Introduction

The various Pleurotus species are tetrapolar heterothallic, nonparasitic ligninolytic fungi. Only P. eryngii complex and P. nebrodensis are weakly parasitic and can live on the root or base stem of living plants in the Umbelliferae family. P. eryngii complex is widespread in the Mediterranean, Central Asia, central Europe, and North Africa (Lewinsohn et al. 2002). However, distribution of P. nebrodensis is restricted to Xinjiang in China (Mao 2001) on Cachrys ferulacea and Sicily in Italy (Venturella et al. 2000) on Ferula sinkiangensis. In Xinjiang, Pleurotus mushrooms growing on F. sinkiangensis are all called ferula mushrooms. There is considerable diversity in the fruiting body morphology of wild ferula mushroom and ferula mushroom cultured from isolates.

Research has indicated abundant genetic diversity in P. eryngii complex (Venturella 2000). Various genera belonging to family Umbelliferae are hosts of P. eryngii complex. They are named, respectively, as various species or variants according to their host. Pleurotus mushroom growing on Cachrys was named as P. nebrodensis by Venturella (2000) and as P. eryngii var. nebrodensis by Bresinsky et al. (1987). Pleurotus mushroom occurring on F. sinkiangensis was first reported as P. ferula and later described as P. eryngii var. tuoliensis (Mu et al. 1987), P. eryngii var. ferulae, P. eryngii var. nebrodensis (Huang 1996), and P. nebrodensis (Mao 2001), respectively. The classification has been equivocal due to the parallel existence of morphological similarities and differences in culture characteristics. The fruiting body morphology is susceptible to environmental influences. Thus, problems frequently arise if the classification is based entirely on morphological characteristics. Molecular biology techniques provide a useful theoretical basis and methodology for systematic classification and analysis of genetic polymorphism in mushrooms. These techniques have enabled the disclosure of the structure and function of ribosomal DNA. The internal transcribed spacer (ITS) and intergenic spacer (IGS) domains have become important DNA fragments for the analysis of biodiversity. ITS and IGS are rDNA domains that evolve at a faster rate. The ITS domain is a molecular marker for comparing different species of the same genus of fungi (Mitchell and Bresinsky 1999). IGS domain is a highly variable domain (Paule and Lofquist 1996), usually containing the effective variable sequence for determining interspecies or individual genetic relationship in fungi (Guidot et al. 1999). ITS and IGS are important molecular markers for extraspecies comparison in the same fungal genus and for analysis of interspecies mutation and genetic polymorphism (Bunyard et al. 1996).

In the present investigation, 17 isolates were prepared from 17 samples collected from F. sinkiangensis and reconfirmed by cultivation. They were divided into five groups according to morphological features and then subjected to antagonism test and rapid analysis of polymorphic DNA (RAPD). Isolates with no antagonism response, but with similar morphological characteristics (Huang 1996) and RAPD profiles (spectra), were eliminated. Those with antagonism response and dissimilar morphological characteristics and RAPD profiles were selected and used for a mating test. Intergenic spacer 1–restriction fragment length polymorphism (IGS1-RFLP), IGS2-RFLP, and sequence analysis of ITS and IGSI domains were carried out with P. eryngii (ACCC50894) with gray pileus produced in Italy as reference. The aim was to investigate its genetic diversity and, at the same time, provide a molecular biology basis for the classification of Pleurotus mushroom on F. sinkiangensis. The reasons for using P. eryngii (ACCC50894) are as follows: (1) it is far from Xinjiang geographically; (2) P. eryngii is a complex close to P. nebrodensis and P. eryngii var. ferulae; (3) to confirm the taxonomic position of P. eryngii var. ferulae that it belongs to Pleurotus; and (4) to confirm the taxonomic position of P. nebrodensis that it does not belong to P. eryngii complex.

Ferula mushroom has economic and also medicinal value. It has been successfully cultivated in China, France, and India. Correct species identification and protection of the rights of breeders of commercial strains are of paramount importance. The intent of the present study is to demonstrate that IGS analysis provides a convenient and quick technology for identification of strains, and that it has promising prospects for application.

Materials and methods

All of the cultures (see Table 1) are kept in the Agricultural Culture Collection of China (ACCC). The specimens of the fruiting bodies are stored in the Microbiology Research Institute of Academia Sinica and in Sanming Mycological Institute, Fujian Province, China.

Table 1 Isolates tested
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Mating tests

The basidiospores were collected at 20°C from fruiting bodies and cultivated in separate houses to prevent contamination. A spore suspension was used to obtain a monokaryon for the mating tests. The mycelium was incubated on potato dextrose agar medium in a Petri dish at 25°C. When small colonies were visible, a monokaryon with nonclamp connection was selected for microscopic examination. Four mating types were determined for various isolates (P. eryngii and P. eryngii var. ferulae have four mating types of basidiospores because Pleurotus is heterothallic with bifactorial control, i.e., exhibits tetrapolarity). The mating tests were then carried out. The existence of clamp connection was used as evidence for compatibility. Compatibility was indicated as “+” and incompatibility was marked as “−.” In general, compatible organisms belong to the same species, whereas incompatible organisms do not.

Extraction of DNA

The mycelium grew on PDA Petri dish with celluloid film at 25°C for 8 days. Then, it was collected, and DNA was extracted as described by Lee and Taylor (1990). The restriction endonucleases used were BsuRI, Hin6I, HpaII, RsaI, and Bsh1236I (Sangon Co., Shanghai, China). IGS1-PCR reaction conditions were 94°C for 1 min (denaturation), 54°C for 1 min (renaturation), and 72°C for 30 min (extension), for a total of 35 cycles, followed by 72°C for 7 min (extension). ITS-PCR and IGS2-PCR reaction conditions were 60°C (renaturation) and 3 min (extension), with other conditions being identical to those for IGS1-PCR. The digestion system comprised 1-μl restriction endonuclease (10 U/μl,), 6 μl PCR product, and 1 μl 10× buffer, and the volume was made up to 10 μl with water. The reaction was carried out at 37°C for 4 h. Agarose gel (2%) was used for electrophoresis. Results of the enzyme digestion were observed under an ultraviolet gel imaging system.

ITS, IGS1-RFLP, and IGS2-RFLP

The amplification system of ITS-, IGS1-, and IGS2-PCR consisted of 4 μl dNTP (2.5 mM each), primers 1 and 2 (2.5 μl each), Ex Taq DNA polymerase (1.25 U), PCR buffer, template (25 ng), and double-distilled water to make up the volume to 50 μl. The sequences of the PCR primers are shown in Table 2.

Table 2 ITS, IGS1, and IGS2 primers
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ITS and IGSI sequence determination

Both ITS and IGSI were amplified as mentioned above. The amplification products were subjected to direct sequencing (Dunham et al. 2003).

Results

Mating test

It was revealed by the mating tests that compatibility existed between P. eryngii var. nebrodensis and all of the isolates designated P. nebrodensis. The isolates of the latter were also compatible. This suggests that all of them belong to the same species, and the differences are just at the strain level, despite the fact that morphological differences in fruiting bodies between the former and the latter were greater than those among all of the latter designated P. nebrodensis. The mating tests showed that P. nebrodensis (ACCC50869) was incompatible with both P. eryngii var. ferulae (ACCC50656) and P. eryngii (ACCC50894). However, P. eryngii var. ferulae (ACCC50656) was compatible with the reference strain P. eryngii (ACCC50894), with which 56.25% dikaryon (9 dikaryons in 16 pairings) was formed (Table 3).

Table 3 Results of mating tests using different strains
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ITS

The results revealed that the size of the ITS of all isolates was identical, being 638 bp, but the nucleotide sequences were different. This difference existed only between incompatible isolates, i.e., only between P. eryngii var. ferulae and P. nebrodensis, and there were no differences in ITS sequence among the compatible isolates of P. nebrodensis. The isolate named P. eryngii var. ferulae was identical to P. eryngii in ITS sequence (GenBank accession no. AY368658). The others were identical to P. nebrodensis in ITS sequence (GenBank accession no. AY311408). The results indicated that the difference of the ITS domain lies mainly in changes in nucleotide sequence and not in variations in length of the region. The ITS sequences showed differences in 19 nucleotides, i.e., a 3% difference between two species.

IGSI-RFLP

All of the tested isolates did not differ in IGS1 size, being about 920 bp and existing in multiple copies. When BsuRI, Hin6I, HpaII, RsaI, and Bsh1236I were used as endonucleases, only the IGS1-RFLP after HpaII digestion indicated a difference between P. eryngii var. ferulae and others (Fig. 1). The IGS1-RFLP profile was identified to reference P. eryngii (data not shown). The IGS1-RFLP profiles of all isolates generated by the other four endonucleases were identical, indicating IGS1 difference only in extraspecies in the present study. It is suggested that no mutations of nucleotide sequence have arisen in various interspecies populations of P. nebrodensis. Sequence analysis disclosed that the estimate of 920 bp for IGS1 length is accurate. P. eryngii var. ferulae had a sequence in IGS1 distinct from P. nebrodensis, and its sequence has been registered in GenBank (accession no. AY463033). The isolates of P. nebrodensis and P. eryngii var. nebrodensis exhibited the same sequence in IGS1. The sequence, as represented by ACCC50869, has also been registered in GenBank (accession no. AY463034). There was a difference in 21 nucleotides (i.e., 2.28% difference) between P. eryngii var. ferulae and P. nebrodensis.

Fig. 1
figure 1

IGS1-RFLP electrophoretic profiles. Results of HpaII cleavage of P. nebrodensis. 1 Isolate number 1, ACCC50656; 2 isolate number 2, ACCC50869; 3 isolate number 3, ACCC51060; 4 isolate number 4, ACCC51452; 5 isolate number 5, ACCC51453. M molecular marker: (λDNA/HindIII and EcoRI). Numbers 1–5 and M also apply to Figs. 2 and 3

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IGS2-PCR and IGS2-RFLP

Intergenic spacer 2 amplification results indicated that the IGS2 domain was longer and variable in length. The length of IGS2 of different isolates manifested polymorphism (Fig. 2). P. eryngii var. nebrodensis generated two bands, 7.8 and 3.5 kb, respectively, and the others gave rise to only one band but of different sizes, which was 3.5 kb for isolate ACCC51453, 7.8 kb for isolates ACCC50869 and ACCC51452 (P. nebrodensis), and 4.4 kb for ACCC50656 (P. eryngii var. ferulae), respectively.

Fig. 2
figure 2

IGS2 amplification results

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With the exception of HpaII, the 17 isolates were divided into five groups based on IGS2-RFLP profiles by digestion of the four endonucleases BsuRI, Hin6I, RsaI, and Bsh1236I. They acted on different restriction sites in the domain, and this resulted in a polymorphic IGS2-RFLP profile after electrophoresis (Fig. 3, Table 4). This indicates that IGS2 represents the domain with the most abundant polymorphism in ribosomal DNA for ferula mushroom population that belongs to two species in Xinjiang, China.

Fig. 3
figure 3

IGS2-RFLP electrophoretic profiles. aBsh1236I; bBsuRI, cHin6I, dRsaI

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Table 4 Genetic polymorphism of IGS1-RFLP and IGS2-RFLP for ferula mushroom
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Discussion

Based on the mating test results obtained in the present study and the Linnaeus concept of species separation, there should be two Pleurotus species growing on F. sinkiangensis, taking into consideration ecological and morphological classification (Venturella 2000; Venturella et al. 2000; Mao 2001), namely, P. eryngii var. ferulae and P. nebrodensis. The difference between them represents extraspecies or species level and not variety level. The sample shaped like a goof (ACCC51060) is the same species as that one shaped like a palm. Both of them should belong to P. nebrodensis and not to the P. eryngii complex due to their incompatibility with P. eryngii and P. eryngii var. ferulae. P. eryngii var. ferulae growing on F. sinkiangensis should be included in the P. eryngii complex due to their compatibility with each other.

Results of ITS sequence analysis concur with the mating test results. ITS sequences are identical between compatible strains. ITS sequence difference is as high as 3% between incompatible strains, much higher than the previously reported interspecies differences of less than 1% (Sugita et al. 1999). Isolates with different ITS sequences are incompatible with each other. Closely related species growing on the same host F. sinkiangensis can be distinguished using one or both of the mating tests and ITS sequence analysis.

The IGSI domains of all strains were relatively conserved, but the IGS2 showed abundant polymorphism. Although all of the P. nebrodensis strains were identical in ITS and IGS1 sequences, there were obvious differences in IGS2 that exhibit size or RFLP profile. IGS2 is a useful domain for investigating interspecies polymorphism in the population of P. nebrodensis. IGS2-RFLP could be used as a DNA molecular marker for identification and distinction between P. nebrodensis strains.

The ITS domain is a ribosomal rDNA region that evolves at a relatively fast pace. It has been used as one of the most efficacious markers of phylogeny for comparing species of the same genus. Álvarez and Wendel (2003) found that in the past 5 years, ITS sequence analysis was conducted in plants in 66% of the papers, and that 34% of the papers were completely based on the analysis of ITS sequence for phylogeny investigation. Similarly, ITS sequence analysis plays an important role in phylogeny studies at or below the genus level and in the identification of macrofungi (Dunham et al. 2003).

For populations of the same species, ITS sequence variation is smaller than IGS domain, especially much smaller than IGS2 region because of a lot of repeat sequences and subrepeat sequences in IGS2. The variation of repeat sequence or subrepeat sequence generates the polymorphism in IGS2 length. It was demonstrated that ITS sequence difference was smaller than 1% in populations of the same species (Sugita et al. 1999). There was only a difference of three nucleotides in the ITS sequences of three pathogenic strains of Cryptococcus neoformans (Xu et al. 2000). There were greater differences in the nucleotide sequence of the IGS1 and IGS2 domains in the three strains (Diaz et al. 2000), which can be used effectively to distinguish the three strains. ITS difference between strains could not be found, and it just existed in different species in the present study. IGS2 domain is a highly variable region in ribosomal DNA as evidenced by the abundant polymorphism of IGS2-RFLP in populations of P. nebrodensis. The difference was much more remarkable than the difference in fruiting body morphology. It is an effective domain for analysis of genetic polymorphism in Pleurotus species, in agreement with reports on other species (Bunyard et al. 1996; Saito et al. 2002). P. nebrodensis is a delicious cultivated mushroom with great economic value. The IGS2-RFLP will probably become a DNA marker for patented strains and protection of breeder's right. It is much easier and much more accurate to use for the identification of cultivars than fruiting body morphology.

Interestingly, two closely related species, P. eryngii var. ferulae and P. nebrodensis, grow on the same species of host plant under the same climatic conditions. These two species manifest obvious differences in ribosomal DNA despite similar fruiting body morphology. The former was separated from the ferula mushroom population and was included in P. eryngii complex based on incompatibility with P. nebrodensis by mating tests or difference in ITS sequence. However, its fruiting bodies are not gray like regular P. eryngii var. ferulae as previously reported by taxonomists, but are nearly white, more closely resembling P. nebrodensis growing on the same host plant. It is probably attributed to gene flow of the dominant species P. nebrodensis growing in the same host under the same ecological environment. It is possible that some genes linked with the fruiting body coloration flow from P. nebrodensis into P. eryngii var. ferulae, bringing it close to the dominant population. Nevertheless, genetically, ITS and IGS1 sequence and IGS1-RFLP of P. eryngii var. ferulae were identical to those of P. eryngii (data not shown). However, the length of its IGS2 was also similar to both the reference strains P. eryngii from Italy and some of P. nebrodensis. Young fruiting bodies of P. eryngii var. ferulae and some of those of P. nebrodensis are both light gray and become milky white, nearly white, or white, respectively, when they grow up. It can thus be surmised that P. eryngii var. ferula with gray cap might exist in Xinjiang, China. However, to this date, it has not yet been confirmed.

The prevailing consensus has been that there is only one Pleurotus species, P. eryngii, growing on F. sinkiangensis. It is disclosed in this study that there are, in fact, two Pleurotus species, P. eryngii and P. nebrodensis. The pileus of Pleurotus mushroom found here is nearly white and not the classical gray color. The fruiting body morphology of macrofungi is affected by environmental conditions. Thus, it is difficult to distinguish between different strains based on fruiting body morphology. Various methods including ITS, IGS1, and ISSR have been used in analyzing the polymorphism of Pleurotus mushroom. However, the results are not satisfactory. ITS can only distinguish between two species; IGS1 cannot distinguish between two species and between different strains of the same species. No clear results are obtained by using ISSR. Clear results were obtained by using IGS2 in the present study. The results were consistent with those based on physiological characteristics and morphological features.

Thus, IGS2 can be used as a means of identification to protect the rights of people engaged in cultivating commercial cultivars of ferula mushroom on F. sinkiangensis that grows in crowded and acid places. As a consequence, the fruiting bodies of Pleurotus species on F. sinkiangensis are very few in number and difficult to collect. The Pleurotus species exhibit polymorphism and grow only on F. sinkiangensis and have good prospects for commercial cultivation. The other Pleurotus species growing in Xinjiang are found in rotten wood of woody plants but have not been found on F. sinkiangensis.