Recognition and differentiation of various Penicillium species , Penicillium citrinum , Penicillium notatum , Penicillium oxalicum and Penicillium frequentans , using random amplified polymorphic DNA-polymerase chain reaction technique

Molecular methods have been established to make better sensitivity and specificity for differentiation of pathogenic and allergenic fungi. The purpose of this study was to obtain an appropriate molecular pattern for differentiation of Penicillium species understudy. In this study, 30 isolates of four Penicillium species including Penicillium citrinum, Penicillium notatum, Penicillium oxalicum and Penicillium frequentans were used. First, isolates were cultured. Then, for obtaining the fungal mats, fungi were grown into Sabouraud glucose broth. Freeze and Thaw, and glass beads were used for cell disruption, and DNA was extracted by phenol-chloroform method. RAPD-PCR technique was performed using three primers. The results showed that each primer produced a different reproduction pattern. A total of 2 to 17 bands were observed in all the isolates of Penicillium, except P. citrinum C2 and P. frequentans F7. According to primer 1, molecular patterns of Penicillium species were approximately similar, but multiplied patterns were not completely similar. Having used primer 2, multiplied patterns in P. oxalicum and P. notatum were to some extent similar, but there were some observed differences between P. frequentans and two other species P. oxalicum and P. notatum. P. citrinum, P. oxalicum and P. notatum had approximately the same patterns using primer 3, but some differences were observed in P. frequentans with three other species. In conclusion, the interspecies differences were demonstrated with all the primers. Primers 1 and 2 were able, to some extent, to differentiate these species, while primers 1 and 3 separated P. frequentans from the others.


INTRODUCTION
Penicillium species classified in subgenus Penicillium are among the most common fungi which spoil food and contaminate indoor environments (Carlile et al., 2001).These organisms are commonly considered as contaminants *Corresponding author.E-mail: sabokbar@kiau.ac.ir.Tel: 0121 2271057.Fax: 0121 2271054.but may cause infections, particularly in immunocompromised hosts.In addition, the wide spread distribution of Penicillium conidia in the air can cause allergic asthma in atopic individuals (Hoffman, 1984).
Species identification relies on evaluation of macromorphological characters, microscopic observation of reproductive structures (De Hoog et al., 2000), easily recognizable secondary metabolite production (Smedsgaard and Frisvad, 1997) and isoenzyme profiling (Pitt and Cruickshank, 1990;Stolk et al., 1990).However, many authors have shown the high variability of some of these characteristics and that morphological criteria do not always allow unambiguous classification (Pitt, 2000).More recently, genotypic characterization has proven very useful to identify Penicillium species and several methods have been used to assess intraspecific and interspecific variation in Penicillium (Dupont et al., 1999;Lund et al., 2003).
These methods include amplification of the internal transcribed spacers (ITS1 and ITS2), restriction fragment length polymorphism (RFLP), random amplified polymorphic DNA (RAPD) and amplified fragment length polymorphism (AFLP).Amplification of ITS regions is effective in identifying the genus Penicillium (White et al., 1990;Peterson, 2000), and some primer combinations allow selective amplification at the subgenus level (Pedersen et al., 1997).Restriction of amplicons generated by PCR (RFLP) can be used to differentiate Penicillium isolates at the species level (Pianzzola et al., 2004).
The use of RAPD markers in studying intra-and interspecific variation in Penicillium has also been reported (Geisen et al., 2001;Pianzzola et al., 2004).Cladistic analyses of gene sequences have been used in the molecular studies of this group, first using ribosomal genes (Skouboe et al., 1996;Peterson, 2000) and later using protein coding genes (Seifert and Louis-Seize, 2000;Peterson, 2004).The aim of this study was to provide the appropriate molecular patterns using three different primers for differentiation of Penicillium species.

Fungal isolates
A total of 30 Penicillium isolates, obtained from the air of different regions of Iran, were chosen from Fungal Collection of Mycology Research Center, University of Tehran (Table 1).

Preparation of fungal mats
The Penicillium isolates were cultured in Sabouraud glucose agar (Merck Co., Darmastdt, Germany), and subsequently were grown in Czapek's agar (Merck Co., Darmastdt, Germany) at 30°C for 48 to 72 h.For obtaining mass-production, some fragments of each fungus were transferred to Erlens Mayer flasks containing 600 ml of sabouraud glucose broth and stored in shacking incubator at 25°C for 10 to 12 days.Fungal colonies were separated from medium using Whatman paper no. 1 and washed three times with sterile distilled water.

Fungal cells disruption
For cells disruption, freeze and thaw method was used and subsequently mechanical triturating with glass beads accompanying with breaking buffer containing 62.5 mol/L Tris, 1 mmol/L Dithioteritol, 0.2 mg/ml PMSF and 15% glycerol, pH = 6.8, was performed.

DNA extraction
Phenol-chloroform method was used for extracting DNA using TES Buffer containing 10% SDS, 20 mg/ml Proteinase k, saturation phenol, 24/1 v Isoamyle alcohol-chloroform, 3 M sodium acetate, pH = 5.2, Isopropanol 70%.For perception of the purity degree of DNA sample, the ratio of absorption of 260 to 280 was calculated.

RAPD-PCR technique
Three primers were randomly used; primer 1: 5' GTA TTG CCC T, primer 2: 5' GCT GGT GG, primer 3: 5' TCA CCC TGC A. Each reaction contained 10x PCR Buffer, 50 mM MgCl2, 10 mM dNTP mix, 20 pm primer and I U Taq DNA polymerase.Micro tubes containing above materials were transferred to thermocycler (Techne, Camlab UK).Amplification was performed for 39 cycles as follow: an initial denaturation of 30 s at 94°C, 30 s at 45°C and 1 min, followed by cycle of 94°C for 30 s, 45°C for 30 s and 72°C for 1 min with a final extension at 72°C for 10 min.

Electrophoresis of PCR productions on agarose gel
Electrophoresis of PCR productions was performed on 1.5% agarose gel and standard markers were run in parallel.The gels were stained with ethidium bromide and transilluminator UV was used for observation of the bands.
Results revealed that multiplied patterns in Penicillium oxalicum and P. notatum were to some extent similar, but  there were some differences between P. frequentans with two other species including P. oxalicum and P. notatum.There were some differences between the molecular patterns of P. citrinum and that of the three other species as well.According to primer 3, 19 of 30 isolates reacted with 1 to 5 bands.The bands were as follows: 500, 700, 800, 1000 and 1500 bp.Band of 1000 bp had the maximum frequency (53.3%), while 500, 700 and 1500 bp bands had the minimum frequency (3.3%).500, 700 and 1500 bp bands were seen only in isolates 22, 27 and 1, respectively.Band of 1000 bp was observed in all the species, while1500 bp band only in P. citrinum and 500, 700 and 800 bp bands only in P. frequentans were demonstrated (Table 4).According to obtained results, P. citrinum, P. oxalicum and P. notatum had approximately the same molecular patterns, but P. frequentans had differences in multiplied patterns with three other species.The present study indicated interspecies differences with all of the primers used.Specific band for each species have been observed with comparing molecular patterns in which caused by each primers (Tables 2 to 4).

DISCUSSION
Saprophytic fungi have got a large variation of species, and in nature, they grow on any kind of organic materials.Spores of these fungi are distributed in the air, causing human and animals to be in contact with them through inhalations (Lehrer et al., 1983;Horner et al., 1995).There are numbers of Penicillium species which grow in the form of saprophyte and on the spoiled foods and fruits.In addition to affecting these materials (with generation of poisons and some of enzymes), their conidia spread in the air.Because of the smallness of the conidia diagonal, they often reach the pulmonary alveolus through the upper respiratory tracts of human and animal.In this condition, they can result in infections and allergic reactions like asthma (Yssel et al., 1998;Kurup et al., 2000).In recent years, molecular methods have been used to diagnosis the fungal infections and their species.PCR technique is one of the most sensitivity and specificity methods for recognition of important medical fungi and diagnosis of fungal infections (Mending and Pinto, 2000;Renhsueh and Jeneteg, 2000).RAPD-PCR is one kind of PCR, which involves random and multiplication of genome segments.Primers will bind with locations which have the highest degree of determined homology according to PCR condition.
Recently, this method is applied for molecular diagnosis and classification of the microorganisms (Ajello and Hay, 1998;Ahmadi, 1998).In present study, molecular typing of P. citrinum (4 isolates), P. notatum (8 isolates), P. oxalicum (6 isolates) and P. frequentans (12 isolates) isolated from the air of different regions in Iran was done using RAPD-PCR technique.Our results indicated that each primer produced different reproduced patterns.Among 30 isolates understudy, two isolates were not responded to any of these primers (isolate 2 related to P. citrinum and isolate 25 related to P. frequentans).Of 30 fungal isolates, 9 strains including 2 P. citrinum isolates (C 3 and C 4 ), 3 P. oxalicum isolates (O 3 , O 4 and O 6 ), 2 P. notatum isolates (N 1 and N 2 ) and 2 P. frequentans isolates (F 9 and F 10 ) reacted with 1 to 2 bands, in which species specific bands were observed with primer 1.With primer 2, all the isolates, except 7 isolates including one P. citrinum isolate (C 2 ), one P. oxalicum isolate (O 4 ) and 5 P. frequentans isolates (F 4 , F 6 , F 7 , F 9 and F 10 ), reacted with 1 to 17 bands, the only band which was observed in all the species, was the ~280 bp band (250 to 300 bp); while 250, 500 and ~510 bp bands (500 to 550 bp) were observed in P. frequentans, ~980 bp (900 to 1000 bp) band only in P. notatum and ~380 (350 to 400 bp) and 1100 bp bands in two species of P. notatum and P. frequentans.With primer 3, 19 of 30 isolates including 2 P. citrinum isolates (C 1 and C 4 ), 5 P. oxalicum isolates (O 1 , O 3 , O 4 , O 5 , and O 6 ), 5 P. notatum isolates (N 1 , N 2 , N 5 , N 6 and N 7 ) and 7 P. frequentans isolates (F 1 , F 2 , F 3 , F 4 , F 5 , F 9 and F 10 ) reacted with 1 to 5 bands in which have been observed only the band with molecular weight of 1000 bp in all of species and 1500 bp only in P. citrinum, 500, 700 and 800 bp bands in P. frequentans.Similar studies for molecular characterization based on RAPD markers among Penicillium species were reported by other investigators.Khosravi et al. (2012) demonstrated polymorphic amplification patterns in various P. citrinum strains with 8 primers.These primers generated a total of 105 reproducible RAPD bands, averaging 13.1 bands per primer.In a study conducted by Tiwari et al. (2011), 20 random primers showed polymorphism of 12 Penicillium species, in particular P. citrinum, P. notatum, P. oxalicum and P. frequentans, and generated a total of 252 RAPD fragments of which 83.73% were polymorphic.The number of amplification products produced by each primer varied from 4 to 16 with an average of 10.55.The sizes of amplified fragments were ranged from 218 to 2939 bp.
In summary, our results showed interspecies differences with all of the primers tested.Primers 1 and 2 were able to some extent differentiate these species, while primers 1 and 3 separated P. frequentans from the others.In fact, none of these primers showed a sufficient capability for separation of the species.Among these three primers, primer 2 was much better than others in differentiation of species.Using more primers and Penicillium species, it would be a great hope to use the RAPD method for evaluating the differentiation of Penicillium isolates in the future.

ACKNOWLEDGEMENT
This study was funded by Research Council of Islamic Azad University, Karaj Branch, Karaj, Iran.

Table 1 .
List of various Penicillium species understudy.

Table 2 .
The results of RAPD-PCR using primer 1 in Penicillium species understudy.

Table 3 .
The results of RAPD-PCR using primer 2 in Penicillium species understudy.

Table 4 .
The results of RAPD-PCR using primer 3 in Penicillium species understudy.