Journal of Threatened Taxa |
www.threatenedtaxa.org | 26 November 2021 | 13(13): 20118–20123
ISSN 0974-7907 (Online) | ISSN 0974-7893
(Print)
https://doi.org/10.11609/jott.6967.13.13.20118-20123
#6967 | Received 09 December 2020 | Final
received 09 November 2021 | Finally accepted 12 November 2021
A new record of psychrotrophic Paecilomyces
formosus (Eurotiales:
Ascomycota) from India: morphological and molecular characterization
Skarma Nonzom
1 & Geeta Sumbali
2
1,2 Department of Botany, University
of Jammu, B.R. Ambedkar Road, Jammu, Jammu & Kashmir 180006, India.
1 skarmanunzom@yahoo.com
(corresponding author), 2 geetasumbalippl@yahoo.co.in
Editor: Richard Mibey,
Nairobi, Kenya. Date of publication: 26
November 2021 (online & print)
Citation: Nonzom,
S. & G. Sumbali (2021). A new record of psychrotrophic Paecilomyces
formosus (Eurotiales:
Ascomycota) from India: morphological and molecular characterization. Journal of Threatened Taxa 13(13): 20118–20123. https://doi.org/10.11609/jott.6967.13.13.20118-20123
Copyright: © Nonzom
& Sumbali 2021. Creative Commons Attribution
4.0 International License. JoTT allows unrestricted use, reproduction, and distribution
of this article in any medium by providing adequate credit to the author(s) and
the source of publication.
Funding: UGC-Rajiv
Gandhi National Fellowship (RGNF) F117.1/2012-13/RGNF-2012-13-ST-JAM18891/
Competing interests: The authors
declare no competing interests.
Acknowledgements: The first author is grateful to
the University Grants Commission (UGC), New Delhi for the financial assistance
in the form of Rajiv Gandhi National Fellowship (RGNF), which facilitated the
study. The authors are also thankful to UGC SAP DRS-II for providing laboratory
facilities.
Abstract: A filamentous fungus
Paecilomyces formosus
(Eurotiales, Ascomycota) was detected for the first
time from the region while surveying fungal diversity of a cold arid
high-altitude pass (4,000 msl) located in Kargil district (Ladakh), India.
The fungal isolate was characterized morphologically with camera lucida drawings and microphotographs, and identified using
internal transcribed spacer (ITS) ribosomal DNA sequences. P. formosus has not been reported from India, or
from arid/semi-arid/cold regions before, thus this represents a new record of
Indian hot/cold desert mycoflora that is psychrotrophic in contrast to the more common thermophilic
fungi.
Keywords: Fungal diversity, Kargil district, Indian mycoflora,
internal transcribed spacer, new record, taxonomy.
The genus Paecilomyces
(Eurotiales, Ascomycota) was first
described by Bainier in 1907, and established as
closely related to the genus Penicillium. Nevertheless, these genera
differ in many aspects, such as colony and spore colour (green in Penicillium,
white, pink, buff or other colours besides green in Paecilomyces),
phialide shape and form of conidiophores. Later, based on morphological
characters, Brown & Smith (1957) and Samson (1974) provided comprehensive
monographs of Paecilomyces with a number of
additions including the sexual stages of several species. Luangsa-ard
& Hywel-Jones (2004) used molecular approaches with 18S rDNA sequencing in
phylogenetic studies of Paecilomyces sensu lato. Similarly, Samson et al. (2009) combined data
from the internal transcribed spacer (ITS) region and β-tubulin and calmodulin
genes and extrolite profiles, and provided detailed
taxonomy and comprehensive description of nine accepted taxa (five sexual
morphs and four asexual morphs).
Sapi La (34.3710N, 76.1970E)
is a high altitude pass (4,000 m) between two villages located in Kargil district in the trans-Himalayan region that is well
known for the Sapi glacier (34.3520N,
76.0760E) and lake (34.3520N 76.0760E; Image 1
a–d). During a mycological survey of this barren pass, which experiences
continual strong winds, low temperatures (below 20 ºC during summer and 0 to
-35 oC in winter) and high UV radiation
throughout the year, more than 30 psychrotolerant
fungi were recovered. Of these, a rare microfungus
belonging to the genus Paecilomyces (P. formosus syn. P. maximus) was
detected, which is being reported for the first time from India. In this
report, we describe the characteristics of this cold desert isolate.
Materials and Methods
Isolation of fungal isolates
For fungal isolation, soil
samples were collected by scraping the superficial layer, not exceeding 3–5cm
in depth, with sterilized spatulas in pre-sterilised polythene bags, and
brought to the laboratory within 18 hours. Prior to fungal isolation the
samples were stored in refrigerator at -4 oC
for 24–48 hours. Fungal isolation was performed using the dilution pour plate
method with modified Czapek Dox Agar (CDA)
supplemented with Rose Bengal (0.1mg/100ml) and streptomycin sulphate
(5mg/100mL). The plates were incubated at 25 oC
for 7–14 days in a BOD incubator. The morphologically different fungal isolates
were further plated on potato dextrose agar (PDA) and malt extract agar (MEA)
plates, and then incubated for 3–7 days at 25°C. The pure fungal isolates of
DI1A (Desert Isolate 1A) were preserved and maintained on PDA slants at 4 °C
for further use.
Morphological characterization
The fungal isolate described was
cultured on MEA and PDA plates for three to four days at 25 °C. For microscopic
observations, the fungal cultures were either teased directly by using
dissecting needles and mounted on glass slides using lactophenol cotton
blue/lactophenol, or by using a transparent adhesive tape. Microscopic line
drawings were made with the aid of camera lucida
(Erma, Japan) at 400x and 1000x magnifications. Dimensions (average of at least
20 measurements) were determined for conidiophores, phialides and conidia using
an ocular micrometer. Microphotography was done using
Sony N50 camera attached to an Olympus CH 20i binocular microscope.
The isolate was identified
morphologically by following the description given by Samson et al. (2009).
Molecular characterization
DNA extraction and sequencing was carried out
at the sequencing facility of the National Centre for Microbial Resource
(NCMR), National Centre for Cell Science, Pune, India. Genomic DNA was isolated
by the standard phenol/chloroform extraction method of Sambrook et al. (1989).
This was followed by PCR amplification of the ITS regions using universal
primers ITS1 [5’-TCC GTA GGT GAA CCT GCG G -3’] and ITS4 [5’-TCC TCC GCT TAT
TGA TAT GC-3’] (White et al. 1990). The amplified PCR product was purified by
PEG-NaCl precipitation and directly sequenced on an
ABI® 3730XL automated DNA sequencer (Applied Biosystems, Inc., Foster City, CA)
sequencing was carried out from both ends so that each position was read at
least twice. Assembly was carried out using Lasergene
package followed by NCBI BLAST against sequences from type material for
tentative identification (Boratyn et al. 2013). The
confirmed sequences were submitted to Genbank,
National Centre for Biotechnology Information (NCBI), Maryland, USA to obtain
GenBank accession number-MK255020.
The construction of phylogenetic
trees was accomplished by maximum-likelihood method implemented in the program
MEGA version 6 with 500 bootstrap replicates (Figure 1). Sequences were
retrieved from GenBank based on their closest related species showing maximum
identity.
Colonization index of the
recovered fungal species
Percentage colonization frequency
(CF%), A/F ratio, abundance and cfu /g were
calculated for the isolated fungal species using formulae given in Table 1.
Results and
Discussion
Taxonomic notes
Paecilomyces formosus Sakag., May. Inoue & Tada ex Houbraken
& Samson, in Samson, Houbraken, Varga & Frisvad, Persoonia 22:
21 (2009)
Ecology and distribution of the
species: Tropical soil, subtropical soil, sponge, wood, air and pot plant soil
in Denmark (Samson et al. 2009); current isolate examined: India,
trans-Himalaya, Kargil district, Sapi
La, isolated from a high altitude extreme habitat, July 2017
Characteristics of the cold
desert isolate- Asexual stage; sexual stage-not observed
Morphological
identification
Colony characters
Colony characters of the fungal
isolate Paecilomyces formosus
desert isolate 1 (DI1A) are depicted in Image 2a. Colonies on PDA show fast
growth, initially light buff, plane, later turning golden yellow to dark
yellow, becoming powdery as spores are produced, reaching a diameter of 25 to
30 mm within 3–4 days at 25 oC; reverse
pale buff.
Micromorphology
Hyphae branched, hyaline, 2.8–5.6
µm in width; conidiophores simple to irregularly branched, Penicillum-like,
arising from simple or funiculose hyphae; metulae 7.0–8.4 × 4–2.8 µm; phialides cylindrical, slightly
swollen at the base with a long tapering narrow zone, sometimes tapering
slightly at the extreme apex, measuring 9.8–21 × 2.5–2.8 µm (Image 2b–e);
conidia variable in shape and size, ovate to fusoid,
hyaline and small when young; large, yellow, mostly with pointed to rounded
apex and truncate base when mature, measuring 4.9–9.1 × 2.1–4.2 µm,
smooth-walled, in exceedingly long chains (Image 2b–e, shown in arrows).
Molecular identification
Blast analysis of the ITS region
(700 bp) showed its closest similarity to the type
material Paecilomyces formosus
Samson et al. (2009) (GenBank: NR_149329.1; E-value 0; identity: 96.36%
and coverage: 100%). Phylogenetic analysis of the sequences of the current
isolate DI1A (GenBank: MK255020) based on combined sequences of 15 selected
isolates of closest type strains confirmed that our isolate forms a strongly
supported clade (99% bootstrap value) with P. formosus
(Figure 1). Aspergillus was used
as outgroup.
Discussions and
Recommendation
During a mycological survey of a
high altitude pass located in the trans-Himalayan region, a psychrotrophic
Paecilomyces isolate DI1A was recovered which
represents a new record to Indian and desert fungi. P. fusisporus was detected earlier from cold desert in
Himachal Pradesh by Sagar et al. (2007). Similarly,
Kotwal & Sumbali (2011) reported three species of
Paecilomyces, viz., P. lilacinus,
P. marquandii and P. variotii
from a similar high altitude pass (5,359 m) located in Ladakh.
From Sapi La high-altitude region, Nonzom & Sumbali (2019) have
also reported another microfungus, Geosmithia rufescens,
of rare occurrence.
This identified Paecilomyces
species, described by Samson et al. (2009) as Paecilomyces
formosus (Sakag., May.
Inoue & Tada) Houbraken & Samson, comb. nov., wherein they illustrated and revised many
sexual and asexual morphs of Byssochlamys and Paecilomyces, respectively. Previously, the
genus constituted a single species, P. variotii
(Bainer, 1907). However, later a number of species
were added to this genus with some revisions and reshufflings (Brown &
Smith 1957; Samson 1974; Houbraken et al. 2006;
Samson et al. 2009). As such, P. variotii
is considered as a variable species, which has been described under diverse
names from the beginning. As discussed earlier, Samson et al. (2009) presented
an elaborated description of Paecilomyces and Byssochlamys, the latter comprising of five species
with known sexual morphs, i.e., B. fulva, B. nivea, B. spectabilis, B. zollerniae, and B. lagunculariae,
while the former included four species with only asexual morph known, i.e., P.
divaricatus, P. formosus,
P. saturatus, and P. brunneolus. Further, based on the ITS sequences and
partial β-tubulin genes, they suggested that, P. formosus
may constitute three distinct species, viz., P. formosus,
P. lecythidis, and P. maximus.
However, these three taxa appear morphologically similar and could not be
identified on the basis of microscopic and analysis of extrolites,
whereas molecular phylogeny data can prove helpful. One of the distinguishing
feature observed by Samson et al. (2009) for the P. maximus clade
and the other members of this diverse group was the rapid growth of this
species at 37˚C than at 30 ˚C and based on their study they proposed P. lecythidis and P. maximus as synonyms
of P. formosus.
As observed by Samson et al.
(2009), on growth tests on PDA and MEA, colonies of the current isolate were
also fast growing, reaching a diameter of 15–25 mm within 3–4 days of
incubation. Morphologically, the conidia were resembling the isolate described
by Samson et al. (2009) in terms of truncate shape (dominant), length, size
range (3–10 µm) and variable shapes exhibited. However, the isolate from this
study had larger conidia and were exhibiting slightly more diameter (up to 4.2
µm) compared to the results of Samson et al. (2009) (up to 3.5 µm) and in
contrast to the formation of chlamydospores that were observed to be produced
on short stalks, no such structures were observed in the current desert isolate
DI1A. In other related asexual Paecilomyces
morphs, Samson et al. (2009) observed the presence of chlamydospores (P. brunneolus and P. saturatus)
but their absence in P. divaricatus.
Formerly, P. maximus was
described to be associated with tropical and subtropical soils, wood and human
bone marrow (Samson et al. 2009). Later, this species was found to be plant
pathogenic in Iran causing dieback diseases in oak and Pistachio (Heidarian et al. 2018; Sabernasab
et al. 2019). So far, there are no reports on the incidence of this isolate
from arid or semi-arid regions and particularly from cold arid soils. Moreover,
Paecilomyces species are usually
considered thermophilic (Samson et al. 2009; Houbraken
et al. 2010; Heidarian et al. 2018). However, in
contrast, the current Paecilomyces isolate is
a psychrotolerant or psychrotrophic
with the capability to survive and surmount extremely low temperatures (up to
-35oC) along with other harsh conditions such as intense UV
radiation (4,000 m altitude), strong wind currents, low oxygen concentration,
and oligotrophic environments. Therefore, through the present investigation we
report that Paecilomyces can thrive and
sustain their activities at temperature ranging from -35oC to 50oC.
This indicates that further research in extreme habitats may unveil the
diversity and distribution of described and undescribed fungal species.
Table 1. Formulae used.
Number of soil samples colonized by a
specific fungus CF(%) = ––––––––––––––––––––––––––––––––––––––––––––––
x 100 Total number of samples studied A/F ratio =
Abundance/Colonization frequency
Total
number of colonies of a specific fungus where, abundance = –––––––––––––––––––––––––––––––––––––––––––––– Number of soil
samples colonized by a specific fungus A/F ratios describe the
distribution pattern of each fungal species into one of the following three
categories: A/F ratio of <0.025 depicts
that the fungal species has regular distribution. A/F ratio between 0.025 &
0.05 depicts that fungal species has random distribution. A/F ratio of >0.05 depicts
that the fungal species has contagious distribution. |
Reference |
Raunkiaer 1934 |
|
CFU/g = a×d/s where, a = average number of colonies on the petriplate; d= dilution factor (10,000) & s= dry weight of the soil
sample |
Parikh & Shah 2006 |
Table 2. CF%, cfu/g
calculated for P. formosus DI1A
Number of soil
samples analysed= 25 |
||||
Number of
samples detected positive |
CF (%) |
Abundance |
A/F ratio |
cfu/g |
3 |
12 |
1.00 |
0.083 |
0.3 × 103 |
For
figure and images - - click here
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