et al. 1992); Eucalyptus nitens (Fisher et al. 1993); Dactylis glomerata
(Sanchez Marquez et al. 2007); Herbivore dung (Lytvynenko & Hayova 2018); Intestinal contents of dead American
bullfrog larvae (this study).
Known Distribution: England (Petrini & Fisher 1998,1990); Balearic Islands (Fisher et al. 1992); Australia
(Fisher et al. 1993); Spain (Sanchez Marquez et al. 2007); Ukraine (Lytvynenko & Hayova 2018); China (This
study).
Material examined: China, Yunnan Province, Qujing Normal University, on intestinal contents of dead American
bullfrog larvae, GPS: 103°44’35”E, 25°30’46”N, 1856.6 m, Wen-hua Lu, ER3, (Herb. HKAS 125766), living culture
22-12509.
Notes: Sordaria macrospora is a coprophilic homothallic pyrenomycete, a fungal model organism in biology that
was first described in 1866 by Auerswald. Our isolate fits the concept of S. macrospora by having elongated neck,
oblong asci with a “J-” apical ring, enwrap eight hyaline to golden to dark brown ascospores, ascospores uniseriate,
visible distinct oil droplets when its young, present verruculose surface when it is mature, without gelatinous sheath
(Lord & Read 2011). The BLASTn results of ITS, and LSU showed our isolate (KUNCC22-12509) are highly similar
to Sordaria species (Sordaria macrospora CBS:346.62, S. humana CBS:416.82, S. fimicola CBS:566.67, S. lappae
CBS:154.97, S. tamaensis and S. nodulifera NBRC:32551), with 99–100% similarity. However, the tub2 gene is
mostly similar to Sordaria macrospora (FGSC 4818) and S. superba (CBS 784.96) with 99% similarity, the tub2
gene of other species is not available (Table 1). From morphology, Sordaria superba is different, as the ascospores are
wrapped by a distinct gelatinous sheath with a single basal germ pore (Yul et al. 2010), our isolate is more similar to S.
macrospora (ascomata: 370–400 (500) × 250–300 µm VS. 340–420 × 270–380 μm; and asci: 160–175 ×20 VS. 20–30
× 15–20 μm) (Ivanová et al. 2015). The phylogenetic tree indicated that our Sordaria macrospora KUNCC22-12509
is complex with the other species that have high similarity in ITS, and LSU genes (Figure 2), and this problem may be
caused by lacking tub2 genes, therefore, our isolate KUNCC22-12509 is identified based on morphological features
(Table 2) coupled with BLASTn results and phylogenetic analyses as Sordaria macrospora which is a new host and
country record.
TABE 2. The ascospore comparison of Sordaria species.
Species Ascospore Reference
S. macrospora 20–31 × 13–22 μm, with gelatinous sheath, brown to dark brown, without a distinct
basal germ pore. Ivanová et al. (2015, 2018)
S. superba 23–27 × 15–17 μm, dark brown, with a single basal germ pore and surrounded by a
gelatinous sheath and smooth-walled ascospores. Yul et al. (2010)
S. humana
23–27 × 17–19 μm, uniseriate, without gelatinous sheath, hyaline to golden when
unmature, atro-viridibus to dark brown later stages, sometimes visible a distinct oil
droplet in each ascospore.
Uecker (1976)
S. tamaensis 15–20 × 7.2–12.5 μm, obliquely uniseriate or occasionally biseriate, dark brown,
surrounded by a gelatinous sheath, apiculate and/or with a germ pore at one end. Watanabe (1989)
S. nodulifera 17–24 × 14–22 μm, obliquely uniseriate, ellipsoidal, black, usually with a single basal
germ pore and surrounded by a gelatinous sheath, often with one to four nodules. Watanabe (1989)
THREE INTERESTING FUNGI FROM AMERICAN Phytotaxa 587 (3) © 2023 Magnolia Press • 261
FIGURE 5 Sordaria macrospora (HKAS 125766). a–c Colony on PDA for one month. d Mature ascomata. e Undeveloped ascomata. f,
g Close-up of ascomata. h Peridium. i Paraphyses. j “J-” apical ring. k Ascus. l–p Ascospores. Scale bars: d = 200 μm, e, h, i, k = 50 μm,
l–q = 20 μm, j = 10 μm.
Hypocreaceae De Not., Giornale Botanico Italiano 2: 48 (1844)
Trichoderma Pers., Neues Magazin für die Botanik 1: 92 (1794)
Index Fungorum number: IF 10282
Type species: Trichoderma viride Pers., Neues Magazin für die Botanik 1: 92 (1794)
Notes: Trichoderma has been known since at least the 1920s for their antagonistic properties to act as biocontrol
agents against plant pathogens (Harman 2006). Recently, 495 records have been listed in the index Fungorum (2023).
Trichoderma are soilborne, green-spored ascomycetes, these fungi are often isolated from forest or agricultural soils
worldwide and present typical green sporulation in vitro (Brotman et al. 2010, Zhang et al. 2022). Some strains of
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262 • Phytotaxa 587 (3) © 2023 Magnolia Press
Trichoderma are opportunistic plant symbionts, that effectively colonize roots, and have evolved multiple mechanisms
to increase plant growth and productivity (Harman 2000, 2006). Moreover, Trichoderma is a strong mycoparasite,
and shows excellent antagonistic properties to other fungi to produce antibiotics that affect other microbes (Weindling
1934, Weindling & Fawcett 1936). Therefore, Trichoderma is an ideal biocontrol agent in agriculture. Especially,
Trichoderma reesei is famous as an industrially important cellulolytic filamentous fungus that produced second-
generation biofuels from cellulosic waste (Schuster & Schmoll 2010).
Trichoderma virens (J.H. Mill., Giddens & A.A. Foster) Arx, Beih. Nova Hedwigia 87: 288 (1987) (Figure 6)
Index Fungorum number: IF 128198
FIGURE 6 Trichoderma virens (HKAS 125765). a Colony on PDA for one month. b–d Close-up of colonies. e, f Undeveloped conidia
and conidiogenous cells. g, h Mature conidia with conidiogenous cells. i Chlamydospores stained by congo red agents. j Conidia. Scale
bars: g, h = μm; i = 20 μm; e, f = 15 μm; j = 10 μm.
THREE INTERESTING FUNGI FROM AMERICAN Phytotaxa 587 (3) © 2023 Magnolia Press • 263
Isolated from intestinal contents of dead American bullfrog larvae. Sexual morph: Undetermined. Asexual morph
on PDA: Aerial mycelium abundant on PDA, fast-growing, forming cream-yellow to green sporulation with maturity,
with a distinctive odour, sometimes producing a farinose to granular mat. Conidiophores 20–30 μm high, irregularly
branched in a dendriform structure. Vegetative hyphae 4–8 μm wide (x = 6 μm, n = 30), branched, hyaline, smooth
and thick-walled, septate, narrow and flexuous, terminal branched, often curved. Conidiogenous cells 5.5–8 × 3–5
μm (x = 6.5 × 4 μm, n = 20), occurring in lateral and terminal clusters, pyramidal. Conidia 4–6 × 3–4 μm (x = 5 ×
3.5 μm, n = 20), catenated, obovoid to globose, hyaline to olivaceous, delicately roughened, aseptate, smooth-walled.
Chlamydospores 8–11 × 7–9 μm (x = 9.5 × 8.5 μm, n = 20), hyaline, thick-walled, globose to subglobose, terminal.
Culture characteristics: Colonies growing on PDA reach 70–80 mm in diameter after one week at 27 °C, forming
the hyaline to olivaceous to green sporulation in PDA. Obverse: aerial, fluffy, hyaline mycelium, peripheral fertile,
creamy green to dark green. Reverse olivaceous to pale brown. Without pigments produced in PDA.
Known substratum: Theobroma cacao (Hanada et al. 2010), Betula pendula & Pinus sylvestris (Mulenko et al.
2008), Soil samples (Arx 1987, Kindermann et al. 1998, Chaverri et al. 2001, Zeng et al. 2016), intestinal contents of
dead bullfrog larvae (This study).
Known distribution: Unites States (Chaverri et al. 2001, Arx 1987), New Zealand (Kindermann et al. 1998),
Poland (Mulenko et al. 2008), Brazil (Hanada et al. 2010), China (Zeng et al. 2016, This study).
Material examined: China, Yunnan Province, Qujing Normal University, intestinal contents of dead American
bullfrog larvae, GPS: 103°44’35”E, 25°30’46”N, 1856.6 m. Wen-hua Lu, ER2 (Herb. HKAS 125765) living culture
KUNCC22-12508.
Notes: Morphologically, our isolate KUNCC22-12508 well fits the concept of Trichoderma virens by fast-
forming typical green sporulation in vitro, with obovoid, hyaline to olivaceous, slightly roughened conidia (Arx 1987,
Chaverri et al. 2001). In addition, its chlamydospores were easily formed between the hyphae or formed terminally in
the hyphae tip, similar results were also observed by Abd-Aziz et al. (2008). Phylogenetically, KUNCC22-12508 has
placed within Trichoderma virens strains, and separated well with neighbor species T. crassum and T. neocrassum with
high statistical supports (100%ML/1 BYPP) (Figure 3). Moreover, the BLASTn results of ITS, LSU, tef1-α and rpb2
expect our isolate KUNCC22-12508 100% similar to Trichoderma virens strains (LZ002, LZ012, Z051). Therefore,
our isolate KUNCC22-12508 is identified as Trichoderma virens, a new host record based on morphological features
and phylogenetic evidence.
Discussion
The American bullfrog is a common carrier of the fungal pathogen Batrachochytrium dendrobatidis. Batrachochytrium
dendrobatidis is a causative agent of chytridiomycosis and is the key driver in the extinction of amphibians globally
(Goraya et al. 2000). In addition, frogs are susceptible to bacterial diseases which could cause high mortality, such as
the red-leg syndrome provoked by Aeromonas hydrophila and Proteus vulgaris (Glorioso et al. 1974, Cunningham
et al. 1996, Pasteris et al. 2006). The other bacteria also commonly infect bullfrogs like Edwardsiella tarda and
Flavobacterium spp. (Mauel et al. 2002). Currently, many strategies have been proposed to alleviate Batrachochytrium
dendrobatidis (Bd) and bacterial diseases outbreaks, while few have been successful, the use of probiotic formulations
has shown as an effective control strategy by killing or inhibiting Bd and bacteria (Walke et al. 2015). Niederle et al.
(2019) found skin-associated lactic acid bacteria (Enterococcus gallinarum, Lactococcus garvieae and Pediococcus
pentosaceus) from American bullfrogs show as potential biocontrol agents of Bd. In addition, Lee et al. (2009)
investigated American bullfrog-related microbes and isolated 40 bacteria (Aeromonas spp., Edwardsiella spp.,
Flavobacterium spp. and Vibrio spp.) from the internal organs of the American bullfrog, and properties of antibiotic
resistances and heavy metal resistances among these isolates were screened.
In this study, we isolated the Boothiella tetraspora (KUNCC22-12510) from intestinal contents of American
bullfrog larvae. Boothiella tetraspora has been found in soil samples, and morphologically, the species is easily
distinguished from other genera in Sordariaceae by non-ostiolate ascomata and four-spored asci, with ocular chamber
(Lodhi & Mirza 1962). One new isolation of Sordaria macrospora was isolated from the intestinal contents of American
bullfrog larvae. Sordaria is the second largest group in Sordariaceae, which is normally isolated from dung, soil, and
seed pods. Eighty-four records are found in fungal databases, and those isolates were mostly reported from European
countries, India, and Japan, and without any record from China, while seven records of S. macrospora were described
from various hosts in Australia, England, Spain, and Balearic Islands (Farr & Rossman 2022). Sordaria species are
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264 • Phytotaxa 587 (3) © 2023 Magnolia Press
easily sporulated in vitro, forming dark black ascomata, eight-spored asci, and form hyaline ascospores that turn
from golden to brown black. Therefore, Sordaria species were considered an ideal model for studying the molecular
mechanisms that regulate fruiting body development (Teichert et al. 2020). However, further molecular studies on the
complex group are necessary, as many species of this genus lack tub2 gene.
In this study, Trichoderma virens from intestinal fungi of American bullfrog larvae was isolated and is the first
report from American bullfrog. Previously, Trichoderma are widely isolated from soil, plants, or as plant endophytes
(Farr & Rossman 2022). Trichoderma as functional fungi have been applied to control plant diseases caused by
pathogens and pests (Woo et al. 2014, Zin et al. 2020). In addition, Trichoderma easily colonizes the plant root
ecosystem, and it can potentially promote Arabidopsis growth (Brotman et al. 2010, López-Bucio et al. 2015).
Acknowledgements
We are grateful to the Center for Yunnan Plateau Biological Resources Protection and Utilization, College of
Biological Resource and Food Engineering, Qujing Normal University for providing the facilities for morphological
and molecular experiments. Er-fu Yang and Itthayakorn Promputtha thank the Faculty of Science and Graduate School,
Chiang Mai University for supporting M.Sc. Scholarship. Dong-Qin Dai would like to thank the National Natural
Science Foundation of China (No. NSFC 31760013) and High-Level Talent Recruitment Plan of Yunnan Provinces
(“Young Talent” Program) for support. Samantha C. Karunarathna thanks National Natural Science Foundation of
China grant number 32260004 for the support.
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