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 %.