Ann Microbiol (2014) 64:397–402 DOI 10.1007/s13213-013-0646-5

SHORT COMMUNICATION

Aspergillus penicillioides—a true halophile existing in hypersaline and polyhaline econiches

Sarita Nazareth & Valerie Gonsalves

Received: 6 December 2012 /Accepted: 2 April 2013 /Published online: 25 April 2013 # Springer-Verlag Berlin Heidelberg and the University of Milan 2013

Abstract Aspergillus penicillioides is a true halophile, 2012; Nayak et al. 2012; Gonsalves et al. 2012), as well as present in diverse econiches from the hypersaline in foods such as grains, dried fruit, baked goods, salted fish, athalassohaline Dead Sea and the thalassohaline solar spices, as well as on binocular lenses and human skin salterns, to the polyhaline estuaries and mangroves of (Andrews and Pitt 1987;Tamuraetal.1999;Pittand Goa-India. Thirty-nine isolates from these environments Hocking 2009). were seen to be moderate halophiles, stenohaline or euryha- Organisms able to grow under conditions of low aw below line in nature, with comparable salt tolerance indices. They 0.85, imposed by high levels of soluble solids such as salts or had an obligate need for a low water activity and were sugars, or due to dry conditions, have been termed as unable to grow on a regular defined medium such as xerotolerant or xerophilic (Andrews and Pitt 1987; Grant Czapek Dox Agar, or on various nutrient rich agar media 2004; Pitt and Hocking 2009; Tamura et al. 1999). However, such as Malt Extract, Potato Dextrose and Sabouraud Agar; while xerophiles grow in relatively dry conditions, organisms however, growth was obtained on all these media when that grow in high osmotic environments of sugar solutions are amended with 10 % solar salt. In the absence of added salt, termed as osmophiles (Tucker and Featherstone 2011)and the conidia either did not germinate, or when germinated, those that require salt, mainly in the form of NaCl or any other distortions and lysis were seen in the short mycelial forms; salt along with a small amount of NaCl, are known as halo- on media with salt, the mycelia and vesicles appeared philes (Kushner 1978). Hence, microorganisms growing in normal. saline environments are adapted to low aw levels as well as high levels of ions, and are described as halotolerant or halo- Key words Aspergillus penicillioides . Dead Sea . Estuary . philic, rather than merely xerotolerant or xerophilic (Grant Mangroves . Salterns 2004). Gymnascella marismortui (Buchalo et al. 1998), Wallemia ichthyophaga (Zalar et al. 2005; Gunde-Cimerman et al. 2009), Trichosporium (El-Meleigy et al. 2010), Findings Aspergillus penicillioides and Aspergillus unguis (Nazareth et al. 2012) have thus far been reported to be obligate Aspergillus penicillioides was first described by Spegazzini halophiles. in 1896 and is strictly asexual (cited in Tamura et al. 1999). This paper reports Aspergillus penicillioides as a true

Its growth is favoured by low water activity (aw), and it can halophile, present in diverse econiches from hypersaline to grow even at an aw of 0.68, which is inhibitory for most polyhaline systems, from athalassohaline to thalassohaline fungi (Tamura et al. 1999;PittandHocking2009). A. environments, and covering a longitudinal distance of ap- penicillioides has been found in diverse habitats of low aw, proximately 38.5° on the Asian continent. such as the Dead Sea, solar salterns, mangroves, estuary In this study, 39 strains of A. penicillioides isolated (Wasser et al. 2003; Butinar et al. 2011; Nazareth et al. previously from the Dead Sea—2 from water (DSw) and 12 from sediment (DSs) samples (Nazareth et al. 2012), from the estuary of Mandovi, Goa, on the West Coast of * : S. Nazareth ( ) V. Gonsalves the Indian peninsula; 16 from surface and bottom waters Department of Microbiology, Goa University, Taleigao Plateau, Goa 403206, India (EMws and EMwb); 5 from sediment (EMs) samples e-mail: [email protected] (Gonsalves et al. 2012); 3 from water samples from 398 Ann Microbiol (2014) 64:397–402 mangroves of Ribander, Goa (MRw) and 1 from solar by MRw207. The other 19 isolates are shown individually. salterns at Santa Cruz (SCw), Goa, India (Nayak et al. The salinity of the water or sediment sample from which the 2012)—were tested. strains had been isolated, as recorded earlier (Nazareth et al. The salt tolerance index of the isolates was obtained from 2012; Gonsalves et al. 2012; Nayak et al. 2012), is shown in halotolerance curves performed in triplicate on CzA at Fig. 1b. 30 °C, as given earlier (Nazareth et al. 2012). As the stan- The results indicate that most of the isolates tested had a dard deviation in the salt tolerance curves was negligible, an minimum salt requirement of 5 % for growth, while a few average of the readings obtained was used to determine the could grow in the presence of 2 % salt, and a few required tolerance index, calculated as the ratio of growth in terms of 10 %, which clearly demonstrated their true halophilic na- colony diameter after 7 days of incubation at 2 %, 5 %, ture. The salt tolerance indices of most of the isolates were 10 %, 15 %, 20 %, 25 % and 30 % concentrations of solar above 0.5 at salt concentrations of 5–20 %, with the major- salt, to the maximal growth obtained at a salt concentration ity growing optimally at a salt concentration of 10 % and a of 5 % or 10 %. few at 5 %, irrespective of the econiche from which they Conidial suspensions of the selected isolates were pre- were isolated, and were therefore termed as moderate halo- pared in 10 % saline containing 0.05 % Tween 80 and 103 philes, as defined by Kushner (1978). Some of these were spores in 5 μl were spot inoculated, in triplicate, on Czapek euryhaline in nature, able to adapt to a wide range of salt Dox Agar (CzA), Malt Extract Agar (MEA), Potato concentrations, while a few were stenohaline, showing Dextrose Agar (PDA) and Sabouraud Agar (SA), (HI growth over only a short range of salt concentrations. Media, Mumbai, India), each without and with 10 % solar A significant difference (P<0.05) was obtained in the salt (S), to confirm the obligate requirement of salt for tolerance index of each isolate at different salt concentra- growth. Growth was measured in terms of colony diameter tions. While there was similarity (P>0.05) in the tolerance after 7 days incubation at 30 °C. index from amongst isolates of a given econiche such as the Conidial suspensions were spot-inoculated on CzA with- Dead Sea and the mangroves, differences were obtained out salt and on CzA+10 % solar salt (S-CzA) and incubated among isolates from different points along the estuary, al- at 30 °C for 15 days. Wet mounts of the isolates prepared in though there was similarity within a given station. This was 1:1 lactophenol cotton blue dye (HI Media) were then due to the fact that the isolates from Stations 5–9 along the viewed microscopically for morphological changes in the estuary showed a better salt tolerance, up to 25 % salt, or mycelia and conidiating structures; where growth was not even 30 % salt by one isolate, although the salinity was not visible on agar media without salt, an agar plug was used for very high. The estuary at these stations is bordered with microscopic examination. luxuriant mangroves. In estuarine ecosystems, the detritus Gene sequence analysis was performed on the 18S rRNA and marsh vegetation constitute a major part of the organic partial sequence; ITS1, 5.8S rRNA gene and ITS2, complete content (Manoharachary et al. 2005). This would serve as a sequence; 28S rRNA, partial sequence (Merck-GeNei source of nutrients for uptake and/or synthesis of compatible Services, Bangalore, India). The primers used were ITS1-F: solutes, thus leading to a high tolerance to salt (Gonsalves et CTT GGT CAT TTA GAG GAA GTA A and ITS4 R: TCC al. 2012). This higher level of salt tolerance was also seen in TCC GCT TAT TGA TAT GC. The PCR conditions were 1 isolates obtained from the mangroves, but not in isolates cycle of denaturation at 94 °C (5 min), followed by 35 cycles from the Dead Sea or from salterns, which would be com- of denaturation at 94 °C (1 min), annealing at 55 °C (45 s) and paratively nutrient depleted. It has been reported that higher extension at 72 °C (90 s) and holding at 72 °C (10 min). The nutritional levels appeared to overcome the inhibitory effect sequences were deposited with GenBank and accession num- of salinity (Borut and Johnson 1962). bers were obtained. The entire sequence was used to acquire The isolates tested were selected on the basis of the sequence similarities using NCBI BLAST and the nucleic acid econiche from which they were isolated and/or variations databases. in their characteristics of salt tolerance: DSw22 and DSs 40 The salt tolerance indices of the isolates are shown in were from the Dead Sea water and sediment, respectively, Fig. 1a; where there was a close similarity in the salt toler- stenohaline in nature, with a narrow range of salt tolerance, ance curve, these isolates were grouped and are represented and with a different minimal solar salt concentration re- by a graph of one of the isolates. Thus DSs56 and DSs30 quired for growth of 5 % and 10 %, respectively. were represented by DSs30; DSs32, DSs38 and DSs42 by EM6s137 and EM8ws146, both from the Estuary of the DSs38; DSs28 and DSs46 by DSs46; EM5ws130, Mandovi, and MRw207 from mangroves, were euryhaline, EM6ws133, EM8wb149, EM9wb155 by EM5ws130; having a wider range of salt tolerance from 5 % to 25 %, EM7wb142 and EM7wb143 by EM7wb143; EM7ws144 with EM8ws146 able to tolerate up to 30 % salt—the only and EM7ws145 by EM7ws145; EM4wb121, EM7wb141 isolate obtained with this range of salt tolerance—and and EM9wb153 by EM9wb153; MRw201 and MRw207 MRw207 requiring a minimal salt concentration of 2 %. Ann Microbiol (2014) 64:397–402 399

Fig. 1 a Salt tolerance index of 1 isolates of Aspergillus A penicillioides from different econiches; b Salinity of the 0.5 water and sediment sample 0 DSw6 DSw22 DSs30 DSs34 DSs36 DSs38 DSs40 DSs44 DSs46 DSs54

1

0 EM2wb107 EM4ws118 EM4ws120 EM4wb125 EM5ws130 EM6s137 EM7ws139

1

0.5 Salt Tolerance Index Salt Tolerance

0 EM7wb143 EM7s145 EM8ws146 EM8ws147 EM8ws148 EM8s153 EM9s156

1

0.5

0 MRw 204 MRw 207 SCw 255 Salt concentration (%) Isolate number B Sampling site and Isolate number *Salinity ‰ Dead Sea (DS) Water: DSw6, DSw22 370 Sediment: DSs28, DSs30, DSs32, DSs34, DSs36, DSs38, DSs40, DSs42, DSs44, 450 DSs46, DSs54, DSs56 Estuary of Mandovi (EM)

Station 2 water: EM2wb107, 35

Station 4 water: EM4ws118, EM4ws120, EM4wb121, EM4wb125 33

Station 5 water: EM5ws130, 31

Station 6 water: EM6ws133, 24 sediment: EM6s137, 05

Station 7 water: EM7ws139, EM7wb141, EM7wb142, EM7wb143, 25 sediment: EM7s144, EM7s145, 10

Station 8 water: EM8ws146, EM8ws147, EM8ws148, EM8wb149 16 sediment: EM8s153, 10

Station 9 water: EM9wb155, 13 sediment: EM9s156 10 Mangrove: MRw201, MRw204, MRw207 32 Salterns: SCw255 230 *Nazareth et al. 2012, Gonsalves et al. 2012, Nayak et al. 2012

Growth of the A. penicillioides isolates on various nutri- was further noted that those media with a greater carbon ent media in the presence or absence of added solar salt is content, when amended with salt, supported the growth of shown in Fig. 2. In the absence of added salt, the isolates the isolates better than CzA with salt (Fig. 2a, b). The higher could not grow on CzA, or on the more nutrient-rich media carbon content could have served better in the synthesis of of MEA, PDA and SA. However, growth was visible on all osmolytes to combat the low aw environment. Each of the these media with addition of 10 % salt within 4 days of isolates from the various sites showed significant dissimi- incubation, thus ascertaining their absolute requirement for larity (P<0.05) in growth on different media, but no varia- salt and, consequently, their obligate halophilic nature. It tion (P>0.05) was seen between the isolates. 400 Ann Microbiol (2014) 64:397–402

Fig. 2 a Colony characteristics of A. penicillioides isolates on A Medium DSw22 DSs40 EM6s137 EM8ws146 MRw207 agar media (CzA CzapekDox, MEA Malt Extract, PDA Potato S-CzA Dextrose, SA Sabouraud) each with 10 % solar salt (S); b Growth of isolates recorded after a 7-day incubation on different media each without and with 10 % salt (S); c Micromorphology of the S-MEA isolates after a 7-day incubation on CzA and S-CzA: a conidia ungerminated, swollen and distorted; b conidia germinated but distorted; c conidia S-PDA germinated and mycelia distorted, with little cytoplasm or lysis

S-SA

B DSw22 DSs40 EM6s137 EM8ws146 MRw207 2

1 Growth Growth

(colony diameter, cms) diameter, (colony 0 CzA S-CzA MEA S-MEA PDA S-PDA SA S-SA Growth medium

C Medium DSs40 EM6s137 MRw207 b c a CzA c

S-CzA

Although A. penicillioides has been described as an utilise energy for compartmentalisation or exclusion of Na+ osmophilic fungus (Wasser et al. 2003), its capacity to grow ions, direct synthesis of compatible osmolytes and synthesis in an environment in which lowering of the aw is due to of proteins conferring salt tolerance (Redkar et al. 1996). sodium chloride ions establishes these isolates as halophilic Obligate halophiles, by means of their absolute require- (Grant 2004), and consequently as true halophiles. The slow ment for salt, are indigenous to saline environments. growth rate, yielding small colonies, may be related to the Obligate halophiles can be termed as specialists, with their increase in energy demands under stress, wherein cells may growth optimum shifted towards extreme values, and have a Ann Microbiol (2014) 64:397–402 401 narrow ecological amplitude (Gostincar et al. 2010). A. estuarine environment of Goa, India. The isolates also penicillioides species do not have a sexual life cycle exhibited dissimilarity in their salt tolerance index as shown (Tamura et al. 1999), which consequently inhibits gene flow. above, and a different colony morphology when grown on the This will have caused a rapid fix of genetic information in test medium of CzA with 10 % salt (Fig. 2a). Using the these populations that have managed to adapt to saline program NCBI BLAST it was observed that the two strains habitats (Gostincar et al. 2010). from the Dead Sea showed a similarity of 97 % with each Micromorphological examination of the selected isolates other, and 95–96 % with strains from the estuary. This indi- (Fig. 2c) when grown in the absence of salt, showed that the cated that the isolates were not identical, but showed some conidia of DSs40 appeared swollen and distorted without ger- similarity to each other, and that the similarity between iso- mination, those of EM6s137 germinated and then got distorted, lates from the same econiche of the Dead Sea was higher than while MRw207 conidia germinated and formed distorted that between isolates from different polyhaline estuarine en- mycelia with very little cytoplasm, and lysis at some portions vironments at widely spaced locations. with oozing of the cytoplasm. At 10 % salt concentration, which The A. penicillioides isolates DSw22 and DSs40, showed supported maximal growth, the mycelia and vesicles appeared a higher similarity of 99 % with the strains ATCC 16910, normal. It was seen therefore, that a low aw was required not South-west0025 and South-west0056, and 98 % similarity only for the growth of the halophile, but also for conidial with the strains NRRL 4548 and NRRL 4550. A 99 % simi- germination and germ tube elongation and branching. The larity was also seen between DSw22 and OUCMBIII101055 osmoadaptation mechanism in true halophiles forms an intrinsic obtained from sea sand, and between DSs40 and KH00256 part of its metabolism. A mitogen-activated protein kinase obtained from tropical sea grass. The isolate EM6s137 showed (MAPK) pathway involved in germ tube elongation, branching, 99 % similarity with an uncultured fungus from a deep sea and hyphal fusion events between conidial germlings (Pandey sediment that was found to have 94 % similarity with A. et al. 2004) has also been shown to be responsible for transcrip- penicillioides NRRL 4550; EM6s137 showed 97 % similarity tion of enzymes involved in glycerol synthesis and intracellular with an A. penicillioides strain from a solar saltern, accession glycerol accumulation in response to osmotic stress (Jin et al. number DQ336711, and similarity of 96 % with the two strains 2005). Therefore, it appears that, in true halophiles, the MAPK NRRL 4548 and NRRL 4550. However, the isolates obtained pathways require stimulation by salt or conditions of low aw, in this study had only 90 % similarity with strain CBS 540.65 and hence, in the absence of such stimulatory conditions, ger- obtained from a non-marine source, namely skin. mination and germ tube elongation does not occur. Aspergillus penicillioides was the only obligate halophile The sequence similarity of the isolates from the Dead Sea, common to the different saline habitats such as the DSw22, DSs40, and from the Estuary, EM6s137, having hypersaline thalassohaline Dead Sea and athalassohaline GenBank numbers HQ702383, HQ702385 and JQ240645, salterns, the polyhaline estuary of Mandovi and brackish respectively (Table 1), was studied to examine the similarity water of mangroves, the rest all being moderate halophiles. of isolates from the same econiche, namely that of the Dead This similarity in the obligate, moderate halophily, was seen Sea, as well as to compare isolates from different widely between isolates from different econiches as well as distant spaced locations and from different econiches, namely, from geographical coordinates. This finding implies the ubiquitous the hypersaline Dead Sea and from the polyhaline or euhaline distribution of this true halophile in varied environments.

Table 1 Sequence similarity of Aspergillus penicillioides A. penicillioides strain No. Accession No. Sample site DSw22 DSs40 EM6s137

DS22 HQ702383 Dead Sea – 97 96 DS40 HQ702385 Dead Sea 97 – 95 EM6s137 JQ240645 Estuary of Mandovi 96 95 – ATCC 16910 AY373862 Unknown 99 99 95 South-west0025 FJ537087 Unknown 99 99 95 South-west0056 FJ537129 Unknown 99 99 95 NRRL 4548 EF652036 Unknown 98 98 96 NRRL 4550 EF652037 Unknown 98 98 96 OUCMBIII101055 HQ914935 Sea sand 99 98 95 KH00256 GU017496 Tropical sea grass 98 99 95 Uncultured fungus AB507845 Deep sea sediment 95 94 99 – DQ336711 Solar saltern 96 96 97 CBS 540.65 JF922039 Skin 90 90 90 402 Ann Microbiol (2014) 64:397–402

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