A new record of psychrotrophic Paecilomyces formosus ( Eurotiales : Ascomycota ) from India : morphological and molecular characterization

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.


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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 (34.371 0 N, 76.197 0 E) 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 (34.352 0 N, 76.076 0 E) and lake (34.352 0 N 76.076 0 E; 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 o C 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.

Isolation of fungal isolates
For fungal isolation, soil samples were collected by scraping the superficial layer, not exceeding 3-5cm J TT 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 o C 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 o C 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.

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)   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.   , 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.

Reference
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,

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the current Paecilomyces isolate is a psychrotolerant or psychrotrophic with the capability to survive and surmount extremely low temperatures (up to -35 o C) 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 -35 o C to 50 o C. This indicates that further research in extreme habitats may unveil the diversity and distribution of described and undescribed fungal species.

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